Schrodinger eUGM Berlin Sept19
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This document discusses the discovery of small molecule inhibitors of Fascin 1 by Cancer Research UK's Drug Discovery Unit. It describes how fragment-based drug discovery was used to identify two binding sites on Fascin 1 and optimize compounds that bind to these sites. In silico screening methods helped progress compounds from the low millimolar range to the low nanomolar range for binding and inhibition of actin bundling. The best compound showed promising pharmacological properties and modest activity in cell invasion assays. The document emphasizes exploring protein flexibility and following the literature to advance drug discovery projects.
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Schrodinger eUGM Berlin Sept19
- 1. DISCOVERY OF SMALL MOLECULE INHIBITORS OF FASCIN 1 Angelo Pugliese Ph.D. Schrödinger EUGM - Berlin 2019
- 2. CANCER RESEARCH UK (CRUK) • World’s largest independent charity dedicated to cancer research with many partners (academic, government and pharma) from across the world • More than 30 agents in pre-clinical and clinical development, two drugs in phase III trials and six drugs already on the market AP2
- 3. CRUK BEATSON INSTITUTE - DRUG DISCOVERY UNIT • A purpose built facility opened in 2008 in Glasgow with state-of-the-art equipment and capabilities - 303 scientists • The DDU is an integrated, industry-standard drug discovery group translating basic biology research from the Beatson and other CRUK centers into anti-cancer medicines • The targets we identify are usually highly challenging from a druggability and/or validation perspective • This “high-risk/high reward” approach to translational research is at the core of CRUK’s drug discovery approach AP3
- 4. Synthetic, medicinal and computational chemistry Biology Biophysics Crystallography DRUG DISCOVERY UNIT - CAPABILITIES AP4 • Fragments approach to hit identification and optimization Purpose-built fragment screening libraries constantly evolving Property Mean value MW (HAC) 202.8 (14.3) clogD (Sol. exp.) 0.76 (>2mM) HBD (HBA) 0.95 (3.52) TPSA 49 Rotatable bonds 1.87 Fsp3 0.4 Bruker NMRs AVANCE NEO 400 and 600. GE SPR instruments - T200 - Biacore 4000 - Biacore 8k Malvern PEAQ-ITC Design & synthesis of chemical tools, leads & candidate drugs – CADD Machine learning Enzymology, Biochemistry, Cell-biology, in vivo, siRNA & Bioinformatics
- 6. INVASION AND METASTASIS AP6 • Cancer metastasis (how cancer spreads) is responsible for over 90% of cancer-related deaths
- 7. FASCIN FUNCTION • During cell migration and invasion cellular protrusions (e.g. filopodia and invadopodia) are formed and actin bundles are required for this process • Actin-bundling protein, cross-linking filamentous actin (F-actin) into parallel bundles • Expression is low or absent in normal epithelia but dramatically increased in a variety of invasive and metastatic tumor types (e.g. pancreatic) • There are still no effective therapies for metastasis, most current cancer therapies target primary disease AP7
- 8. GOAL • Develop an anti-metastatic agent blocking the actin- fascin interaction (Kd 150-300nM) • Highly challenging drug target due to its mechanism of protein-protein interaction and the lack of knowledge around the critical actin-binding sites • Initially a blank canvas regarding binding sites and function • Fascin proteins are composed of 4 β-trefoil domains (55 kDa - length 493 AAs) AP8
- 9. TARGET ASSESSMENT - LIGANDABILITY - SITEMAP AP9 Only best site shown We consider 0.90 and above “good” Dscores for a tractable PPI Type PROTEIN TARGET Comments Dscore Volume PDB ID: 3LLP Apo Fascin close to functionally important residues 1.002 472.997 Apo Fascin 0.947 158.123 Apo Fascin 0.857 229.81 Apo Fascin 0.783 142.345 Apo Fascin 0.602 59.682
- 10. Fragment Screen by SPR single point (500µM) then dose response 53 (5.4%) cross- validated hits with millimolar affinity X-ray crystallography SCREENING CASCADE AP10 His8-His8-Fascin 1050 frags Initially 7 yielded crystal structures (17 in total) Functional Biochemical Assay Actin Bundling Assay Ligand fascin engagement in cells Cellular Thermal Stability Assay (CETSA) Functional Cell Assay: Incucyte Invasion Assay MDA-MB-231 cells (Breast) Kd >1mM Kd >1mMKd >2mM Kd >1mMKd >1mM Kd >1mM Kd >200mM Site 1 Site 2
- 11. FASCIN FRAGMENT BINDING SITES AP11 KD (M) SE(KD) Rmax (RU) 8.623E-4 1.2E-4 28.3 180˚ Site 1 Domain 1 Domain 2 Domain 3 Domain 4 Site 4 Site 2Site 3 Site 1 - Cleft between domains 1 and 4 surrounded by functionally important residues (mutant studies) Ligandable - Initially Prioritised Site 2 - small enclosed very hydrophobic pocket between domains 1 and 2, scope for efficient binding ligands but functional scope initially unknown Site 1
- 12. SITE 1: FRAGMENT TO COMPOUND • Initial effort on phenyl piperazine: increase potency and introduce functional activity 12 N N H N N H O N OH Cl • Abundance of higher MW commercial analogues (rapidly address potency) • Clear vectors to important functionally-critical residues (biological activity) AP Lys41 His392 Ser39 Lys43
- 13. • Where to focus commercial analogue search ? - Structure-based approach AP13 SITE 1: FRAGMENT TO COMPOUND Substructure search on commercial DB MW ≤ 400, cLogD ≥ -1 ≤ 4 Unwanted Functionalities Focused library 14478 unique compounds Ligprep Site 1 docking Glide SP, XP 300 compounds Hydrophobic patches: bury more surface and gain some affinity?
- 14. • Where to focus analogue search ? - Interactive Focused Library Enumeration and Docking AP14 SITE 1: FRAGMENT TO COMPOUND Growing from 3 positions Built-in 43 Fragments 79507 structures in comb. library Library expansion (LigPrep) + Glide docking CombiGlide 43 * 43 * 43
- 15. • Best in-silico optimized fragment used for shape similarity searching AP15 docking score -9.5 GLE -0.35 Focused Library ≥ 0.75 Hits Phase Shape ≤ 0.75 Diversity-based Selection200 compounds Optimized Fragment template 1K Molecules Sim. Range: 0.86 – 0.77 SITE 1: FRAGMENT TO COMPOUND
- 16. AP16 SITE 1: OVERALL IN SILICO SCREENING 334 unique cmpds Shape and pharmacophore 200 cmpds Structure-based virtual screening 300 cmpds Chemical similarity 94 cmpds Med chem eye-balling Availability/cost 130 cmpds acquired/tested 3 in SPR dose response ECFP4 - 0.8 sim. threshold MW ≤ 400, cLogD ≥ -1 ≤ 4 • >10 folds improvement in affinity over fragment • Valid design hypothesis of approaching the hydrophobic area • After one round of optimization affinity improved further: Kd=38µM (still exploiting the pocket) >2mM ~500µM 38µM Fragment VS Synthesis
- 17. AP17 SPR Kd (µM) Actin Bundling IC50 (µM) Binding Site 1.8 23.2 1 2.5 64.2 1 • Progress slow, best compounds modest affinity (Kd~2µM) with limited functional activity • No apparent structural opportunities • Site 2 opportunities? • And 219 crystal structures later………… SITE 1 ENDG ME
- 18. SITE 2: COUPLING FRAGMENT & HTS DATA AP18 Site 2 - small enclosed very hydrophobic pocket between domains 1 and 2, scope for efficient binding ligands but functional scope initially unknown Domain 1 Domain 2 Domain 2 Domain 1 Apo-Fascin (PDB id 3LLP) SPR Kd 30 mM (LE 0.24) Actin Bundling IC50 ~30 mM SPR Kd 93 mM (LE 0.42) SPR Kd >2000 mM
- 19. SITE 2: FUNCTIONAL ACTIVITY RATIONALE AP19 This conformational change leads to inhibition of binding and bundling of filamentous actin Site 2 Domain 2 Domain 1
- 20. AP20 SITE 2: Trp101 Phe216 Glu215 Arg217 BDP-00010300 SPR KD 30 mM (LE 0.24) Actin Bundling IC50 >30 mM • The induced conformational changes create access to the protein surface creating opportunities for compound elaboration
- 21. AP21 SITE 2: IN SILICO SUCCESS (AGAIN) 62 BDP-10300 Shape Analogues ~2M In-house Virtual Library 18 binders – 3 with Kd values <100 μM 110 Tested - Single Point SPR (100μM) Duplicates Removed - Visual Check 136 Selected for Purchasing 181 Pareto-ranked compounds 200 Pareto-ranked compounds 62 BDP-10300 Shape analogues ~2M In-house Virtual Library ~1K BDP-10300 eMolecules Structural Analogues ~2.7M In-house Virtual Library Structure-Based Structure-Based Ligand-Based ~2.7M In-house Virtual Library 809 BDP-10300 Commercial Analogues “Best compromise” - Pareto efficiency as the ranking technique Parameters used: • calculated free energy of binding (from docking – largest weight) • QED • interaction pattern similarity (based on BDP-10300 structural interaction fingerprints) BDP-00010300 Kd (SPR) ~30μM Solubility ~50μM WO2014031732A2 Isoquinolone – Virtual hit Kd (SPR) ~25μM Solubility >500μM
- 22. AP22 SITE 2: ANALYSING THE “NEW COMER” Energetic analysis of this water suggests it is relatively tightly bound (ΔG = -3.9 kJ/mol) and that displacing it would likely be unfavorable Any changes to the amide (N-alkylation, carbonyl deletion) abolish fascin binding Trp101 Phe216 Glu215 Arg217 Isoquinolone – Virtual hit Kd (SPR) ~25μM Solubility >500μM Actin Bundling IC50 55 μM
- 23. SITE 2 ELABORATION AP23 Kd (SPR) 1.5μM (LE 0.27) Actin Bundling IC50 1.2 μM Kd (SPR) 0.60 μM (LE 0.24) Actin Bundling IC50 0.7 μM Virtual hit (isoquinolone) Kd (SPR) ~25μM (LE 0.24) Actin Bundling IC50 55 μM BDP-00010300 Kd (SPR) ~30μM (LE 0.24) Actin Bundling IC50 >30 mM
- 24. AP24 SITE 2: CHANGES TO THE HETEROAROMATIC AMIDE SPR Kd 0.60 mM (LE 0.24) Actin Bundling IC50 0.7 mM SPR Kd 1.5 mM (LE 0.27) Actin Bundling IC50 1.2 mM • A number of compounds with alternative heterocycles were made • Pyridin-4-yl yielded the largest improvement in terms of both affinity and inhibition. • QM calculations suggest that at least for (hetero)aryl derivatives improved stacking with Trp101 may be a major factor in this increase in potency LMP2/cc-pVTZ(-f)++ The interaction energies between the amino acid and the different ligands were calculated by subtracting the single- point energy of the two monomers, as suggested by the Supermolecular approach: E-interaction = E-complex – E-monomerA – E-monomerB Ab initio single-point calculations were carried out within the Jaguar program at the LMP2/CC-pVTZ++ level of theory. As LMP2 is fundamentally free of basis set superposition errors (BSSE), counterpoise corrections were not performed.
- 25. AP25 SITE 2 BEST COMPOUNDS (BDP13176) Kd SPR (nM) (LE 0.41) 29 Kd FP (nM) 171 IC50 Actin Bundling (nM) 180 LogD7.4/MW 1.8/497 Turbidometric Solubility (midpoint, µM) 65 Caco-2 Papp (x10-6 cm/s)/Efflux ratio 0.15/38 Mic. Stab. Clint /Hep. Stab. Clint (µL/min/mg protein) 6.4/7.8 CYP450 IC50 (µM) 7.0 (CYP3A4) 1.6 (CYP2D6) 22.1 (CYP2C19) hERG IC50 (µM) >25 Assessment of compounds in the Incucyte invasion assay demonstrates (modest) cell activity
- 26. TAKE-HOME MESSAGES AP26 • Closely follow the literature regarding your project - (the benefit of the doubt) • Protein flexibility can be an obstacle but also an opportunity • Crystal structures usually only reveal one form of a protein • Always worth making a few compounds that don’t look like they’ll fit
- 27. Drug Discovery Unit Justin Bower Heather McKinnon