resistance and binding profile of cabozantinib, a …...ii resistance and binding profile of...
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Resistance and Binding Profile of Cabozantinib, a ROS1 Inhibitor
and Design of Peptidomimetic Inhibitors of STAT5 Protein
by
Rahul Rana
A thesis submitted in conformity with the requirements for the degree of Masters of Science in Chemistry
Department of Chemistry University of Toronto
© Copyright by Rahul Rana 2016
ii
Resistance and Binding Profile of Cabozantinib, a ROS1 Inhibitor
and Design of Peptidomimetic Inhibitors of STAT5 Protein
Rahul Rana
Masters of Science in Chemistry
Department of Chemistry
University of Toronto
2016
Abstract
Genes encoding for tyrosine kinases that undergo rearrangement at chromosomal
breakpoints have been associated with constitutively activated transcript products. Repressor of
silencing 1 (ROS1) tyrosine kinase fusions have been shown to drive cellular proliferation and
survival signaling pathways in numerous human cancers. First generation kinase inhibitors of
ROS1 suffer from acquired resistance due to a point mutation in the ROS1 kinase domain. This
work looks to determine the structural features of cabozantinib, a potent inhibitor of both wildtype
and mutant ROS1. Inhibitor binding to the ROS1 kinase domain and specific scaffold contributions
with synthesized analogues will be covered.
Additionally, a peptidomimetic strategy was employed to design inhibitors of the SH2
domain of the signal transducer and activator of transcription 5 (STAT5) protein, a transcription
factor constitutively phosphorylated in hematological malignancies and inflammatory diseases.
Native receptor peptide sequences that interact with the STAT5 SH2 domain were used for the
initial library.
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Table of Contents
Chapter 1: An introduction to Repressor of Silencing 1 (ROS1)
1.1 ROS1 tyrosine kinase gene fusions............................................................................... 1
1.2 Oncogenic Activity of ROS1 ........................................................................................ 7
1.3 Inhibiting ROS1 fusion kinases .................................................................................. 10
1.4 Conclusion .................................................................................................................. 17
Chapter 2: Resistance and selectivity profiles of cabozantinib and derivatives with structural
insight
2.1 Computational studies of TKI interaction with ROS1................................................ 19
2.2 Proposed library of cabozantinib derivatives.............................................................. 26
2.3 Synthesis of small-molecule TKIs of ROS1 ............................................................... 31
2.4 In vitro evaluation of preliminary library ................................................................... 37
2.5 Binding constant determination using biophysical assay .......................................... 38
2.6 Concluding remarks .................................................................................................... 44
Chapter 3: Design of peptdiomimetic inhibitors of signal transdcuer and activator of transcription
factor 5 (STAT5)
3.1 Introduction to signal transducer and activator of transcription factor 5 (STAT5) .... 46
3.2 Structure and signaling of STAT5 .............................................................................. 47
3.3 Role of STAT5A and STAT5B in disease.................................................................. 51
3.4 Therapeutic strategies towards the STAT5A/B signaling pathway ........................... 53
3.5 Proposed isoform-selective peptidomimetic inhibitors of STAT5A/B ...................... 57
iv
3.6 Initial FP analysis of proposed phosphopeptides ....................................................... 64
3.7 Conclusion ................................................................................................................. 70
Chapter 4: Conclusions and Future Directions ................................................................. 71
References ......................................................................................................................... 73
v
List of Abbreviations
Akt protein kinase B
ALL acute lymphoblastic leukemia
Alk anaplastic lymphoma kinase
AML acute myelogenous leukemia
Bcl-XL B-cell lymphoma-extra large
Bcr-abl Philadelphia chromosome
CCD coiled-coil domain
CDK cyclin-dependent kinase
CML chronic myelogenous leukemia
C-Met hepatocyte growth factor receptor kinase
EGFR Epidermal growth factor receptor
EPO erythropoietin
EpoR Epo receptor
Erk Extracellular signal-regulated kinases
FI fluorescence intensity
FIG fused in glioblastoma
FISH fluorescence in situ hybridization
FLT3 Fms-like tyrosine kinase
FP fluorescence polarization
FRET fluorescence resonance energy transfer
GAS gamma-activated sequence
GH growth hormone
GM-CSF granulocyte macrophage colony stimulating factor
gp-130 glycoprotein 130 HTS high-throughput screen
IL interleukin
IFN interferon
ITC isothermal titration calorimetry
Jak Janus kinase
K562 a CML cell line
Lck lymphocyte-specific protein tyrosine kinase
MAPK mitogen-activated protein kinase
Mcl-1 induced myeloid leukemia cell differentiation protein
MV-4-11 an AML cell line
NGF nerve growth factor
NSCLC Non-small cell lung cancer
NTD N-terminal domain
PI3K Phosphatidylinositol 3 kinase
PIAS protein inhibitors of activated STAT
PLC phospholipase C
PKB Protein kinase B
PRL prolactin PTP
phosphatase pY
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phosphotyrosine
RET rearranged
during transfection
kinase
ROS1 repressor of silencing 1
RSK ribosomal S6 kinase
RTK receptor tyrosine kinase
SAR structure-activity relationship
SCCHN squamous cell carcinoma of the head and neck
SH2 Src-homology 2
Shp Src homology-2 protein phosphatase
SOCS suppressors of cytokine signaling
SPPS solid-phase peptide synthesis
SPR surface plasmon resonance
STAT signal transducer and activator of transcription
TAD transactivation domain
TKI tyrosine-kinase inhibitor
TPO thrombopoietin
TRF time resolved fluorescence
TR-FRET time resolved fluorescence resonance energy transfer Y
tyrosine
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List of Schemes
Scheme 2-1. Amide coupling synthesis of cyclopropylcarboxylic acid and aniline: (i) SOCl2,
reflux, 2 h; then aniline, THF, DIPEA, rt, 16 h; (ii) aniline, HBTU, DIPEA, DMF, rt, 18 h; (iii)
LiOH.H2O, THF/H2O, 0 oC to rt, 2 h.
Scheme 2-2. Alkylation of catechol with numerous alkyl halides and subsequent preparation
of aniline: (i) 1,3-dibromopropane, K2CO3, ethylene glycol, 75-110 oC for 8-10 h; (ii) H2(g), Pd/C
(10%), THF/MeOH.
Scheme 2-3. Cyclization of quinolone using microwave-assisted conditions, chlorination
and subsequent coupling: (i) 90 oC, 10 min, microwave assisted; then aniline 6a-l, EtOH,
reflux, 2h; (ii) Ph2O, 230 oC, 10-20 min, microwave assisted; (iii) POCl3, reflux, 2 h; then cold
H2O, Na2CO3; (iv) NaH, 4-aminophenol, DMSO, 10 minutes; then chloroquinoline 12a-l
DMSO, 100 oC, 12h.
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List of Tables
Table 2.1- Summarized KD values for cabozantinib and analogues determined by LanthaScreen
TR-FRET assay.
Table 3.1- Summary of pY-containing ligands and negative control analogs
Table 3.2- Calculated Ki values for EpoR and GM-CSFR derived peptide ligands
ix
Acknowledgements
x
Contribution of Authors
The work reported in this dissertation includes contributions from several workers. The initial
computational work and method development was performed by Dr. Nadeem Vellore (Huntsman
Cancer Institute, University of Utah). Dr. Thomas O’Hare’s laboratory conducted the biological
work, specifically the cell cytotoxicity assay. Finally, Daniel Ball contributed significantly
towards the computational methods, docking simulations, chemical synthesis. He also developed
the LanthaScreen assay parameters for the inhibitor library and optimized the protocol.
Furthermore, he carried out the calibration and competition assays with data analysis.
xi
List of Figures
Figure 1.1- Postulated extracellular domain of ROS1 determined from sequencing and homology
comparison. Three β-propeller domains (purple) are dispersed over β-sandwich structures (blue).
(Image from ref 7).
Figure 1.2- Tertiary structure of ROS1 kinase domain (PDB code: 3ZBFf) with amino residues
in the catalytic site highlighted red and represented by stick models.[PT1] [RR2] Image
generated using MacPyMol 2009-2010.
Figure 1.3- Possible downstream signaling cascades transduced by ROS1. (Figure reproduced
from reference 6)
Figure 1.4- ROS1 fusion genes with corresponding receptor partner and breakpoint regions
indicated. Rearranged genes are categorized by disease prominence.[PT1] (Figure taken from
Stumpfova, M.; Janne, P.A. Zeroing in on ROS1 Rearrangements in Non-Small Cell Lung Cancer.
Clinical Cancer Research. 2012. 18, 4222-4224.)
Figure 1.5- A) Sanger sequencing of RT-PCR products from various tissue samples of the NSCLC
patient indicating the G2032R mutation. B) Western blot analysis of cell lysates prepared from
293T cells expressing CD74-ROS1 or CD74-ROS1G2032R post crizotinib and TAE684 treatment.
C) Crystal structure of native ROS1 with crizotinib on the left. The right panel illustrates the
predicted steric clash between crizotinib and R2032. D) First-generation small molecule TKI,
crizotinib and E) TAE684. (Figure taken from reproduced from reference 39)
Figure 1.6- Second-generation TKI inhibitors, foretinib and cabozantinib, both originally
developed by Exelixis.
Figure 2.1- A) Active and inactive dynamic equilibrium of RTK enzymes controlled by
phosphorylation of the DFG motif in the A-loop; B) Anatomy of kinase domain with specific
regions highlighted. ‘Hydrophobic region I’ is the cleft adjacent to the adenine regions which gets
exposed upon dephosphorylation of the kinase to the inactive state. (Figure from reference 50).
Figure 2.2- Overlap of the active and inactive conformations of the ROS1 kinase domains with
the DFG motif phenylalanine 2103 represented by a stick model. Image generated using
MacPyMol 2009-2010.
Figure 2.3- Docking score histograms for active ROS1-cabozantinib (top), inactive
ROS1cabozantinib (bottom left) and inactive ROS1-cabozantinib (bottom right).
Figure 2.4- Lowest energy conformation of cabozantinib bound to ROS1 kinase domain. Panel A
shows exposed methoxy groups and the quinoline ring occupying the ATP binding site (green).
The tethering 4-aminophenol and cyclopropyl carboxamide motifs reside in the hinge region,
highlighted yellow. Panel B shows a few of the interacting amino acids. Panel C illustrates
xii
occupancy of the hydrophobic groove (adjacent to the adenine site) by the 4-fluorobenzene
appendage. Image generated using MacPyMol 2009-2010.
Figure 2.5- Alternative, reversed binding pose of foretinib with ROS1 compared to predicted
cabozantinib-ROS1 complex. Image generated using MacPyMol 2009-2010.
Figure 2.6- Proposed structural modifications to cabozantinib for fragment-binding computational
studies.
Figure 2.7- Tabulated Ki values (nM) for alkyl group derivatives of 6,7-dimethoxyquinoline
position from docking screen.
Figure 2.8- Tabulated Ki values (nM) for alkyl group derivatives of 6,7-dimethoxyquinoline
position from docking screen.
Figure 2.9- Proposed mechanisms for decarboxylation-mediated cyclization reactions of imines
9k and 9h. Mechanisms A and B illustrate how 9k, can give two regioisomers, 10k(i) and 10k(ii).
Similarly, diverging mechanisms C and D outline the formation of 10h(i) and 10h(ii).
Figure 2.10- Preliminary library of synthesized analogues of cabozantinib, 13a-t.
Figure 2.11- Graphical representation of IC50 values of preliminary library of small-molecule
inhibitors 13a-t and crizotinib in Ba/F3 CD74-ROS1 and CD74-ROS1G2032R cells.
Figure 2.12- Illustration of principle of LanthaScreen (Invitrogen) TR-FRET assay. (Figure taken
from reference 74).
Figure 2.13- Calibration curves of Alexa Fluor-647 tracer titration with ROS1 kinase with
100μs, 50 μs, 25 μs and 20 μs time delay between excitation and measurement of emission, in the
absence (black trace) and presence (red trace) of staurosporine.
Figure 2.14- Emission ratio plot with increasing concentrations of tracer titrated with
Eu(III)chelate antibody in the absence of ROS1 protein.
Figure 3.1- Schematic illustration of STAT5A/B domains with critical Y694 (STAT5A) and Y699
(STAT5B) highlighted. (Image generated using MacPyMol 2009-2010)
Figure 3.2- Brief schematic of normal JAK/STAT5 signaling with cytokine/growth factor
receptors dimerized and bound to activated JAK2. Latent STAT5 is recruited via the SH2-pY
interaction and undergoes phosphorylation. Dimerization in the cytosol is followed by nuclear
translocation and regulation of transcription of target genes. Negative regulators, SOCS, PIAS and
PTP are represented as well. (Image generated using ChemBioDraw 14)
Figure 3.3- First generation BCR-ABL TKI imatinib for CML treatment. Second-generation TKIs
from imatinib, nilotinib and dasatinib are shown below.
Figure 3.4- First-generation FLT-3 TKI lestaurinib and following second-generation derivative
sorafenib. Both drugs are used for FLT-3 mutation positive AML. Ruxolitinib is the widely used
xiii
JAK2 inhibitor in AML treatment, given its efficacy against JAK2V617F
Figure 3.5- Traditional SH2 domain tertiary structure ribbon illustration. The central anti-parallel
β sheet is flanked by two α-helices. Interacting pY-containing peptide is shown in black, stick
figure representation, with pTyr, +1, +2 and +3 sites labeled. (figure from ref 60).
Figure 3.6- A) Overlap of STAT5A (orange) and STAT5B (blue) ribbon figures. The five
differing residues in the SH2 domain in the vicinity of the pY binding motif are represented by
stick figures. B) Shared Ser622, Arg618 and Lys600 in STAT5A/B SH2 domains. On comparison
with conventional SH2 domain structure, these three residues contribute to recognition of pY
functional group. (Image generated using MacPyMol 2009-2010)
Figure 3.7- Calibration curve for EpoR derived 5-FAM-GpYLVLDKW fluorescent probe with
STAT5A protein.
Figure 3.8- Calibration curve for EpoR derived 5-FAM-GpYLVLDKW fluorescent probe with
STAT5B protein.
Figure 3.9- Normalized FP inhibition curve for EpoR derived peptide, QDTpYLVLDKWL for
STAT5A. Ki = 522.3 nM
Figure 3.10- Normalized FP inhibition curve for EpoR derived peptide, QDTpYLVLDKWL for
STAT5B. Ki = 426.0 nM
Figure 3.11- Normalized FP inhibition curve for GM-CSFR derived peptide, QQDpYLSLPPWE
for STAT5B. Ki = 876.6 nM
Figure 3.12- Normalized FP inhibition curve for GM-CSFR derived peptide, QQDpYLSLPPWE
for STAT5A. Ki = 652.4 nM
1
Chapter 1: An introduction to repressor of silencing 1 (ROS1)
1.1 ROS1 tyrosine kinase gene fusions
Structural chromosomal aberrations have been studied extensively for their effects on the
genome at the cellular level and the resultant biological phenotype. This specific category of
chromosomal aberrations is defined as the physical breakage of chromosomal segments in the
DNA.1 This results in numerous possible mutations including, deletions, duplications,
translocations, inversions, and insertions.2 Specifically, translocations involve partitions in two
different chromosomes and eventual exchange of chromatin material. Chromosomal translocations
of exon DNA segments ultimately compromise the function and regulation of the natural
geneencoding product. Concurrently, it has been established that several key translocation genetic
events contribute towards the initiation of carcinogenesis. The first chromosomal translocation
identified in human cancer was in chronic myeloid leukemia (CML) where the fragment exchange
resulted in the fusion gene product, BCR-ABL. The fusion was characterized as the association of
the ABL1 and BCR genes where the expressed product is a tyrosine kinase (TK). The discovery of
the BCR-ABL fusion gene represents an important breakthrough in recognition of TK fusion genes
as a result of chromosomal translocations and their oncogenic potential.3
There are approximately 90 TK genes in the human genome, out of which, 58 encode for
receptor tyrosine kinases (RTKs).4 Repressor of Silencer 1 (ROS1) is a RTK of the insulin receptor
family that has been characterized as a partner of multiple fusion oncoproteins responsible for
driving numerous epithelial cancers including glioblastoma, cholangiocarcinoma, gastric cancer,
ovarian cancer, and non-small cell lung cancer (NSCLC).5
The human ROS1 gene was initially discovered as the homolog of the UR2 avian sarcoma
virus.6 The ROS1 protein shares 49% sequence homology with the anaplastic lymphoma kinase,
ALK, with more than 64% identical residues in the kinase domain.7, 8 ROS1 is a 264 kDa protein
which spans over 44 exons on chromosome 21.7 The tertiary structure of ROS1 protein follows the
conventional RTK arrangement: an N-terminus extracellular receptor region (residues 1-1861), a
single transmembrane-spanning domain (residues 1862-1882) and a C-terminus region (residues
2
1882-2346) which contains the catalytic active site.9 The extracellular segment consists of three β-
propeller domains distributed along nine β-sandwich domains,7 shown in figure 1.1.
Figure 1.1- Postulated extracellular domain of ROS1 determined from sequencing and homology
comparison. Three β-propeller domains (purple) are dispersed over β-sandwich structures (blue).
(Image from ref 7).
Despite possessing an extensive extracellular domain, a ligand for the human ROS1 protein
has yet to be identified. This ‘orphan RTK’ status has impeded investigations concerning the ROS1
activation and resultant signaling pathways. However, sequence comparison with known receptor
sequences suggests that the extracellular domain is involved in mediating cell-cell adhesion. These
peptides resemble fibronectin type III proteins, which exhibit high affinity for extracellular
adhesion molecules.6 Thus, cellular attachment and similar processes are postulated to be trigger
events for the activation of the tyrosine phosphorylation activity of the kinase with following
intracellular signaling cascades.10 The kinase domain, situated at the intracellular domain of ROS1
comprises of the traditional tyrosine kinase elements: an ATP biding pocket, a substrate binding
pocket, an activation loop (A loop) and finally, the catalytic loop (C loop)11 with the catalytic
K1980 residue, figure 1.2.
3
Figure 1.2- Tertiary structure of ROS1 kinase domain (PDB code: 3ZBFf) with amino
residues in the catalytic site highlighted red and represented by stick models. Image generated
using MacPyMol 2009-2010.
Unfortunately, the definitive function of wild type ROS1 in humans remains elusive. Northern blot
studies of various human organs conducted by Acquaviva and co-workers in 2009 indicated
highest ROS1 RNA expression occurs in the lungs.10 Reitchmacher et al. were able to study the
biological effects upon loss of the receptor in vivo. They discovered that male ROS1knockout mice
were viable but suffered from loss of normal reproduction. The infertility was attributed to defects
in the epididymis epithelium.12
Through the use of its tyrosine phosphorylation activity, ROS1 kinase is responsible for the
regulation of signaling pathways that control cell proliferation and survival. The precise series of
events that switch ROS1 into the active conformation from its own inactive state are currently
unknown. This can be attributed to an unidentified native ligand as well as the difficulty in
expressing the full-length extracellular domain of ROS1 in vitro. However, similarities can be
drawn from analogous RTKs, including ALK and RET (rearranged during transfection). In the
case of ALK, ligand–receptor binding induces dimerization of the receptor via oligomerization
motifs. This orients the intracellular kinase domains in an ideal positionfor an autophosphorylation
event. In the inactive conformation, the Asp-Phe-Gly triad in the activation loop block substrate
L 2 0 8 6 &
K 1 9 8 0 &
F 2 1 0 3 &
L 2 0 2 8 & E 2 0 2 7 &
D 2 1 0 2 &
4
binding. Upon phosphorylation, however, the kinase A loop undergoes a conformational shift
forcing the same amino acid residues to protrude outwards. This phosphorylation-triggered
structural change permits binding of the substrate and subsequent ATP transfer. In human ROS1,
Tyr2274 has been shown to be the site of phosphorylation and mutation of this residue to Phe
abolished ROS1 activity.13 Like all tyrosine kinases, the C-terminal tail serves as a docking site for
native substrates. With the limitations mentioned earlier, detailing the ROS1 downstream signaling
mechanisms have been hindered. One approach to circumvent these challenges of studying this
orphan RTK is the use of artificially engineered ROS1 fusions with known ligand binding
receptors. Examples include the insulin receptor14, epidermal growth factor receptor (EGFR)15,
nerve growth factor (NGF) receptor16, and CD74 receptor.17 Utilizing known receptor partners
with the ROS1 kinase domain allows one to regulate enzyme activity in a liganddependent manner
and potentially study the interacting proteins. These chimeric ROSI fusion models have shed some
light on the ambiguous, yet diversified set of signaling cascades stimulated upon ROS1 activation.
The proteins activated upon ROS1 phosphorylation include: the mitogenactivated protein kinases
(MAPKs), ERK1 and ERK2, Src-homology region 2 domain phosphatase 2 (SHP-2),
phosphatidylinositol 3-kinase (PI3K), protein kinase B (PKB or Akt), and signal and transducer
and activator of transcription 3 (STAT3). All of the mentioned proteins function as signaling
factors that regulate cellular growth, differentiation, mitosis, and apoptosis. The signaling
mechanisms are shown in figure 1.3 below.
5
Figure 1.3- Possible downstream signaling cascades transduced by ROS1. (Figure reproduced
from reference 6)
The MAPK/ERK pathway is an established oncogenic signaling cascade. The pathway begins
with binding of mitogens to cellular receptors which are eventually translated to regulation in
transcription of oncogenic genes. The pathway involves the phosphorylation of multiple proteins
with MAPKs, ERK1, and ERK2 as pivotal contributors. These extracellular signal-related kinases
phosphorylate factors such as ribosomal s6 kinase (RSK), c-MYC, and c-FOS. Upon
phosphorylation, these oncoproteins are rendered active, and upregulate the transcription of genes
involved in cell proliferation and survival. Xiong and co-workers observed activation of ERK1/2
in mouse fibroblasts upon treating EGFR-ROS1 fusions with ligand.15 Additionally, Davies et al.
reported increase in ERK phosphorylation in Ba/F3 cells expressing with SD-C4-ROS1 protein.17
Alternatively, Gu and colleagues conducted experiments with Ba/F3 cells transfected with other
ROS1 fusions and noted a decrease in ERK phosphorylation levels upon treating with potential
ROS1 inhibitors.18
SHP-2 (encoded by PTPN6 gene) is a member of the protein tyrosine phosphatase (PTP)
family. This enzyme contains two independent SH2 domains that recognize phosphotyrosine (pY)
6
motifs. It is believed that recruitment of SHP-2 to ROS1 is mediated by the pY-SH2 domain
interaction.19 Phosphorylated SHP-2 has been shown to activate MAPKs and thus, the subsequent
MAPK/ERK pathway.20 Charest et al. studied glioblastoma mouse derived cells harbouring ROS1
fusions which resulted in the increase levels of SHP-2 protein phosphorylation.19 Further evidence
of SHP-2 mediated ROS1 signaling was reported by Rikova and co-workers as they successfully
isolated peptide sequences containing the tyrosine-phosphorylation motifs of SHP-2 from solid
NSCLC tumors.21
Protein kinase B (PKB) or Akt, is a serine/threonine kinase that phosphorylates key
regulators of cell apoptosis and mitosis. Akt is known to disrupt the Bcl-2/Bcl-X multimeric protein
in the mitochondrial membrane, which in turn compromises the cell’s pro-apoptosis mechanism.
Cyclin-dependent kinases 1 and 2 (CDK1 and CDK2) regulate the cell-cycle check phases, G1 and
G2. Activation of Akt via phosphorylation is known to overcome cell cycle arrest junctures, hence
increasing the chances of mutagenesis and uncontrolled cellular division. Nguyen and co-workers
demonstrated inhibition of Akt significantly diminished the ability of ROS1 fusions to promote
colony growth in NIH3T3 fibroblasts.22 Glioblastoma mouse models incorporating ROS1 fusions
also exhibited increased levels of phosphor-Akt.19
The PI3K signaling pathway is a crucial link between several cellular trafficking events
spanning from metabolism to ligand-independent proliferation. PI3K is responsible for the
phosphorylation and subsequent activation of Akt, whose oncogenic kinase activity is described
above. Validating the direct interaction between ROS1 and PI3K has been difficult, given the
complexity of the involved signaling mechanisms. Also, with Akt being an important protein in
PI3K signaling pathway, all evidence indicating increase in phospho-Akt levels can be correlated
to ROS1 activation of PI3K. One interesting study conducted by Uttamsingh and colleagues
suggested that knockdown of the PI3K pathway reduced anchorage independent growth in ROS1
activated fibroblasts.
STAT3 belongs to a family of cytoplasmic transcription factors that undergo
phosphorylation by kinases stimulated by cytokine binding to cell surface receptors.
Phosphorylated STAT3 monomers (pSTAT3) dimerize via reciprocal SH2 domain-pY interactions
and translocate into the nucleus. After recognizing specific DNA sequences, the STAT3 dimer
promotes transcription of genes that induce angiogenesis, metastasis, and proliferation. In tandem
7
with their study of ROS1 induced PI3K/Akt signaling, Gu et al. also studied effects on STAT3.
They noted a marked increase in pSTAT3 protein levels when Ba/F3 cells where transformed with
a chimeric ROS1 fusion. In a separate finding, Zong and co-workers observed fibroblast colony
growth was markedly reduced when cells expressed dominant negative STAT3 allele. Rimkuna and
colleagues also conducted experiments trying to solve the signaling cascades elicited upon ROS1
activation in 2012 and reported decrease in pSTAT3 levels in Ba/F3 cells transfected with ROS1
fusion when treated with ROS1 inhibitor.
1.2 Oncogenic activity of ROS1
Overall, numerous studies indicate that ROS1 can engage multiple signaling pathways upon
activation. One common theme across these cascades is the phosphorylation of a protein substrate,
usually a kinase, and eventual downstream transcription of genes promoting cell growth and
survival. The specific targets mentioned above could be constitutively activated by aberrant ROS1
activity. On a molecular level, this is the case when the RTK is in the active conformation, which
is considered the “switched on’ state. RTK fusions are susceptible to adapting their catalytic
conformations through binding of cytokines and growth factors specific to the fused receptor.
Ideally, the downstream tyrosine containing protein targets of ROS1 fusions are identical to that
of the wild type protein mentioned above, and tyrosine-phosphorylating activity is independent of
the receptor motif. Thus, ligand sensitive ROS1 fusions that force the intracellular kinase domain
into a constitutively active mode have the potential to deregulate conventional cell growth
mechanisms and inhibit apoptosis. These ligand-transmitted events, which would otherwise be
absent with wild type ROS1, are believed to be responsible for transformation to the oncogenic
phenotype.
To date, seven ROS1 fusion genes have been reported and sequenced (figure 1.4).
Birchmeier et al. reported the first instance of a ROS1 fusion in a tumor model in 1987. The workers
isolated rearranged ROS1 cytogenically with the use of fluorescence in situ hybridization (FISH)
and RT-PCR from the U118MG glioblastoma cell line.23 FISH is a powerful technique used to
identify location of breakpoints in chromosomal rearrangements with the help of fluorescently
labeled oligonucleotide probes. Two different coloured DNA probes that are complimentary to the
8
regions flanking the gene of interest are designed. In the absence of translocation events involving
the target gene, the two probes remain adjacent to one another, resulting in a fusion colour. When
chromosomal translocation disrupts the gene of interest, the probes are separated and consequently,
the primary coloured probes are observed individually.
This specific application of FISH, often referred to as “break-apart” FISH offers high specificity
with minimal false positive outcomes.
Figure 1.4- ROS1 fusion genes with corresponding receptor partner and breakpoint regions
indicated. Rearranged genes are categorized by disease prominence. (Figure taken from
Stumpfova, M.; Janne, P.A. Zeroing in on ROS1 Rearrangements in Non-Small Cell Lung Cancer.
Clinical Cancer Research. 2012. 18, 4222-4224.)
The genetic fusion partner was hence termed ‘fused in glioblastoma’ (FIG), and the
resultant hybrid kinase was eventually discovered in cholangiocarcinoma, ovarian cancer, and
NSCLC patient samples.6 Furthermore, the FIG-ROS1 fusion has been expressed in murine cell
lines, murine epithelium tissues and even mouse models to study its role to drive oncogene
character independently. Consistently, the experiments resulted in uncontrolled cellular
proliferation, carcinogenesis, and rapid tumor growth. Since then, numerous ROS1 fusion partners
have been characterized and sequenced in various human cancers. In all ROS1 rearrangements
9
known to date, the kinase domain of ROS1 is conserved and the fusion partner contributes its
extracellular receptor domain.
Interestingly, there are numerous instances of ROS1 fusions in NSCLC throughout
academic literature and clinical data wherein the ROS1 derived oncogene most frequently reported
is the CD74 rearrangement. The first CD74-ROS1 hybrid was discovered recently in 2007 by
Rikova et al. in a non-smoking, female NSCLC patient.21 The fusion kinase profiling investigation
was part of a comprehensive RTK signaling study in 41 NSCLC cell lines and more than NSCLC
150 Chinese patient tumor samples. Additionally, transforming capability of CD74-ROS1 has now
been confirmed both in vitro and in vivo.17 Fused CD74-ROS1 occurs when exon 6 of CD74
(chromosome 18) is juxtapositioned to exon 34 of ROS1 resulting in the N-terminal extracellular
domain of CD-74 fused to the transmembrane and C-terminal kinase domain of ROS1. As a result,
CD-74 functions as the extracellular motif which relays ligand binding into protein structural
changes that prompt the constitutively active conformation of the ROS1 kinase. More specifically,
CD74 recognizes the macrophage migration inhibitory factor (MIF), an inflammatory cytokine
involved in inflammation responses. Through MIF signaling, CD-74 plays a crucial role in the
expression and transport of the major histocompatibility complex II (MHCII).24
In a subsequent study, Bergethon and co-workers screened 1073 NSCLC patients and noted
18 occurrences of ROS1 gene rearrangements. However, only 6 samples were sufficient for RT-
PCR analysis.25 Five CD74-ROS1 fusions were sequenced successfully, while other samples were
inconclusive in identifying the receptor partner. Interestingly, in their panel of NSCLC tumors, the
breakpoint of ROS1 was observed to occur at exon 32, highlighting another rearrangement pattern.
Three more groups reported the frequencies of CD74-ROS1 fusions while probing ALK and ROS1
translocations in a large panel of lung cancer patient-derived cell lines and tumors. The independent
studies consistently found approximately 1.2-2.4% of NSCLC patient tumors with CD74-ROS1
rearrangements.26,27,28 Generally, ROS1 rearranged positive patients are neversmokers and tended
to be younger than the median age of patients with NSCLC. In addition, NSCLC patients
harbouring translocated ROS1 are devoid of other oncogenic RTK-receptor hybrids. Overall, ROS1
rearrangements have been identified in approximately 2% of patients with NSCLC. Although this
is seemingly a low percentage, with an estimated 1.5 million new cases of NSCLC worldwide each
year, this corresponds to more than 20,000 patients with oncogenic ROS1 fusions.29,30 This
evidence undoubtedly highlights the prevalence of ROS1 rearrangements as oncogenic drivers in
10
NSCLC and new data continues to support this trend. As a result, this RTK is considered in the
class of oncoproteins that can independently drive transformation in NSCLC both in vitro and in
vivo.
1.3 Inhibiting ROS1 fusion kinases
Given the rise of ROS1 fusion positive NSCLC cases in the clinic, there has been substantial
effort in therapeutic intervention of the ROS1-relevant pathways. A traditional strategy is to inhibit
the aberrant kinase activity of the protein itself. This strategy involves preventing the constitutive
tyrosine phosphorylating activity of ROS1. Directly inhibiting the enzyme would diminish the
aberrant anti-apoptotic signaling and potentially take advantage of the ‘oncogene addiction’ state
of the tumor. The principle of oncogenic addiction refers to the dependency of tumors on specific
oncoproteins to sustain malignant growth.31 Gene mutations that switch on a certain oncogene
typically drive aberrant survival processes in the cell that would otherwise be controlled under
biological cues. Once the function of the oncoprotein is constitutively activated, it plays a much
more involved role in survival, promoting conversion to the neoplastic state. The cancer cells then
become reliant on the translated product of the same oncogene to sustain its tumor phenotype.32
From a molecular perspective, the output of the hyperactive oncoprotein tends to outcompete
natural pro-apoptosis cascades and cell cycle checks. Furthermore, the identical mutated genome
and resulting ‘addictive’ oncogenic mechanism is present in progeny cells. Therapeutic
intervention abolishing the aberrant function of the oncoprotein in question can result in cancer
cell death and eventual tumor regression or ‘reversal’ of the malignant phenotype. With oncogene-
specific inhibition, the anti-survival and cell division checks are no longer dominated by the
hyperactive function of the oncoprotein. This notion is supported by experimental data where
therapeutics targeting an individual activated oncoprotein have led to apoptosis in cancer cells.33
This specific hypothesis is termed “oncogene shock” and has formed the fundamental principle
underlying target-specific therapeutics. Appropriately, ROS1-targeted inhibition is the primary
approach for therapeutic treatment in the relevant set of diseases, including NSCLC. Previous and
contemporary strategies with the aim of inhibiting the ROS1 protein are discussed below.
In the 1990’s, significant strides were made in identifying tyrosine kinases as major role
11
players in oncogenic transformation. The human kinome was thoroughly examined and potential
oncokinases were labeled as important targets for drug development programs. Genetically
rearranged kinases became a crucial part of the kinase inhibitor discovery umbrella because of the
growing information on their role in cancer. Approaches to inhibit RTK fusions include: ligand
modulation, monoclonal antibodies to inhibit the receptor domain, siRNA therapeutics, anti-sense
oligonucleotides, peptidomimetics, and direct small molecule inhibitors.34 The category of small
molecule tyrosine kinase inhibitors (TKIs) comprises of natural and synthetic chemicals that inhibit
the kinase activity through a variety of mechanisms, all of which involve direct interaction with
the protein.35 Possible mechanisms include: allosteric inhibition, substrate competitive inhibition,
and ATP competition. TKIs targeting the ATP binding site in the catalytic domain have
substantially stood out as the most well-studied and efficacious subset of inhibitors with
considerable success in the clinic. Given these small molecules were designed to compete for the
well-defined ATP pocket, most TKIs possess a scaffold that mimics ATP. Initially, the challenge
of achieving selective inhibition of RTK fusions with ATP competitive binders seemed
monumental as the ATP binding site was universal amongst all proteins in the human kinome.
Nonetheless, advances in small molecule screening, computational chemistry, protein X-ray
crystallography, and in silico guided structure-based drug design have contributed considerably in
the discovery of selective and potent TKIs. Amino acids in the catalytic site of the kinase are now
easily identifiable and interactions with ATP on the molecular level can be elucidated. Binding
clefts surrounding the sugar, phosphate, and adenine binding sites of the target RTK are now easy
to characterize and serve as the basis for inhibitor scaffold design. An important result of TKI drug
design was imatinib, the first case of a selective, RTK small molecule inhibitor with an outstanding
clinical profile. Imatinib (trade name: Gleevec) has been approved as the first-line treatment of
BCR-ABL positive CML by the F.D.A.36
The first series of small molecule inhibitors of ROS1 fusion proteins originated from the
ALK drug discovery program. As mentioned earlier, ROS1 and ALK share approximately 64%
overall sequence homology in the kinase domain and 84% in the ATP binding site. This high
degree of similarity in the linear amino acid residue sequence can be extrapolated to considerable
overlap in the tertiary structure of the ALK and ROS1 kinase domains. Thus, small molecule
inhibitors that bind appreciably to the ALK kinase domain should exhibit a similar inhibitory
12
profile against ROS1. McDermott and coworkers were the first to recognize the sensitivity of
ROS1 to ALK TKIs in vitro in 2008.37 They observed potent activity of the ALK inhibitor,
TAE684, in the HC778 NSCLC cell line, which was positive for only ROS1 rearrangements.
Promising results followed this work with Gu et al. reporting reduction in phospho-ROS1 (pROS1)
and subsequent apoptosis of Ba/F3 cells harbouring FIG-ROS1, when treated with TAE684.18 In
addition, when performing western blot analysis, there was concomitant with decrease in levels of
p-STAT3, p-ERK, p-Akt and p-SHP-2 in a dose-dependent manner. In parallel with the
breakthrough work validating use of ALK inhibitors to inhibit ROS1 fusion proteins, clinical trials
with the ALK inhibitor, crizotinib (developed by Pfizer), as a NSCLC therapeutic were underway.
The impressive inhibitory activity of crizotinib against rearranged ALK kinases warranted
investigations probing the efficacy of crizotinib in ROS1 fusion positive NSCLC tumors. In 2012,
three separate studies by Yasuda, Davies, and Bergethon concluded that crizotinib was indeed a
potent ROS1 inhibitor. Their collective work showed crizotinib induced apoptosis in ROS1 fusion
harbouring NSCLC cell lines with concomitant reduceed levels of pROS1 and downstream protein
targets. More importantly, tumor growth in NSCLC patient tissue samples was reduced and upon
dosing a ROS1 positive NSCLC patient, tumor burden decreased with no symptoms of recurrence.
Critzotinib (figure 1.5.D) became the first line treatment for patients diagnosed with ROS1
translocation positive NSCLC. NSCLC patients can be easily assessed for the presence of ROS1
fusions using break-apart FISH. Following this, RT-PCR is employed for gene amplification and
Sanger sequencing to identify the fusion partner. Initial stages of a Phase I/II clinical trial in 50
NSCLC patients carried out by Awad et al. (funded by Pfizer; ClinicalTrials.gov number,
NCT00585195) looked promising and ROS1 tumors (with the CD74 being the most prominent
fusion partner) showed extreme sensitivity to crizotinib treatment. Unfortunately, tumor growth
and associated symptoms redeveloped in a subset of patients, after initial response. Continued drug
administration proved to be ineffective and it was revealed the patients had acquired resistance to
crizotinib.39
13
Figure 1.5- A) Sanger sequencing of RT-PCR products from various tissue samples of the NSCLC
patient indicating the G2032R mutation. B) Western blot analysis of cell lysates prepared from
293T cells expressing CD74-ROS1 or CD74-ROS1G2032R post crizotinib and TAE684 treatment.
C) Crystal structure of native ROS1 with crizotinib on the left. The right panel illustrates the
predicted steric clash between crizotinib and R2032. D) First-generation small molecule TKI,
crizotinib and E) TAE684. (Figure taken from reference 39)
Tissue biopsy of a resistant tumor from a patient confirmed a point mutation in the kinase
domain of ROS1. Sanger sequencing of RT-PCR products from tissues from the chest wall, right
and left lungs, pleural fluid, and lymph nodes showed mutation of guanine to adenine at position
6094, which in turn, corresponded to a Gly2032Arg (G2032R) substitution in the kinase domain
illustrated in figure 1.5.A. This specific mutation was not detected in the tumor prior to crizotinib
administration and was the only such case retrieved from the resistant specimen. The investigators
conducted a series of experiments to determine the molecular basis of the acquired mutation in
vitro. First, cell lysates were prepared from 293T human embryonic kidney cells transfected with
CD74-ROS1 and G2032 mutant CD74-ROS1 after crizotinib treatment. Western blot analysis of
pROS1 levels confirmed crizotinib was ineffective against the G2032 mutant with IC50 values >
14
1000 nM, compared to IC50 = 30-50 nM for the non-mutant fusion, figure 1.5.B. An enzymatic
activity assay showed an increase in the Ki value of the G2032 mutant kinase by a factor of 270
with respect to non-mutant ROS1 (Ki = 570 nM cf. 2.1 nM). Finally, to delve into the specific
inhibitor-protein interaction, the workers crystallized the phosphorylated ROS1 kinase domain
bound to crizotinib. As expected, the crystal structure showed crizotinib bound to the ATP binding
site where Gly2032 is solvent exposed, positioned towards the distal end of the kinase hinge. In
terms of molecular interactions, this specific glycine is situated such that it can participate in van
der Waal’s interactions with the pyrazole ring crizotinib. Modeling studies that replace the G2032
residue with arginine in silico, place the substituted side chain guanidinium substituent in close
proximity to the piperidine functional group in crizotinib, figure 1.5.C. It is predicted that the
bulkier amino residue sterically clashes with the inhibitor to a considerable extent, thus decreasing
its binding affinity for ROS1.
Despite initial responses in TKI chemotherapy regimes, acquired drug resistance is a
recurring theme universally recognized in the clinic, not only limited to NSCLC.40 Targeted
therapy using small molecule inhibitors eventually results in genetic alterations that confer loss of
sensitivity to inhibitor activity.41 Continued therapy is ineffective as the resistant cells successfully
survive and pass on their mutated genetic material and outcompete non-resistant cells. In essence,
interference with TKI targeted therapy serves as an artificial selection pressure which allows the
tumor to ‘evolve’. In the case of crizotinib inhibition of ROS1, the molecular basis of acquired
resistance was a mutation in the oncoprotein of interest. Rapidly dividing cancer cells are highly
susceptible to point mutations. These single codon modifications operate by reducing the binding
affinity of the TKI for the target kinase domain. A weak binding inhibitor does not effectively
abrogate the catalytic activity of the kinase, hence allowing constitutive phosphorylation of
substrates. Alternate mechanisms of drug resistance include amplification of the target oncogene
or activation of independent signaling cascades that restore the hyperactivity of the downstream
signaling pathways while the original oncoprotein is inhibited.40,42
Initial strategies to overcome the acquired resistance paradigm observed in clinical TKI
therapy revolve around the design of next-generation kinase inhibitors. This requires designing
ATP-mimetic small molecules that do not suffer loss in binding interactions with the mutant
oncoprotein. This strategy has yielded success in other secondary cancer treatments, most notably,
15
in CML.43 Upon continuous exposure to imatinib treatment, it was observed the BCR-ABL fusion
kinase developed point mutations that confer resistance to against small molecule inhibitors. The
most common mutation was observed with Thr315, the gatekeeper residue to Ile. New TKIs were
developed with the aim of improved binding interactions with the mutant ABL kinase domain from
the previous generation. A series of second and third generation inhibitors including dasatinib,
nilotinib, bosutinib, and ponatinib had an improved activity profile against imatinib resistant
BCRABL while sustaining desirable inhibition against the non-mutated fusion.40 This has seen
successfully translated to the clinic where next generation TKIs are now part of the chemotherapy
regime for imatinib resistant CML.
Drawing inspiration from the progress of leukemia therapy using next-generation TKIs,
several groups screened a series of ALK, RET, and MET multi-kinase inhibitors for their activity
against rearranged ROS1 positive NSCLC and the reported G2032 mutant. A breakthrough was
made when Davare et al. identified foretinib (developed by Exelixis, XL-880, figure 1.6) as a more
potent and selective ROS1 and ROS1G2032 inhibitor than crizotinib in 2013.44 In vitro cell growth
assays coupled with immunoblot analysis pROS1 and downstream targets in Ba/F3 transformed
wild type CD74-ROS1 and CD74-ROS1G2032 cell lines confirmed the mutant kinase is insensitive
to crizotinib. Ba/F3 CD74-ROS1G2032 cells were highly resistant to crizotinib treatment (IC50 =
2200 nM) when compared with wild type CD74-ROS1 (IC50 = 14 nM). Foretinib also decreased
phosphorylation levels of ROS1 and its downstream targets, ERK1/2 and SHP-2 in a
dosedependent fashion across both wild type and G2032 mutant cell lines.
Figure 1.6- Second-generation TKI inhibitors, foretinib and cabozantinib, both originally
developed by Exelixis.
Another high-throughput screen of established TKIs (approved by the F.D.A. or under
clinical trials) by Katayama and colleagues in 2014 led to the discovery of cabozantinib as a potent
inhibitor of cell survival in CD74-ROS1 and CD74-ROS1G2032 transformed Ba/F3 cell lines.45
16
Originally developed by Exelixis, cabozantinib (XL-184, figure 1.6) is a multikinase domain
inhibitor of cMET and VEGFR-246 and is currently approved for treatment of refractory medullary
thyroid cancer.47,48 Firstly, the workers examined viability of Ba/F3 cells either expressing
CD74ROS1 and CD74-ROS1G2032 fusion protein. Ba/F3 CD74-ROS1 cells exhibited significant
reduction in cellular growth when exposed to either crizotinib or cabozantinib with IC50’s of 2 nM
and 2.12 nM, respectively. However, CD74-ROS1G2032 Ba/F3 cells did not respond to crizotinib
treatment (IC50 = 253.7 nM, 126-fold increase), whereas cabozantinib was quite potent in reducing
cell viability with IC50 = 13.53 nM. Next, they compared the extent of ROS1 autophosphorylation
in Ba/F3 cell lines expressing either CD74-ROS1 or CD74-ROS1G2032. Upon incubation with
increasing concentrations of cabozantinib and crizotinib, immunoblotting revealed that both
inhibitors were efficient in suppressing pROS1 protein levels in non-mutated CD74-ROS1
constructs. Not surprisingly, only cabozantinib retained its anti-pROS1 activity in the
G2032mutated CD74-ROS1 Ba/F3 cells. Immunoblotting studies were extended to known
downstream substrates of ROS1 phosphorylation. Treatment with either TKI suppressed pSTAT3,
pAkt and pERK proteins, but this trend failed to carry over to CD74-ROS1G2032 mutants with
crizotinib remaining inactive against the G2032 mutated fusion kinase. It is important to note the
encouraging toxicity profile of these inhibitors as negative control experiments involving parent
Ba/F3 cells treatment with both TKIs did not inhibit cell growth (IC50 = 10,000 nM). The same
workers assessed the efficacy of both inhibitors in crizotinib resistant NSCLC patient-derived cell
lines harbouring the G2032 mutation (MGH047 cells). As expected, cabozantinib potently
inhibited growth of MGH047 cells whereas crizotinib did not show any anti-proliferative potency.
Finally, a comparison of pROS1 levels and its associated signaling partners replicated the
observations seen in the Ba/F3 cell lines where only crizotinib effectively suppressed
phosphorylation of ROS1, ERK and Akt. In light of these findings, strong emphasis was placed on
cabozantinib’s selectivity and resistance profile against the CD74-ROS1 fusion kinase and the
G2032 variant. Following from their initial work on foreitinib in CD74-ROS1 driven NSCLC,
Davare and colleagues also found cabozantinib to be a highly potent inhibitor of Ba/F3 CD74-
ROS1 and CD74-ROS1G2032 cell proliferation and viability with IC50’s of 1.1 nM and 15.3 nM
respectively. Both foretinib and cabozantinib demonstrated high selectivity for CD74-ROS1 as
Ba/F3 cells expressing rearranged EML4-ALK fusion kinase were insensitive to inhibition when
treated with concentrations of up to 2,500 nM for both TKIs. This degree of selectivity is
17
considered remarkable considering the selectivity profile of other kinase inhibitors and their loss
in potency upon acquired resistance. The same workers also observed foretinib suffers from a
greater loss in activity against ROS1G2032R (IC50 = 50.1 nM) compared to cabozantinib (IC50 = 15.3
nM). In conclusion, it is clear that cabozantinib can overcome the point mutation of the solvent-
front Gly2032 to Arg in the kinase domain of ROS1 following initial crizotinib treatment. This
provides a promising alternative therapeutic strategy for CD74-ROS1 positive NSCLC patients
suffering from acquired crizotinib resistance.
1.4 Conclusion
Both foretinib and cabozantinib have shown encouraging results in both wild type and
genetically modified G2032 CD74-ROS1 models. The structural variations between these
inhibitors and crizotinib must contribute to the observed differences in CD74-ROS1G2032R
inhibition in vitro. More specifically, the overall scaffold of the molecule offers a specific binding
conformation within the ATP recognition pocket, and this particular binding event is evidently
perturbed in the case of crizotinib-CD74-ROS1G2032. The specific interacting residues in the ROS1
kinase domain can help elucidate which functional groups/substituents contribute towards the
second-generation ROS1 TKIs in retaining their binding potency for the G2032R mutated kinase.
Furthermore, we can even look to gain more insight of the structural effects of the G2032R
substitution. This direction would involve designing cabozantinib/foretinib analogues with
incorporation of functional groups that interact with specific residues in the G2032R mutated
ROS1 kinase domain. Such a study would help decipher the exact mechanism of TKI binding to
the ROS1 kinase domain, an important question that has yet to be answered. We can even take a
step back and try to identify the structure-based resistance liabilities in crizotinib. The clinically
relevant G2032R mutation in CD74-ROS1 arises only upon exposing ROS1 fusion positive
NSCLC patients to crizotinib, suggesting the resistance pathway of CD74-ROS1 is induced upon
exposure to this specific TKI. Analyzing differences between the inhibitor-catalytic site interaction
for crizotinib and second generation TKIs will pinpoint if substituents on these molecules
contribute to amino acid substitutions in the first place. This will definitely help improve future
18
efforts in TKI design as we can seek to chemically modify drug scaffolds that offer less resistance
liabilities.
The work presented in the following chapter details our efforts in identifying structural
contributions into the selectivity and resistance profiles of ROS1 TKIs, with special focus on
cabozantinib. We have employed computational modeling to study the lowest energy binding
conformation of relevant TKIs and the interactions they generate with the ATP pocket of both wild
type and G2032 mutated ROS1. We then designed a diverse library of analogues and performed
docking simulations with the ATP binding site of ROS1 to probe for any trends in predicted
binding affinities. From these results, we deduced a representative set of inhibitors which were
taken forward and synthesized. In vitro evaluation was conducted using biophysical and cell-based
assays.
19
Chapter 2: Resistance and selectivity profiles of cabozantinib and
derivatives with structural insight
2.1 Computational studies of TKI interaction with ROS1
The X-ray crystal structure of the ROS1 kinase, PDB ID: 3ZBF, reported by Awad et al.
in 2013 was used for our computational studies (obtained from the Research Collaboratory for
Structural Bioinformatics Protein Data Bank). The reported crystal structure was incomplete and
required several in silico modifications. Firstly, there were 5 and 11 amino residues omitted from
the P- and A-loops, respectively. These key residues were introduced into the protein using the
Prime program of Schrödinger’s Suite (2012, Maestro 9.3). This protein-structure prediction
program allows insertion or substitution of residues and translates the modified linear sequence
into an accurate 3-dimensional structure. All the coordinates of the backbone atoms and side chains
of conserved residues in the protein were retained. Newly installed residue side chains were
inserted from a library of known peptide dihedral angles and side chain residues. In any tertiary
protein, however, the structural orientation, ionization state, and conformational rigidity of every
amino acid are governed by its microenvironment. Thus simple addition of the newly inserted
residues was not sufficient. Loop refinement is a task in Prime that calculates all local contacts and
generates multiple conformations of the loop.49 The conformations were clustered and energy
minimization was conducted using the OPLS_2005 force field. The output with the lowest total
energy of the system was selected as the final model.
All ATP competitive TKIs are known to interact with their target kinase either in a type I
or type II orientation.50 Type I kinase inhibitors recognize the catalytically active conformation of
the kinase where the DFG amino residues in the activation loop adopt the ‘in’ state. On the other
hand, type II TKIs engage the kinase in the inactive state where the DFG motif out from the kinase
ATP pocket, as shown in figure 2.1A.51 When the active loop is not phosphorylated, type II kinase
inhibitors are able to take advantage of the unique hydrophobic site that is adjacent to the ATP
binding region, highlighted in blue in figure 2.1.B.
20
Figure 2.1- A) Active and inactive dynamic equilibrium of RTK enzymes controlled by
phosphorylation of the DFG motif in the A-loop; B) Anatomy of kinase domain with specific
regions highlighted. ‘Hydrophobic region I’ is the cleft adjacent to the adenine regions which gets
exposed upon dephosphorylation of the kinase to the inactive state. (Figure from reference 50).
Upon examining the inhibitor-bound ROS1 crystal structure, (see Chapter 1, figure 1.5)
crizotinib can be classified as a type I TKI. As expected, the 3-isopropoxypyridin-2-amine rests in
the adenine site, lined with A1978, E2027, and L2028. Hydrophobic contacts are predicted
between the 1,3-dichloro-4-fluorobenzyl ring of the inhibitor and V1959, L2086, and L2010 of the
protein. Most significantly, the piperidine substituent faces the solvent, surrounded by G2032,
D2033, and T2036 and the bridging pyrazole is positioned flat with respect to the
3isopropoylpyridin-2-amine ring, lying below L1951.
In the absence of an inactive ROS1 crystal structure, Prime was employed to produce a
suitable ROS1 model where the DFG triad in the activation loop is projecting out from the kinase
domain. The inactive ALK crystal structure (PDB code: 4FNY) was used as a template and
homology modeling was performed to construct the inactive ROS1 protein. The deviation in the
21
position of the DFG motif in inactive ROS1 from its active state was comparable to that of ALK
(approximately ~10 Å with respect to the alpha carbon in Phe2103), see figure 2.2.
Figure 2.2- Overlap of the active and inactive conformations of the ROS1 kinase domains with
the DFG motif phenylalanine 2103 represented by a stick model. Image generated using
MacPyMol 2009-2010.
With the inactive and active states of the native ROS1 kinase domain model in hand, we
then proceeded to study the lowest energy binding poses of cabozantinib and foretinib in the ROS1
catalytic site using molecular docking simulations. The ligand-receptor docking program, GLIDE
(Grid-Based Ligand Docking with Energetics, version 6.1, Schrödinger Suite 2014) was employed
to run the docking simulations for both TKIs. Docking simulations required three steps before
running the docking algorithm: 1) preparation of the protein, 2) preparation of the ligand, and 3)
establishing docking parameters.
First, all waters excluding bridging between a co-factor and protein were deleted. The
ROS1 kinase structures were then individually optimized using restrained minimization, which
reorients any amino acid side-chain hydroxyl groups and readjusts steric clashes. Formal charges,
bond orders, all hydrogen atoms, and protonation states were added sequentially. An OPLS_2005
I n c $ v e ' R O S 1 '
A c $ v e ' R O S 1 '
22
force field was used for optimization. Secondly, LigPrep, a ligand preparation module available
through Maestro (version 9.2, Schrödinger Suite 2014)52 produced single low-energy 3D structures
of cabozantinib and foretinib. From a 2D structural input, multiple functions within Ligprep were
utilized. Relevant chiralities and possible tautomers were inserted. In addition, ionization states of
the TKIs were generated within pH range of 7.0 ± 0.4. Finally, low-energy ring conformation
sampling and geometry optimization, using OPLS_2005 force field was performed to relax the
cabozantinib and foretinib 3D structures. A docking grid with dimensions of 15 Å for the ROS1
receptor was generated using the binding site residues defined from the crizotinib-bound ROS1
crystal structure. This precise location in the kinase domain fully encompassed the ATP binding
site with specific residues L1951, A1978, K1980, E1997, M2001, L2028, G2032, L2086, and
D2102. The dimensions of the grid box were selected such that all possible conformations and
rotations of the ligands could be accommodated within the ATP cleft.
Docking simulations were then carried out with a rigid protein structure with a flexible
ligand (includes acyclic torsion bonds, pyramidal nitrogen inversions of amides and sample ring
conformations) using a Lamarckian Genetic Algorithm (LGA).53,54 The GLIDE extra-precision
(XP) mode is a comprehensive sampling function that offers many advantages over the GLIDE
standard precision (SP) module. Not only does the XP mode account for all the energetically
favourable and non-favourable contacts, it incorporates a much more rigorous treatment of
solvation and hydrophobic interactions with the protein.55 GLIDE XP interprets interactions
between the ligand and protein involving charged and/or polar groups by comparing the overall
energy if these same functional groups were solvated, as in a natural biological system. By
assessing the difference in energies, GLIDE XP is able to assess any solvation inadequacies and
assign corresponding penalties in the scoring algorithm. Furthermore, the GLIDE XP tool looks to
reward contacts between the ligand and receptor involving hydrophobic substituents.
Lipophiliclipophilic interactions, pi-pi stacking, and even potential pi-cation interactions are
recognized and rewarded.55 This component in the GLIDE XP scoring algorithm includes the
significant non-polar contributions towards ligand-protein binding. Incorporation of the buried
hydrophobic sites in a receptor and its enclosure of lipophilic atoms in a ligand are crucial for
binding and simple nonpolar surface area analysis is not sufficient. We assessed the validity of the
molecular docking protocol by conducting an initial docking study with crizotinib and the
ROS1G2032 and comparing the obtained results with the published inhibitor-protein bound crystal
23
structure (PDB code: 3ZBF). Fittingly, the conformation of the most populated conformation was
almost identical with that of the crystal structure. The superimposed crizotinib structures revealed
minimal root-meansquare deviation (RMSD) between the atoms. This control computational
analysis confirmed the accuracy and precision of our docking protocol.
Figure 2.3- Docking score histograms for active ROS1-cabozantinib (top), inactive
ROS1cabozantinib (bottom left) and inactive ROS1-cabozantinib (bottom right).
Analysis of the lowest energy binding poses for foretinib and cabozantinib revealed both
TKIs exhibited type II binding with the ROS1 kinase domain, with predicted binding energies of -
10.10 kcal/mol and -12.00 kcal/mol, respectively for the highest docking score, figure 2.3. In the
case of cabozantinib, the active state of ROS1 was not preferred as the energy for the highest
ranked binding pose was approximately -7.5 kcal/mol. As shown in in figure 2.4.A, the
6,7dimethoxyquinoline ring occupied the adenine pocket (coloured green) forming hydrogen
bonds with residues E2027 and M2029 amide backbone (figure 2.4.B). Interestingly, both
dimethoxy groups are solvent exposed and the rest of the scaffold projects in towards the tunnel-
shaped sugaradenine-linker/hinge-hydrophobic series of pockets. The tethering 4-aminophenol
aromatic ring participates in pi-pi stacking with F2013 of the DFG motif. Positioning of the
quinoline ring follows typical type II TKI binding profile wherein numerous inhibitor-RTK crystal
a ct i ve - co n f
R O S 1 ' : ' i n a c $ v e '
R O S 1 ' : ' a c $ v e '
R O S 1 ' : ' i n a c $ v e '
24
structures confirm nitrogen-containing aromatic groups occupy the adenine-binding site. The
catalytic K1980 residue forms a hydrogen bond with the dicarboxamide functional group.
Importantly, the hydrophobic cleft adjoining the ATP recognition pocket captures the terminal 4-
fluorobenzene substituent through T-shaped and pi-pi aromatic interactions with F2004 and F2075,
respectively (figure 2.4.C). This specific set of molecular interactions involving ROS1 and a type
II TKI has never been reported and hence presents a focal point of our study. Finally, the
cyclopropyl appendage provides hydrophobic contacts with M2001 and L2070 in the linker/hinge
region (figure 2.4.B, coloured yellow), which is slightly above the plane of the quinoline and
4fluorobenzene.
Figure 2.4- Lowest energy conformation of cabozantinib bound to ROS1 kinase domain. Panel A
shows exposed methoxy groups and the quinoline ring occupying the ATP binding site (green).
The tethering 4-aminophenol and cyclopropyl carboxamide motifs reside in the hinge region,
highlighted yellow. Panel B shows a few of the interacting amino acids. Panel C illustrates
occupancy of the hydrophobic groove (adjacent to the adenine site) by the 4-fluorobenzene
appendage. Image generated using MacPyMol 2009-2010.
Foretinib is a very similar structural analog of cabozantinib with the fundamental quinoline,
carboxamide linker, and 4-fluorobenzene-containing scaffold retained.Foretinib has an additional
morpholine ring attached to the 3-phenoxy of the quinoline through a 3-C linker along with a
2fluoro-4-aminophenol bridging system. Analysis of the lowest-energy docked pose of foretinib
E 2 0 2 7 *
M 2 0 2 9 * *
D 2 1 0 2 * L 2 0 8 6 *
L 2 0 7 0 *
B *
A *
C *
25
revealed a molecular conformation analogous to cabozantinib. Similarly, the morpholine-branched
6,7-dimethyoxyquinoline sits in the ATP site with the morpholine lying solvent exposed and
interacting with residues K1976 and E2030. The remainder of the inhibitor traverses the narrow
ROS1 kinase domain tunnel in the identical cabozantinib-manner without much variance in the
interacting protein amino residues. Closer examination of the distribution cluster of the
foretinibROS1 docking simulation however showed a possible reverse binding conformation,
figure 2.5. The type II inhibitor model is retained, however, the orientation of the molecule in 32%
of the poses, is completely inverted with respect to the anatomy of the ROS1-cabozantinib
complex. The morpholine group engages the hydrophobic cavity whereas the 4-fluorobenzene ring
recognizes the adenine-binding region.
ROS1@CabozanBnib* ROS1@ForeBnib*
Figure 2.5- Alternative, reversed binding pose of foretinib with ROS1 compared to predicted
cabozantinib-ROS1 complex. Image generated using MacPyMol 2009-2010.
With a well-characterized inhibitor-protein interaction blueprint, both cabozantinib and
foretinib were good primary candidates to use in our study of the molecular mechanism of mutation
and subsequent acquired resistance in the ROS1 kinase domain. While we were conducting our
docking studies, it was reported that the ongoing clinical trial for foretinib as a therapeutic for
NSCLC was withdrawn. Novartis had acquired GlaxoSmithKline’s oncology portfolio and
terminated development of foretinib partially due to possible its toxicity issues in the trial and
26
cabozantinib’s F.D.A. approved status. With foretinib being dropped as an inhibitor for
CD74ROS1 positive NSCLC in the clinic, only cabozantinib was taken forward in the project.
There would be no potential drug resistance due to inhibitor-induced point mutations in the case
of foretinib and all therapeutic efforts for ROS1 were being directed towards cabozantinib.
Additionally, as the docking studies indicated, approximately one-third of the foretinib binding
conformations adopted a reversed orientation, suggesting a dual binding mode in presence of the
G2032R mutation. This system could be further resolved with molecular dynamic simulations to
probe for any induced binding effects and their corresponding energy calculations. Unfortunately,
such studies were out of the scope of this project, and thus, cabozantinib was the ideal candidate
given its affirmed interaction with the ROS1 kinase domain and higher four-fold higher potency
against ROS1G2032 relative to foretinib.
2.2 Proposed library of cabozantinib derivatives
We segmented cabozantinib based on the different pockets recognized by the TKI and its
corresponding interactions. The 6,7-dimethoxyquinoline and 4-fluorobenzene groups provided
maximum scope for a structure-based inspection on the inhibitor binding mechanism. As
previously mentioned, the methoxy groups are solvent-exposed, protruding from the
adeninebinding site. We postulated this could be due to hydrophobic driven effect since the
methoxy groups are surrounded by G2031 and L2034 and most importantly, G2032. This
hypothesis could certainly be tested by comparing the docking and accompanied in vitro binding
activity of cabozantinib analogues with various alkyl groups on the 6,7-diphenoxyquinoline motif.
We decided to replace the methyl groups with ethyl, propyl, iso-propyl, and tert-butyl along with
the parent non-substituted 6,7-diphenoxyquinoline. Installing bulkier alkyl groups and comparing
their binding profiles with cabozantinib would allow us to deduce the impact of the hydrophobic
effect and any trends derived from sterics. Furthermore, probing the substituents in close proximity
to the crucial 2032 residue will lend insight in to its role in inhibitor binding.
With regards to the 4-fluorobenzene appendage, numerous substituents are synthetically
feasible including: different sized-aromatic rings, numerous heteroaromatic systems, variable
27
regioisomeric, and atomic substitutions of the parent 4-fluorobenzene group. The cavity
surrounding this motif is quite spacious, lined with non-polar residues M2001, F2004, L2070,
F2075, H2077, and I2010. Our proposed derivatives included furan, pyrrole, pyridine, 5-fluoro-
2pyridinyl, and 6-fluoro-3-pyridinyl rings. For the furan and pyrrole, connectivity to the
carboxamide through C2 and C3 were considered. We generated a comprehensive first round
library of cabozantinib analogues with each permutation of both ends of the scaffold. Every
molecule was subjected to molecular docking simulations with the inactive ROS1G2032R kinase
domain. The derivatives proposed are summarized in figure 2.6 below.
Figure 2.6- Proposed structural modifications to cabozantinib for fragment-binding computational
studies.
At the time of proposing the library of cabozantinib structural derivatives, Schrödinger
Suite was unavailable to conduct the computational studies. We opted to use the AutoDock 4.2
(Scripps Research Institute, 2013) program to study the receptor-ligand interactions of our system.
Just like Glide, AutoDock 4.2 utilizes the LGA for its search methods and follows an identical
protocol where the protein, ligand and receptor grid are prepared independently.56
28
First, all the polar and non-polar hydrogens of the protein were introduced with partial
charges (using Gasteiger method) applied to each atom. Following this, three-dimensional
structures of cabozantinib and its derivatives were prepared using Chem3D Pro 12.0 with
energyminimization using an MM2 force field calculation with a minimum RMS gradient of 0.000.
The coordinates of the receptor grid box used for Glide was duplicated and implemented for
AutoDock 4.2 purposes. Docking simulations were then executed using global and adaptive local
search parameters through 100 trials of the “long” LGA runs. All calculations were performed with
a rigid protein structure and a flexible ligand. Clusters of conformations were populated based on
their free energy of binding with ROS1.
To validate the consistency of Autodock 4.2, we first docked cabozantinib with the
ROS1G2032R kinase domain and looked to compare the output with the results from Glide. To our
satisfaction, Autodock 4.2 produced a lowest energy conformation with a structural orientation and
corresponding predicted binding energy (ΔG = -11.20 kcal/mol) very similar to that of Glide (ΔG
= -12.00 kcal/mol).
From the docking results, we observed that increasing steric bulk on the
6,7diphenoxyquinoline confers more favourable binding affinity (Ki). The predicted Ki for
cabozantinib was approximately 1 nM whereas for the 6,7-di-tert-butoxyquinoline analogue, Ki <
1 x 10-2 nM. The ethyl, propyl, and iso-propyl containing alkyl derivatives had similar Ki’s to that
of the original inhibitor. With regards to the lowest energy conformation of the proposed
molecules, no significant deviations were observed. The 4-fluorobenze retained its occupancy of
the adjacent hydrophobic site while the quinoline ring remained in the ATP pocket. This trend can
only be supported by entropically driven hydrophobic effects during binding. The interaction of a
lipophilic group or surface in the receptor with a bulky substituent in the ligand is entropically
favoured by the displacement of ordered water molecules, hence an overall beneficial contribution
to ΔG of binding. Calculated Ki values (nM) for representative compounds are summarized in
figure 2.7 below.
29
R&Group&
Figure 2.7- Tabulated Ki values (nM) for alkyl group derivatives of 6,7-dimethoxyquinoline
position from docking screen.
Docking results of molecules containing the 4-fluorobenzene substitutions did not indicate
any meaningful trends in terms of ring size, identity of heteroatom, nor presence of fluorine. The
predicted Ki values of all the derivatives were within 4-9 nM, with cabozantinib (4-fluorobenzene)
exhibiting the most favourable binding profile, Ki ≈ 1 nM figure 2.8. Similar to the trend with the
analogues mentioned above, the aromatic rings did not affect the conformation of the lowest energy
pose. All of the appendages occupied the cavity adjacent to the ATP site while the
6,7dimethoxyquinoline motif recognized the adeninecleft..
110$
1 0.1$ R&Group&
30
10$ 1$
Figure 2.8- Tabulated Ki values (nM) for alkyl group derivatives of 6,7-dimethoxyquinoline
position from docking screen.
Since our first library of proposed molecules did not provide any promising candidates for
examining the ROS1 binding mechanism, we looked to other derivatives for our purposes. We
continued our focus on the two terminal ends of the structure as all lowest energy conformations
consistently showed the bridging aminophenol and cyclopropylcarboxamide residing in their
respective well-defined cavities. We looked to substitute the para-fluorine group with other
halogens: Cl, Br, and I along with other isosteres: H, CH3, and CF3. This initial small set of
derivatives could potentially help delineate any specific contribution of the para substituted
benzene ring towards cabozantinib’s unprecedented type II inhibition of ROS. In addition, the
revised 6,7-dimethoxyquinoline-structure activity relationship (SAR) would encompass the
originally proposed ethyl, propyl, iso-propyl, and tert-butyl, as well as new substituents including,
iso-butyl, n-butyl, bridging 2C, 3C and 4C alkyl chains and finally, the monomethoxy analogues.
With this library, we intended to looktowards second-generation analogues and compare selectivity
and resistance profiles for both wt ROS1 and ROS1G2032R through in vitro assays.
O
R O
F
F
O
O
N
O H N H
N
O O
H N
H N
N
N F
N
31
2.3 Synthesis of small-molecule TKIs of ROS1
We embarked on the synthesis of cabozantinib and its select derivatives using the synthetic
procedure outlined in the original patent (U.S. Patent No. 8,067,436 B2, Nov. 29, 2011)57 with
appropriate deviations to incorporate necessary changes. We approached the molecule through a
convergent synthesis by preparing the 4-fluorobenzene modified cyclopropane-1,1-dicarboxylic
acid and the 6,7-dimethoxyquinoline synthon incorporating the bridging 4-aminophenol. The
commercially available monomethylester of cyclopropane-1,1-dicarboxylic acid 1, was
transformed to its corresponding acid chloride, using excess thionyl chloride and reflux conditions.
Amide coupling (THF with DIPEA) with various para-substituted anilines afforded amides 2a-g
and subsequent saponifaction using LiOH.H2O in THF/H2O produced the modified carboxylic
acids 3a-g, scheme 2. We later discovered that simple amide coupling using (2-(1H-benzotriazol1-
yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) proved to be equally efficient but
could be performed with milder conditions than acid-chloride activation. The addition step with
numerous para-substituted anilines provided a synthetically feasible divergence point necessary
for the library.
Scheme 2-1. Amide coupling synthesis of cyclopropylcarboxylic acid and aniline: (i) SOCl2,
reflux, 2 h; then aniline, THF, DIPEA, rt, 16 h; (ii) aniline, HBTU, DIPEA, DMF, rt, 18 h; (iii)
LiOH.H2O, THF/H2O, 0 oC to rt, 2 h.
Synthesis of the remaining half of the inhibitor scaffold required a more elaborate
procedure as a majority of the alkyl derivatives, the corresponding amines were unavailable. We
32
looked to synthesize the desired amines by first alkylating 4-nitrocatechol (4), followed by
reduction of the nitro group to produce the amine substrate. Rigorous method development was
conducted to obtain a facile and robust alkylation protocol. We initially used 1,3 dibromopropane
as the substrate to survey ideal reaction conditions. Attempts included using DIPEA (2.2 equiv.),
and 1.5 equivalents of the dibromide in numerous solvents: CH3CN, DMSO, DMF and THF. With
no significant conversion to the desired product, we opted to use Cs2CO3 base in the same set of
solvents. However, product yields were quite low and further synthetic improvements were
required. We considered a much stronger base, NaH, however the alkylation did not proceed in
desirable yields at room temperature and even upon increasing the temperature to 80 oC. Addition
of AgNO3 to force the reaction forward via formation of AgBr salt also proved to be futile.
Ultimately, we were able to successfully carry out the alkylation of the 4-nitrocatechol with
1,3dibromopropane using K2CO3 in ethylene glycol at 110 oC for 8 h. This protocol was applied
to various alkyl bromides to synthesize the corresponding alkylated intermediates, 5a-h.
33
Scheme 2-2. Alkylation of catechol with numerous alkyl halides and subsequent preparation
of aniline: (i) 1,3-dibromopropane, K2CO3, ethylene glycol, 75-110 oC for 8-10 h; (ii) H2(g), Pd/C
(10%), THF/MeOH.
Next, we explored two sets of reduction conditions for conversion of the nitro group in 5ah
to the amine. First, we attempted SnCl2 reduction in methanol with refluxing conditions.
Unfortunately, there was minimal conversion to product even with addition of concentrated
HCl/AcOH forcing us to resort to another reduction procedure. As a test reaction, we discovered
catalytic hydrogenation of 4-methoxynitrobenzene with palladium on carbon proved to be
promising, with a convenient work-up and purification (92% yield). Upon extending these
conditions to all nitro-substrates, we were pleased to see efficient 75%-96% yields conversion to
the corresponding anilines, 6a-h. For the 6,7-dimethoxy, 6-methoxy, 7-methoxy, and
nonsubstituted quinolone systems, the commercially available anilines 6i, 6j, 6k and 6l were
purchased.
Entry! R1! R2! Entry R1 R2
a! !O
O !
h(ii)!
!
O
b!
c!
i! H ! H !
d!
j! O ! H !
k(i)!
H O
e!
H3CO f!
N O O
O ! O
! O
!
O !
O
! O
! O
! O
! O
N
34
O O
k(ii)!
H O
g! O O
CH O
h(i)! O O
O
l O O
Scheme 2-3. Cyclization of quinolone using microwave-assisted conditions, chlorination and
subsequent coupling: (i) 90 oC, 10 min, microwave assisted; then aniline 6a-l, EtOH, reflux, 2h;
(ii) Ph2O, 230 oC, 10-20 min, microwave assisted; (iii) POCl3, reflux, 2 h; then cold H2O, Na2CO3;
(iv) NaH, 4-aminophenol, DMSO, 10 minutes; then chloroquinoline 12a-l DMSO, 100 oC, 12h.
The remainder of the quinoline ring was installed in a three-step procedure. First, enolate
alkylation of 2,2-dimethyl-1,3-dioxane-4,6-dione (7), with triethylorthoformate (8), under
microwave-assisted conditions (90 oC for 10 min) afforded the 1,3-dioxane-4,6-dione intermediate.
This crude reaction mixture was taken forward to imine condensation with anilines 6a-l in refluxing
EtOH. For all the imine products 9a-l, filtration of the reaction mixture and subsequent EtOH
washes were sufficient as further purification was not required. The third and final step was a
decarboxylation-mediated ring closure performed in diphenyl ether at 230 oC for 10 min in
microwave-accelerated conditions. In some cases, if there wasn’t sufficient conversion to the
quinolin-4-ols product (10a-l), the reaction was setup again with identical conditions for 20
minutes.
Interestingly, we detected the presence of two regioisomeric products when subjecting 9h
and 9k to the quinoline ring closure conditions mentioned above. The presence of two isomers can
be attributed to the first step in the mechanism of the reaction, where attack at the 1,3-dioxane-
4,6dione ester group can proceed either through C8, as shown in mechanism A, or through C6,
indicated in mechanism B for imine 9h, figure 2.9. Para-directed mechanism A yields quinolin-
4ol 10h(i), whereas the ortho-selective attack in mechanism B produces 10h(ii). Similarly, in the
case of 9k, nucelophilic attack is plausible through either C2 (see mechanism C) or C6 (mechanism
D). The two regioisomers 8-methoxyquinolin-4-ol 10h(i), and 5-methoxyquinolin-4-ol 10h(ii),
were easily separated using silica gel chromatography. Unfortunately, complete purification of
!
!
!
!
N
3
!
! O N
35
10k(i) and 10k(ii) from the product mixture was not possible as each intermediate retained slight
impurity of its regioisomeric partner (~10%). This was carried forward in the remainder of the
synthesis with the possibility that the resulting final compounds could be separated with the use of
much more comprehensive purification techniques such as HPLC.
Mechanism*D* O
Figure 2.9- Proposed mechanisms for decarboxylation-mediated cyclization reactions of imines
9k and 9h. Mechanisms A and B illustrate how 9k, can give two regioisomers, 10k(i) and 10k(ii).
Similarly, diverging mechanisms C and D outline the formation of 10h(i) and 10h(ii).
With the set of substituted quinolin-4-ols 10a-l in hand, we looked to add the bridging
4aminophenol motif using nucleophilic aromatic substitution (SNAr). The 4-position alcohol was
chlorinated in the presence of excess POCl3 and reflux conditions, furnishing 4-chloro-
6,7dialkoxyquinolines, 11a-l, in essentially quantitative yields. To drive the chemoselectivity of
the SNAr for the free -OH, 4-aminophenol was initially treated with NaH, followed by addition of
the 4-chloroquinoline substrate. For all intermediates 12a-l, this methodology proved to be
successful in selecting the phenol as the nucleophile in the SNAr. The final coupling step was
attempted using traditional peptide coupling reagents such as HBTU or N,N′-
36
Diisopropylcarbodiimide (DIC) but with little success as significant quantities of both starting
materials were recovered. We then elected to pursue convert the acids 3a-g to produce their
corresponding acid chlorides using thionyl chloride. Cabozantinib (13a) was successfully
synthesized by coupling the acid chloride of 3a with aniline 12l under mild conditions (1.5 equiv.
DIPEA, 1.1 equiv. K2CO3, THF, room temperature).
This final coupling procedure was adapted to synthesize the library of cabozantinib analogues 13at,
shown in figure 2.10. Due to synthetic challenges associated with the alkylation of the
4nitrocatechol in the early stages of the scheme, 19 final molecules were prepared. Synthesis of
the 6,7-di-tert-butyl derivative proved to be quite challenging and hence was omitted.
F N
O
H N
O
H N
O F
O O
H N
O
H N
O O
O
N
37
O 13s 13t
Figure 2.10- Preliminary library of synthesized analogues of cabozantinib, 13a-t.
2.4 In vitro evaluation of preliminary library in cell models
The preliminary cabozantinib derivatives were screened for anti-proliferative activity in
Ba/F3 cells expressing native CD74-ROS1 or CD74-ROS1G2032. Cabozantinib and crizotinib were
used in the MTS cell viability assay as a control. Parental Ba/F3 cells were cultured in RPMI
medium 1640 with additional 10% (v/v) FBS, L-glutamine, penicillin/streptomycin. Cells were
supplemented with 15% (vol/vol) WEHI-3– conditioned media as a source of IL-3 and maintained
at densities of 0.5 × 106 to 1.5 × 106 per mL. The cell cultures were infected with retrovirus either
encoding native or G2032R mutant human CD74-ROS1. Stable cells were confirmed using
puromycin-based selection by GFP expression using a FACS Aria flow cytometer. Stable cell lines
were then washed to remove exogenous IL-3. Cells exhibiting healthy proliferation and survival
post IL-3 removal were maintained and carried forward for cell proliferation assays.
All inhibitors were prepared as 1 mM stocks in DMSO before each experiment. Ba/F3 cells
expressing CD74-ROS1 and CD74-ROS1G2032R constructs were seeded (800 cells per well; 25 μL)
into 384-well plates. Final compounds were added with 25 μL per well of complete medium.
Twofold dilution series format was setup with maximum concentration of 500 nM of inhibitor (12
concentrations per compound). The plates were incubated for 72 h at 37 °C, 5% CO2. Viability was
measured using a methanethiosulfonate-based assay (CellTiter96 Aqueous One Solution;
Promega) read on a Biotek Synergy 2 plate reader (emission wavelength = 490 nm). All
experiments were performed at least two independent times in triplicate. Data were normalized
using Microsoft Excel, and absolute IC50 values were calculated for each inhibitor with GraphPad
Prism software using a nonlinear curve fit equation and results are tabulated below, figure 2.11.
38
Figure 2.11- Graphical representation of IC50 values of preliminary library of small-molecule
inhibitors 13a-t and crizotinib in Ba/F3 CD74-ROS1 and CD74-ROS1G2032R cells.
Unfortunately, the preliminary library of TKIs did not reveal any noticeable trends in terms
of cell viability inhibitory activity. Most notably, there were no inhibitors that were more potent
than cabozantinib. For the majority of the compounds tested, 50% of cell death was observed only
at concentrations close to the maximum drug dose, 500 nM, for Ba/F3 CD74-ROS1 cells. Activity
against Ba/F3 CD74-ROS1G2032R cells was significantly diminished with only inhibitors 13b and
13f having IC50 values comparable to cabozantinib, 13a. We are currently pursuing analysis of
levels of pROS1 and its downstream signaling proteins, pSTAT3, pAkt, pERK and pSHP-2 by
Western blot in both cell lines.
2.5 Binding constant determination using biophysical assay
We considered a variety of biophysical assays that would allow us to characterize the
potency and selectivity of our compounds for the wild type ROS1 kinase domain and its G2032
mutant in vitro. Ideal experiments would provide inhibitory constant (Ki) values that would define
the concentration of TKI required in order to inhibit the maximal enzymatic activity by 50%. Direct
modulation of an enzyme’s activity by an inhibitor is an important approach to study the system of
interest without interfering biological cascades. We can effectively adapt such an assay for a high-
throughput screening (HTS) format and use this as a benchmark to rank inhibitors in terms of
1 3 f * 1 3 a * 1 3 g * 1 3 c * 1 3 e * 1 3 b * 1 3 d * 1 3 h * 1 3 i * 1 3 j * 1 3 k * 1 3 l * 1 3 m * 1 3 n * 1 3 o * 1 3 p * 1 3 q * 1 3 r * 1 3 s * 1 3 t *
39
potency. Academic groups and industry have extensively utilized a host of kinase enzymatic assays
given the popularity of TKI drug development programs.58 These kinase assay technologies
generally require quantification of a species in the phosphoryl transfer reaction catalyzed by the
enzyme. Methods include detection of ATP consumption, measuring ADP accumulation or
tracking the formation of the phosphopeptide/phosphoprotein product.59 Fluorescence-based
detection assays are the most commonly used approach for TKI profiling given their HTS
compatibility, relative ease of use and wide applicability to numerous kinases.60,61 Techniques
relying on fluorescence measurements, however, are liable to false-positive and false-negative
results due to interfering or non-specific fluorescence output. The contribution of fluorescent
tracers, labeled substrates or fluorescent compounds to high background and interfering signals is
an important consideration for assay design including FI, FP and even FRET experiments.71
In light of the susceptibilities of the previously mentioned fluorescence-based procedures,
we looked to another assay to attain valuable binding data for our TKI-ROS1 kinase domain study.
Time-resolved-fluorescence resonance energy transfer (TR-FRET) provided a suitable alternative
as it offers the advantages of both FRET and time-resolved fluorescence (TRF) spectroscopy. TRF
is considered an extension of fluorescence spectroscopy where the emission of a sample is
monitored over time after the initial excitation. TR-FRET looks to eliminate the hampering
background fluorescence observed in conventional FRET by introducing a time delay between
excitation of the acceptor chromophore and signal acquirement.72 Given the background
fluorescence signals have a typical lifetime in the range of nanoseconds, time-resolved
measurements this short-lived emission can be cleared from the measured fluorescence. To achieve
suitable FRET with a time delay, the donor species must have a fluorescence lifetime that is much
higher than that of any background or undesirable emission. Lanthanide ion complexes such as
europium (III) and terbium (III) chelates are commonly used for these purposes, given their long
emission lifetimes (10-6 to 10-3 seconds). 73 A comparatively longer lifetime will allow the decay
of transient signals and the observed emission to be a result of only energy transfer from the donor
to the acceptor. The combination of FRET and TRF results in a much more sensitive and reliable
assay. Specifically, we elected to use the LanthaScreen TR-FRET assay developed by Invitrogen.
A brief representation of the assay is presented below in figure 2.12.
40
Figure 2.12- Illustration of principle of LanthaScreen (Invitrogen) TR-FRET assay. (Figure taken
from reference 74).
The principle of this assay is based on FRET occurring between a europium(III) labeled
antibody bound to the kinase and a proprietary chromophore (Alexa Fluor 647) conjugated
ATPcompetitive tracer.74 The ROS1 protein is modified with a GST tag recognized by the Eu(III)
containing anti-GST antibody in solution. Binding of the Alexa Fluor 647 containing tracer to the
ROS1 kinase domain is preferred at the ATP recognition pocket since the tracer is derived from
scaffolds of various ATP mimetic TKIs. Excitation of the Eu(III) chelate complex with light is
succeeded by energy transfer to the proximal Alexa Fluor 647-tracer species and its associated
emission. Thus, concurrent binding of the Eu(III)-antibody and Alexa Fluor 647 labeled tracer to
ROS1 results in a high degree of FRET since both species would be in ideal molecular proximity
when bound. A TKI specific for the ROS1 catalytic site would compete for binding with the tracer,
thus disturbing the proximity of the FRET pair. As the inhibitor is titrated in solution at increasing
concentrations, the Alexa Fluor 647 labeled tracer would continue to get displaced, thus decreasing
overall FRET signal. In further support, Invitrogen’s LanthaScreen TR-FRET assay is appropriate
for both type I and type II TKIs with high sensitivity. We attempted to assess the in vitro potency
of our inhibitors and cabozantinib for the native ROS1 protein using the LanthaScreen assay.
Prior to any inhibitor competition experiments, a calibration experiment had to be
performed to calculate the KD of the tracer. The KD value calculated from this binding experiment
would be used to approximate the concentration of the tracer required in the inhibitor titrations.
Ideally, we would like to select tracer concentrations that extract the maximum dynamic range
from the experiment from completely bound tracer (high FRET) to free, unbound tracer (low
41
FRET). The calibration experiment required a final concentration of 3 nM ROS1 kinase protein
and 6 nM of the Eu(III) chelate antibody in each well. Titration of increasing concentrations of
tracer would lead to increases in concentration of the antibody-ROS1-tracer complex, thereby
generating a dose-response curve with respect to observed FRET. The concentration of tracer
ranged from 0.0153 to 500 nM (2 fold dilutions). An additional experiment was performed in the
presence of 0.150 nM of staurosporine, a universal kinase inhibitor. In this negative control, excess
staurosporine occupies the ATP site in ROS1, thus negating any binding of the tracer. Any potential
energy transfer from the excited Eu(III) antibody to unbound tracer in solution can be monitored
with this setup and accounted for in the calibration curve and subsequent inhibitor experiments.
GST-tagged ROS1 protein, 5X kinase buffer A (1X buffer contains: 50mM HEPES pH 7.5, 10
mM MgCl2, 1 mM EGTA, 0.01% Brij-35), kinase tracer 236 (50 μM stock in DMSO),
LanthaScreen Eu-anti-GST antibody and staurosporine were obtained from Invitrogen. Assays
were conducted in triplicate in clear low-volume 384-well plates (Greiner). Addition of all the
components was followed by a 60-minute incubation at room temperature. All fluorescence
measurements were performed with the BioTek Cytation3 plate reader instrument. Acceptor
LanthaScreen Eu(III)-anti-GST antibody excitation was at 340 nm, emission of the Eu(III)-chelate
bound antibody was 615 nm and kinase tracer emission was measured at 665 nm. A delay time of
100 μs was inserted between excitation and fluorescence measurement. The emission ratio was
calculated by dividing the emission of the Alexa Fluor 647 conjugated tracer (acceptor, 665 nm)
by the emission of the Eu(III)-tagged antibody (donor, 615 nm). Concentration of tracer was plotted
against emission ratio in the presence and absence of staurosporine.
We were able to retrieve an ideal calibration curve for binding of the tracer with ROS1 protein
with complete saturation of the kinase, figure 2.13. Based on the tracer-ROS1 saturation
experiment, the KD was determined to be 1.272 nM with 95% confidence interval of 1.123 to 1.421
nM.
42
Figure 2.13- Calibration curves of Alexa Fluor-647 tracer titration with ROS1 kinase with 100μs,
time delay between excitation and measurement of emission.
Competitive inhibitor experiments were then performed to determine the KD values for the
preliminary library based on the quadratic mathematical model describing competitive binding of
two different ligands to a protein molecule described by Wang, 1994.66 Experimental procedure
was identical to that of the calibration experiment described earlier, except each well had a final
10 nM of tracer, 1 nM of ROS1 protein and 4-fold inhibitor concentration range from 0.024 nM to
100 𝛍M. Emission ratios obtained were plotted against concentration of inhibitor and were fit using
GraphPad Prism 5.0 to obtain dissociation constants. The KD values obtained are summarized in
table 2.1 and figure 2.14 below (binding curves are included in supplemental information).
Table 2.1- Summarized KD values for cabozantinib and analogues determined by
LanthaScreen TR-FRET assay.
Compound ROS1 KD (nM)
13a 21.43 ± 1.642
13b 16.8 ± 1.563
13c 30.5 ± 4.467
13d 341.2 ± 25.29
13e 196.7 ± 19.44
13f 56.22 ± 3.829
43
13g 412.92 ± 38.37
13h 413.8 ± 43.62
13i 966.3 ± 162.8
13j 5433 ± 1832
13k 7359 ± 1907
13l 63.88 ± 7.324
13m 237 ± 29.27
13n 61.43 ± 7.247
13o 2850 ± 374.6
13p 8056 ± 1273
13q 5095 ± 611.4
13r 4518 ± 585.6
13s 941.5 ± 89.28
13t 1526 ± 249.9
Figure 2.14- Graphical representation of calculated KD values for initial library in addition with
crizotinib and staurosporine controls.
44
Cabozantinib had a KD value of 21.43 ± 1.642 nM, whereas there was no significant improvement
in binding potency with the analogues. Interestingly, in the MTS cell proliferation assay,
cabozantinib’s IC50 value of 8.4 nM (in Ba/F3 transformed CD74-ROS1 cells) is lower than its KD
for the protein, indicative of off-target binding and slight non-selective profile. Trends between the
inhibitor binding potency correlate strongly with that of the cell cytotoxicity results in section 2.5.
Small isostere replacements of the para-fluorine position rendered a similar in vitro activity profile
both in CD74-ROS1 transformed cells and the LanthaScreen assay with the chloroderivative, 13b
(KD = 16.8 ± 1.563 nM), bromo-derivative, 13c (KD = 30.5 ± 4.467 nM) and methyl group, 13f,
(KD = 56.22 ± 3.829 nM). There was eventual loss in activity with the bulkier iododerivative (13d)
and simple phenyl derivative 13e (KD = 30.5 ± 4.467 nM). An incremental loss in affinity for ROS1
kinase was seen with bulkier alkyl substituents on the quinoline ring (13l-p) and activity was
completely lost with the cyclized analogues bearing bridged alkyl units (13q-t). The trends
observed in both the cellular and biophysical in vitro results for the bulky alkyl analogues such as
the ethyl, propyl, iso-propyl, n-butyl, iso-butyl heavily contrast with the Ki values predicted by the
docking model in GLIDE (section 2.3). This disparity cannot be directly attributed to the entropic
contribution towards binding as water molecules are removed from the protein structure for the
docking simulations. As a result, displacement of water molecules from the binding pocket cannot
be deemed as a contributor to ΔGbinding for the more hydrophobic analogues in the series. It is
possible that the favourable hydrophobic contacts and corresponding surface area calculated by
GLIDE in silico do not translate in vitro when considering a dynamic protein free in solution or
even in the context of a cellular environment.
Concluding remarks
The in vitro evaluation of our library of cabozantinib analogues is still in its early stages. We are
currently looking to use the LanthaScreen assay first and rank compounds based on their binding
affinity with the native ROS1 protein. Furthermore, we have considered additional experiments
such as a thermal-shift assay to gain further information on inhibitor and protein interaction. Other
typical experiments that monitor a binding event including surface plasmon resonance (SPR)
spectroscopy and isothermal calorimetry (ITC) are being considered as well.
45
From the thermodynamic and kinetic binding data generated from these experiments, we can
investigate any potential trends and elucidate the substituents that contribute to recognition of the
ATP site in the native ROS1 kinase domain and its G2032 mutant. Ideally, these results can
corroborate with our computational modeling work and highlight the scaffold features of
cabozantinib that contribute to its unique in silico predicted binding mode and in vitro potency and
selectivity for ROS1 and ROS1G2032. Additionally, we are also interested in the effect on the levels
of pROS1 and downstream signaling proteins by western blot analysis in Ba/F3 CD74-ROS1 and
Ba/F3 CD74-ROS1G2032 cells to ensure target selectivity.
46
Chapter 3: Design of peptdiomimetic inhibitors of signal transdcuer and
activator of transcription factor 5 (STAT5)
3.1 Introduction to Signal Transducer and Activator of Transcription Factor 5
(STAT5)
Signal transduction pathways serve as the basis of biological communication to efficiently
regulate cellular cycle growth, survival, and apoptosis. These molecular pathways begin with the
interaction of an external molecule with its specific target followed by the succeeding downstream
cascade. Generally, the event of exogenous ligand-receptor binding is relayed to protein-protein
interactions (PPIs) that eventually modulate the level of transcription of specific genes.75 One
specific protein mediated signalling network of physiological interest is the signal transducer and
activator of transcription (STAT) associated pathway. The study of this network of signaling
molecules is of significance due to its relevance with numerous human cancers: blood, prostate,
breast, pancreas, lymphatic system, and liver.76,77,78 The presence of excessive growth factors,
hyperactive kinase activity, oncogenic mutations or a decrease in activity of the negative regulators
of the STAT cascade leads to over-expression of anti-apoptotic and proliferation inducing genes.
Collectively, these factors lead to the aberrant activity of the STAT transcription factors and
eventually drive the oncogenic phenotype.
The STAT family of signal transducers consists of seven members, namely: STAT1,
STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6. STATs have been characterized as
90115 kDa proteins consisting of approximately 750-900 amino acids.79 Genes encoding for the
various STAT amino acid sequences map to three different chromosomes. STAT3, STAT5A, and
STAT5B map to chromosome 17; STAT1 and STAT4 map to chromosome 2; finally, STAT2 and
STAT6 map to chromosome 12. Out of the seven members of STAT transcription factors, STAT5A
and STAT5B have been profoundly associated with cellular transduction mechanisms that mediate
hormone-related responses and cellular growth events. More importantly, STAT5A and STAT5B
signaling is heavily involved in the regulation the growth and maintenance of the hematopoietic
and lymphoid systems. 80
47
3.2 Structure and signaling of STAT5
The STAT5 protein was originally isolated in mouse mammary glands in 1991 by
SchmittNey et al. and was identified as a mammary gland factor (MGF), responsible for the
transcription of the β-casein gene.81 Further studies conducted by Gouilleux et al. identified STAT5
as a signaling molecule which mimicked the effects of prolactin.82 Upon finding a significant
degree of sequence homology with the STAT transcription factor family, MGF was renamed,
STAT5. In 1995, an isoform of STAT5 was characterized in murine mammary glands and was
confirmed as a separate gene product from MGF.83 This homolog was named STAT5B whereas
the original MGF/STAT5 was termed as STAT5A. Consequently, the proteins share a high degree
of similarity in amino acid sequence (approximately 93%). The structural and functional units of
both homologs are shown below, figure 3.1:
Figure 3.1- Schematic illustration of STAT5A/B domains with critical Y694 (STAT5A) and
Y699 (STAT5B) highlighted. (Image generated using MacPyMol 2009-2010)
The STAT5A and STAT5B proteins consist of six well-defined domains that are
structurally and functionally conserved amongst the STAT family. The N-terminal domain (NTD)
contributes to stabilization of the STAT-dimer and even the STAT-DNA complex. Neighbouring
the NTD, is the coiled-coil domain (CCD), a four α-helical motif rich in hydrophilic amino
acids.84,85 The CCD recognizes native binding partners including nuclear transport proteins,
receptor α-helix motifs, chaperones, and intranuclear co-transcription proteins.86,87 Adjacent to the
CCD is the DNA-binding domain (DBD), an arrangement of β-sheet units that bind to the
N " t e r m i n a l + + N H 2 " + c o i l e d " c o i l + + D N A + b i n d i n g + L i n k e r + S H 2 + T r a n s a c : v a : o n + " C O O H +
Y 6 9 4 % %
S T A T 5 A + ( 7 9 4 + a a , + 9 4 + k D a ) + +
Y 6 9 9 % %
S T A T 5 B + ( 7 8 8 + a a , + 9 2 + k D a ) + +
48
TTCNNNGA γ-interferon activation sequence (GAS) sites in the regulatory elements of the target
genes.88 The linker domain is a short sequence that stabilizes the multimeric proteinprotein/protein-
DNA complexes and connects the DBD to the Src-homology 2 (SH2) domain. In both STAT5A/B
isoforms, the SH2 domain of STAT5A/B is an significant site contributing to receptor recruitment
and protein dimerization by recognizing conserved phosphotyrosine (pY) residues. In the signaling
mechanism, STAT5A and STAT5B dimerization occurs in a reciprocal fashion, wherein the SH2
domain of one partner recognizes the phosphorylated tyrosine of the other.84,89 Finally, the C-
terminal transactivation domain (TAD) is highly specialized for STAT5A (aa 722- 794) and
STAT5B (aa 727-787), owing to the association with isoform-specific transcription co-regulators.
Furthermore, the TAD region is heavily involved in co-regulatory localization. Structural
divergence originates at the NTD and TAD, whereby the difference in residue sequence and
resulting tertiary structure may contribute to interaction with different transcription factors and
recognition of varying DNA sequences.84 Interestingly, there are no differences in the transcription
promoter elements recognized by both isoforms. Thus, any nonredundant functions of STAT5A
and STAT5B are a result of difference in tissue distribution or divergence in their TAD regions.90
In healthy cells undergoing normal division and differentiation, the signaling activity of
STAT5A/B is transiently activated by the JAK/STAT pathway and tightly regulated by multiple
negative regulatory mechanisms. The activation of the both STAT5 isoforms signaling is initiated
by binding of the cytokines or growth factors to their respective receptors. The interleukin family
(IL-2, IL-3, IL-5, IL-7, IL-12 and IL-15), interferons (IFNs), granulocyte-macrophage colony
stimulating factor (GM-CSF), erythropoietin (EPO) and thrombopoietin (TPO) have been reported
ligands that induce STAT5A/B signaling.91,92 Glycopeptides including Growth Hormone (GH),
prolactin, epidermal growth factor (EGF) and even insulin have been discovered as ligands that
regulate STAT5 activity upon binding to their extracellular receptors.84
Cytokine receptors are devoid of kinase activity themselves, and therefore are dependent
on the JAK (Janus kinase) family of tyrosine kinases for activation and subsequent protein target
phosphorylation. In mammals, four enzymes constitute the JAK family, JAK1, JAK2, JAK3, and
TYK2.93 These kinases are ubiquitously associated with cytokine receptors on the membrane
proximal domain and are activated on the receptor following ligand association. Other cellular
49
tyrosine kinases have been associated with STAT5 signaling such as the Src kinase family and
Etk/Bmx (from the Tec family kinases). However, conclusive studies establishing their respective
signalling mechanisms remain elusive. Upon extra-cellular cytokine binding, the receptors undergo
homo- or hetero-dimerization and adopt an activated conformation. This conformational change
forces the receptor associated JAKs within close proximity to allow transphosphorylation,
producing a catalytically ‘active’ kinase. Next, active JAK kinases then catalyze phosphorylation
of conserved tyrosine residues on the intracellular domain of the receptor.16,6 Latent cytoplasmic
STAT5 monomers are recruited to these receptor pY motifs via their SH2 domains. Following
STAT5-receptor binding, JAK kinases phosphorylate specific tyrosine residues in the C-terminus
of STAT5A and STAT5B. In the case of STAT5A, the phosphoryl group is transferred to Y694,
whereas in STAT5B, the tyrosine acceptor is Y699. Phosphorylated STAT5A/B dissociate from
the receptor and undergo hetero- or homo-dimerization in the cytosol through reciprocal pY-SH2
recognition. Homo-dimerization of STAT5A/B isoform units is favoured over heterodimerization
of STAT5A and STAT5B. Active STAT5-dimer complex dimers are able to translocate into the
nucleus through the nuclear pore complex (NPC) in an energy dependent process. Importin
proteins in the nuclear membrane mediate transportation of phosphorylated STAT5 dimers across
the NPC by recognizing a specific nuclear localization signal (NLS) sequence on the STAT5A/B
TAD domains.94
Once in the nucleus, phosphorylated STAT5 dimers recognize specific oligonucleotide
GAS regions (TTCNNNGA) through the DBD and induce transcription of target genes. Numerous
operational transcription factors and chromosomal-associated proteins are involved in regulation
of gene expression and thus interact with intranuclear STAT5. The STAT5A/B transcription
machinery is responsible for increase in transcription levels of genes controlling cell proliferation
such as BCL-XL (B-cell lymphoma-extra large; member of apoptotic Bcl-2 proteins), pim-1
(protooncogene serine/threonine kinase Pim-1)80 and C-MYC (proto-oncogene promoting cyclin
production, downregulates Bcl-2).95 STAT5 activity also controls the transcription elements
responsible for inflammation and differentiation include OSM (encodes Oncostatin-M, an
interleukin) and c-fos (encodes Fos protein) respectively.95 STAT5 signaling has also been reported
to activate expression of the MCL-1 pro-survival gene.96
50
The principle inhibitory mechanisms associated with the regulation of the JAK/STAT
pathway include protein tyrosine phosphatase (PTP) enzymes, suppressor of cytokine signalling
(SOCS) proteins and the protein inhibitors of activated STAT (PIAS) family of regulators.97 The
direct route of deactivation of activated STAT5 is conducted by PTP enzymes, which catalyze the
dephosphorylation of pY694 and pY699 in STAT5A and STAT5B, respectively. Known PTPs that
cleave the phosphate group of pSTAT5A/B are PTP1B98 and SH2 domain containing
phosphatases, SHP-1 and SHP-2.99,100 Protein tyrosine phosphatase non-receptor 2 (PTPN2 or TC-
PTP) is believed to carry out the dephosphorylation of STAT5A/B in the nucleus.101
PIAS proteins are known transcription co-regulators that inhibit transcription by either
binding to the transcription factor directly, disrupting binding with DNA or labelling such factors
for protein degradation.102 More specifically, PIAS proteins have been shown to conjugate SUMO
(small ubiquitin related modifier) to target transcription factors such as STAT5 with their E3 ligase
activity.103 Upon SUMO modification, STAT5A/B are labelled for degradation by the 26s
proteasome.
Finally, the SOCS family consists of CIS (cytokine-inducible SH2 domain protein) and
members SOCS1 – 7. Collectively, they modulate the JAK/STAT pathway via a negative feedback
loop. SOCS indirectly decreases the phosphorylation of STAT5 monomers by binding to the active
receptor associated JAKs, inhibiting the enzyme activity.104 Alternatively, CIS proteins deactivate
STAT5 signaling by interacting with the cytokine/growth-factor receptors, to inhibiting access to
the STAT5 binding site.105 These binding interactions are mediated through the SOCS SH2 domain
and pY residues on the target proteins. The overall signaling cascade of STAT5 is presented below,
figure 3.2.
51
Figure 3.2- Brief schematic of normal JAK/STAT5 signaling with cytokine/growth factor
receptors dimerized and bound to activated JAK2. Latent STAT5 is recruited via the SH2-pY
interaction and undergoes phosphorylation. Dimerization in the cytosol is followed by nuclear
translocation and regulation of transcription of target genes. Negative regulators, SOCS, PIAS and
PTP are represented as well. (Image generated using ChemBioDraw 14)
3.3 Role of STAT5A and STAT5B in disease
Given STAT5’s key role as a transcription factor for genes that control crucial cell
processes such as proliferation, survival and apoptosis, the signaling cascade needs to be tightly
regulated to ensure normal cell function and morphology. An in vivo study conducted by Grimley
et al. revealed genetic knockout of STAT5A, STAT5B or both isoforms in mice did not
compromise viability. Both STAT5A and STAT5B murine knockout models showed deficiencies
in immune responses.106 In addition, STAT5A null adult mice (STAT5-/-) exhibited defects in
mammary gland development and lactogenesis.107 On the other hand, STAT5B -/- mice showed
reduced growth and other morphological defects characteristic of insufficient growth hormone
levels.108
52
Contrastingly, constitutive STAT5 activity has been linked to several diseased states
including inflammation, auto-immunity and most importantly, tumorgenesis and metastasis. 109,110
The resulting phenotype is dependent on the specific tissue, cell types and even the isoform of the
STAT5 protein. The role of STAT5 in hematological malignancies such as CML and AML has
been extensively documented. In the CML myeloproliferative disease, the BCR-ABL fusion kinase
constitutively phosphorylates STAT5 even in the absence of cytokine or growth-inducing
stimuli.111 This leads to hyperactivity of STAT5 protein, whereby transcription of downstream
anti-apoptotic and pro-survival genes is elevated. On the other hand, in the case of AML which is
characterized by excessive immature white blood cells, mutations in receptor-associated JAK2 and
FLT3 (Fms-like tyrosine kinase 3) kinases lead to increased levels of active STAT5.112 These
kinase mutations that drive hematological cancers through constitutive activation of STAT5 do not
show any isoform specificity and are dependent only on the hematopoietic system development.
As previously mentioned, the non-redundant physiological functions of STAT5A and STAT5B are
attributed primarily due to difference in expression levels in varying tissues. Specific cell types
exhibit varying levels of expression of each isoform and thus maintain their required thresholds.
High levels of STAT5A are found in mammary tissue whereas STAT5B is more prevalent in the
muscle, prostate, and hepatic tissues. Thus, deregulations in the form of isoform specific point
mutations that contribute to hyperactivity of STAT5A and STAT5B individually can lead to
isoform-driven tumors. More specifically, hyperactivated STAT5A drives proliferation of human
mammary carcinoma cells, promoting tumor development and progression
113 Constitutively active STAT5B has been shown to increase in tumor volume in
breast cancer.
and invasion in squamous cell carcinoma of the head and neck114 (SCCHN) and colorectal
cancer.115 Other diseased phenotypes associated with sustained STAT5B signaling include growth
hormone insensitivity116, IL-2 based immunodeficiency and autoimmune disease.117 Mutations
within the STAT5A/B isoforms which themselves confer to its oncogenic activity, independent of
up-stream mutated kinases are also possible. Several in vitro studies have established
isoformspecific point mutations in STAT5A and STAT5B that render the proteins capable of
malignant transformation on their own.
53
Kitamura and co-workers discovered a STAT5A mutant through a mutagenesis screen
identifying transformed Ba/F3 cell lines capable of IL-3 cytokine independent growth.118 They
identified the point mutation N642H in the SH2 domain, in close proximity to the pY-recognition
site. It is believed this particular mutation provides extra stability to the STAT5-dimer complex,
particularly at the pY-SH2 domain site, hence sustaining its signaling in the absence of IL-3.
Interestingly, prolonged pSTAT5 activity upon IL-3 stimulation was observed. The same workers
discovered another potential oncogenic STAT5A mutation using the same mutagenesis screen. The
amino acid substitution reported was S710F in the TAD domain. Expression of the STAT5AS710F
mutant in STAT5 null Ba/F3 cells led to transformation with increase in transcription levels of
STAT5 target genes inducing proliferation.119
Numerous STAT5B variants harbouring point mutations have been proposed to promote
leukemogenesis in T-lymphocytes in patients who relapsed from lymphocytic leukemias. The most
commonly occurring mutation observed in patient samples was substitution of Asn642 by His,
similar to STAT5A.120 In vitro, it was observed that leukemia cell populations expressing
STAT5BN642H protein were able to induce cytokine-independent survival with the STAT5B
transcriptional machinery retaining sustained activity.121
Given the growing body of evidence supporting the significance of the aberrant STAT5
signaling pathway in multiple diseases, with strong clinical emphasis on human malignancies, such
as CML and AML, therapeutic intervention is highly critical. In particular, effective inhibition of
the STAT5-mediated cascade can supress transcription of genes that promote immature
differentiation and uncontrolled proliferation ultimately leading to malignant growth and
maintenance. Furthermore, with studies revealing the oncogenic capacity of STAT5A and
STAT5B in several isoform specific malignancies and other hormone-related diseases, strategies
that can achieve isoform-selective inhibition would be of utmost importance as diagnostic tools or
potential therapeutics.
3.4 Therapeutic strategies towards the STAT5A/B signaling pathway
The majority of therapeutic approaches to combat hyperactive signal transduction have
revolved around the identification and optimization of small molecule inhibitors that target
54
upstream kinases. Tyrosine kinase inhibitor (TKI) drug programs have yielded substantial success
in the treatment of multiple cancers by increasing survival rate, and prolonging patient remission.
The BCR-ABL TKI, imatinib has been successful in the clinic for CML treatment with high rates
of complete patient remission. Imatinib effectively induces apoptosis in leukemic cells harbouring
the BCR-ABL chromosomal rearrangement and decreases overall tumor burden. However, it was
later discovered that prolonged treatment of imatinib eventually resulted in acquired resistance
through point mutations in the BCR-ABL fusion kinase.122 The most frequently identified mutation
was the gatekeeper mutation, T315I. These amino acid substitutions occur in the BCR-ABL kinase
domain and compromise imatinib binding. It is also suggested that cells are capable of bypassing
inhibition of BCR-ABL kinase activity via a compensatory mechanism whereby other kinases
phosphorylate STAT5, thus maintaining constitutive expression of downstream pro-survival
genes.123 Increased dosing regimes become difficult to tolerate due to toxic side effects and the
multi-targeted nature of most TKIs. As a result, second-generation TKIs have been developed as a
means to address the acquired resistance observed in the clinic. Nilotinib and dasatinib were
second-line TKIs developed for imatinib resistant CML patients with higher potency than
firstgeneration ABL inhibitors, figure 3.3. Unfortunately, these small-molecules are not able to
diminish leukemic cell viability against all BCR-ABL mutant variants, thus limiting their use to a
small class of CML patients.124
Figure 3.3- First generation BCR-ABL TKI imatinib for CML treatment. Second-generation TKIs
from imatinib, nilotinib and dasatinib are shown below.
55
Based on the success of BCR-ABL TKIs as effective CML therapeutics, a similar drug
development method was adapted for AML treatment wherein mutated FLT3 kinase activity results
in constitutive phosphorylation of STAT5A/B. Notable first generation FLT3 TKIs include
nanomolar inhibitors lestaurtinib and sorafenib, both with promising initial success in clinical
trials, figure 3.4.125 One common theme amongst FLT3 small molecule inhibitors is poor
pharmacodynamic and pharmacokinetic profiles. This serves as a major limitation to advancement
of potential candidates towards clinical application. Another concern is potential loss of sensitivity
to TKIs by FLT-3 kinase domain mutations.
Therapeutic intervention of the JAK2/STAT5 pathway has also been attempted in treatment
of myeloproliferative neoplasms where JAK2 reportedly harbours somatic mutations. The most
commonly occurring mutation is a B1859T base pair inversion, resulting in the V617F substitution.
This mutation is localized at the autoinhibitory domain of JAK2, and disrupts the modulation of
the catalytic activity of the kinase.126 The loss of the autoinhibitory function of JAK2 leads to
constitutive phosphorylation of STAT5A/B, inducing increased levels of antiapoptotic
transcription products. The JAK2 inhibitor ruxolitinib has faired quite successful in the clinic with
potent inhibition of JAK2V617F at therapeutic concentrations, figure 3.4. Similar to BCR-ABL and
FLT-3 TKIs, small molecule inhibitors of JAK2 lack specificity, given their reactivity against
multiple off-targets.127
Figure 3.4- First-generation FLT-3 TKI lestaurinib and following second-generation derivative
sorafenib. Both drugs are used for FLT-3 mutation positive AML. Ruxolitinib is the widely used
JAK2 inhibitor in AML treatment, given its efficacy against JAK2V617F
In summary, therapeutic strategies aimed at inhibition of STAT5 signaling have focused
on the discovery and optimization of inhibitors targeting upstream mutant kinases. Aberrant kinase
56
activity, as seen in BCR-ABL, FLT-3 and JAK2-driven disease, leads to upregulation of cell
growth and survival via hyperactive STAT5A/B signal transduction. Although TKIs have had the
most success, they have several drawbacks, including adverse toxic side effects and acquired drug
resistance. Cardiac toxicity, hepatotoxicity, hemorrhaging, nausea, and immunodeficiency are a
few examples of side effects associated with TKI administration in patients. Generally, these
symptoms are a result of drug interaction with off-target proteins, a pronounced trend with TKIs,
given their multikinase nature.128,129 Acquired drug resistance is a multifaceted drawback
extensively covered in Chapter 1. Reduction in TKI binding affinity as a result of upstream kinase
mutations diminishes inhibitor efficacy, requiring increased dosing. Additionally, the cell can
resort to complimentary bypass mechanisms through other signaling cascades or kinases to
constitutively activate STAT5. Most importantly, since TKIs work against an upstream protein,
there is no possibility of inhibition of STAT5A/B isoform selective function. Reduction in the
hyperactive signaling of one specific isoform in particular diseases is not attainable with current
therapies. Furthermore, given that mutations in STAT5A and STAT5B can render the proteins as
the primary source of oncogenic transformation themselves, upstream TKIs will be ineffective in
suppressing tumor growth and survival. One strategy to resolve both shortcomings is the targeted
inhibition of STAT5A and STAT5B proteins directly.
To date, little progress has been made towards STAT5A/B isoform selective inhibitors.
Only Berg et al. have identified a small molecule capable of isoform selective inhibition of
STAT5A/B proteins.130 The workers reported Stafib-1, as the first small molecule which inhibits
the STAT5B SH2 domain, (Ki = 44 ± 1 nM determined by FP) with more than 50-fold selectivity
over STAT5A. Treatment of K562 CML cell lines with a prodrug analogue of Stafib-1 showed
dose-dependent decrease in pSTAT5B levels with minimal suppression of pSTAT5A across the
same range of concentrations. Despite the high affinity binding profile of Stafib-1 and its
derivatives, there is an opportunity to determine structural differences in the SH2 domains of
STAT5A and STAT5B that contribute to recognition of the isoform selective inhibitors.
STAT5A/B specific amino residues and their interaction with specific functional groups in the
proposed inhibitors is an interesting investigation in its own.
57
3.5 Proposed isoform-selective peptidomimetic inhibitors of STAT5A/B
We decided to approach the challenge of designing direct STAT5 isoform selective
inhibitors with the use of phosphopeptides and subsequently derived peptidomimetics.
Peptidomimetic inhibitors represent an important field in medicinal chemistry and have had
monumental impact in the discovery and advancement of numerous drugs targeting protein-protein
interactions.131 Peptidomimetic design originates form the identification of native peptide
sequences that interact with the target protein. Then, an alanine scan mutagenesis study is usually
conducted wherein each amino acid in the inherent peptide is substituted for an alanine. By
comparing the difference in binding affinity of each alanine-derivative in the combinatorial library
with the original sequence, residues important for interaction with the target are identified.132 The
alanine scan screening allows a series of logical truncations and chemical modifications are
implemented to remove the pharmacokinetic liabilities of peptides while retaining the original
pharmacophore. Peptidomimetic inhibitors seek to incorporate drug-like properties into the
scaffold of a biologically active peptide with minimal loss in activity. Overall, a peptidomimetic
strategy encompassing the use of high affinity STAT5A/B phosphopeptides with subsequent
chemical modification to potent and isoform selective peptidomimetic inhibitors presents a novel
therapeutic route to suppress aberrant STAT5A/B signaling.
As previously mentioned, the SH2 domain is an important module as it mediates critical binding
events. Recruitment to cytokine and growth factor receptors and homo/heterodimerization
represent critical junctures in STAT5A/B signal transduction. Accordingly, both phosphorylation
of STAT5 monomers and formation of the dimeric species that regulates transcription depend on
the SH2 domain’s ability to recognize selective pY protein substrates. Given its role in recognizing
specific pY containing motifs in various cellular receptors through PPIs, the SH2 domain is an
ideal hotspot region in both STAT5A and STAT5B. There is an abundance of information
concerning the interaction of STAT5 with several phosphorylated cellular receptors, which can be
used to select the preliminary library of SH2 domain-binding phosphopeptides. McMurray et al.
have successfully employed this approach to inhibit the STAT3 SH2 domain, using a peptide
sequence derived from the STAT3-native cellular receptor, gp130.133 The SH2 assembly is the
largest class of domains dedicated to selective recognition of pY motifs with 111 proteins in the
58
human proteome containing at least one SH2 domain.134 Usually consisting of 100 amino acids,
SH2 domains possess an evolutionary conserved tertiary structure that must recognize a pY residue
generally located on signal transduction factors participating in PPIs.135 They must derive
significant binding energy from the phosphorylated tyrosine residue so that binding is dictated by
the pY/Y (“active/inactive”) state. Furthermore, SH2 domains gain substrate specificity from the
residues flanking the pY site. The recognition of adjacent residues not only provides additional
energetically favourable contacts, but also dictates the selectivity of the SH2 domain-
phosphopeptide interaction. For example, studies investigating the Src and Lck SH2 domains have
indicated that although 50% of binding affinity is obtained from the phosphate group of the pY
residue, residues -2 to +4 relative to the phosphotyrosine contribute towards binding specificity
through maximizing their interactions with the surface of the SH2 domain.136 Additionally,
numerous co-crystal structures of SH2 domains bound to their respective ligands suggest a larger
contact surface is plausible.137 The traditional SH2 domain fold comprises of a central anti-parallel
β-sheet flanked by two α-helices providing two chemically unique environments on each side of
the β-sheet, figure 3.5.134 On one side, there is a positively charged pocket consisting of arginine,
serine, lysine/arginine and histidine that recognize the pY moiety. This group of amino acids is
highly invariable across all SH2 domains and participates in coordination of the oxygen atoms on
the phosphate functionality. On the other hand, the secondary pocket adjacent to the central β-sheet
provides an extended cleft for the interaction of residues Cterminal to the pY. We are particularly
interested in the molecular structure of this extended surface as it can provide a desirable degree
of selectivity for pY-containing peptide ligands.
59
Figure 3.5- Traditional SH2 domain tertiary structure ribbon illustration. The central anti-parallel
β sheet is flanked by two α-helices. Interacting pY-containing peptide is shown in black, stick
figure representation, with pTyr, +1, +2 and +3 sites labeled. (figure from ref 60).
Despite sharing overall 93% sequence homology, STAT5A and STAT5B differ in 6 amino acids
in their respective SH2 domains. This lends further support of targeting the SH2 domain as this
specific interface of the STAT5A/B transcription factors has differences in molecular structure that
can govern binding affinity for phosphopeptide ligands and generate selectivity. Unfortunately,
only the STAT5A protein crystal structure has been reported.138 To conduct a thorough comparison
of the SH2 domains of both isoforms, we engineered a STAT5B protein 3D model using homology
modeling with STAT5A as the sequence template. The STAT5A crystal structure was retrieved
from the Research Collaboratory for Structural Bioinformatics Protein Data Bank (PDB ID code:
1Y1U) and the methodology described in chapter 2 used to generate the inactive ROS1 structure
was employed. Briefly, using the Prime 3.9 module from Schrödinger Suite, the STAT5A protein
sequence was used as a template and the STAT5B amino acid sequence was inserted. The differing
amino residues were replaced using a library of side chain residues and was subjected to energy
minimization using the OPLS_2005 force field. Finally, loop refinement and side chain
optimization generated the final STAT5B model. Upon careful examination, we observed the
conventional SH2 domain arrangement in both isoforms. We proposed that the positively charged
60
pY recognition site encompasses residues Arg618, Lys600 and Ser622 allowing electrostatic and
hydrogen bond interactions with the phosphotyrosine group originating from either a receptor or a
STAT5 binding partner, see figure 3.6. The six variable residues between STAT5A and STAT5B
were in moderate neighbouring distance to the pY-binding cleft. Amino acids Pro636, Leu639 and
Asn640 occupying the α-helix flanking the antiparallel β-sheet in
STAT5A were replaced by Gln, Phe and Met respectively in STAT5B. Located on the terminal
βstrand, the residue within closest proximity to the critical Arg618 was Lys644 in STAT5A
whereas the identical position was occupied by Met in STAT5B as shown in figure 3.6. Thus, there
was some promising variability between the molecular architectures of the STAT5A and STAT5B
SH2 domains that could be utilized by different pY-containing peptide ligands. The remaining
substitution in STAT5A, for Asn in STAT5B at position 654 is situated out of the scope of the
pYbinding site.
B+
Figure 3.6- A) Overlap of STAT5A (orange) and STAT5B (blue) ribbon figures. The five differing
residues in the SH2 domain in the vicinity of the pY binding motif are represented by stick figures.
B) Shared Ser622, Arg618 and Lys600 in STAT5A/B SH2 domains. On comparison with
conventional SH2 domain structure, these three residues contribute to recognition of pY functional
group. (Image generated using MacPyMol 2009-2010)
With a hot-spot target in hand, we then explored receptor sequences known to bind to
STAT5. In the early stages of peptidomimetic design, it is always preferable to use native binding
peptides for the target given their naturally high binding affinity and selectivity.
P r o 6 3 6 + G l n 6 3 6 +
P h e 6 7 9 + T y r 6 7 9 +
L y s 6 4 4 + M e t 6 4 4 +
A s n 6 3 9 + M e t 6 3 9 +
L e u 6 4 0 + P h e 6 4 0 +
S T A T 5 A % S T A T 5 B %
A +
A r g 6 1 8 +
L y s 6 0 0 +
S e r 6 2 2 +
61
Erythropoietin (Epo) is a cytokine whose signaling is necessary for the proliferation,
differentiation, and survival of erythrocyte precursor cells in erythropoiesis.139 Epo elicits its
cellular response by binding to the Epo receptor (EpoR), triggering receptor dimerization and
subsequent phosphorylation by receptor associated JAK2.140 STAT5 is then recruited to the
intracellular domain of EpoR, triggering the STAT5 signaling pathway. Silva et al. first reported
the survival function of Epo, through its induced expression of Bcl-xL. Upon Epo stimulation in
Ba/F3 cells transfected with EpoR, increased levels of Bcl-xL expression were concurrent with
activation of STAT5, along with expression of another STAT5 target gene, oncostatin M.141 In
1996, Klingmüller and colleagues verified the role of EpoR pY in STAT5 recruitment and were
able to deduce the exact site in the 96-amino acid intracellular domain. Through mutation studies
of all eight tyrosine residues in the intracellular domain of Epo, immunoblotting for pSTAT5
suggested that Y343 or Y401 are sufficient for maximal activation of STAT5 whereas Y429 and
Y431 can partially activate STAT5.142 A following investigation by Quelle and co-workers in 1996
confirmed the significance of residue Y343 in localization of STAT5 and its activation to EpoR.143
Cells transfected with Y343 truncated-EpoR could not induce STAT5 phosphorylation mediated
signaling. A competition gel-based assay showed that pY343 containing peptide successfully
prevents the binding of DNA:STAT5 by disrupting the formation of STAT5 dimers. In this
competition assay, the peptide was inclusive of residues flanking pY343 on the N- and C-terminus,
with an overall sequence: QDTpY343LVLDKWL. Berg et al. used this EpoR derived sequence to
design a fluorescence polarization assay for STAT5B.144 Through their method development, they
discovered that amino acids at the N-terminus of the pY should be omitted and the insertion of a
glycine linker between the pTyr and the fluorophore, 5-carboxyfluorescein (5-FAM) prevented any
interference with binding to STAT5B. Using the 5-FAM modified peptide as their fluorescent
probe, a Ki value of 0.21 ± 0.03 μM for the QDTpYLVLDKWL ligand was obtained. Thus, the
EpoR phosphopeptide is an appropriate candidate to screen for potency for STAT5A, followed by
a thorough combinatorial alanine scanning.
Another ligand known to activate the STAT5 signaling pathway is the
granulocytemacrophage colony-stimulating factor (GM-CSF). The GM-CSF glycoprotein was
first reported for its capacity to modulate the production of specific leukocytes including
granulocytes, macrophages, eosinophils and megakaryocytes in mice, through process termed as
62
myelopoiesis.145 The sequencing of the human GM-CSF factor revealed its pivotal role in regulate
the survival, proliferation and differentiation of monocytes and macrophages.146 The GM-CSF
receptor (GM-CSFR), is a heterodimer, consisting of a ligand-binding α-subunit and the βc-subunit
which is a chain common to the cytokine receptors, IL-3 and IL-5. Upon GM-CSF binding, the
two subunits undergo dimerization, prompting the transphosphorylation of two βc-subunits via
receptor-associated JAK2.147 The GM-CSF induced activation of STAT5 is know to upregulate the
transcription of target gene elements related to inflammation and survival, pim-1 and
cytokineinducible SH2-containing protein (CIS).146 Numerous investigations in monocytes,
macrophages, and Ba/F3 cells transfected with GM-CSFR have pointed out STAT5 activation is a
key event in GM-CSF signaling.148 All evidence strongly suggests that pSTAT5 levels and
downstream transcriptional markers increase upon GM-CSF stimulation. In terms of a specific pY
containing sequence responsible for STAT5 recruitment, van Dijk et al. found Y612 of the βc-
subunit was independently capable of inducing activation of endogenous STAT5 upon GM-CSF
stimulation.149 While there were two other tyrosine residues reported for GM-CSF mediated
STAT5 activation150,
May and co-workers confirmed that Y612 of the βc-subunit, or Y882 of the entire GM-CSFR was
responsible for STAT5 recruitment using hybrid receptors.151 Their chimeric receptor construct
composed of the extracellular Epo domain and the gp130 transmembrane and intracellular regions.
The intracellular domain of gp130 presents a motif negative for STAT5 localization. Hence,
addition of various tyrosine modules to the gp130 fold can provide insight on their ability to recruit
STAT5, upon receptor stimulation. An EMSA readout was used to determine the extent of pSTAT
levels after stimulating COS-7 cells expressing chimeric receptors containing the QDYLSLP
sequence in the intracellular domain representative of the module QDpY882LSLP. We decided to
expand the phosphopeptide sequence for our studies to maintain consistency with EpoR with
regards to sequence length flanking the pY motif. Upon exploring the receptor sequence of the
GM-CSF, we obtained QQDpYLSLPPWE.
The STAT5 signaling cascade is known to transmit horomone-related responses to the
nucleus. Prolactin is a peptidic hormone that has been established as a ligand responsible for
STAT5 phosphorylation.152 Not only does prolactin balance lactogenesis, it also plays a profound
role in lymphocyte development and proliferation. Prolactin is believed to be involved in positively
effecting the uncontrolled division of immature granulocytes, activation of malignant B
63
lymphocytes and lymphoma cells.153,154 In addition, several autoimmune diseased states are
believed to be caused by prolactin activity. Specific transcription products of prolactin-STAT5
signaling include c-myc in lymphoid tissue.155 Prolactin relays external stimuli to the
phosphorylation of STAT5 using the prolactin receptor (PRL-R) and receptor associated JAK2.
Prolactin ligand binding to PRL-R follows the exact same cascade mentioned for EpoR and
GMCSFR. Pezet and colleagues conducted selective mutation studies of all tyrosine groups in the
PRL-R intracellular domain. Immunoprecipitation studies with 293 fibroblasts expressing rat
PRLR revealed upon substitution of all tyrosines to phenylalanine residues, STAT5 activity was
completely lost. Upon narrowing down the mutation studies to individual tyrosine motifs, three
positions were determined as potential STAT5 docking sites: Y473, Y479 and Y580.156 However,
when comparing the rat PRL-R sequence to the human analog, only Y479 was conserved,
translating to Y509. Thus, we proceeded with this phosphotyrosine site and ensured that the amino
acids spanning from Y-3 to Y+7 were identical. Similar to their observation of GM-CSFR-STAT5,
May et al. observed similar activation of STAT5 with the Y509 module. The final phosphopeptide
taken forward was PLDpYVEIHKVN.
Finally, we looked into deriving a high affinity pY containing ligand from the IL-2 receptor.
Interleukin 2 (IL-2) is a signaling cytokine molecular utilized by the innate immune system to
promote the differentiation of immature T cells into helper T cells, memory T cells and suppressor
T cells. These lymphocytes also rely on cytokine signaling for proliferation, including IL-2. IL-2
uses transactivation of the JAK/STAT5 mechanism to induce C-MYC, BCL-2 and BCLXL gene
expression.157 The IL-2 receptor (IL-2R) consists of three distinct chains, IL-2Rα, IL-2Rβ and IL-
2Rγ. While IL-2Rα is not involved in signaling, IL-2 binding initiates the heterodimerization of
the β and γ chains.158 The IL-2Rγ subunit is shared amongst other IL receptors, whereas the IL-
2Rβ polypeptide is unique to IL-2 and IL-5 as it is associated with JAK1.159,160 Amongst relevant
tyrosine sites prone to phosphorylation, Y338 is required for activation of SHC-1 protein while
phosphorylation of Y392 and Y510 is known to activate STAT5. Lord et al. demonstrated complete
loss of STAT5 activation levels when all tyrosines except Y510 in IL-2Rβ were deleted in CTLL-
2 T cells and Ba/F3 transformed cells.160 Activated STAT5 levels and T cell proliferation were
comparable with the non-mutant receptor constructs, suggesting Y510 is the most potent activating
residue. The expression levels of downstream STAT5 target genes were also rescued upon using
64
the Y510 containing IL-2Rβ receptor chain. Given the strong activity of Y510 in IL-2Rγ in the
phosphorylation of STAT5, this sequence was an ideal choice for our initial pY peptide screening.
The overall sequence considered was TDApYLSLQELQ.
3.6 Initial FP analysis of proposed phosphopeptides
The biologically active peptides known to recruit STAT5 upon external stimulation are
summarized below. We first decided to screen these pY peptides and their non-phosphorylated
counterparts against both STAT5A and STAT5B using FP. Berg and co-workers had initially
reported a high-throughput FP assay for STAT5B in 2008 and a complementary assay for STAT5A
in 2015 using the EpoR derived 5-FAM-GpYLVLDKW as their fluorescent reporter. We first
conducted saturation experiments with the fluorescent probe with both STAT5A and STAT5B to
determine KD values for both proteins.
Fluorescence assays were performed in black, flat bottomed, non-treated, 384-well plates
(Corning #3573) and FP measurements were taken with the Infinite M1000 machine (Tecan,
Crailsheim, Germany). The excitation wavelength was set at 475 nm and the emission wavelength
was observed at 525 nm, characteristic of 5-carboxyfluorescin. Measurements were taken with an
optimal gain, 50 flashes and a G-factor of 1. The buffer conditions for all assays were 10 mM
HEPES, 25 mM NaCl, 1mM EDTA, 2 mM dithiothreitol, pH 7.5 and the final DMSO
concentration in the wells was kept constant at 10%. DMSO was used to improve the solubility of
the inhibitor compounds in the buffer system, as well as the fluoresceinated-phosphopeptide. A
calibration curve for STAT5A and STAT5B protein was derived by incubating a 10 nM final
concentration of the EpoR derived fluorescent-phosphopeptide, 5-FAM-GpYLVLDKW, which is
known to bind STAT5A/B’s SH2 domains. Increasing concentrations of STAT5 protein were
titrated ranging from 0 to 2.5 µM. The raw data was normalized in excel and fitted with a 1:1
binding model using GraphPad Prism 6.0. Saturation curves are shown below, figures 3.7 and 3.8
for STAT5A and STAT5B respectively.
65
Figure 3.7- Calibration curve for EpoR derived 5-FAM-GpYLVLDKW fluorescent probe with
STAT5A protein.
Figure 3.8- Calibration curve for EpoR derived 5-FAM-GpYLVLDKW fluorescent probe with
STAT5B protein.
The 5-FAM-EpoR probe showed saturation with both STAT5A and STAT5B as expected.
C a l i b r a ti o n C u r v e ST A T 5 B
66
The KD values obtained for both proteins were comparable with reported literature values, KD =
123 nM cf. 130 nM for STAT5A and KD = 126 nM cf. 125 nM for STAT5B. Next, we conducted
competition experiments with all four phosphopeptides and their non-phosphorylated analogs
listed in Table 1. However, peptides were ordered from CanPeptide, and only the EpoR and GM
CCSFR peptides were synthesized and delivered. The non-phosphorylated negative controls of
EpoR and GM-CSFR and remaining peptide library will be synthesized using solid-phase peptide
synthesis (SPPS).
Table 3.1- Summary of pY-containing ligands and negative control analogs
Receptor pY Derived Sequence Non-phosphorylated analog
EpoR QDTpYLVLDKWL QDTYLVLDKWL
GM-CSFR QQDpYLSLPPWE QQDYLSLPPWE
PRL-R PLDpYVEIHKVN PLDpYVEIHKVN
IL-2Rβ TDApYLSLQELQ TDAYLSLQELQ
For the STAT5A and STAT5B competitive-binding fluorescence polarization assays, the
5-FAM-GpYLVLDKW peptide and wildtype STAT5A/B protein (purchased from SignalChem)
were first incubated for 20 minutes at room temperature. Inhibitors were titrated at concentrations
ranging from 1 nM – 500 µM and incubated for a further 15 minutes. The fluorescence polarization
measurements were then taken in triplicate (λex = 470 nm, λem = 525 nm). The final well
concentration of the fluoresceinated-phosphopeptide was 10 nM after addition of the inhibitor
component. Final concentrations of STAT5A and STAT5B proteins were 130 nM and 125 nM,
respectively. The resulting fluorescence polarization measurements were normalized and plotted
against inhibitor concentration. The raw data was fitted with a standard dose response inhibition
curve with four parameters using GraphPad Prism 6.0 software. The IC50 values were converted to
Ki values using Equation 1, the Nikolovska-Coleska equation.161
Equation 1
There were major discrepancies in the initial set of data obtained, particularly pertaining to the
experiments with STAT5A. Fluorescence polarization values observed were not consistent with
67
any binding event under the given experiment conditions. The overall change in fluorescene
polarization (mP units) for the STAT5A experiments (~100 units) was much smaller relative to
STAT5B (~300 units). Upon retaking measurements after 1 minute and 5 minutes delay after the
initial measurement, the observed fluorescence anisotropy values drastically changed across all
concentrations ranging from no inhibitor to expected complete displacement of probe. We
attributed this to the quality or the stability of the STAT5A protein given the consistency of the
results from the experiments with STAT5B protein. Competition binding experiments for STAT5A
were then altered by removing the incubation times so that protein stays at 0 oC. This gave much
more reliable data with a larger range of fluorescence anisotropy (~150 units). The competition FP
curves for EpoR and GM-CSF derived peptides against STAT5A (0 oC, no incubation) and
STAT5B (room temperature) are shown below with Ki values summarized in Table 2.
Figure 3.9- Normalized FP inhibition curve for EpoR derived peptide, QDTpYLVLDKWL for
STAT5A. Ki = 522.3 nM
Ep o R ST A T 5 A
Log[ Ep o R i n µ M]
68
Figure 3.10- Normalized FP inhibition curve for EpoR derived peptide, QDTpYLVLDKWL for
STAT5B. Ki = 426.0 nM
Figure 3.11- Normalized FP inhibition curve for GM-CSFR derived peptide, QQDpYLSLPPWE
for STAT5B. Ki = 876.6 nM
Ep o R ST A T 5 B
Log[ Ep o R i n µ M]
G M-C SF R ST A T 5 A
L o g [ G M-C SF R i n µ M]
69
Figure 3.12- Normalized FP inhibition curve for GM-CSFR derived peptide, QQDpYLSLPPWE
for STAT5A. Ki = 652.4 nM
Table 3.2- Calculated Ki values for EpoR and GM-CSFR derived peptide ligands
pY Peptide STAT5A Ki STAT5B Ki
QDTpYLVLDKWL (EpoR) 522.3 nM 426.0 nM
QQDpYLSLPPWE (GM-CSFR) 876.6 nM 652.4 nM
Both EpoR and GM-CSFR derived phosphopeptides bind with much higher affinity to the
STAT5B SH2 domain in comparison to the STAT5A SH2 domain. However, given the observed
discrepancy in the FP experiments with the STAT5A protein and pending Ki values from other
ligands, there are no conclusive trends. The data also indicates incomplete displacement of the
fluorescent tag from the STAT5A SH2 domain for both EpoR and GM-CSFR derived peptides.
The measurements taken at the higher concentration of the inhibitor are not completely reliable
given the temporal instability of STAT5A and may consequently affect the calculated Ki values.
To circumvent this, we will possibly look to another source for STAT5A protein given its lack of
application for a high-throughput assay. Complete binding saturation of the fluorescent probe was
observed since there was no incubation time, but the range of anisotropy values are still not
consistent with those reported literature by Berg et al. The workers utilized a different plasmid
vector for expression of STAT5A and an N-terminal maltose protein binding tag. Upon obtaining
functional STAT5A protein, we will look to redo the calibration curves and inhibitor experiments.
G M-C SF R ST A T 5 B
L o g [ G M-C SF R i n µ M]
70
3.7 Conclusion
The development of STAT5A/B isoform selective peptidomimetic inhibitors is still in its primitive
stages. We are attempting to narrow down the SH2 domain as the “hot-spot” of STAT5A/B and
examine the structural differences in this region between the two isoforms in silico. Through
exhaustive literature research, we deduced a novel peptidomimetic strategy involving
pYcontaining peptides that are known to recruit STAT5A/B transcription factors and cause their
eventual activation. Using FP as our initial assay to determine the binding affinities of the proposed
peptides, we will look to study the efficacy of each receptor-derived ligand. Important trends and
contribution of each amino acid in the phosphopeptide sequence will be confirmed with an alanine
scan mutagenesis study.
71
Chapter 4 Conclusions and Future Directions
Chromosomal aberrations involving structural changes in the original genome lead to
dysregulation of affected genes or a hybrid genetic product. In the case of cellular receptor tyrosine
kinases, fusion genes often result in a kinase domain juxtapositioned to a foreign extracellular
receptor. ROS1 is an RTK that has been reported as a fusion partner with several receptors. ROS1
rearrangements are capable of enforcing oncogene addiction and numerous clinical studies have
highlighted ROS1 fusions as molecular drivers in numerous malignancies, most notably, NSCLC
(CD74-ROS1). Conventional therapy involves small-molecule TKIs that recognize the ATP site of
the enzyme. Despite initial success with the first-line therapeutic crizotinib, cases of acquired drug
resistance have been reported. Specifically, in ROS1 fusion positive NSCLC patients, a G2032R
point mutation in the ROS1 kinase domain reduces sensitivity to crizotinib and has warranted the
use of other TKIs. With the emergence of cabozantinib and foretinib as highly potent and selective
ROS1 and ROS1G2032 inhibitors, we were interested in the structural properties of the scaffolds that
contribute to their privileged resistance profiles. We designed in silico models of the
ROS1and ROS1G2032 kinase domains and conducted computational docking to study how
cabozantinib and foretinib interact with the relevant binding pockets. Our initial work suggests that
both TKIs occupy an unprecedented binding conformation with ROS1, with access to a
hydrophobic region adjacent to the ATP site. Furthermore, cabozantinib exhibited a much more
consistent binding profile with ROS1 over foretinib, prompting us to explore the effect of the major
functional groups that distinguish cabozantinib as a unique ROS1 inhibitor. We screened a large
library of cabozantinib derivatives encompassing iterative changes in the buried 4-fluorobenzene
appendage with numerous fluorine isosteres, 5 and 6-membered aromatic rings including
heteroatoms. We even probed the solvent-exposed 6,7-dimethoxyquinoline region of cabozantinib
with bulkier alkyl groups and bridged cyclic structures. With no initial trends, we focussed on the
4-fluorobenzene by preparing a preliminary library of cabozantinib analogues with various
substituents at the para position. Along with this, we synthesized another library of molecules in
which the 4-fluorobenzene group was kept constant and the 6,7-dimethoxyquinoline was modified
to investigate the entropic contributions to cabozantinib-ROS1 binding. The initial library was
found to be not as active as cabozantinib when screened for anti-proliferation activity against Ba/F3
cells transfected with CD74-ROS1 or CD74-ROS1G2032R. More interested in the inhibitor
72
interaction with the ROS1 kinase domain, we decided to employ a FRET-based LanthaScreen
assay to derive KD values for our library. The assay results were in corroboration with the trends
observed from the cellular viability experiment. The KD value of cabozantinib was calculated to be
21.43 ± 1.642 nM with no marked improvement in ROS1 binding affinity for the synthesized
derivatives. Collectively, the results obtained from both in vitro experiments contradict the binding
affinities predicted by GLIDE in silico. With our continued efforts, we hope to study the structural
and resistance profile of cabozantinib for ROS1 and the clinically relevant ROS1G2032 mutant and
decipher how this unique TKI interrogates the kinase domain.
This thesis also covered the work towards the design of peptidomimetic inhibitors for a
transcription factor downstream of aberrant kinase activity. We focussed specifically on STAT5,
a signaling protein critical in the JAK/STAT pathway driving cell differentiation, growth and
apoptosis in lymphoid and hematopoietic systems. Considering the drawbacks of conventional TKI
chemotherapy, we explored the therapeutic potential of STAT5 with emphasis on the two isoforms,
STAT5A and STAT5B. The pY-recognizing SH2 domain is the major hot-spot for
STAT5’s protein-protein interactions. We designed an in silico model to investigate the differing
residues in the STAT5A and STAT5B SH2 domains and concluded that peptidomimetic based
inhibitors developed from the critical pY would be an effective strategy for isoform-selective
inhibition. Native receptor sequences that interact strongly with STAT5A/B’s SH2 domains were
deduced and taken forward for competitive binding experiments against STAT5A and STA5B. We
performed FP experiments with each receptor-derived sequence to determine Ki values that would
allow us to elucidate any trends or critical residues amongst the peptide ligands. The initial set of
inhibitor competition experiments revealed the instability of STAT5A and other sources the protein
are being investigated. Finally, we will look to examine the contribution of each amino acid in the
most potent pY-peptide through alanine mutagenesis combinatorial screening. Selective
truncations and eventual ‘drug-like’ modifications will be imposed after the alanine scan. We hope
to develop potent and isoform selective STAT5A/B peptidomimetic inhibitors for use in the study
and potential treatment of isoform specific STAT5 associated diseases.
73
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