Structural Perspectives on Drug Resistance

Transcription

1 Structural Perspectives on Drug Resistance Irene Weber Departments of Biology and Chemistry Molecular Basis of Disease Program Georgia State University Atlanta, GA, USA

2 What have we learned from 20 years and hundreds of crystal structures of HIV protease? A. Insights into Molecular Mechanisms of Resistance High to atomic resolution crystal structures of mutants reveal structural alterations that correlate with altered protease activity, stability and inhibition. with John Louis; Jozsef Tozser B. Strategy to Combat HIV Drug Resistance Antiviral inhibitors designed to target resistant protease by introducing new polar interactions with conserved structural elements. with Arun Ghosh; Hiroaki Mitsuya

3 Database of ~60 HIV Protease Structures HIV-1 PR and Mutants of L24I, M46L, G48V, I50V, F53L, I54V/M, G73S, V82A, I84V, N88D, L90M, R8Q, K45I, D25N, combinations Structures Resolution R-factor (Å) % 20 Complexes with substrate analogs Unliganded structure Complexes with reaction intermediates Complexes with Antiviral Inhibitors 12 with Darunavir with Saquinavir with Indinavir with GRL with GRL-06579A, -0105A, -0255A HIV-2 PR Complexes with Darunavir, GRL-06579A, GRL SER-CAT

4 Advantage of High Resolution X-ray Crystallography Highly accurate atomic positions at ~1 Å resolution Increasing significance of changes in distances > 1 Å > 0.3 Å > 0.1 Å Asp60 Gln58 Val56 Tyr59 Arg57 1.6σ 2.6σ 3.6σ 2F o -F c 2.2 Å 1.5 Å 0.84 Å improving resolution

5 Multiple Conformations in Atomic Resolution Structures Asp Asp25 V82A/Darunavir at 1.1Å Resolution ccupancy: 0.6/0.4 Asp Catalytic Aspartates in V82A/Saquinavir at 0.97Å Resolution DRV

6 Strategy for Drug Design Derived From Conserved Hydrogen Bond Interactions Important main chain-main chain interactions connect inhibitor with protease flaps and residues Gustchina & Weber, FEBS Lett., 1990; Gustchina et al., Protein Eng., 1994 Met46 Gly48 Ile50 Ile50 Gly48 Lys45 P3 P2 P1 P3 Asp30 Asp29 Gly27 P1 P2 Gly27 Asp29 Asp30 Asp25 Asp25 PR-p2/NC at 1.4Å Tie et al., FEBS J. 2005

7 Verification of Darunavir Design Saquinavir designed as substrate mimic to target wild type PR Darunavir PR/SQV structure Saquinavir forms 3 H-bonds with the PR main chain atoms - nm affinity. (Tie et al., Proteins, 2007.) Saquinavir Darunavir designed with more H-bonds with main chain PR atoms to target resistant PR mutants PR/DRV structure Darunavir forms 6 H-bonds with the PR main chain atoms - pm affinity Collaboration with Arun Ghosh (Koh et al., Antimicrob. Agents Chemother 2003)

8 Designs for Antiviral Inhibitors based on Darunavir backbone P2 H N Ph Vary P2 NH 2 Vary P2 H N S P2 Darunavir Ki 16pM, antiviral IC50 4nM H N Ph H 3k N S Me GRL-0255K Ki 6 nm, IC50>1000nM H N Ph H N S GRL-06579A Ki 4.5pM, IC50 1.8nM H H N Ph H N S GRL-0255A Ki 26pM, IC nm Me H N Ph H N S GRL IC50 0.3nM Ghosh et al., JMC 2006; 2008; Amano et al., Antimicrob. Agents Chemother 2007; Wang et al., JMC 2007)

9 New Antiviral Inhibitors GRL-0255A Ki = 26pM, IC 50 = 5nM New interactions Asp30 Gly48 3.4Å 3.0Å 3.2Å 2.9Å 2.9Å 2.8Å 3.3Å 3.2Å Ile50 3.0Å 2.9Å Ile50 3.6Å 3.3Å PR/GRL0255A crystal structure solved at 1.0 Å resolution Gly48 GRL Å 3.4Å 3.4Å Asp30 Asp29 Gly27 Gly27 Asp29 Asp25 (Ghosh et al., J. Med. Chem, 2008) Asp25 H-bond dashed lines; C-H dotted lines

10 Sites of Drug Resistant Mutations Distal Flaps Active Site Dimer Interface L23I L24I D30N V32I V33F M46IL I47VA G48VM I50VL F53L I54VTALM G73ST L76V V82ATFSL I84VAC N88DS L90M Resistance mutations from hivdb.stanford.edu, 2008

11 Darunavir Inhibition of HIV-1 PR Mutants Relative Ki WT D30N V32I M46L I50V V82A I84V L90M D30N, V32I, M46L and I50V predicted to be resistant to darunavir Tie et al. JMB 2004, 338, Kovalevsky et al. JMedChem 2006, 49, Kovalevsky et al. JMB 2006, 363,

12 Active Site Mutants Lose Interactions with Inhibitor Val50 mutant has lost hydrogen bonds with Darunavir. Relative Ki = Asp Val Ala82 Darunavir Pro Ile Val Darunavir Val Ala82 has moved to reform favorable interactions with Darunavir. Relative K i = 3 Val84 has lost van der Waals interactions with Darunavir. Relative K i = 5

13 Mutants Vary in Activity on Substrates and Stability Catalytic activity varies (kcat/km) % kcat/km UC50 Stability in urea varies (UC50 is Urea Concentration at ½Vmax) WT L24I I50V F53L G73S Kd < <5 nm Reduced dimer stability and loss of intersubunit interactions L24I, I50V, F53L Ile Ile47 (Liu et al., J Mol Biol 2005; 2006) Val50 Ile50

14 Mutant F53L has Altered Intersubunit Interactions in Absence of Inhibitor open flaps Very open flaps F53 I50 L53 no interaction + I50 WT F53L F53L had more open flaps than unliganded WT and has lost intersubunit interactions of side chains of F53 and I50. F53L has 15% of WT kcat/km, reduced stability: 60% of WT stability in urea, Kd~20nM, and 10-fold worse Ki for indinavir

15 Mutant I54V has lost flap interactions with reaction intermediate I54V showed 40% WT kcat/km, ~10-20-fold reduced inhibition by DRV or SQV, and no significant change in stability Gly48` Ile50 Ile50` Gly48 Gly48` Ile50 Ile50` Gly48 P3` P3 P2` P1 P1` P2 Asp30` Asp29` Gly27` Gly27 Asp25` Asp25 Asp29 Asp30 Asp30` Asp29` Gly27` Asp25` Gly27 Asp25 Asp29 Asp30 PR-TI I54V-TI Structures at 1.5Å resolution, R factor of 0.15 [Kovalevsky et al., Biochem. 2007]

16 Hydroxyls of Darunavir and Reaction Intermediate TI Superimpose H of darunavir in similar position to H of TI Fo-Fc map at 3.6 σ level shows the TI hydroxyl oxygens. Asp25 ~ 0.7 Å ~ 0.5 Å Asp25 WT/TI, WT/DRV and unliganded F53L. Arrows show shifts relative to the unliganded structure. Catalytic Asps change orientation with ligands.

17 0.84 Å Resolution Crystal Structure of PR V32I -darunavir Darunavir binds at active site and flap site DMS 60% species: PR V32I /(DRV) 2 40% species: PR V32I /DRV Crystal contains two molecular species Kovalevsky et al. JMB 2006, 363,

18 Darunavir Interactions with Flap Residues Arg57 Trp Val Val Lys55 H-bonds C-H. Lys43 Pro44 Lys45 Met46 The flap site provides a novel target for drug designs But, no resistant mutations found here after >2 years of darunavir therapy.

19 Diverse Mechanisms of Drug Resistance Structural Differences in PR Mutants Correlate with Variation in Catalytic Activity, Inhibition and Stability 1. Reduced interactions with inhibitor and lower inhibition: D30N, V32I, M46L, I50V, I54M, I84V 2. Shift of main chain to accommodate inhibitor: V82A 3. Reduced interactions with reaction intermediate: I54V 4. Reduced intersubunit contacts and stability: L24I, F53L, I50V Guide designs of next generation of inhibitors for resistant PR

20 Structural Perspectives on Drug Resistance Crystal structures refined at Å resolution form a unique resource for structural variation due to mutations or ligands. Mechanisms of Drug Resistance Diverse effects of mutations: altered interactions with inhibitor or substrate; reduced protease stability. Structure-guided Drug Designs Darunavir and new antiviral inhibitors to combat resistance. N H N S NH 2 H N Ph H N S Me

21 Collaborators Johnson Agniswamy Brian Shen Ying Zhang Xiaxia Yu Alexander Chumanevich Andrey Kovalevsky Fengling Liu Yunfeng Tie Ting-Yi Chiu Yuan-Fang Wang Irene Weber, Biology Robert Harrison, Computer Science Georgia State University, Atlanta, GA Arun Ghosh Purdue University Lafayette, IN John Louis Hiroaki Mitsuya NIH, Bethesda, MD Jozsef Tozser Debrecen University Debrecen, Hungary Supported by the National Institutes of Health, the Georgia Cancer Coalition, the Georgia Research Alliance, and the Georgia State University Molecular Basis of Disease Program

22 Andrey Kovalevsky Alexander Chumanevich Brian Shen Johnson Agniswamy Ting-Yi Chiu Tracy Tie Yuan-Fang Wang