1. Copyright © 2007 Merck & Co., Inc., Whitehouse Station, New Jersey, USA All rights Reserved 14 th CROI February 27, 2007 Next Generation Inhibitors of HIV-1 Integrase Strand Transfer: Structural Diversity and Resistance Profiles John Wai Merck Research Laboratories West Point, PA

2. 2 Copyright © 2007 Merck & Co., Inc., Whitehouse Station, New Jersey, USA All rights Reserved HIV Life Cycle

3. 3 Copyright © 2007 Merck & Co., Inc., Whitehouse Station, New Jersey, USA All rights Reserved Potent integrase strand transfer inhibitor (IC 50 ~ 10 nM) Antiviral IC 95 ~ 33  23 nM (50% NHS) Retains potency across diverse clinical isolates, HIV-2 Potent antiviral effect in treatment naïve patients (Markowitz et al IAC 2006) Robust activity in HIV-1 patients with triple class resistance – Phase II 24 week data (Grinsztejn et al CROI 2006) – Phase III data (Cooper et al & Steigbigel et al CROI 2007) Lipid neutral in naïve patients (Mascolini et al ICAAC 2006) Few drug interactions; Metabolized via glucuronidation MK-0518: Raltegravir

4. 4 Copyright © 2007 Merck & Co., Inc., Whitehouse Station, New Jersey, USA All rights Reserved MAINTAIN favorable properties of Raltegravir with respect to efficacy, tolerability, lipid profile few drug interactions and ACHIEVE Once daily dosing (stand alone) Activity against InSTI resistant isolates Question: Clinical resistance profile of InSTIs??? Objectives for 2nd generation InSTI

5. 5 Copyright © 2007 Merck & Co., Inc., Whitehouse Station, New Jersey, USA All rights Reserved E152 D64D116 Mg In Vitro Selection for Mutants Catalytic Residues

6. 6 Copyright © 2007 Merck & Co., Inc., Whitehouse Station, New Jersey, USA All rights Reserved S153 I151 F121 T125 V72 M154 N155 T66 E152 D64D116 Mg In Vitro Selection for Mutants Catalytic Residues Diketoacid Naphthyridine L-810

7. 7 Copyright © 2007 Merck & Co., Inc., Whitehouse Station, New Jersey, USA All rights Reserved Q148 S153 I151 F121 T125 V72 M154 N155 T66 E152 D64D116 G140 E138 Mg In Vitro Selection for Mutants Bind to catalytic site Different inhibitors selected for different sets of mutants Multiple possible binding orientations Mutation points are spread around the catalytic site Catalytic Residues Diketoacid Naphthyridine L-810 Pyrimidine MK-518

8. 8 Copyright © 2007 Merck & Co., Inc., Whitehouse Station, New Jersey, USA All rights Reserved Enabling Tool: Mutant Panel Q148 S153 F121 T125 V72 N155 T66 G140 E138 Mg Mutant viruses constructed using site directed mutagenesis Activity assessed with single cycle infectivity assay Catalytic Residues Diketoacid Naphthyridine L-810 Pyrimidine MK-518

9. 9 Copyright © 2007 Merck & Co., Inc., Whitehouse Station, New Jersey, USA All rights Reserved C 2 symmetry Diversification of DKA Isosteres O OH OHO Diketoacid Pharmacophore N O N N OH O

10. 10 Copyright © 2007 Merck & Co., Inc., Whitehouse Station, New Jersey, USA All rights Reserved Optimization Against InSTI Mutants.. Spread T66I/ S153Y F121YQ148K N O F N N OH O 156 22x 76x 20x 31x ClC 95 nM 90x Ratio of IC 50 ’s in Infectivity assay N155S E138A/ G140A/ Q148K 0.81x N N O F N N OH O Cl N N S O O N O N N OH O MK-2048 Benchmark

11. 11 Copyright © 2007 Merck & Co., Inc., Whitehouse Station, New Jersey, USA All rights Reserved T66I/S153Y N155S Q148K E138A/G140A/Q148K F121Y MK-2048 MK-518 GS-9137 0 20 40 60 80 100 116X 169X 395X 388X IC 50 ratio of mutants vs. WT HIV-1 in infectivity assay Comparison with Clinical Candidates Screening Mutant Panel

12. 12 Copyright © 2007 Merck & Co., Inc., Whitehouse Station, New Jersey, USA All rights Reserved Comparison with Clinical Candidates MK-518 Clinical Isolates IC 50 ratio of mutants vs. WT HIV-1 in infectivity assay N155H E92Q/N155H Q148R G140S/Q148H G140S/Q148R MK-2048 MK-518 GS-9137 0 100 200 300 399 / 2194X

13. 13 Copyright © 2007 Merck & Co., Inc., Whitehouse Station, New Jersey, USA All rights Reserved Weeks Primary Mutation Selection of Resistance in cell culture at IC 95 Time taken for the first emergence of mutant in vitro correlates with mutation profiles of inhibitors Time to Selection of Resistance in Cell Culture 7 11 21 >37 N155H Q148K S153Y T112I Compound B MK-518 Compound A Numbers of weeks of incubation for the first emergence of mutation in all isolates # of Clones 5/5 6/6 7/7 5/12 MK-2048 >37 wk 0 10 20 30 40 >37 wk

14. 14 Copyright © 2007 Merck & Co., Inc., Whitehouse Station, New Jersey, USA All rights Reserved Summary: Second Generation InSTIs Resistance profiling leads to the identification of inhibitors with activity against broad spectrum of integrase resistance isolates. Provides Proof of Concept for developing second generation InSTIs. T66I/S153Y N155S Q148K E138A/ G140A/ Q148K F121Y MK-2048 MK-518 GS-9137 0 20 40 60 80 100 116X 169X 395X 388X IC 50 ratio of mutants vs. WT HIV-1 in infectivity assay

15. 15 Copyright © 2007 Merck & Co., Inc., Whitehouse Station, New Jersey, USA All rights Reserved 2nd Generation HIV-1 InSTIs Discovery Team Medicinal Chemistry Thor Fisher Mark Embrey Bo-young Kim Melissa Egbertson Wei Han Debbie Perlow Rowena Ruzek Peter Williams Marie Langford Janetta Pellicore Donnette Staas Vanessa Sherman Kelly Savage Cathy Wiscount Lee Tran Melody McWherter Linghang Zhuang Kyle Robinson Matt Morrissette Lou Anne Neilson Richard Isaacs Linda Payne Rick Pracitto Vanessa Obligado Wayne Thompson Peter Munson Brian Phillips John Wai Terry Lyle Joe Vacca Biological Chemistry Daria Hazuda Mike Miller Jay Grobler Pete Felock Kara Stillmock Amy Simcoe Marc Witmer Alysha Day Robert Danovich Yuxiong Ke Viral Vaccine Research Jessica Flynn Linda Ecto Sinouen Touch Lori Gabryelski Bill Schleif Drug Metabolism Chuck Thompson Prasad Kari Joan Ellis Kim Michel Anne Taylor Reza Anari Chris Kochansky Pharm R&D Dave Dubost Wei Xu Scale-up Lab John Swestock Rick Woodward Randy Newton Kim Strauss Jim Guare NMR & MS Groups Steve Pitzenberger Sandor Varga Mike Bogusky Janine Brouillette Chuck Ross Joan Murphy Molecular Modeling Kate Holloway Chris Culberson Chromatography Group Carl Homnick Todd Anderson Christy Olson Analytical Group Kristi Hoffman Ken Anderson Matt Zrada Amanda Cortes Deanne Rudd