A Computational perspective on the concerted cleavage mechanism of the natural targets of HIV-1 protease.

Doctoral Degree. University of KwaZulu-Natal, Durban.One infectious disease that has had both a profound health and cultural impact on the human race in recent decades is the Acquired Immune Deficiency Syndrome (AIDS) caused by the Human Immunodeficiency Virus (HIV). A major breakthrough in the treatment of HIV-1 was the use of drugs inhibiting specific enzymes necessary for the replication of the virus. Among these enzymes is HIV-1 protease (PR), which is an important degrading enzyme necessary for the proteolytic cleavage of the Gag and Gag-Pol polyproteins, required for the development of mature virion proteins. The mechanism of action of the HIV-1 PR on the proteolysis of these polyproteins has been a subject of research over the past three decades. Most investigations on this subject have been dedicated to exploring the reaction mechanism of HIV-1 PR on its targets as a stepwise general acid-base process with little attention on a concerted model. One of the shortcomings of the stepwise reaction pathway is the existence of more than two TS moieties, which have led to varying opinions on the exact rate-determining step of the reaction and the protonation pattern of the catalytic aspartate group at the HIV-1 PR active site. Also, there is no consensus on the actual recognition mechanism of the natural substrates by the HIV-1 PR. By means of concerted transition state (TS) structural models, the recognition mode and the reaction mechanism of HIV-1 PR with its natural targets were investigated in this present study. The investigation was designed to elucidate the cleavage of natural substrates by HIV-1 PR using the concerted TS model through the application of computational methods to unravel the recognition and reaction process, compute activation parameters and elucidate quantum chemical properties of the system. Quantum mechanics (QM) methods including the density functional theory (DFT) models and Hartree-Fock (HF), molecular mechanics (MM) and hybrid QM/MM were employed to provide better insight in this topic. Based on experience with concerted TS modelling, the six-membered ring TS structure was proposed. Using a small model system and QM methods (DFT and HF), the enzymatic mechanism of HIV-1 PR was studied as a general acid-base model having both catalytic aspartate group participating and water molecule attacking the natural substrate synchronously. The natural substrate scissile bond strength was also investigated via changes of electronic effects. The proposed concerted six-membered ring TS mechanism of the natural substrate within the entire enzyme was studied using hybrid QM/MM; “Our own N-layered Integrated molecular Orbital and molecular Mechanics” (ONIOM) method. This investigation led us to a new perspective in which an acyclic concerted pathway provided a better approach to the subject than the proposed six-membered model. The natural substrate recognition pattern was therefore investigated using the concerted acyclic TS modelling to examine if HIV-1 (South Africa subtype C, C-SA and subtype B) PRs recognize their substrates in the same manner using ONIOM approach. A major outcome in the present investigation is the computational modelling of a new, potentially active, substrate-based inhibitor through the six-membered concerted cyclic TS modelling and a small system. By modelling the entire enzyme—substrate system using a hybrid QM/MM (ONIOM) method, three different pathways were obtained. (1) A concerted acyclic TS structure, (2) a concerted six-membered cyclic TS model and (3) another sixmembered ring TS model involving two water molecules. The activation free energies obtained for the first and the last pathways were in agreement with in vitro HIV-1 PR hydrolysis data. The mechanism that provides marginally the lowest activation barrier involves an acyclic TS model with one water molecule at the HIV-1 PR active site. The outcome of the study provides a plausible theoretical benchmark for the concerted enzymatic mechanism of HIV-1 PRs which could be applied to related homodimeric protease and perhaps other enzymatic processes. Applying the one-step concerted acyclic catalytic mechanism for two HIV-1 PR subtypes, the recognition phenomena of both enzyme and substrate were studied. It was observed that the studied HIV-1 PR subtypes (B and C-SA) recognize and cleave at both scissile and non-scissile regions of the natural substrate sequences and maintaining preferential specificity for the scissile bonds with characteristic lower activation free energies. Future studies on the reaction mechanism of HIV-1 PR and natural substrates should involve the application of advanced computational techniques to provide plausible answers to some unresolved perspectives. Theoretical investigations on the enzymatic mechanism of HIV-1 PR— natural substrate in years to come, would likely involve the application of sophisticated computational techniques aimed at exploring more than the energetics of the system. The possibility of integrated computational algorithms which do not involve partitioning/restraining/constraining/cropped model systems of the enzyme—substrate mechanism would likely surface in future to accurately elucidate the HIV-1 PR catalytic process on natural substrates/ligands

Role of Protein Flexibility in Function, Resistance Pathways and Substrate Recognition Specificity in HIV-1 Protease: A Dissertation

In the 30 years since the Center for Disease Control\u27s Morbidity and Mortality Weekly Report published the first mention of what later was determined to be AIDS (Acquired immunodeficiency syndrome) and HIV (Human immunodeficiency virus) recognized as the causative pathogen, much has been done to understand this disease’s pathogenesis, development of drugs and emergence of drug resistance under selective drug therapy. Highly Active Antiretroviral Therapy (HAART), a combination of drugs that includes HIV-1 reverse transcriptase, protease, and more recently, integrase and entry inhibitors, have helped stabilize the HIV prevalence at extraordinarily high levels. Despite the recent stabilization of this global epidemic, its dimensions remain staggering with estimated (33-36 million) people living with HIV-AIDS in 2007 alone. This is because the available drugs against AIDS provide treatment for infected individuals, but HIV evolves rapidly under drug pressure and develops resistant strains, rendering the therapy ineffective. Therefore, a better understanding underlying the molecular mechanisms of viral infection and evolution is required to tackle drug resistance and develop improved drugs and treatment regimens. HIV-1 protease is an important target for developing anti-HIV drugs. However, resistant mutations rapidly emerge within the active site of the protease and greatly reduce its affinity for the protease inhibitors. Frequently, these active site drug resistant mutations co-occur with secondary/ non-active site/ associated or compensatory mutations distal to the active site. The role of these accessory mutations is often suggested to be in maintaining viral fitness and stability of protease. Many of the non-active site drug resistant mutations are clustered in the hydrophobic core in each monomer of the protease. Molecular dynamic simulation studies suggest that the hydrophobic core residues facilitate the conformational changes that occur in protease upon ligand binding. There is a complex interdependence and interplay between the inherent adaptability, drug resistant mutations and substrate recognition by the protease. Protease is inherently dynamic and has wide substrate specificity. The PI (protease inhibitor) resistant mutations, perhaps, modulate this dynamics and bring about changes in molecular recognition, such that, in resistant proteases, the substrates are recognized specifically over the PIs for the same binding site. In this thesis research, I have investigated these three complementary phenomena in concert. Chapter II examines the importance of hydrophobic core dynamics in modulating protease function. The hydrophobic core in the WT protease is intrinsically flexible and undergoes conformational changes required for protease to bind its substrates. This study investigated if dynamics is important for protease function by engineering restricted vs. flexible hydrophobic core region in each monomer of the protease, using disulfide chemistry. Under oxidizing conditions, disulfide bond established cross-link at the interface of putative moving domains in each monomer, thereby, restricting motion in this region. Upon reduction of the disulfide bond, the constraining influence was reversed and flexibility returned to near WT. The disulfide cross-linked protease showed significant loss of function when tested in functional cleavage assay. Two protease variants (G16C/L38C) and (R14C/E65C) were engineered and examined for changes in structure and enzymatic activity under oxidizing and reducing conditions. (R14C/E65C) was engineered as an internal control variant, such that cysteines were engineered between putative non-moving domains. Structurally, both the variants were very similar with no structural perturbations under oxidizing or reducing conditions. While significant loss in function was observed for (G16C/L38C) only under oxidizing conditions, (R14C/E65C) did not show any loss of function under oxidizing or reduced conditions, as expected. Successful regain of function for cross-linked (G16C/L38C) was obtained upon reversible reduction of the disulfide bond. Taken together, these data demonstrate that the hydrophobic core dynamics modulates protease function and support the hypothesis that the distal drug resistant mutations, possibly causing drug resistance by modulating hydrophobic core dynamics via long range structural perturbations. Since protease recognizes and cleaves more than 10 substrates at different rates, our further interest is to investigate if there is a differential loss of activity for some specific substrates over the others, and whether the order of polypeptide cleavage is somehow affected by restricted core mobility. In order to better answer these questions it is essential to understand: what determines the substrate binding specificity in protease? A two-pronged approach was applied to address this question as described in chapter III and IV respectively. In chapter III, I investigated the determinants of substrate specificity in HIV-1 protease by using computational positive design and engineered specificity-designed asymmetric protease (Pr3, A28S/D30F/G48R) that would preferentially bind to one of its natural substrates, RT-RH over two other substrates, p2-NC and CA-p2, respectively. The designed protease was expressed, purified and analyzed for changes in structure and function relative to WT. Kinetic studies on Pr3 showed that the specificity of Pr3 for RT-RH was increased significantly compared to the wild-type (WT), as predicted by the positive design. ITC (Isothermal Titration Calorimetry) studies confirmed the kinetic data on RT-RH. Crystal structural of substrate complexes of WT protease and Pr3 variant with RT-RH, CA-p2 and p2-NC were further obtained and analyzed. The structural analysis, however, only partially confirmed to the positive design due to the inherent structural pliability of the protease. Overall, this study supports the positive computational design approach as an invaluable tool in facilitating our understanding of complex proteins such as HIV 1 protease and also proposes the integration of internal protein flexibility in the design algorithms to make the in-silico designs more robust and dependable. Chapter IV probed the substrate specificity determining factors in HIV-1protease system by focusing on the substrate sequences. Previous studies have demonstrated that three N-terminal residues immediate to the scissile bond (P1-P3) are important in determining recognition specificity. This work investigated the structural basis of substrate binding to the protease. Catalytically active WT protease was crystallized with decameric polypeptides corresponding to five of the natural cleavage sites of protease. The structural analyses of these complexes revealed distinct P side product bound in all the structures, demonstrating the higher binding affinity of N terminal substrate for protease. This thesis research successfully establishes that intrinsic hydrophobic core flexibility modulates function in HIV-1 protease and proposes a potential mechanism to explain the role of non-active site mutations in conferring drug resistance in protease. Additionally, the work on specificity designed and N terminal product bound protease complexes advances our understanding of substrate recognition in HIV protease

Multivalent biological interactions for the detection and inhibition of HIV-1 protease

Several diseases including cancer and pathogen infection are mediated by protease activity. In HIV infection, the viral protease plays a central role in the virus lifecycle, which has made it a clear therapeutic target. The dominant approach for the treatment of HIV is heavily dependent on inhibitors of this enzyme, but no new drugs have reached the market since 2006. There is thus a need for new design principles for the development of anti-retroviral therapies. Traditional methods of HIV detection are also limited in their use at point-of-care in resource-limited settings due to their reliance on highly trained laboratory personnel, cold-chain transport and expensive reagents. This thesis examines the role of peptide-protein interactions for the inhibition and detection of HIV-1 protease. Phage display is used to isolate heptameric peptide sequences which interact specifically with the enzyme. These peptides are then utilised as sensors for the detection of the enzyme through Forster Resonance Energy Transfer (FRET). The inhibitory properties of the peptides, both in isolation and through multivalent conjugates are also investigated. Finally, insights into the nature of these peptide-protein interactions are explored through molecular docking and all-atom classical molecular dynamics simulations. The expression of recombinant HIV-1 protease in E. coli is also discussed. The peptide based systems described here are expected to be more stable to environmental effects than protein based therapies and it is hoped that this work will provide new pathways for the design of peptide-based therapeutics and diagnostics for protease related diseases which do not rely on traditional methods.Open Acces

Testing the Substrate-Envelope Hypothesis with Designed Pairs of Compounds

Acquired resistance to therapeutic agents is a significant barrier to the development of clinically effective treatments for diseases in which evolution occurs on clinical time scales, frequently arising from target mutations. We previously reported a general strategy to design effective inhibitors for rapidly mutating enzyme targets, which we demonstrated for HIV-1 protease inhibition [Altman et al. J. Am. Chem. Soc. 2008, 130, 6099–6113]. Specifically, we developed a computational inverse design procedure with the added constraint that designed inhibitors bind entirely inside the substrate envelope, a consensus volume occupied by natural substrates. The rationale for the substrate-envelope constraint is that it prevents designed inhibitors from making interactions beyond those required by substrates and thus limits the availability of mutations tolerated by substrates but not by designed inhibitors. The strategy resulted in subnanomolar inhibitors that bind robustly across a clinically derived panel of drug-resistant variants. To further test the substrate-envelope hypothesis, here we have designed, synthesized, and assayed derivatives of our original compounds that are larger and extend outside the substrate envelope. Our designs resulted in pairs of compounds that are very similar to one another, but one respects and one violates the substrate envelope. The envelope-respecting inhibitor demonstrates robust binding across a panel of drug-resistant protease variants, whereas the envelope-violating one binds tightly to wild type but loses affinity to at least one variant. This study provides strong support for the substrate-envelope hypothesis as a design strategy for inhibitors that reduce susceptibility to resistance mutations.National Science Foundation (U.S.) (NSF grant 0821391)National Institute of General Medical Sciences (U.S.) (NIH (GM066524))National Institute of General Medical Sciences (U.S.) (GM065418)National Institute of General Medical Sciences (U.S.) (the NIH (GM082209)National Institute of General Medical Sciences (U.S.) (AI41404)National Institute of General Medical Sciences (U.S.) (AI43198

Computational chemistry studies of subtypes B and South African C HIV proteases.

Master of Medical Science in Pharmacy. University of KwaZulu-Natal, Durban, 2016.HIV/AIDs is a prevalent disease infecting millions of people throughout the world. Although a lot of improvement has been achieved over the year in regard to the reduction of AIDs related deaths, a huge task lies ahead as the HIV/AIDs global epidemic keeps spreading annually. It is therefore paramount to discover and develop more and efficient drug inhibitors against HIV. The HIV protease (HIV PR) is a C2-symmentric homodimer and consisting of 99-amino acids in each monomer and because of the important role it plays in the HIV mutation, it became a major HIV drug target for the past three decades. It is on this basis that various effective antiretroviral protease inhibitors have been designed and approved for application in HIV therapy.The HIV subtype B strain is prominent in Europe and North America and is the most researched virus. The majority of the antiretroviral drugs were designed and tested against HIV subtype B. However, non-subtype B strains of the HIV virus makes up most of these infections in Southern and Eastern Africa, which are highly affected regions in the world. In South Africa, subtype C HIV-1 is the dominant strain and little research has been done regarding drug design for this subtype or testing of the effectiveness of the HIV approved antiretroviral drugs against these non-subtype B strains. Two potentially devastating mutations of subtype C-SA HIV PR were recently reported by our group. These were designated I36T↑T and L38L↑N↑L HIV PR. The I36T↑T PR mutant includes an extra amino acid, the mutation occurs at position 36 (isoleucine to threonine) and is followed by an insertion at the second threonine indicated by the upward arrow. The L38L↑N↑L PR mutant involves two amino acids insertions that is completely different from the usual 99-amino acids HIV PR, as well as five point mutations occur at the E35D, I36G, N37S, M46L and D60E. The two insertions occur at position 38 (asparagine and leucine) indicated by the two upward arrows. Therefore, the I36T↑T and L38L↑N↑L mutations consist of 100 and 101-amino acids in each monomer of the proteases respectively.In this thesis, a hybrid computational model (QM: MM) using the ONIOM approach was followed. The selected FDA inhibitors were complexed with the various proteases in the active pocket interacting with Asp 25/25' catalytic residues using the same pose in the subtype B PR as a reference X-ray structure. The HIV PR inhibitors and Asp 25/25' were treated at a high-level with quantum mechanics (QM) theory using B3LYP/6-31G(d), and the remaining HIV PR residues were considered at a low layer using molecular mechanics (MM) with the AMBER force field. This method was applied to calculate the binding free interaction energies of the selected FDA approved HIV PR drugs complexed to the HIV protease enzyme. The aim was to create and test this computational model that will reflect the experimental binding energies against subtype B, C-SA HIV PR and also a mutant from the subtype C-SA PR designated L38L↑N↑L HIV PR. The calculated binding free interaction energies results from the subtype B follow a satisfactory trend with the experimental data. However, the C-SA HIV PR inhibitor―enzyme complexes showed some discrepancies and this was ascribed to the simplified computational model that omitted water in the active site of the enzyme. The calculated binding free interaction energies for L38L↑N↑L PR as well as experimental results, showed reduced binding affinities for all the selected FDA approved inhibitors in comparison with the subtype C-SA HIV PR. The deviation could be as a result of the insertion and mutation of the subtype C HIV-1 PR that is expected to have a significant effect in altering either the binding affinity of the HIV PR inhibitors and or characteristics of the parent protease. The computational model used in this research will be improved by introducing water into the active pocket of the Asp 25/25' catalytic residues that will be treated at least at semi-empirical level. Optimization of the different ONIOM levels will be attempted in order to accurately predict activities of new potential HIV PR inhibitors

A Look Inside HIV Resistance through Retroviral Protease Interaction Maps

Retroviruses affect a large number of species, from fish and birds to mammals and humans, with global socioeconomic negative impacts. Here the authors report and experimentally validate a novel approach for the analysis of the molecular networks that are involved in the recognition of substrates by retroviral proteases. Using multivariate analysis of the sequence-based physiochemical descriptions of 61 retroviral proteases comprising wild-type proteases, natural mutants, and drug-resistant forms of proteases from nine different viral species in relation to their ability to cleave 299 substrates, the authors mapped the physicochemical properties and cross-dependencies of the amino acids of the proteases and their substrates, which revealed a complex molecular interaction network of substrate recognition and cleavage. The approach allowed a detailed analysis of the molecular–chemical mechanisms involved in substrate cleavage by retroviral proteases

Structure-based Development of Secondary Amines as Aspartic Protease Inhibitors

As novel promising scaffold for HIV protease inhibition pyrrolidine-derived inhibitors have recently been reported. In this thesis the stepwise improvement of this compound class to potent inhibitors of wildtype as well as selected mutant proteases utilizing rational drug discovery methods is reported. Based on the crystal structure of a (rac)-3,4-dimethyleneamino-pyrrolidine in complex with HIV-1 protease symmetric pyrrolidine-diesters possessing the same stereochemistry were synthesized following a chiral-pool approach. The most potent compounds of the series achieve one-digit micromolar inhibition towards wild type as well as two mutant proteases (Ile50Val and Ile84Val). The cocrystal structure of one derivative in complex with the Ile84Val HIV protease revealed that two inhibitor molecules are bound in the large active site cavity comprising an area encompassed by the catalytic dyad and the flaps in the open conformation. This is the first HIV protease cocrystal structure in which the open-flap conformation of the enzyme is stabilized by an inhibitor that concomitantly addresses the catalytic dyad. As an alternative approach towards HIV protease inhibitors, the development of symmetric 3,4-bis N-alkyl sulfonamide-pyrrolidines is described. The initial lead structure possessing benzene sulfonamide groups and benzyl substituents exhibited a Ki of 2.2 µM. The X-ray structure in complex with the HIV protease enabled the rational design of a second series of inhibitors and revealed three promising symmetric substitution patterns for further lead optimization: (A) Elongation of the P1/P1’-benzyl moieties with hydrophobic substituents in para-position, (B) ortho-substitution at the P2/P2’-phenyl ring systems, and (C) para-substitution at the P2/P2’-phenyl moieties. All three strategies were pursued and resulted in inhibitors with improved affinities up to 260 nM. To elucidate the underlying factors accounting for the SAR, the crystal structures of four representatives, at least one of each modification type, in complex with HIV protease were determined. These structures provided deeper insights into the protein–ligand interactions and the underlying principles of the SAR thus enabling to choose the most promising combination of substituents in the next design cycle. The combination of these substituents rendered a final inhibitor showing a significantly improved affinity of Ki = 74 nM and the cocrystal structure in complex with the HIV protease confirmed the successful application of the pursued optimization strategy. Subsequently the influence of the active site mutations Ile50Val and Ile84Val on these inhibitors is investigated by structural and kinetic analysis. Whereas the Ile50Val mutation leads to a significant decrease in affinity for all compounds in this series, they retain or even show increased affinity towards the crucial Ile84Val mutation. By detailed analysis of the crystal structures of two representatives in complex with wild-type and mutant proteases the structural basis of this phenomenon was elucidated. Inhibitors bearing smaller N-alkyl substituents revealed a selectivity profile not being explicable with the initial SAR. By cocrystallization of the most potent derivative of a small series with HIV-1 protease, astonishingly two different crystal forms, P2(1)2(1)2(1) and P6(1)22, were obtained. Structural analysis revealed two completely different binding modes, the interaction of the pyrrolidine nitrogen atom to the catalytic aspartates being the only similarity. Encouraged by the successful utilization of cyclic secondary amines as anchoring group in the development of HIV protease inhibitors, this strategy was expanded into a general approach for lead structure identification for aspartic proteases. An initial library comprising eleven inhibitors based on easily accessible achiral linear oligoamines was developed and screened against six selected aspartic proteases (HIV-1 protease, plasmepsin II, plasmepsin IV, renin, BACE-1, and pepsin). Several hits could be identified, among them selective as well as rather promiscuous inhibitors. The design concept was consecutively confirmed by determination of the crystal structure of two derivatives in complex with HIV-1 protease. The binding modes exhibit high similarity to the binding orientation of substrates as well as to that of peptidomimetic inhibitors. Using this information, a generalization of this binding situation to other aspartic proteases appears reasonable, thus providing a first insight into the observed structure-activity relationships

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

Off-target-based design of selective hiv-1 protease inhibitors

The approval of the first HIV-1 protease inhibitors (HIV-1 PRIs) marked a fundamental step in the control of AIDS, and this class of agents still represents the mainstay therapy for this illness. Despite the undisputed benefits, the necessary lifelong treatment led to numerous severe side-effects (metabolic syndrome, hepatotoxicity, diabetes, etc.). The HIV-1 PRIs are capable of interacting with “secondary” targets (off-targets) characterized by different biological activities from that of HIV-1 protease. In this scenario, the in-silico techniques undoubtedly contributed to the design of new small molecules with well-fitting selectivity against the main target, analyzing possible undesirable interactions that are already in the early stages of the research process. The present work is focused on a new mixed-hierarchical, ligand-structure-based protocol, which is centered on an on/off-target approach, to identify the new selective inhibitors of HIV-1 PR. The use of the well-established, ligand-based tools available in the DRUDIT web platform, in combination with a conventional, structure-based molecular docking process, permitted to fast screen a large database of active molecules and to select a set of structure with optimal on/off-target profiles. Therefore, the method exposed herein, could represent a reliable help in the research of new selective targeted small molecules, permitting to design new agents without undesirable interactions