Robust inhibition of hepatitis c viral propagation

ROBUST INHIBITION OF HEPATITIS C VIRAL PROPAGATION PRADEEP ANAND RAVINDRANATH (Master of Science, University of Edinburgh Bachelor of Technology, Anna University) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN COMPUTATION AND SYSTEMS BIOLOGY SINGAPORE-MIT ALLIANCE NATIONAL UNIVERSITY OF SINGAPORE 2012 Declaration I hereby declare that this thesis is my original work and it has been written by me in its entirety I have duly acknowledged all the sources of information which have been used in the thesis This thesis has also not been submitted for any degree in any university previously Pradeep Anand Ravindranath August 24, 2012 Acknowledgement First I would like to thank my parents and my brother for the support and encouragement they have been providing me throughout my educational journey I would also like to thank my friends who were with me through all my ups and downs motivating and not letting me give up at anytime Further I would like to thank my undergraduate mentor Dr Sharmila Anishetty for her support and guidance that helped me in shaping my career, and Prof Kannian, my tutor during higher secondary education, for motivating me to always the best in things that I aspire to I would like to express my sincere gratitude to Prof Bruce Tidor for instilling in me the approach to solve problems in a systematic way, to ask properly framed scientific questions and to answer questions scientifically, through his mentorship Further I would like to thank him for providing me an opportunity to learn and work in the area of protein and small molecule research, and for being supportive throughout my candidature I would also like to thank Prof Chen Yu Zong for his support Initial training and guidance are very essential when starting any work I would like to thank Dr Nathaniel W Silver for introducing me to the techniques involved in inverse drug design and for assisting me with my questions regarding the project as well in problems concerning technical issues, in spite of overseas time differences I would also like to thank Dr Yang Shen for useful discussions regarding electrostatics Further I would like to extend my thanks to Dr Animesh Samanta, Dr Krishna Kanta Ghosh and Dr Hyung Ho Ha James from the Chang lab for assisting me with the chemistry behind the possible synthesis of triazine-based compounds i I would like to thank the current and previous lab members from MIT (Dr Bracken M King, Dr Jared E Toettcher, Dr Josh Apgar, Dr Nathaniel W Silver, Mr Gil Kwak, Dr Yang Shen, Mr Jason Biddle, Dr Yuanyuan Cui, Dr Tina Toni, Ms Nirmala Paudel, Mr Ishan S Patel and Mr David R Hagen) and SMART (Dr Sudipta Samanta, Dr Devanathan Raghunathan and Dr Jessie Lie) for being extremely supportive I want to specially thank Dr Sudipta Samanta, Dr Devanathan Raghunathan and Dr Jessie Lie for being my excellent colleagues providing useful discussions in molecular and quantum mechanics I would specially like to thank Dr Senthil Raja Jayapal for proof reading my thesis and providing me valuable comments and suggestions Finally I would like to thank Singapore-MIT Alliance for the funding and its previous and current office members (in particular Ms Carol Cheng and Ms Juliana Chai) for assistance I would also like to thank SMART and its members (in particular Dr Balasubramanian Narayanan, Ms Jocelyn Sales, Ms Regina Chan Siak Choo and Dr Ali Asgar Bhagat) for providing a wonderful work environment, support and assistance ii Contents Summary v List of Figures vii List of Tables ix Preface x Hepatitis C Virus and Inhibitor design 1.1 Significance 1.2 Life cycle 1.3 Target selection 1.4 Complexities associated with HCV drug development 1.5 NS3/4A serine protease inhibitors 1.6 Drug resistance 10 1.7 Substrate envelope hypothesis 11 1.8 Inverse Design Methodology for Small Molecule Drug Discovery 12 Inverse Drug Design Methodology with conformational change 17 2.1 Definition - rigid and non-rigid binding 18 2.2 HCV NS3/4A protease – structure selection, substrate modeling and protease preparation 19 2.2.1 Importance of the HCV NS3 helicase 21 2.2.2 Substrate modeling and protease preparation 24 2.3 Fixed shape constraint 28 2.3.1 Dynamic substrate envelope 29 2.4 Grid-based potentials 30 2.4.1 Thermodynamic process for calculation of change in total electrostatic energy upon intermolecular binding 30 2.4.2 Electrostatic binding potential calculation considering conformational change 33 2.4.3 Calculation of grid-based potentials 35 iii 2.5 2.6 2.7 2.8 Scaffold search and placement 2.5.1 Scaffold preparation and ensemble generation Side group library preparation Pairwise decomposition of the scoring function considering conformational change 2.7.1 Functional group attachment and pairwise energy evaluation Computational inverse inhibitor design 2.8.1 Design considering the conformational change 2.8.2 Hierarchical re-scoring of top structures 38 39 41 41 43 45 45 46 Design results and Analysis of designed inhibitors 3.1 Validation of non-rigid binding design implementation 3.1.1 Protein preparation, substrate envelope construction and grid-based potentials 3.1.2 Side group preparation 3.1.3 Scaffold library preparation 3.1.4 Scaffold placement and design 3.1.5 Combinatorial search, hierarchical re-scoring and results 3.2 Combinatorial search results and hierarchical rescoring 3.3 Computational analysis of designed inhibitors for HCV NS3/4A protease 3.3.1 Current inhibitors, substrate envelope and resistance mutations 3.3.2 Computational methods 3.3.3 Comparison to known inhibitors 3.3.4 Expected behavior with known mutants 48 48 Conclusion 76 Bibliography 81 49 49 50 50 51 53 60 62 63 66 67 iv Summary The design of inhibitors subject to the constraint that they only bind within the shape occupied by the substrate has been identified as a useful strategy to avoid drug resistance (termed the substrate envelope hypothesis) This effectively limits the interactions a ligand makes with the receptor to those also made by the substrate or substrates, which reduces the opportunity for mutations that disrupt inhibitor binding without also disrupting substrate binding We have developed a systematic inverse design approach that searches for optimal binders that satisfy the substrate-geometry constraint using a hierarchical re-scoring procedure; it includes an initial fast gridbased evaluation followed by use of more sophisticated energy functions to improve energetic accuracy This approach has been applied to design high-affinity human immunodeficiency virus (HIV-1) protease inhibitors with subnanomolar binding affinities and relatively flat binding profiles when tested against a panel of resistant variants Here we have applied the inverse method to design robust inhibitors for the shallow, solvent exposed, substrate-binding groove of hepatitis C virus (HCV) NS3/NS4A protease, using a serine trap warhead to covalently anchor the inhibitor scaffold to the protease This work introduces novel methodology for the covalent ligand attachment incorporated into the design procedure using a thermodynamic-cycle framework to treat the conformational change and covalent bond accompanying binding The design resulted in a collection of inhibitors that make substrate-like interactions The binding energy calculations revealed that they remained minimally affected by known prevalent resistance mutations (Arg 155 and Ala 156) losing only a maximum of kcal · mol−1 for Arg 155 and less than 15 kcal · mol−1 for Ala 156 In comparison, the inhibitors Boceprevir, ITMN-191, and TMC-435 lost nearly 15 kcal · mol−1 for Arg 155 and 35 kcal · mol−1 for Ala 156 Furthermore, our analysis validates the substrate envelope hypothesis by demonstrating that v systematic design approaches can lead to high-affinity inhibitors computed to be less susceptible to resistance than ordinary candidates, even when considering this shallow, solvent-exposed binding site vi List of Figures 1.1 1.2 1.3 1.4 1.5 Life cycle of HCV HCV proteins – Topology and function NS3 viral protein Substrate envelope hypothesis Inverse drug design 13 15 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 2.10 2.11 2.12 2.13 2.14 Inverse drug design with conformational change Rigid and nonrigid binding Substrate bound to NS3 helicase/protease Substrate bound to NS3 helicase mimic/protease Substrate residue by residue analysis Helicase mimic Modelling of substrtate Bound and Unbound envelope Thermodynamic process for rigid binding Thermodynamics process for nonrigid binding Ketoamide inhibition mechanism Triazine core Ketoacid to ketoamide synthesis Scaffold ensemble 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 3.10 3.11 Validation Low Vs Medium resolution calculation - vdW and electrostatics Low Vs Medium resolution calculation - Total energy Medium Vs High resolution calculation - vdW and electrostatics Medium Vs High resolution calculation - Total energy Designed inhibitor Preferred side groups Inhibitors inside substrate envelope Individual energy terms Substrate - Receptor interaction Inhibitors with modeled mutations 18 19 23 24 25 25 27 30 33 34 39 39 39 40 52 56 57 58 59 61 61 63 68 69 70 vii 3.12 Mutation analysis 71 3.13 Robust acting preferred side groups 73 viii reflects the established claims about the existing types of inhibitors and the mutations, and stands to support our results One limitation of the method includes requirement of the same atom centers for both bound and unbound states, except at the place where conformational change occurs to avoid ”grid errors” in grid-based calculations If the designed compounds were synthesized and experimentally tested, the findings could help to improve the design As done for the HIV protease, the experimental binding energies and the predicted binding energies can be checked for correlation Further crystal structures could be obtained and checked for the interactions and binding mode In summary, we have applied the inverse method to design robust inhibitors 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Blue – NS4A cofactor; Red – Helicase mimic; Licorice - substrate Protease; Blue – NS4A cofactor; Pink – Pink – Helicase mimic; Licorice - substrate %" c Covalent bonding &" ''" $" Scaffold and... change can be accounted for with the change in the molecule’s coulombic energy upon conformational change Hence the thermodynamic process to calculate the total electrostatic free energy change... Thermodynamic process for calculation of change in total electrostatic energy upon intermolecular binding 30 2.4.2 Electrostatic binding potential calculation considering conformational change