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Fragment-based Drug Discovery of the Synthetic Small Molecule HSP90 Inhibitor AT13387 A211 Christopher W. Murray, Maria G. Carr, Gianni Chessari, Miles Congreve, Joseph E. Coyle, Philip J. Day, Lynsey Fazal, Martyn Frederickson, Brent Graham, Jonathan Lewis, Rachel McMenamin, M. Alistair O’Brien, Sahil Patel, Glyn Williams, Andrew J. Woodhead and Alison J.-A. Woolford. . Astex Therapeutics Ltd., 436 Cambridge Science Park, Milton Road, Cambridge, CB4 0QA, UK. INTRODUCTION HSP90 is a molecular chaperone that directs the folding and maturation of its client proteins, many of which are oncogenes regulating tumour cell growth, survival and activation Here we describe the identification of the clinical candidate, AT13387, discovered by applying fragment-based drug design to the N-terminal domain of HSP90 Fragment-based drug discovery is a rapidly growing alternative to high throughput screening in which very small molecules are screened by specialised techniques such as NMR and X-ray Only relatively small libraries of fragments are required and observed fragments often possess high potency when normalised to their size (high ligand efficiency) FRAGMENT-BASED DISCOVERY OF AT13387 Figure 1. NMR screening and X-ray identify fragment 1 binding in the ATP site of N-terminal HSP90 Figure 2. Experimental binding mode of compound 2 represented as a surface O Fragment 1, was identified as having a Kd of 790μM for HSP90 and the experimental binding mode indicated a clear pathway to optimise the molecule After three iterations of structure-guided medicinal chemistry, compound 4 was identified which is just six heavy atoms larger than fragment 1 but is over 1,000,000 fold more potent Lead optimisation led to AT13387 which is currently in Phase I clinical trials for the treatment of cancer Early preclinical data on the biological properties of AT13387 are presented here The accompanying poster (A217 by Lyons et al) gives more preclinical data and discusses the unusually long pharmacodynamics exhibited by AT13387 relative to other HSP90 inhibitors Figure 3. Experimental binding mode of fragment 3 Figure 4. Binding mode of lead molecule 4 superimposed on fragment 1 N O O N Figure 5. Lead optimisation yielded the clinical candidate AT13387 whose chemical structure and experimental binding are shown below N (4) (2) HO O (3) N N N HO OH OH OH Kd (ITC) = 8.6μM LE = 0.38 N (1) O OH Kd (ITC) = 790μM LE = 0.26 Fragment screening of 1500 fragments identified many hits including fragment 1, a known respiratory drug Despite its poor initial ligand efficiency and potency, the X-ray structure indicated two good design ideas Superimposition on the natural product radicicol (top middle) suggested conversion to a resorcinol Replacement of the methoxy group with non-planar hydrophobes should provide better fit to the proximal lipophilic pocket (top right) Figure 6. Client protein knock down with AT13387 in breast, melanoma and lung cancer cell lines BT474 (Breast) 10 30 100 300 1000 NCI-H1993 (NSCLC) [AT13387] nM: C HER2 C-MET HSP70 HSP70 CDK4 CDK4 GAPDH GAPDH A375 (Melanoma) [AT13387] nM: C 10 30 100 300 1000 NCI-H1975 (NSCLC) [AT13387] nM: C B-RAF EGFR HSP70 HSP70 CDK4 CDK4 GAPDH 10 30 100 300 1000 10 30 100 300 1000 GAPDH AT13387 knocks down known oncogenic protein clients of HSP90 (HER2, C-MET, B-RAF and EGFR). In all cases, AT13387 induces HSP70 and knocks down protein client CDK4. Amide replacements were synthesised based on examination of the experimental binding mode Tertiary amides were prioritised to preserve the conformational twist observed in fragment 1 Isoindoline 3 was one of the most potent offering a 30-fold improvement over compound 2 Figure 8. Efficacy of AT13387 in A375 mutant B-raf melanoma xenograft model The resorcinol 4 is over 100-fold more potent than the corresponding phenol, and shows good cell activity with a confirmed mechanism of action The binding mode was conserved during the fragment optimisation (above left) Lead molecule 4 gave weak but encouraging biomarker response Figure 9. Biomarker knock down after dosing of AT13387 in A375 tumour bearing mice 100000 Tumour Plasma Blood Muscle Brain 10000 Concentration (ng/mL or ng/g) [AT13387] nM: C Small lipophilic replacements of the methoxy group of fragment 1 were synthesised Isopropyl (7μM) and t-butyl (9μM) were the best examples showing approximately 100-fold improvement Both analogues give superior hydrophobic contact in the proximal lipophilic pocket as illustrated for compound 2 (above left) Figure 7. Tumour levels of AT13387 in HCT116 tumour bearing mice Cells were incubated with varying concentrations of AT13387 for 18 hours, lysates harvested and analysed by western blotting. Kd (ITC) = 0.25μM LE = 0.41 1200.0 Raf1 1000.0 Tumour volume (mm 3) O Kd (ITC) = 0.00054μM LE = 0.57 cLogP = 3.3 MW = 297 Da Cell IC50 = 0.031μM 1000 100 10 Matrix Half-life Tumour 65hr Plasma 4hrs Blood 4.6hr Muscle control 800.0 AT13387 55 mg/kg 2qw 24 48 72 AT13387 60 mg/kg (qd x 2) 400.0 CDK4 200.0 MWM 0.0 0 3hr 96 Cleaved PARP AT13387 90 mg/kg 1qw 5 10 15 20 25 30 0 3 6 8 16 24 48 72 hours post dose 35 Day 1 0 HSP70 AT13387 70 mg/kg 2qw 600.0 120 144 168 192 216 240 264 288 312 336 Time (hr) Pharmacokinetic profiling of AT13387 in plasma, blood, brain muscle and tumour after a single 60mg/kg IP dose shows compound retention in only tumour suggesting an opportunity for a greater therapeutic window. AT13387 was dosed weekly or twice weekly by the ip route up to day 21. A treatment group consisted of 8 animals. AT13387 shows good efficacy in this and other models. Presented at the EORTC-NCI-AACR Meeting in Boston on Monday November 16th 2009. This poster can be downloaded from the Astex website at www.astex-therapeutics.com Pharmacodynamic studies show that a single ip dose of 90mg/kg of AT13387 resulted in loss of client proteins Raf1 and CDK4 for 72 hours or more. There was also a concomitant increase in HSP70 and cleaved PARP levels, the latter being indicative of apoptosis. Kd (ITC) = 0.00072μM LE = 0.41 cLogP = 3.5 MW = 410 Da Cell IC50 = 0.048μM OH AT13387 Initial lead optimisation focussed on improving in vivo properties Addition of basic groups off the isoindoline was aimed at improving the volume of distribution and led to several compounds with good efficacy and extended pharmacokinetics in tumours Some of these compounds possessed activity against hERG (patch clamp) and further SAR showed it was possible to obtain compounds with good efficacy and little or no activity in the hERG assay AT13387 was chosen as a clinical candidate after further profiling during a candidate selection phase CONCLUSIONS The phenol fragment 1 was identified using fragment screening Structure-guided medicinal chemistry was used to identify the highly ligand efficient lead molecule 4 Only 6 heavy atoms were added during Hits to Leads yet the potency increased by over 6 orders of magnitude making this one of the most ligand efficient fragment optimisation campaigns so far described Only 60 compounds were synthesis in going from fragment 1 to lead 4 AT13387 was identified after a lead optimisation campaign centred on increasing the volume of distribution and reducing off-target activities AT13387 shows good efficacy and biomarker response in a number of models and exhibits a particularly long half-life in tumours The accompanying poster (A217 by Lyons et al) gives more details on the favourable biological profile of AT13387 AT13387 is currently in Phase 1 clinical trials for the treatment of cancer © 2009 Astex Therapeutics Limited. 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