Selective SH2 Domain Proteomimetics As Potent Disruptors of SH2 Domain-Mediated

Cell Signalling

Joel A. Drewry* and Patrick T. GunningDepartment of Chemistry, University of Toronto; Department of

Chemical and Physical Sciences, University of Toronto

1. Background on SH2 Domains and SH2 Domain Mediated Cell Signalling• Stat3 as an example

2. Rationale for Development of SH2 Domain Proteomimetics• Mask the phosphopeptide vs block the binding domain

3. Current and Future Work• Biphenyl-based scaffolds• Benzothiazoles

Outline

• ~120 SH2 domains across 115 human proteins• STATs, SOCSs, Src, GRB etc

• Highly selectively recognize specific phosphotyrosine-containing sequences on the surface of proteins• pY, pY-1, pY+1 through pY+5 can be important to selectivity

• Mediate protein-protein interactions and mediate cell signalling• SH2 domain on protein A is specific for phosphorylated sequence on

protein B

SH2 Domain Mediated Cell Signalling

SH2 Domain Mediated Cell Signalling

SH2 domains recognize short disordered phosphotyrosine containing peptide sequences on the surface of other proteins – regulatory module of signalling cascades

Translocation to Nucleus

Gene Expression

Inactivation of Stat3 by

Intranuclear Phosphatas

es

Translocation to Cytoplas

m

Activation by

Upstream Stat3

Regulators

• Constituitively active in many cancer types including breast and lung

• Many cancers are dependant on elevated Stat3 levels

• Activity is critically dependant on phosphopeptide-SH2 domain interaction!

J. E. Darnell, Jr., Nature Medicine 2005, 11, 595S. Fletcher; J.A. Drewry; V.M. Shahani; B.D. Page; P.T. Gunning, Biochem Cell Biol. 2009, 87, 825

SH2 Domain Interactions in Oncoproteins: Stat3

cytoplasm

nucleus

YY

Stat3

Stat3

Stat3

Stat3

Phosphorylated Monomer ‘Active’ Protein Complex

• Association relies on phosphotyrosine-SH2 domain interaction between monomers

• Metal complexes bind and mask phosphorylated residue, preventing protein-protein association

SH2A SH2

‘Masked’ Monomer

SH2

M2+ =

SH2 Domain Proteomimetic

SH2 Domain Proteomimetics: Rationale

B

B B

A

A

A A

• Family of mono- and di-functionalized Cu(II) complexes synthesized• Minimal selectivity for phosphopeptides, highly toxic

N

N

N

NN

N

NH

NH

2+Cu

Cu2+

O

O

N

N

N

NN

N

NH2

NH2

2+Cu

2+Cu

H2N

H2N

N

N

N

NN

N

NH

NH

2+Cu

2+Cu

NH

NH

O

O

O

O

OTf4

-

N

N

N

NN

N

NH

NH

O

O

OMe

MeO

2+Cu

Cu2+

O

O

N

N

N

NN

N

NH2

NH2

2+Cu

2+Cu

NNN

NN

N

O

O OMe

OMe

2+Cu Cu2+

OTf4

-OTf

4

-

OTf4

-OTf

4

-OTf

4

-

J. A. Drewry; S. Fletcher; H. Hassan; P. T. Gunning. Org. Biomol. Chem. 2009, 7, 5074J. A. Drewry; P. T. Gunning. Chem. Commun. 2010, 6, 892

Early Work – Monotopic pTyr Receptors

• Advanced scaffold for selective phosphopeptide recognition based on ortho-substituted biphenyl core

• Project hydrophobic/hydrogen bonding functionality directly onto flanking (pY+2,3) peptide regions

N

NNZn2+

N

NNZn2+

O

HN

R

OTf-4-

Ditopic Receptors – Enhanced Selectivity?

2

1

Computationally Guided Inhibitor Design

• L-amino acids in the 2’-position predicted to interact primarily with the amino acid in the pY+2 position

• MD-based study of receptor-peptide conjugate for 50 ns

• L-Tyr functionalized biphenyl bound to Stat3 derived sequence (GpYLKTK)

• Strong electrostatic and hydrophobic interactions predicted

R =

N

NNZn

N

NNZn

O

RNH

O

O

NH

O

O

NH

OH

O

NH

O

O

O

OH

NH

O

O

HO O

NO

ONH

O

O

NH

O

O

NH

OH

O

NOH

O

NH

OH

O

NH

OH

O

NH

O

O

NH

OH

O

NH

O

O

NH

OH

O

NH

NH2

ONH

NH2

O

NH

OH

O

NH2

NH

OH

O

HO

OTf -4

Biphenyls SH2 Domain Proteomimetics

Synthesis

O O

Br

O O N N

O O

OMe OMe

NNN N

N N

O

OH

NNN N

N N

O

NHR

NNN N

N

NNZn2+

N

NNZn2+

O

NHR

B(OH)2

O

OMe

, K2CO3R B(OH)2

R =

Pd(PPh3)3Cl

DMF, 100oC

15hr

81-89%

NH

N

N, NaBH(OAc)3

1,2-DCE

22oC, 3hr

85-92%

NaOH

3:1:1 THF:MeOH:H2O

40oC, 24hr

84-90%

HBTU, DIPEA, NHR

DMF, 23oC, 15hr

75-90% yield

X(OTf)2

MeOH, 23oC, 15hr

99% yield

6 7

8

9 10 Final Receptor

OTf 4

• Fluorescence Intensity of FAM-labelled peptides used to measure binding affinity• Static quenching occurs upon peptide-receptor complexation

Fluorescence Intensity as a Measure of Binding

Receptor vs. Src Sequence

J. A. Drewry; P. T. Gunning. Chem. Commun. 2010, 6, 892

Functionalization Confers Selectivity

J. A. Drewry, P. T. Gunning et al. J. Am. Chem. Soc. 2011, submitted

• FP results (and FI) show diverse and selective binding patters:• Stat3, GP130 selective receptors identified

Functionalization Confers Selectivity

N

NNZn2+

N

NNZn2+

O

R

NH

O

O

NH

OH

O

HO

OTf -4

12 3

4

OH

NH

NH2

ONH

NH2

O

5

J. A. Drewry, P. T. Gunning et al. J. Am. Chem. Soc. 2011, submitted

Variable Cytotoxicity!

• Wide array of selectivity observed for library vs AML-2, MDA 468 and DU145• Points to different cytosolic targets!

N

NNZn2+

N

NNZn2+

O

R

NH

O

O

NH

OH

O

HO

OTf -4

12 3

4

OH

NH

NH2

ONH

NH2

O

5

• Working to develop the first sequence-specific phosphopeptide receptor• Based on benzothiazole scaffold – more rigid, more predictable

Current and Future Work

= His, Asp, Glut, Cys, Leu, Ile

HN

NH

HN

O

O

O

NH

HN

O

OP

HO

OHO

N

N

N

N

N

N

Zn2+

Zn2+

S

NR

R =

NH2 OH O O O

NH

OOH

O ONH

O

NH2

H2N

OOH

O

• Prof. Patrick Gunning• Dr. Aaron Schimmer• Eugenia Duodu• Steven Burger• Diane Kraskouskaya• The Gunning group

Funding Bodies

• The Leukemia and Lymphoma Society of Canada• NSERC• The University of Toronto

Conclusions