Targeting PPIs in Oncology using a Fragment-Based Drug Discovery Approach Justin F. Bower CRUK Beatson Institute

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1 Targeting PPIs in Oncology using a Fragment-Based Drug Discovery Approach Justin F. Bower CRUK Beatson Institute

2 Cancer Research UK Beatson Institute Establish an outstanding basic research programme into the cell biology of cancer Understanding of cell invasion and metastasis Regulation of cancer metabolism, growth and survival Identify critical components of these pathways as novel therapeutic targets Develop strong clinical links to translate research into new therapies and diagnostic/prognostic tools

3 Fragment Approach to Hit Identification & Optimisation Surface Plasmon Resonance (SPR) Multiple orthogonal screening/validation technologies 600 MHz Nuclear Magnetic Resonance (NMR) Computational Chemistry Fragment Library (1040 cpds) Property Mean value MW (HAC) 200 (14.2) clogd (Sol.) 0.53 (>1mM) X-Ray Crystallography HBD (HBA) 1 (2.8) TPSA 47 Rotatable Bonds 1.84 Structure generation to inform design Fsp3 0.42

4 Cancer Metastasis Cancer metastasis (how cancer spreads) is responsible for over 90% of cancer-related deaths It is a complex process We can watch cancer cells in real time! The ability to control metastasis in some way would have a dramatic impact for patients Mierke, CT, Eur. J. Cell. Biol., 2013, 92,

Fascin Fascin proteins belong to a large group of actin binding

Cross links filamentous actin (F-actin) into parallel bundles Required

Filopodium/invadopodium G-Actin monomers Arp2/3 complex Ena/VASP

5 Fascin Fascin proteins belong to a large group of actin binding proteins Fascin s bundling activity regulated by PKCα and Rho GTPases Cross links filamentous actin (F-actin) into parallel bundles Required for membrane protrusions, cell motility and Fascin ECM degradation Filopodium/invadopodium G-Actin monomers Arp2/3 complex Ena/VASP Fascin 3 Actin 1 2 Arp2/3 WASP F-Actin Merge Adapted from Hashimoto et al., 2011 Fascin

Rationale and validation Fascin 1 expression is low or absent in normal epithelia Overexpressed in a variety of tumour types including bladder, colon, lung and pancreas Expression associates with

6 Rationale and validation Fascin 1 expression is low or absent in normal epithelia Overexpressed in a variety of tumour types including bladder, colon, lung and pancreas Expression associates with most aggressive and metastatic tumour types (e.g. Pancreatic cancer) Prognostic indicator of poor clinical outcome Fascin 1 knockdown reduces tumour cell invasion and proliferation Normal Duct PDAC PDAC Karim et al.,

7 Rationale and validation Fascin 1 expression is low or absent in normal epithelia Overexpressed in a variety of tumour types including bladder, colon, lung and pancreas Expression associates with most aggressive and metastatic tumour types (e.g. Pancreatic cancer) Prognostic indicator of poor clinical outcome Fascin 1 knockdown reduces tumour cell invasion and proliferation Normal Duct PDAC KPC-Fascin KO PDAC Cells PDAC Karim et al., Inhibiting Fascin may be effective in the treatment of invasive/metastatic cancers

8 Targeting Fascin a Protein-Protein Interaction Relatively large protein (55kDa) - 4 b-trefoil domains Looking for small molecules that could perturb this actin bundling process

9 Project Status at Outset Rich biological data Collaboration with Prof L. Machesky s group at the Beatson accesses leading edge cell biology Heterologous protein expression system published Crystal structure in PDB complexed with a small molecule 3D druggability assessment (SiteMap) suggests reasonable small molecule binding pockets Challenges: A PPI with very limited information to guide strategy Limited assays - Low-Medium throughput actin bundling assay available Lack of robust Biomarkers. E-cadherin repression? Clinical progression of invasion/metastasis mechanisms

10 Inhibitors of Fascin Chen et al. Nature 2010, 464, 15 Synthesis: Danishefsky et al, JACS, 2004, 126, Screening strategy: Crystal Large structure protein & with known Fascin-1 binder - NMR screen (STD / WaterLOGSY) - Competition

observed in SPR, NMR or crystal soak Competition

11 Optimistically Built Macroketone Limited evidence for macroketone binding in deposited structure No binding observed in SPR, NMR or crystal soak Competition experiments not possible 2mFo-DFc/Fo-DFc electron density, ligand omitted

12 Fascin Screening Strategy Fragment Screen by SPR (single point then dose response) Orthogonal validation by ligand based NMR (STD and WaterLOGSY) X-ray crystallography

a Nickelcharged surface Fragments screened at 500mM and hits confirmed at 500mM and 125mM

13 Surface Plasmon Resonance Fascin - 55kD protein at upper limit for SPR fragment screening (looking for 250Da change in 55000Da) Strategy to use double-his tagged Fascin-1 linked to a Nickelcharged surface Fragments screened at 500mM and hits confirmed at 500mM and 125mM Hits further validated in dose response experiments & NMR ~5% hit rate - Structure Determination

14 Fascin Fragment Screening and Binding Sites Fragment Site SPR Kd / um 1 > (LE 0.42) 3 > >2000

15 Decision-making & Optimisation Prioritising the hits Structure Binding strength / Ligand Efficiency Chemical tractability Likelihood of achieving functional activity Steric blocker? Allosteric? Activator? All the above ideal world Using the biology.

16 Prioritisation Site 1 - druggable pocket within clear vicinity of functionally important residues (mutant studies) - Prioritised Site 2 - small enclosed very hydrophobic pocket, scope for efficient binding ligands but functional scope initially unknown Yang et al, J. Biol. Chem., 2013, 288,

17 Site 1 Elaboration

18 Site 1 Elaboration

19 Site 1 Elaboration

20 Site 1 Elaboration

21 Site 1 Elaboration

22 Site 1 Elaboration

23 Site 1 Elaboration

Fascin X-Ray Crystallography Compound aggregation WaterLOGSY NMR Functional

24 Improved Binders but Functionally Active? Binding Assay: SPR (Single point & DR) Structure/activity based selection Fascin X-Ray Crystallography Compound aggregation WaterLOGSY NMR Functional Biochemical Assay Full length Fascin; SP 100µM >30% inhibition Functional Biochemical Assay Full length Fascin 1-493; 5 Point DR

25 Functional Biochemical Assay Actin Bundling Assay F-actin F-actin F-actin + Fascin Centrifuge F-actin + Fascin 1 Jansen et al., 2011 JBC 286: Remove Sup t Fascin F-actin Fascin P S P S Actin Run on gel and Coomassie stain P S P S % Actin Bundling

26 Opportunities in Site 2? Ligand Efficient but looks to be embedded with limited opportunities (not obviously close to functionally important residues) to grow SPR Kd 93 mm

27 Potential - Coupling Fragment & HTS Data Phe216 Leu214 Phe216 Leu214

28 Using the Literature Structure Inhibition of Bundling (mm) Patent WO2014/031732) X X

29 Potential - Coupling Fragment & HTS Data Phe216 Phe216 Leu214 Leu214

30 Optimisation Opportunities

31 Optimisation Fragments, HTS & Computational Work Trp101 Arg217 Glu215 Phe216

32 Optimisation Fragments, HTS & Computational Work Trp101 Thr213 Glu215 Phe216 Arg217

33 Optimisation Fragments, HTS & Computational Work Ala58 Ser57 Trp101 Gln50 Arg217 Thr213 Glu215 Phe216

34 Optimisation Fragments, HTS & Computational Work Ala58 Ser57 Trp101 Gln50 Thr213 Glu215 Phe216 Arg217

35 Functional Activity Conformational Change

36 Fascin Summary A fragment-based screening approach for fascin led to compounds which bind to either site 1 or site 2 Generation of >500 fascin-ligand complex crystal structures has enabled elaboration of compounds at both sites Compounds identified from a virtual screen create an induced pocket partially aligning with earlier fragments found in the fully enclosed site 2 Identified highly tractable chemical series for optimisation Screening cascade established with excellent accordance between assays Development of a robust and reproducible bundling assay enabled assessment of ligand functional activity in vitro Preliminary assessment of compounds in the CETSA assay has been used to demonstrate cell activity Great collaboration with Laura Machesky s lab Compounds in 3D scratch wound assay Working to identify a suitable biomarker approach for inhibition of fascin activity

Allosteric Inhibition of SHP2: Identification of a Potent, Selective, and Orally Efficacious Phosphatase Inhibitor!

Allosteric Inhibition of SHP2: Identification of a Potent, Selective, and Orally Efficacious Phosphatase Inhibitor Allosteric pocket SHP2 Phosphatase ovel allosteric Phosphatase inhibitor Evan Carder Wipf