Scanning the protein surface to uncover new small molecule binding sites using fragments Gregg Siegal ZoBio
Increasing Interest in Alternative Small Molecule Binding Sites 1. Enhanced specificity 2. Adress generation of resistance 3. Improved efficacy 4. Better physicochemical properties leading to e.g. better PK or brain penetrance Schapira, Cell Chem. Biol., 2016, 23, p. 1067
Case Study 1: Total Surface Screen 1. Target: Dot1L Histone Lysine Methyl Transferase 2. S-Adenosyl Methionine (SAM) Cofactor 3. Approach: a. Screen apo-dot1l TINS & SPR b. Validate hits with NMR & SPR +/- SAM to sort out different possible binding sites c. Analog each series d. Structural Biology to support hit elaboration (X-ray)
Gene Regulation by Histone Methylation Heterochomatin Writers: Methyl transferases Acetyl transferases Kinases Ubiquitin ligases DNA methyl transferases Erasers HDACs Demethylases DUBs Euchromatin Readers Bromo domains PHD finger domains Tudor domains Chromo domains etc
Protein Methylases SET Domain Containing (K) non-set Domain (primarily R)
Challenges in KMT Drug Discovery Ligand discovery Chemical characteristics of SAM site Specificity (?) Structural biology IP space (?) Oral bioavailability
DOT1L as a therapeutic target in MLL-rearranged Leukemia MLL-rearranged (MLL-r) leukemia accounts for ± 10% of acute leukemia of lymphoid and myeloid, and > 70% of cases among infants The gene encoding MLL is incorrectly translocated following a double-strand break The resulting MLL-AF4 fusion product recruits DOT1L to MLL-binding promoters Aberrant transcription activation of homeobox genes (e.g. HOXA09 & MEISI1) causes leukemogenesis (Cell. 2005;121(2):167-178) DOT1L is a validated therapeutic target in MLL-r leukemia Ectopic localization of Dot1 results in hyper methylation of K79 of Histone 3. HOXA09/MEISI1 aberrant expression Leukemogenesis 8
Targeting DOT1L Pinometostat (Epizyme) DOT1L inhibitor in phase 1 trial for MLL-r leukemia patient (Blood 122, 1017 1025 (2013). hdot1l(1-416) PDB:1NW3 Min et al., 2003 SAM Activation loop N C Substrate loop SAM entry site Lysine channel
Targeting DOT1L Can Fragments Find Alternatives? Improve drug properties of SAM competitive inhibitors SAM based inhibitors have low cell penetrance and oral bioavailability: Blood 122, 1017 1025 (2013) & Nat. Commun. 3, 1288 (2012) New binding sites - Improve selectivity among SAM dependent methyl transferase Outside of IP protection Enhanced Structural Biology: Soakable crystal system
Dot1L Project Flow Protein Engineering Fragment Screening Combinatorial Biology Dot1L TINS Biacore Libraries: Diversity 1,600, Mw ~ 220 Da Focused 130, Mw 280 Da Leads, nm K D X-ray Structural Based Design/Testing Structural Biology Gregg Siegal ZoBio Hit characterization Biacore Hit characterization Competition Binding Validated, Prioritized Fragment Hits, M-mM K D
Orthogonal Screening NMR and SPR have opposite k off dependence. SPR LO NMR K 10-9 10-8 D 10 0 10-7 10-6 10-5 10-4 10-3 10 5 10-2 k 10-1 off 10 1 10 2 10 3 10 4 10 6 Assume k on = 10 8 / sec Gregg Siegal ZoBio
Ligand Discovery - TINS
Selecting Hits and Assessing Ligandability * * T R Highly ligandable Poorly ligandable Unligandable
Ligand Discovery - SPR 400 350 300 250 200 150 100 50 0-50 0 200 400 600 800 1000 1200 Compound number Selected hits ZB1713 ZB2125 % occupancy
Sorting Out the Binding Mode SAM analog ZB2125 SAM analog + SAH ZB2125 + SAH TINS screen SPR screen Common Screened 1601 1562 1544 Hits 110 219 11 SPR Binding Defined 30 41 non-sah competitive 6 8 2500 2000 1500 1000 KD (um) 500 0 K D ( M) range SAH COMP SAH COMP NON- COMP NON- COMP 0,5 0,45 0,4 0,35 0,3 0,25 0,2 0,15 SAH COMP LE range SAH COMP NON- COMP NON- COMP LE
Hit Confirmation by Rapid Analoging TINS Hits 18
Three Interesting Scaffolds Compound SAH competitive? Screening Technology ZB2125-1 No NMR ZB514 No SPR B15 Yes SPR
Each Series Targets a Different Site
Structure of a Ternary Complex Loop Remodeling Substrate loop SAM/SAH bound Pinometostat bound ZB2125-1 bound Activation loop SAM/SAH bound Pinometostat bound ZB2125 bound-1 ZB2125-1 - SPR screen hit - K D /LE measurements: 575 M/0.34
Structure of a Ternary Complex An Induced Pocket Activation loop Substrate loop ZB514 180 Da - SPR screen hit - K D /LE measurements: o - SAH 575 M/0.34 o + SAH 530 M/0.37
Focused Library Novel Chemistry in the SAM Pocket
We are not alone...
Case Study 2: Directed Screen 1. Target: Nucleotide binding protein 2. NT binding site unligandable 3. Approach: a. Screen FL target in the presence of saturating NDP (TINS) b. Validate hits with SPR (FL and NBD) c. Analog d. Structural Biology to support hit elaboration
The Importance of Sample Preparation: 1 Combinatorial Biology to Find the Optimal Construct Domain of Interest Generate all combinations of N & C termini Up to 5m clones can be assayed Clone, express and screen for soluble protein Screen with split-gfp Technology Select best clones, scale up and purify Test clones for optimal structural biology characteristics Pedelacq et al., NAR, 2011, Vol. 39, No. 18 e125
The Importance of Sample Preparation: 2 Optimizing Sample Conditions Combinatorial Biology Combinatorial DNA assembly High-throughput transformation & expression High-throughput solubility screening Up to 5m constructs IMAC resin loaded tips Tag DNA fragments and Scale-up and purify interesting constructs Confirm Soluble & Functional ÄKTA purifier ÄKTAxpress Thermal shift assay SEC-MALS 1D/2D NMR
Target 2: Sample Optimization Stable for months Use of SEC-MALS to optimise buffers.
Directed Screening Summary: 14 fragment hits binding fully characterized by SPR 9 Bind FL & NBD K D 0.3 4 mm LE 0.17 0.3 Inhibit NTPase activity
Binding site location (CSP) CSP Large Medium Small G N-H M N-H N N-H K N-H D N- H F N- H R N- H T N- H
NMR is a Powerful Alternative to X-ray Target 2
NMR-Based Structural Elucidation Shah et al., J. Med. Chem, 2012, 55, 10786 10790 Gregg Siegal ZoBio
Intermolecular structure constraints Green no CSP Gregg Siegal ZoBio
Conclusions Carefully designed orthogonal screening is critical to finding (nearly) all interesting hits. Fragment screening usually uncovers multiple binding sites on the target, enabling the development of inhibitors with different MoA. Multiple screening strategies can be used to find allosteric sites (if the screening technology is sufficiently sensitive). Often the ligandability of allosteric sites is less than orthosteric sites. X-ray crystallography can provide structural information for hits from different screening approaches. NMR is an important complement to X-ray crystallography.
Acknowledgments Dot1L Utrecht University: Bas Lamoree, Martin Lutz, Piet Gros ZoBio: Pierre Boronat, Oscar van Linden, Ruta Nachane, Dan Chen Mercachem: Anita Wegert, Gerhard Mueller, Jan Schultz