STD-NMR screening Introduction Case study ...

Mycobacterium tuberculosis InhA focused fragment based drug discovery Federica Prati1,2, Fabio Zuccotto1, Raquel Fernandez-Menendez2, Robert Bates2, Lourdes Encinas2, Chun-wa Chung3, Maire Convery3, Paco De Dios Anton2, Alfonso Mendoza2, Daniel Fletcher1, Simon Green1, Margaret Huggett1, Paul Wyatt1, Peter Ray1 i 1Drug

Discovery Unit, University of Dundee, Dundee, United Kingdom 2Diseases of the Developing World, Tres Cantos Medicines Development Campus, GlaxoSmithKline, Tres Cantos, Spain 3Structural and Biophysical Sciences, PTS, GlaxoSmithKline Medicines Research Centre, Stevenage, United Kingdom

Introduction The enoyl acyl carrier protein reductase (InhA) is involved in the Mtb cell wall synthesis pathway. Isoniazid is a frontline TB drug and targets InhA.1 Resistance to isoniazid is due to mutations in the KatG activating enzyme and not InhA.2 Direct InhA inhibitors are expected to recapitulate the clinical efficacy of isoniazid and maintain activity against isoniazid resistant mutants. Herein we show that STD NMR screening of novel diverse fragments with covalent functional-group complexity,3 provide fragment starting points with multiple interaction against InhA protein and its cofactor (NAD+). We show that fragment actives with ‘built-in’ functional group complexity are more likely to maintain their pose during the optimization process, as a result of the increased protein interactions, which allows for faster elaboration into potent enzyme inhibitors with good ligand efficiency (LE) metrics.

STD-NMR screening

FB screening cascade INV-FRAG

InhA FG INF-FRAG PROJ-FRAG DIV-FRAG HIST-FRAG: 573 cps HIST-FRAG 3D-FRAG 3D-FRAG: 170 cps FG-DIV-FRAG:326 cps InhA-INF-FRAG: 46 cps INV-FRAG: 124 cps PROJ-FRAG: 121 cps

STD for NADH with no ligands

FRAGMENT SCREENING SETS

STD – addition of compound 1

Compound 1 for NMR displacement InhA pIC50 = 7.9

1360 compounds STD active fragment

i) ii)

STD NMR InhA screening Competition experiments

Hit rate ~ 15%: STD signal Hit rate ~ 11% : 60% loss of STD intensity on addition of 1

Cocktail spectrum

149 hits (STEP 1) Priority compounds: • NMR binding • Biochemical potency • Chemical diversity

Hit rate ~ 2.4%: loss of NADH STD intensity on addition of fragments

High concentration @ 500 uM InhA biochemical screening 8 hits (STEP 2)

Hit rate ~ 2.4% vs library source

Hit rate ~ 11% vs library source 1% 6%

6%

9% 48%

29%

15 hits (STEP 3)

HIST-FRAG

13%

34%

3D-FRAG

FG-DIV-FRAG InhA-INF-FRAG

7%

FG-DIV-FRAG

41%

InhA-INF-FRAG

INV-FRAG

PROJ-FRAG

PROJ-FRAG

X-ray crystallography SPR studies

HIST-FRAG

3D-FRAG

6%

High hit rates were obtained from screening the FG-DIV-FRAG library of diverse fragments with ‘built-in’ functional group complexity, envisaged to allow identification of additional interactions within target proteins and facile optimization.3

3 chemical series prioritized for FBLG

FB lead generation Case study: merging and growing FG-DIV-FRAG hit InhA pIC50 < 3

Hist-FRAG hit InhA pIC50 < 3

Fragment merging and growing Two series InhA pIC50 > 6.5

• FG-DIV-FRAG hits showed additional interactions with the InhA protein • FG-DIV-FRAG hits allowed rapid progress • FG-DIV-FRAG hit optimised leads maintained their initial binding poses with InhA

References 1

a) Banerjee, A. et al. Science 1994, 263, 227−230; b) Dessen, A. et al. Science 1995, 267, 1638−1641. 2 a) Schroeder, E. K. et al. Curr. Pharm. Biotechnol. 2002, 3, 197−225; b) Basso, L. A. et al. Med. Chem. Rev. 2005, 2, 393−413. 3 Ray, P et al Drug Discov Today 2016, in press.

FB lead generation targeted criteria FB lead targeted criteria

Series I Compound 2

Series II Compound 3

Series III Compound 4

InhA pIC50 > 6 H37Rv MIC < 10 µM LE > 0.3, LELP < 10 PFI < 6 Kin. solubility > 250 µM MW < 400 Chrom LogD < 5 clogP < 3 HepG2 pIC50 < 4

7.0 80 0.37, 6.21 7.37 192 371 5.37 2.3