supplementary materials for - science · 2013. 7. 3. · (darmstadt, germany) in ddh 2 o. blocking...
TRANSCRIPT
www.sciencemag.org/cgi/content/full/341/6141/84/DC1
Supplementary Materials for
Monitoring Drug Target Engagement in Cells and Tissues Using the Cellular Thermal Shift Assay
Daniel Martinez Molina, Rozbeh Jafari, Marina Ignatushchenko, Takahiro Seki, E. Andreas Larsson, Chen Dan, Lekshmy Sreekumar, Yihai Cao, Pär Nordlund*
*Corresponding author. E-mail: [email protected]
Published 5 July 2013, Science 341, 84 (2013) DOI: 10.1126/science.1233606
This PDF file includes:
Materials and Methods Supplementary Text Figs. S1 to S4 Table S1 References
2
Materials and Methods.
Chemicals and Buffers. TNP-470, inhibitor of MetAP2, was from Takeda
Chemicals (Osaka, Japan). Vemurafenib (PLX4032), SB590885 and AZ628, inhibitors of
BRAF; AZD5438, inhibitor of CDK2 and CDK9; PD0332991, inhibitor of CDK4 and
CDK6; Olaparib (AZD2281) and Iniparib (BSI-201), inhibitors of PARP-1 were from
Selleck Chemicals (Houston, TX, USA). TS inhibitors Raltitrexed (Tomudex) and 5-FU
(5-Fluorouracil); DHFR inhibitor Methotrexate (Abitrexate); transport inhibitors Suramin
and S-(4-Nitrobenzyl)-6-thioinosine (NBMPR); and the TS substrate 2′-Deoxyuridine 5′-
monophosphate (dUMP) were from Sigma-Aldrich (St. Louis, MO, USA). All
abovementioned chemicals were dissolved and diluted using dimethyl sulfoxide (DMSO)
except dUMP that was dissolved and diluted in ddH2O. Phosphate-buffered saline (PBS)
was prepared using 10 mM phosphate buffer (pH 7.4), 2.7 mM potassium chloride and
137 mM sodium chloride. Kinase buffer (KB) (25 mM Tris(hydroxymethyl)-
aminomethane hydrochloride (Tris-HCl, pH 7.5), 5 mM beta-glycerophosphate, 2 mM
dithiothreitol (DTT), 0.1 mM sodium vanadium oxide, 10 mM magnesium chloride) was
from Cell Signaling Technology (Beverly, MA, USA). Tris-Buffered Saline with Tween
(TBST) buffer (150 mM NaCl, 0.05% (v/v) Tween-20, 50 mM Tris-HCl buffer (pH 7.6))
was prepared by dissolving TBS-TWEEN tablets obtained from Merck KGaA
(Darmstadt, Germany) in ddH2O. Blocking buffer was 5% (w/v) non-fat milk (Semper
AB, Sundbyberg, Sweden) diluted in TBST. Complete (EDTA-free) protease inhibitor
cocktail was from Roche (Switzerland). Isothermal calorimetry buffer (ITC buffer) (1%
DMSO, dUMP 100 µM, 20 mM Hepes pH 7.5 and 150 mM NaCl) was prepared prior to
experiments.
3
Heterologous protein expression. Genes (P26-V313 of human TS and K662-
T1011 of human PARP1) were subcloned into the pNIC28-Bsa4 vector (GenBankTM
accession number EF198106), yielding an expression construct with an N-terminal
hexahistidine tag and a TEV protease recognition site. The positive recombinant clone
was retransformed and expressed in T1 phage-resistant BL21(DE3) E. coli strain
(Merck). For expression, cells were grown at 37°C in a LEX system using 0.75 L of
Terrific Broth medium supplemented with 8 g/L glycerol, 50 µg/mL kanamycin, and 34
µg/mL chloramphenicol. When OD600 reached ca. 2, the temperature was reduced to
18°C. After 30–60 minutes, the expression of the target protein was induced by addition
of 0.5 mm isopropyl β-d-thiogalactopyranoside and incubation for 17–20 h. The cells
were harvested by centrifugation and resuspended in lysis buffer (100 mM HEPES, 500
mM NaCl, 10 mM imidazole, 10% (v/v) glycerol, 0.5 mM TCEP, pH 8.0) supplemented
with Protease Inhibitor Mixture Set III, EDTA free (Merck) and 2000 units of benzonase
(Merck), and stored at −80°C. Cells were disrupted by sonication on ice using Vibra-Cell
processor (Sonics & Materials Inc., Newtown, CT, USA). The lysate was clarified by
centrifugation at 47,000 × g for 25 min at 4°C, and the supernatant was filtered through a
1.2-µm syringe filter. Filtered lysates were loaded onto 1 mL of nickel-nitrilotriacetic
acid Superflow resin (Qiagen Inc., Valencia, CA, USA) in IMAC wash buffer 1 (20 mM
HEPES, 500 mM NaCl, 10 mM imidazole, 10% (v/v) glycerol, 0.5 mM TCEP, pH 7.5)
and washed with IMAC wash buffer 2 (20 mM HEPES, 500 mM NaCl, 25 mM
imidazole, 10% (v/v) glycerol, 0.5 mM TCEP, pH 7.5). Bound proteins were eluted with
500 mM imidazole and loaded onto a HiLoad 16/60 Superdex-200 column (GE
4
Healthcare, Waukesha, WI, USA) pre-equilibrated with equilibration buffer (20 mM
HEPES, 300 mM NaCl, 10% (v/v) glycerol, 0.5 mM TCEP, pH 7.5). Fractions containing
the protein of interest were pooled. TCEP was added to a final concentration of 2 mM,
and the sample was concentrated using Vivaspin 20 filter concentrators (15 kDa MW
cutoff) (GE Healthcare) at 15°C. The final protein concentration and yield was 15.5
mg/mL, 1.4 mg (PARP-1) and 20.6 mg/mL, 30 mg (TS). The protein batches were then
aliquoted, frozen in liquid nitrogen, and stored at −80°C.
Cell culture and in vitro experiments. Human cancer cell lines K562 (ATCC
No. CCL-243) and A549 (ATTC No. CCL-185) were cultured in RPMI-1640 medium
(Sigma-Aldrich); A375 cells (ATCC No. CRL-1619) were maintained in Dulbecco’s
Modified Eagle Medium (Sigma-Aldrich), and HEK-293 cells (ATCC No. CRL-1573)
were maintained in Eagle's Minimum Essential Medium (Sigma-Aldrich). All culture
media were supplemented with 0.3 g/L L-glutamine and 10% fetal bovine serum (FBS,
Gibco/Life Technologies, Carlsbad, CA, USA), 100 units/mL penicillin and 100 units/mL
streptomycin (Gibco/Life Technologies). Short-term passages (<15) were used for in
vitro cell experiments. The cell line K562 was used for the CDKs, TS and DHFR
experiments; A549 for MetAP2 experiments; HEK293 for PARP-1 and TS experiments.
A375 (BRAF V600E) and K562 (BRAF wild-type) were used for BRAF experiments
(Table I). Equal numbers of cells (0.6-1.0x106 cells per data point) were seeded in T-25
cell culture flasks (BD Biosciences, San Jose, CA, USA) or 12-well cell culture plates
(Corning Inc., Corning, NY, USA) in appropriate volume of culture medium and exposed
to a drug for 3 hours in an incubator chamber (with 5% CO2) (Memmert GmbH,
Schwabach, Germany). Control cells were incubated with an equal volume of diluent for
5
the corresponding drug. For drug concentrations see Supplementary Table I. Following
the incubation the cells were harvested (either directly or detached from the surface using
Trypsin/EDTA solution (Sigma-Aldrich)) and washed with PBS in order to remove
excess drug. Equal amounts of cell suspensions were aliquoted into 0.2 mL PCR
microtubes, and excess PBS was removed by centrifugation to leave 10 uL or less PBS in
each microtube. These cell pellets were used for CETSA as described below.
For transport inhibition experiments equal numbers of K562 cells (0.6x106 cells
per data point) were seeded in 12-well cell culture plates in appropriate volume of culture
medium and pre-incubated with a transport inhibitor (Suramin or NBMPR) for 30
minutes in an incubator chamber. The appropriate inhibitor concentrations were
determined in preliminary CETSA experiments (data not shown). Control cells were
incubated with an equal volume of DMSO. The cells were then exposed to varying
concentrations of an appropriate drug (Methotrexate or 5-FU, respectively) for 3 hours in
an incubator chamber. Following the incubation the drug-containing media were removed
by centrifugation; the cells were harvested, washed with PBS and prepared for CETSA as
described below.
For time-course experiments equal numbers of K562 cells (0.6x106 cells per data
point) were seeded in T-25 cell culture flasks and exposed to varying concentrations of
Raltitrexed. Cell culture aliquots were removed at specified times; the cells were washed
with PBS and prepared for CETSA as described below.
For re-feeding experiments equal numbers of K562 cells (0.6x106 cells per data
point) were seeded in 12-well cell culture plates in appropriate volume of culture medium
and exposed to varying concentrations of Raltitrexed for 10 minutes, 30 minutes, or 3
6
hours in an incubator chamber. Following the incubation the drug-containing media were
removed by centrifugation; the cells were harvested, washed with PBS and prepared for
CETSA as described above. The removed media were used to resuspend freshly pelleted
non-treated K562 cells (0.6x106 cells per data point). The cell suspensions were
transferred to fresh 12-well cell culture plates and incubated for additional 3 hours. The
cells were harvested, washed and prepared for CETSA as described below.
Trypan blue dye exclusion. Trypan blue dye exclusion was used to evaluate the
integrity of cell membranes in the heat treated cells. Approximately 0.6x106 cells from
different cell lines were heated at different temperatures for 3 minutes and allowed to
cool to RT for 3 minutes. A 10µL cell aliquot from each sample was briefly mixed with
an equal volume of 0.4% (w/v) trypan blue dye solution (Bio-Rad) and analyzed using a
TC20™ automated cell counter (Bio-Rad). Cells with the ability to exclude trypan blue
were considered retaining their cell membrane integrity whereas dye stained cells were
identified as cells with lost cell membrane integrity.
In vivo mice experiment. TNP-470 was dissolved in 100% ethanol, followed by
dilution in PBS containing 5% ethanol. The drug solution was prepared immediately
before use. For the double-blind study, 6-7 week old female C57Bl/6 mice
(Microbiology, Tumor and Cell Biology Animal Facility, Karolinska Institute,
Stockholm, Sweden) were injected subcutaneously with TNP-470 (0 - 20 mg/kg, 0.1 ml)
at the mid-dorsal location. Control animals were injected with the appropriate amount of
ethanol in PBS. All mice were kept in separate cages. After 4 hours, mice were
euthanized; liver and kidneys were removed and stored on dry ice immediately. All
7
animal procedures were carried out in accordance with the Northern Stockholm
Experimental Animal Ethical Committee approved protocols.
Cellular Thermal Shift Assay (CETSA). For the cell lysate CETSA
experiments, cultured cells from abovementioned cell lines were harvested and washed
with PBS. The cells were diluted in KB for BRAF and CDK´s and in PBS for MetAP2,
TS, DHFR and PARP-1. All buffers were supplemented with Complete protease inhibitor
cocktail. The cell suspensions were freeze-thawed three times using liquid nitrogen. The
soluble fraction (lysate) was separated from the cell debris by centrifugation at 20000 x g
for 20 minutes at 4°C. The cell lysates were diluted with appropriate buffer and divided
into two aliquots, with one aliquot being treated with drug (Supplementary Table 1) and
the other aliquot with the diluent of the inhibitor (control). After 10-30 minute incubation
at room temperature the respective lysates were divided into smaller (50µL) aliquots and
heated individually at different temperatures for 3 minutes (Veriti thermal cycler, Applied
Biosystems/Life Technologies) followed by cooling for 3 minutes at room temperature.
The appropriate temperatures were determined in preliminary CETSA experiments (data
not shown). The heated lysates were centrifuged at 20000 x g for 20 minutes at 4°C in
order to separate the soluble fractions from precipitates. The supernatants were
transferred to new microtubes and analyzed by sodium dodecyl sulfate polyacrylamide
gel electrophoresis (SDS-PAGE) followed by western blot analysis. For a schematic
procedure of CETSA see Supplementary Figure S1.
For cell lysate experiments carried out on TS, the dUMP concentration was kept
constant at 100 µM as a binding co-factor to Raltitrexed. Raltitrexed was added from
8
DMSO stocks to the final concentration of 100µM and DMSO concentration 1%. Control
samples were incubated with an equal amount of DMSO.
For cell lysate experiments carried out on PARP1, Iniparib and Olaparib were
added from DMSO stocks to the final concentration of 100µM and DMSO concentration
1%. Control samples were incubated with an equal amount of DMSO.
For the intact cell experiments the drug-treated cells from the in vitro experiments
above were heated as previously described followed by addition of KB (30µL) and lysed
using 2 cycles of freeze-thawing with liquid nitrogen. The soluble fractions were isolated
and analyzed by western blot as described above.
For the in vivo mice experiments, lysates of frozen tissues were used. Since TNP-
470 binds covalently to MetAP2, the drug dissociation from the target after cell lysis
would be limited; the target engagement will therefore be similar to that in the tissue
before lysis. The frozen organs (i.e. liver and kidneys) were thawed on ice and briefly
rinsed with PBS. The organs were homogenized in cold PBS using tissue grinders
followed by 3 cycles of freeze-thawing using liquid nitrogen. Tissue lysates were
separated from the cellular debris and lipids as mentioned above. The tissue lysates were
diluted with PBS containing protease inhibitors, divided into 50µL aliquots and heated at
different temperatures. Soluble fractions were isolated as previously described and
analyzed by western blot.
SDS-PAGE and western blot. NuPage® Novex Bis-Tris 4-12% polyacrylamide
gels with NuPAGE® MES SDS running buffer (Life Technologies) were used for
separation of proteins in the samples. Proteins were transferred to nitrocellulose
membranes using the iBlot® blotting system (Life Technologies) and to polyvinylidene
9
difluoride (PVDF) membranes using Trans-Blot® Turbo™ (Bio-Rad, Hercules, CA,
USA). Primary antibodies anti-MetAP2 (sc-365637), DHFR (sc-81844, sc-377091), TS
(sc-33679), BRAF (sc-9002), CDK2, (sc-6248), CDK4 (sc-601), CDK6 (sc-53638),
CDK9 (sc-13130), PARP-1 (sc-8007), β-actin (sc-69879); secondary goat anti-mouse
HRP-IgG (sc-2055) and bovine anti-rabbit HRP-IgG (sc-2374) antibodies (Santa Cruz
Biotechnology, Santa Cruz, CA, USA) were used for immunoblotting. Rabbit
monoclonal anti-MetAP2 (RabMAb, Epitomics, Burlingame, CA, USA) was used for
western blots for the samples generated from the in vivo mice experiments. All
membranes were blocked with blocking buffer; standard transfer and western blot
protocols recommended by the manufacturers (listed above) were used. All antibodies
were diluted in blocking buffer. The membranes were developed using SuperSignal West
Dura Chemiluminescent HRP-Substrate (Thermo Scientific) according to the
manufacturer’s recommendations. Chemiluminescence intensities were detected and
quantified using a ChemiDoc™ XRS+ imaging system (Bio-Rad) with Image Lab™
software (Bio-Rad). β-actin levels were used to normalize the intensities of the in vitro
cell and in vivo mice experiments. For the CETSA curves the band intensities were
related to the intensities of the lowest temperature for the drug exposed samples and
control samples, respectively. For the ITDRFCETSA experiments the band intensities were
related to control samples.
Statistical analysis. All lysate, in vitro cell and in vivo mice CETSA data were
expressed as means ± SEM. The data (band intensities) from multiple runs (n ≥ 3) were
plotted using Graphpad Prism 6.0 software (GraphPad Software Inc., La Jolla, CA,
USA). The ITDRFCETSA data were fitted using a sigmoidal (variable slope) curve fit.
10
DSLS-experiments. Temperature-dependent aggregation was measured by using
static light scattering (StarGazer, Harbinger Biotechnology) as previously described (12)
at a constant protein concentration of (0.2 mg/mL) using a ramp rate of 1°C/min. Graphs
were plotted with Graphpad and fitted using Sigmoidal dose-response (variable slope).
CETSA-like dot-blot experiments on purified proteins. Purified protein (0.5
µg) was added to the wells of a PCR plate and the volume adjusted to 50 µL by addition
of buffer or cell lysates and ligands depending on the experimental setup. The samples
were heated for the designated time and temperature in a Veriti thermocycler (Applied
Biosystems/ Life Technologies). After heating, the samples were immediately
centrifuged for 15 min at 3000 x g and filtered using a 0.65µm Multiscreen HTS 96 well
filter plate (Merck). 3 µL of each filtrate was blotted onto a nitrocellulose membrane.
Primary antibody Penta-His Antibody BSA-free (Qiagen) and secondary goat-anti-mouse
IgG/HRP conjugate (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA,
USA) were used for immunoblotting. All membranes were blocked with blocking buffer;
standard transfer and western blot protocols recommended by the manufacturers were
used. All antibodies were diluted in blocking buffer. The dot-blot was developed using
SuperSignal West Dura chemiluminescence kit (Thermo Scientific). Chemiluminescence
intensities were detected and imaged using a Fujifilm LAS-3000 imaging system
(Fujifilm, Tokyo, Japan). Raw dot blot images were processed using ImageJ (37). The
background was subtracted and intensities quantified using the MicroArray Profile
(http://image.bio.methods.free.fr/dotblot.html) plugin. Graphs were plotted with
Graphpad software and fitted using sigmoidal dose-response (variable slope).
11
Isothermal titration calorimetry (ITC) of TS. ITC titrations on TS were
performed with Raltitrexed at two different temperatures. Protein and Raltitrexed were
transferred to ITC buffer. The Raltitrexed concentration was 400 µM and protein
concentration in the reaction chamber was 62.5 µM and 61.1 µM for the 25°C and 37°C
experiments, respectively.
12
Supplementary text
Discussion on validation data and theoretical background of CETSA.
Cell and cell membrane integrity: Trypan blue is a dye widely used to
determine integrity of mammalian cells. The blue dye only enters and stains cells when
the cell membrane integrity is lost and pores are formed whereas cells with intact
membranes do not take up this dye and remain unstained when viewed under a
microscope. In order to investigate the integrity of the cells and cell membranes after
heating, a series of dye exclusion experiments were conducted. Cell aliquots of different
cancer cell lines were heated to different temperatures, cooled, mixed with a trypan blue
solution and the cell number and dye exclusion (i.e cell membrane integrity) were
measured. The data demonstrated that for the cell lines studied, most cells are not lysed
even at high temperatures (65°-70°C) (Fig. S2) since the number of cells remains
unchanged compared to the control cells incubated at 37°C. These data confirm that the
cell membranes remain intact at temperatures up to 60-65°C depending on cell line (Fig.
S2).
Correlation of CETSA with TSA on purified proteins and lysate effects.
To determine whether the data obtained by CETSA correlate with data obtained by
conventional TSA, we overexpressed and purified TS and the poly (ADP-ribose)
polymerase domain of PARP-1. We subsequently compared the thermal melt behavior of
the purified proteins using the CETSA protocol to differential static light scattering
(DSLS) data obtained using Stargazer instrument, routinely used for TSA of purified
proteins (12). CETSA of purified PARP-1 with cognate inhibitors replicated the essential
13
features of the DSLS experiment, including the approximate melting temperature of the
ligand-free protein and the shift sizes induced by ligand binding (Fig. S3A).
To allow direct comparison of thermal behavior of a heterologously expressed vs.
endogenous protein from mammalian cells, we performed experiments on purified
polyHis-tagged TS. We obtained DSLS melting curves of the purified TS, as well as its
CETSA profiles upon addition of high concentrations of the folate analog Raltitrexed.
We also performed a CETSA assay on a sample containing an E. coli lysate added to the
purified protein and on a HEK-293 cell lysate containing endogenous TS. The substrate
dUMP is required for Raltitrexed binding and was therefore added at constant
concentration with the purified protein (100µM). Comparative analysis of these two
experiments yielded similar melting temperatures for the unliganded protein as well as
significant shifts for all the proteins upon addition of the compound. The data also
confirmed that addition of the lysate did not have a substantial effect on the experimental
outcome (Fig. S3B).
To shed further light on the lysate effect, we performed ITDRFCETSA on BRAF in
A375 cell lysates. Three cognate inhibitors were used, and each experiment was
performed in a high lysate concentration as well as a low, 10-fold diluted, lysate (Fig.
S3C). Very small effects were caused by two of the inhibitors (AZ628, SB590885), while
PLX4032 was apparently less effective at the higher lysate concentration than at the
lower one, which could be due to protein binding or metabolic factors at the higher lysate
concentration.
Monitoring of parameters in antifolate transport and activation. As for TS
with Raltitrexed (Fig. 2C), a similar shift of ITDRFCETSA was obtained for DHFR with
14
Methotrexate (Fig. S4A). A time-course experiment with Raltitrexed indicated that after
2-3 hours, the ITDRFCETSA of TS was saturated (Fig. 2D); this saturation was not due to
depletion of the drug in the medium (Fig. S4B). By contrast, a similar experiment using
starved cells resulted in a limited accumulation of the drug (Fig. S4C).
Theoretical background for CETSA. Thermal shift assays with purified
proteins are widely used to monitor maximum shifts at high compound concentrations, as
the melting temperature (Tm) correlates to affinities to the target proteins (13, 14). Dose-
response experiments have only been shown to directly yield absolute binding constants
in the specific cases when a fully reversible system can be established (38, 39). Dose-
response dependent shifts start above Tm for the ideally behaving systems. However,
many proteins precipitate upon unfolding; hence the system is not reversible. When
precipitation following unfolding is used for detection, as in DSLS experiments and
CETSA, the systems are clearly not in equilibrium. However the determined aggregation
temperatures at high concentrations of compound, Tagg, are often close to Tm and can be
used as a relative measure to rank affinities (38, 39). When the system is not in
equilibrium, dose-response curves based on Tagg or Tm measurements will be complex
and based on multiple parameters. In these cases the dose-response curves will be
smoother; significantly higher concentrations of compounds will be needed to achieve a
significant response.
To exemplify the temperature dependence of binding constants for one of the targets, we
performed Isothermal calorimetry titrations on TS with Raltitrexed at two different
temperatures. The ITC data was fitted to give a Kd at 25°C and 37°C to 25.8 and 38 nM
respectively. By extrapolation via the integrated van’t Hoff relation, assuming a constant
15
Cp, the Kd at 50°C is estimated to be below 100 nM. Ki values for TS has been reported
to be in the range of 90-1000 nM using different activity assays at 30-37°C (Pubchem;
Raltitrexed CID104758).
Due to the complications discussed above, in the present work we do not attempt to
determine absolute affinities, but instead use an isothermal dose-response curve as a
fingerprint of the target engagement along a range of drug concentrations, ITDRFCETSA.
These experiments are informative as long as a reasonable constant set of measurement
parameters is used (cell or lysate condition, temperature and time), and can be utilized to
compare concentration effects of two different experiments. For example, when it can be
assumed that the target protein is in the same activation state in the two experiments, i.e.,
have the same affinity to the drug, the observed difference in concentration between two
ITDRFCETSA will directly correspond to the difference in the concentration of the added
compound required to establish the same local effective concentration of compound at the
drug target in the lysate or cell. In cases where only the activation state of the protein is
changed, ITDRFCETSA will reflect changes in affinity which will correlate with absolute
affinities. Due to the higher temperatures of the experiment and the continuous loss of
protein due to precipitation during the heating stage, this value will need further de-
convolution to allow better determination of drug affinity at 37°C. We envisage that
detailed studies of the CETSA behavior of a specific protein in lysates at different
conditions, as well as correlation with other data such as IC50 values obtained in lysates
or for purified proteins, could potentially allow direct correlation of CETSA affinities to
absolute affinities in cells. In practice, during drug development programs, the relative
16
ranking of the behavior of compounds within compound series is the main goal, and
ITDRFCETSA should constitute a valuable tool for such prioritizations.
Although CETSA data in many aspects is highly related to the data obtained from
purified proteins (as discussed above), the melting curves in cells and lysates are more
dispersed than for purified proteins. The exact causes for the topology of these curves for
each protein will differ, but it is likely that some of the topology features reflect the
presence of multiple forms of the protein in the cell. Detailed CETSA studies might
therefore allow the convolution of the behavior of different isoforms or activation states
of specific proteins in the cell.
Finally, there is room for significant improvements in the quantification and collection of
CETSA data. To allow rapid establishment of assays for many proteins, and to verify that
the right protein is probed, we used western blots in the present feasibility study.
However, western blots are prone to significant quantification errors. When fully
developed, high-quality and ultra-sensitive immune techniques (40) could assist in
generating improved data whereby smaller samples could be used (currently.
ca. 106 cells). In the present study we used a relatively long temperature exposure time (3
minutes) to make sure that the temperature is equilibrated in the entire sample and that
there is no doubt that the proteins have had time to unfold and aggregate. However, as
most proteins unfold in the second or sub-second time range (41, 42), it should be
possible to shorten the temperature stage significantly in order to minimize other
potential global cellular effects in the cell of the heating stage.
Considering the tissues experiment, which were made after lysis, they are also highly
feasibly to make directly by heating tissue aliquots, to get in cell data for non-covalent
17
binders. However, the establishment of high reproducibility of such experiments requires
some technical developments, which we felt was outside the scope of this initial
feasibility study.
18
Fig. S1.
Figure S1. Schematic illustration of CETSA melt curve and ITDRFCETSA
procedure.
19
Fig. S2.
Figure S2. Cell count and cell membrane integrity. Cell count and dye
exclusion by four different cell lines after heating to different temperatures. Data
presented as mean ± SEM, n ≥ 3.
20
Fig S3.
Figure S3. Correlation of CETSA with TSA on purified proteins and lysate
concentration effects. A) Comparison of thermal shifts (apparent Tm) of
21
purified PARP-1 domain derived from the DSLS light scattering experiment and
the CETSA process. The top graph shows an overlay of melting curves of the
catalytic domain of PARP-1. Two experiments on purified protein were performed
in the same buffer using Stargazer (solid lines) and a CETSA-type denaturation
experiment (symbols and dashed lines) with a set of PARP-1 inhibitors. These
data show that the drop in signal in the centrifugation experiments follows the
increase of Stargazer signal (which monitors the light scattering of the
aggregates formed upon precipitation). B) Correlation of CETSA thermal shifts of
purified proteins in buffer, with lysate added, and in human HEK-293 cells. Bar
graph showing a comparison of TS TAgg data with and without 100uM of
Raltitrexed from Stargazer and CETSA-type denaturation experiments in buffer,
E. coli lysate and HEK-293 lysate. C) ITDRFCETSA at 52°C for BRAF with different
inhibitors in high lysate concentration and in lysate diluted 10 times with buffer
(“Low”) (n=3).
22
Fig S4.
23
Figure S4. Monitoring of parameters in antifolate transport and activation.
A) ITDRFCETSA at 52°C of DHFR with Methotrexate in intact cells vs. lysate (n=4).
B) Saturation of ITDRFCETSA at 52°C of TS. Responses after 10, 30 and 180
minutes of incubation with Raltitrexed. C) The media from these cells were added
to fresh cells followed by incubation for 180 minutes. The response of TS in the
cell shows that Raltitrexed is not depleted in the media. D) ITDRFCETSA at 52°C of
TS in cells grown under starvation conditions (day 2 and 3) indicate decreased
24
transport of Raltitrexed (n=4).
Table S1. Targets, drugs, drug concentrations and cell lines used in this study. ITDRFCETSA Melt curve
Cell lines used
Temperature °C Drug concentration
Target/Drug Lysate Intact Cell
Tissue lysate Lysate
Intact Cell
Tissue lysate
BRAF
A375/K562 PLX4032 52/56 56
100µM -
SB590885 52/56 52
100µM - AZ628 52/56 56
100µM -
CDK2/4/6/9
K562 PD0332991 - 45
100µM 10µM
AZD-5348 52 -
- -
TS
HEK-293/ K562
Raltitrexed 52 52
100µM 10µM Methotrexate - 52
- 10µM
5-FU - 52
100µM 150µM MetAP2
A549
TNP-470 72 - 72 1µM 10µM 0-
20mg/kg DHFR
K562
Methotrexate 52 52
100µM 10µM PARP-1
HEK-293
Olaparib 50 -
100µM - Iniparib 50 -
100µM -
Supplementary Table 1. Temperatures of the isothermal dose-response fingerprint
(ITDRFCETSA) and the drug concentrations used in the saturated CETSA curve
experiments. CDK: Cyclin dependent kinase; BRAF: v-Raf murine sarcoma viral
oncogene homolog B1; MetAP2: methionine aminopeptidase-2; DHFR: dihydrofolate
reductase; TS: thymidylate synthase; PARP-1: Poly [ADP-ribose] polymerase 1. 5-FU: 5-
Fluorouracil.
25
References and Notes
1. K. I. Kaitin, Deconstructing the drug development process: The new face of innovation. Clin. Pharmacol. Ther. 87, 356 (2010).doi:10.1038/clpt.2009.293 Medline
2. J. Orloff et al., The future of drug development: Advancing clinical trial design. Nat. Rev. Drug Discov. 8, 949 (2009). Medline
3. T. A. Yap, S. K. Sandhu, P. Workman, J. S. de Bono, Envisioning the future of early anticancer drug development. Nat. Rev. Cancer 10, 514 (2010).doi:10.1038/nrc2870 Medline
4. M. Rask-Andersen, M. S. Almén, H. B. Schiöth, Trends in the exploitation of novel drug targets. Nat. Rev. Drug Discov. 10, 579 (2011).doi:10.1038/nrd3478 Medline
5. A. Ruiz-Garcia, M. Bermejo, A. Moss, V. G. Casabo, Pharmacokinetics in drug discovery. J. Pharm. Sci. 97, 654 (2008).doi:10.1002/jps.21009 Medline
6. J. P. Gillet, M. M. Gottesman, Mechanisms of multidrug resistance in cancer. Methods Mol. Biol. 596, 47 (2010).doi:10.1007/978-1-60761-416-6_4 Medline
7. R. Barouch-Bentov, K. Sauer, Mechanisms of drug resistance in kinases. Expert Opin. Investig. Drugs 20, 153 (2011).doi:10.1517/13543784.2011.546344 Medline
8. D. S. Auld, N. Thorne, W. F. Maguire, J. Inglese, Mechanism of PTC124 activity in cell-based luciferase assays of nonsense codon suppression. Proc. Natl. Acad. Sci. U.S.A. 106, 3585 (2009).doi:10.1073/pnas.0813345106 Medline
9. C. Schmidt, GSK/Sirtris compounds dogged by assay artifacts. Nat. Biotechnol. 28, 185 (2010).doi:10.1038/nbt0310-185 Medline
10. M. Guha, PARP inhibitors stumble in breast cancer. Nat. Biotechnol. 29, 373 (2011).doi:10.1038/nbt0511-373 Medline
11. B. I. Kurganov, Kinetics of protein aggregation. Quantitative estimation of the chaperone-like activity in test-systems based on suppression of protein aggregation. Biochemistry (Mosc.) 67, 409 (2002).doi:10.1023/A:1015277805345 Medline
12. M. Vedadi et al., Chemical screening methods to identify ligands that promote protein stability, protein crystallization, and structure determination. Proc. Natl. Acad. Sci. U.S.A. 103, 15835 (2006).doi:10.1073/pnas.0605224103 Medline
13. E. Wahlberg et al., Family-wide chemical profiling and structural analysis of PARP and tankyrase inhibitors. Nat. Biotechnol. 30, 283 (2012).doi:10.1038/nbt.2121 Medline
14. O. Fedorov et al., A systematic interaction map of validated kinase inhibitors with Ser/Thr kinases. Proc. Natl. Acad. Sci. U.S.A. 104, 20523 (2007).doi:10.1073/pnas.0708800104 Medline
15. S. Marsh, Thymidylate synthase pharmacogenetics. Invest. New Drugs 23, 533 (2005).doi:10.1007/s10637-005-4021-7 Medline
16. Y. G. Assaraf, Molecular basis of antifolate resistance. Cancer Metastasis Rev. 26, 153 (2007).doi:10.1007/s10555-007-9049-z Medline
17. M. Visentin, R. Zhao, I. D. Goldman, The antifolates. Hematol. Oncol. Clin. North Am. 26, 629, ix (2012).doi:10.1016/j.hoc.2012.02.002 Medline
18. N. Hagner, M. Joerger, Cancer chemotherapy: Targeting folic acid synthesis. Cancer Manag. Res. 2, 293 (2010). Medline
19. M. Huang, Y. Wang, S. B. Cogut, B. S. Mitchell, L. M. Graves, Inhibition of nucleoside transport by protein kinase inhibitors. J. Pharmacol. Exp. Ther. 304, 753 (2003).doi:10.1124/jpet.102.044214 Medline
20. S. Q. Yin, J. J. Wang, C. M. Zhang, Z. P. Liu, The development of MetAP-2 inhibitors in cancer treatment. Curr. Med. Chem. 19, 1021 (2012).doi:10.2174/092986712799320709 Medline
21. J. D. Moore et al., Phase I dose escalation pharmacokinetics of O-(chloroacetylcarbamoyl) fumagillol (TNP-470) and its metabolites in AIDS patients with Kaposi’s sarcoma. Cancer Chemother. Pharmacol. 46, 173 (2000).doi:10.1007/s002800000149 Medline
22. E. Lounkine et al., Large-scale prediction and testing of drug activity on side-effect targets. Nature 486, 361 (2012). Medline
23. J. Cicenas, M. Valius, The CDK inhibitors in cancer research and therapy. J. Cancer Res. Clin. Oncol. 137, 1409 (2011).doi:10.1007/s00432-011-1039-4 Medline
24. M. Malumbres, M. Barbacid, Cell cycle, CDKs and cancer: A changing paradigm. Nat. Rev. Cancer 9, 153 (2009).doi:10.1038/nrc2602 Medline
25. D. W. Fry et al., Specific inhibition of cyclin-dependent kinase 4/6 by PD 0332991 and associated antitumor activity in human tumor xenografts. Mol. Cancer Ther. 3, 1427 (2004). Medline
26. H. Davies et al., Mutations of the BRAF gene in human cancer. Nature 417, 949 (2002).doi:10.1038/nature00766 Medline
27. J. T. Lee et al., PLX4032, a potent inhibitor of the B-Raf V600E oncogene, selectively inhibits V600E-positive melanomas. Pigment Cell Melanoma Res. 23, 820 (2010).doi:10.1111/j.1755-148X.2010.00763.x Medline
28. G. Bollag et al., Clinical efficacy of a RAF inhibitor needs broad target blockade in BRAF-mutant melanoma. Nature 467, 596 (2010).doi:10.1038/nature09454 Medline
29. X. Liu et al., Iniparib nonselectively modifies cysteine-containing proteins in tumor cells and is not a bona fide PARP inhibitor. Clin. Cancer Res. 18, 510 (2012).doi:10.1158/1078-0432.CCR-11-1973 Medline
30. E. Dean et al., Phase I study to assess the safety and tolerability of olaparib in combination with bevacizumab in patients with advanced solid tumours. Br. J. Cancer 106, 468 (2012).doi:10.1038/bjc.2011.555 Medline
31. B. Lomenick, R. W. Olsen, J. Huang, Identification of direct protein targets of small molecules. ACS Chem. Biol. 6, 34 (2011).doi:10.1021/cb100294v Medline
32. J. Luo, N. L. Solimini, S. J. Elledge, Principles of cancer therapy: Oncogene and non-oncogene addiction. Cell 136, 823 (2009).doi:10.1016/j.cell.2009.02.024 Medline
33. G. P. Hussmann, K. J. Kellar, A new radioligand binding assay to measure the concentration of drugs in rodent brain ex vivo. J. Pharmacol. Exp. Ther. 343, 434 (2012).doi:10.1124/jpet.112.198069 Medline
34. B. Lomenick et al., Target identification using drug affinity responsive target stability (DARTS). Proc. Natl. Acad. Sci. U.S.A. 106, 21984 (2009).doi:10.1073/pnas.0910040106 Medline
35. G. M. West, L. Tang, M. C. Fitzgerald, Thermodynamic analysis of protein stability and ligand binding using a chemical modification- and mass spectrometry-based strategy. Anal. Chem. 80, 4175 (2008).doi:10.1021/ac702610a Medline
36. P. M. Matthews, E. A. Rabiner, J. Passchier, R. N. Gunn, Positron emission tomography molecular imaging for drug development. Br. J. Clin. Pharmacol. 73, 175 (2012).doi:10.1111/j.1365-2125.2011.04085.x Medline
37. C. A. Schneider, W. S. Rasband, K. W. Eliceiri, NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671 (2012).doi:10.1038/nmeth.2089 Medline
38. J. K. Kranz, C. Schalk-Hihi, Protein thermal shifts to identify low molecular weight fragments. Methods Enzymol. 493, 277 (2011).doi:10.1016/B978-0-12-381274-2.00011-X Medline
39. D. Matulis, J. K. Kranz, F. R. Salemme, M. J. Todd, Thermodynamic stability of carbonic anhydrase: Measurements of binding affinity and stoichiometry using ThermoFluor. Biochemistry 44, 5258 (2005).doi:10.1021/bi048135v Medline
40. M. Hammond, R. Y. Nong, O. Ericsson, K. Pardali, U. Landegren, Profiling cellular protein complexes by proximity ligation with dual tag microarray readout. PLoS ONE 7, e40405 (2012).doi:10.1371/journal.pone.0040405 Medline
41. A. R. Fersht, V. Daggett, Protein folding and unfolding at atomic resolution. Cell 108, 573 (2002).doi:10.1016/S0092-8674(02)00620-7 Medline
42. U. Mayor, C. M. Johnson, V. Daggett, A. R. Fersht, Protein folding and unfolding in microseconds to nanoseconds by experiment and simulation. Proc. Natl. Acad. Sci. U.S.A. 97, 13518 (2000).doi:10.1073/pnas.250473497 Medline