Molecular dynamics simulations of complex systems including HIV-1 protease

Advances in supercomputer architectures have resulted in a situation where many scienti�fic codes are used on systems whose performance characteristics di�ffer considerably from the platform they were developed and optimised for. This is particularly apparent in the realm of Grid computing, where new technologies such as MPIg allow researchers to connect geographically disparate resources together into virtual parallel machines. Finding ways to exploit these new resources efficiently is necessary both to extract the maximum bene�fit from them, and to provide the enticing possibility of enabling new science. In this thesis, an existing general purpose molecular dynamics code (LAMMPS) is extended to allow it to perform more efficiently in a geographically distributed Grid environment showing considerable performance gains as a result. The technique of replica exchange molecular dynamics is discussed along with its applicability to the Grid model and its bene�fits with respect to increasing sampling of configurational space. The dynamics of two sub-structures of the HIV-1 protease (known as the flaps) are investigated using replica exchange molecular dynamics in LAMMPS showing considerable movement that would have been difficult to investigate by traditional methods. To complement this, a study was carried out investigating the use of computational tools to calculate binding affinity between HIV-1 protease mutants and the drug lopinavir in comparison with results derived experimentally by other research groups. The results demonstrate some promise for computational methods in helping to determine the most eff�ective course of treatment for patients in the future

Characterizing early drug resistance-related events using geometric ensembles from HIV protease dynamics:

The use of antiretrovirals (ARVs) has drastically improved the life quality and expectancy of HIV patients since their introduction in health care. Several millions are still afflicted worldwide by HIV and ARV resistance is a constant concern for both healthcare practitioners and patients, as while treatment options are finite, the virus constantly adapts via complex mutation patterns to select for resistant strains under the pressure of drug treatment. The HIV protease is a crucial enzyme for viral maturation and has been a game changing drug target since the first application. Due to similarities in protease inhibitor designs, drug cross-resistance is not uncommon across ARVs of the same class

Structural insights into HIV-1 capsid assembly, maturation and stability by cryo-electron tomography

Human immunodeficiency virus type 1 (HIV-1) is an enveloped lentivirus from the family Retroviridae which infects CD4+ T-lymphocytes in a human host, leading to Acquired Immunodeficiency Syndrome (AIDS) if untreated. A subset of retroviruses, most notably lentiviruses such as HIV-1, are unique in their ability to infect non-dividing cells. To do this, the reverse transcribed viral genome must be trafficked across an intact nuclear membrane and integrated into the host cell genome. The viral capsid plays a central role in this process. The first stage of capsid assembly is polymerisation of the viral polyprotein Gag via its CA (capsid) domain into a hexagonal immature lattice, forming a truncated sphere. The viral protease cleaves Gag and frees the CA domain, which rearranges to form a conical capsid around the viral genome, built from CA hexamers and pentamers. Despite advances in recent years, many open questions remain about immature Gag lattice assembly, maturation and modulation of capsid stability by host factors upon infection. One key question that has persisted in the field is how the remarkable structural transition between the immature Gag lattice and the mature CA lattice is achieved, which involves breaking almost all of the interactions stabilising the immature lattice. To address this, I applied cryo-electron tomography (cryo-ET) and subtomogram averaging to obtain high resolution structures of immature and mature CA in a panel of HIV-1 constructs containing different combinations of proteolytic cleavage sites inactivated by mutation. Unexpectedly, proteolytic processing directly on either side of CA was sufficient for mature lattice formation at low frequencies. I also show that a beta-hairpin domain at the CA N-terminus, previously proposed to be a structural switch, is dispensable for maturation. Instead, destabilisation of a six-helix bundle between the CA C-terminus and the adjacent SP1 peptide is the main structural determinant of maturation. Viral maturation is tightly linked to immature Gag lattice assembly, but many details such as the basic unit of lattice assembly remain unclear. The immature lattice is maintained by inter- and intra-hexamer interactions but is not a complete sphere, and the structure of Gag at discontinuous lattice edges is unknown. I implemented a new workflow to obtain Gag lattice structures by subtomogram classification of a cryo-ET data set of intact HIV-1 virions. These structures show that Gag forms novel, incomplete hexamers at lattice edges and that the CA-SP1 region forms ordered helical bun- dles in partial hexamers. Molecular dynamics simulations suggest that these partial bundles exhibit increased an tendency to unfold, suggesting a role of partial hexamer structures in initiation of maturation. Capsid stability after cell entry is important to prevent degradation of the viral RNA genome, and is modulated by small molecules such as inositol hexakisphosphate (IP6) and host proteins, including cleavage and polyadenylation specific factor 6 (CPSF6) and nucleoporin 153 (Nup153). A combination of CA pentamers and hexamers that flex to adopt different curvatures provides many different potential interfaces for cofactor binding. I developed a workflow to routinely obtain near-atomic resolution structures of CA hexamers and pentamers, by subtomogram averaging of conical, IP6-stabilised in vitro CA assemblies. These were used to investigate CPSF6 and Nup153 binding to pentamers and the effect of lattice curvature on the common binding pocket for these factors. The structures obtained show that CPSF6 and Nup153 do not bind to pentamers at the concentrations used, and that lattice flexibility can modulate Nup153 binding to hexamers

Crystallographic Analysis and Molecular Modeling Studies of HIV-1 Protease and Drug Resistant Mutants

HIV-1 protease (PR) is an effective target protein for drugs in anti-retroviral therapy (ART). Using PR inhibitors (PIs) in clinical therapy successfully reduces mortality of HIV infected patients. However, drug resistant variants are selected in AIDS patients because of the fast evolution of the viral genome. Structural, kinetic and MD simulations of PR variants with or without substrate or PIs were used to better understand the molecular basis of drug resistance. Information obtained from these extensive studies will benefit the design of more effective inhibitor in ART. Amprenavir (APV) inhibition of PRWT, and single mutants of PRV32I, PRI50V, PRI54M, PRI54V, PRI84V and PRL90M were studied and X-ray crystal structures of PR variants complexes with APV were solved at resolutions of 1.02-1.85 Å to identify structural alterations. Crystal structures of PRWT, PRV32I and PRI47V were solved at resolutions of 1.20-1.40 Å. Reaction intermediates were captured in the substrate binding cavity, which represent three consecutive steps in the catalytic reaction of HIV PR. HIV-1 PR20 variant is a multi-drug resistant variant from a clinical isolate and it is of utility to investigate the mechanisms of resistance. The crystal structures of PR20 with inactivating mutation D25N have been determined at 1.45-1.75 Å resolution, and three distinct flap conformations, open, twisted and tucked, were observed. These studies help understand molecular basis of drug resistance and provide clues for design of inhibitors to combat multi-drug resistant PR. The evaluation of electrostatic force in MD simulations is the computationally intensive work, which is of order theta(N2) with integration of all atom pairs. AMMP invokes Amortized FMM in summation of electrostatic force, which reduced work load to theta(N). A hybrid, CPU and GPU, parallel implementation of Amortized FMM was developed and improves the elapsed time of MD simulation 20 fold faster than CPU based parallelization

Structure and Dynamics of Viral Substrate Recognition and Drug Resistance: A Dissertation

Drug resistance is a major problem in quickly evolving diseases, including the human immunodeficiency (HIV) and hepatitis C viral (HCV) infections. The viral proteases (HIV protease and HCV NS3/4A protease) are primary drug targets. At the molecular level, drug resistance reflects a subtle change in the balance of molecular recognition; the drug resistant protease variants are no longer effectively inhibited by the competitive drug molecules but can process the natural substrates with enough efficiency for viral survival. Therefore, the inhibitors that better mimic the natural substrate binding features should result in more robust inhibitors with flat drug resistance profiles. The native substrates adopt a consensus volume when bound to the enzyme, the substrate envelope. The most severe resistance mutations occur at protease residues that are contacted by the inhibitors outside the substrate envelope. To guide the design of robust inhibitors, we investigate the shared and varied properties of substrates with the protein dynamics taken into account to define the dynamic substrate envelope of both viral proteases. The NS3/4A dynamic substrate envelope is compared with inhibitors to detect the structural and dynamic basis of resistance mutation patterns. Comparative analyses of substrates and inhibitors result in a solid list of structural and dynamic features of substrates that are not shared by inhibitors. This study can help guiding the development of novel inhibitors by paying attention to the subtle differences between the binding properties of substrates versus inhibitors

Molecular Mechanics Studies of Enzyme Evolutionary Mechanisms

In the current dissertation, closely related studies to quantify the mechanism underlying enzyme evolution have been discussed. The HIV-1 protease and beta-lactamase enzymes were used as model systems for these studies. These are well known enzymes that are associated with drug resistance and are associated with the pathogenic diseases, and therefore, developing molecular level understanding of drug resistance through these enzymes has fundamental as well as practical importance. In chapter 2, the relationship between errors in modeled protein structures and associated binding affinity predictions to small molecules is established. The results of this study are applicable in addressing a wide range of biological questions including enzyme evolutionary mechanisms. The next three chapters discuss different aspects of HIV-1 protease evolution. In chapter 3, the role of substrate binding in manipulating the catalytic activity of HIV-1 protease during evolution has been examined. The results suggest that HIV-1 protease can optimize its catalytic activity by manipulating its substrate binding affinity. This part of study also emphasizes the importance of considering the in-vivo environment while studying physical-chemical aspects of enzymatic evolution. In chapter 4, the role of dynamics as a constraint on the evolution of HIV-1 protease has been examined. Low frequency motions (dynamics) of an enzyme have been suggested to be critical for its function. It has been further suggested that any mutation that disrupts these low frequency motions may have an adverse affect on the catalytic function of the enzyme. In this part of study, the role of dynamics as a constraint on the evolution of HIV-1 protease has been examined by comparing experimental activity data for over 90 mutants of HIV-1 protease to correlated motion data obtained from molecular dynamics simulations of a Michaelis complex. The results of this study suggest that dynamics do not impose a significant constraint on the evolution of HIV-1 protease. In chapter 5, the role of fold stability as a constraint on the evolution of HIV-1 protease is examined. A significant tradeoff between evolvability and fold stability for HIV-1 protease was observed in our study. The results of this study suggest that fold stability imposes a significant constraint on the evolution of HIV-1 protease, and in future attempts to predict evolutionary outcomes (drug resistant mutations), fold stability should also be taken into consideration. In chapter 6, the evolution of cefotaximase activity within beta-lactamase is described. beta-lactamase is a bacterial enzyme that catalytically hydrolyzes the beta-lactam antibiotic, and therefore inactivates these drugs. Five point mutations are, however, required in the gene of this enzyme in order to develop cefotaximase activity. In this part of our study, we have studied the effect of four drug resistant amino acid mutations [A42G, E104K, G238S, and M182T] on the structural properties and cefotaximase activity of beta-lactamase. Along with the successful identification of evolutionary beneficial mutations, our analyses suggest structural rearrangement within active site as a possible mechanism for increasing the activity against cefotaxime

Hiv Integrase Mechanisms Of Resistance To Raltegravir, Elvitegravir, And Dolutegravir

ABSTRACT HIV INTEGRASE MECHANISMS OF RESISTANCE TO RALTEGRAVIR, ELVITEGRAVIR, AND DOLUTEGRAVIR by KYLA ROSS December 2015 Advisor: Dr. Ladislau Kovari Major: Biochemistry and Molecular Biology Degree: Master of Science HIV-1 integrase (HIV-1 IN or IN) is a multimeric enzyme that integrates the HIV-1 genome into the chromosomes of infected CD4+ T-cells. Currently there are three FDA approved HIV-1 IN strand transfer inhibitors (INSTIs) used in clinical practice: raltegravir (RAL), elvitegravir (ELV), and dolutegravir (DTG). The [Q148H], [Q148H, G140S], [Q148R], [Q148R, G140A] and [N155H, E92Q] mutations decrease IN susceptibility to RAL and ELV and may result in therapeutic failure. As an indicator of protein flexibility, the root mean square deviation (RMSD) of each HIV-1 IN residue in the last 5 ns of a 40 ns molecular dynamics simulation was calculated for HIV-1 IN catalytic core domain as an apoprotein and in complex with RAL, ELV, and DTG to study how the mutations affect HIV-1 IN flexibility. In addition, we studied the relationship between HIV-1 IN flexibility and resistance. We found that the mutants reduced overall HIV-1 IN flexibility relative to the WT IN apoprotein. We also observed that the catalytic 140s loop in the HIV-1 IN-INSTI complexes were more flexible in mutants that displayed higher reported EC50 FC (fold change) values. To further investigate the mutations effect on the more complexed full length HIV-1 IN structure, we used molecular dynamics simulations to study the impact of the mutants on binary (IN-viral DNA complex) and ternary (IN-viral DNA- INSTI) IN flexibility. RMSD analyses revealed that that the mutants have a rigid structure relative to the WT IN. Furthermore, mutant IN showed transient changes in the secondary structure of the 140s loop compared to the WT. In addition to these reduced flexibility and structural changes, resistance mutations alter the binding mode of RAL, ELV, and DTG to IN and viral DNA. This study is the first to identify a structural basis of IN mechanism of resistance to INSTIs that develops under treatment pressure in HIV-1 IN

Computational Approaches: Drug Discovery and Design in Medicinal Chemistry and Bioinformatics

This book is a collection of original research articles in the field of computer-aided drug design. It reports the use of current and validated computational approaches applied to drug discovery as well as the development of new computational tools to identify new and more potent drugs