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

Molecular analysis of human immuno-deficiency virus-1 (South African subtype C) protease drug resistance mutations emerging on Darunavir therapy.

Masters Degree. University of KwaZulu-Natal, Durban.Human immunodeficiency virus as the causative agent of acquired immune deficiency syndrome remains a serious infectious disease and the leading cause of deaths worldwide. According to UNAIDS, approximately 37 million individuals are living with HIV/AIDS and 770,000 AIDS related deaths. HIV-1 subtype C strain is responsible for approximately 70 % of individuals living with HIV. Even with this staggering statistic, not many studies have been conducted on this subtype. Currently, there exist no treatment that completely eradicates the virus from an infected individual. Although, three enzymes required by the virus to undergo intracellular replication have been targeted to delay the progression of the disease, these enzymes include; reverse transcriptase crucial for completion of the initial stages of HIV replication, integrase essential for the integration of pro-viral DNA into the host chromosomal DNA and finally the enzyme for which this study will focus only is protease which is vital for the development and assembly of infectious viral progeny. The HIV aspartyl protease plays a major role in the life cycle of the virus and has long been a target in antiviral therapy. This advancements in the knowledge of HIV biology, pathogenesis and pharmacology has led to unprecedented efforts to interpret basic findings in the development of novel antiviral drug therapies. Nonetheless, the emergence of drug resistant mutations has hampered the efficacy of HIV-1 protease inhibition therapy. These mutations reduce the binding affinity of inhibitors while maintaining viable catalytic activity and affinity for the natural substrate. In HIV-1 protease, mutations at the following positions V32I, I50V,154M, and I84V are associated with subtle structural changes that confer resistance to protease inhibitors especially darunavir. These mutations located at or adjacent to the active site cavity, compromise drug susceptibility due to weak Van der Waals interaction and binding site distortion resulting in treatment failure. In this study we analysed the functional effects of these mutations on the HIV-1 South African subtype C protease. To understand how these mutations influence drug susceptibility in HIV1 CSA protease, the mutations were introduced by site directed mutagenesis and confirmed by DNA sequencing. Over-expression and purification of wild-type and mutant protease. Followed by enzyme kinetics, inhibition (Ki) and thermodynamics studies carried out against six clinically approved drugs. Significant difference was not observed in the substrate affinity of the variant protease compared to the wildtype C-SA protease with a Michaelis constant (Km) values of 104 and 124 µM and turnover number (Kcat) of approximately 2.2 and 0.2 s-1 for variant and wildtype protease respectively. The six clinically approved drugs used in this study demonstrated reduced binding affinities and weaker inhibition towards the variant protease in comparison to the wild-type HIV-1 protease. Atazanavir, amprenavir, darunavir and saquinavir exhibited the weakest inhibition towards the variant protease with Ki ratio values of 163, 232, 465 and 247 respectively. Thermodynamic data showed less favourable Gibbs free binding energy in selected protease inhibitors towards the variant protease, largely due to decreased binding entropy. Vitality values for the variant protease against the selected protease inhibitors, confirm the impact of these mutations on the HIV-1 CSA protease. In the presence of these drug resistant mutations V32I, I50V,154M, and I84V the efficacy of the selected protease inhibitors used in this study is significantly reduced. Future studies would involve crystallization and structure determination. This will give an in-depth understanding on the structural interaction of the variant protease towards the protease inhibitors

Molecular analysis of HIV-1 resistance: biosynthesis, kinetic, and thermodynamic study of resistant HIV-PROTEASE (C-SA) mutants.

Doctoral Degree. University of KwaZulu-Natal, Durban.Abstract available in pdf

Molecular dynamics study of HIV-1 protease inhibitors and their effects on the flap dynamics of the HIV-1 subtype-C (C-SA).

Masters Degree. University of KwaZulu-Natal, Durban.The aspartyl protease human immunodeficiency virus type 1 (HIV-1) is a 99-amino acid-long homodimer responsible for processing the Gag and Gag-Pol polyproteins into functional constituent proteins necessary for development of infectious HIV particles. Of global infections recorded, subSaharan African region is represented by 56 % where nearly 25 million people are living with HIV. South Africa has been shown to carry the heaviest HIV burden in sub-Saharan Africa where the HIV-1 subtype C (C-SA) is the prominent strain. Most of the HIV-1 scientific research has been done specifically for subtype B and this has been highlighted by the weaker binding affinity displayed by the South African HIV-1 subtype C for most of the clinically approved protease inhibitors when compared to the HIV-1 subtype B protease. The two flaps of the HIV-1 PR are very essential in functioning of the enzyme as their conformations control entry of the substrate into the catalytic site of the enzyme and also to release product. It is very important to explore and understand the dynamics of these flaps in binding of different inhibitors with different binding affinities. In addition, studies have highlighted the focus on inhibiting the cleaving function of HIV-1 PR with protease inhibitors (PIs) by competing with the natural substrate for the enzyme’s active or catalytic site and thus rendering its ineffective. It has been shown that in addition to the active site, more regions of the enzyme can be possible targets for inhibition process by developing drugs that can hinder the opening of the flaps or disrupt the stability of the dimer interface. This study involved the use of computational techniques to explore the major contributing factors other than interactions with the binding site, in binding affinity of FDA approved second generation PIs complexed to HIV-1 C-SA PR. In pursuance of this objective, molecular dynamics simulations, binding-free energy calculations and dynamic analyses were utilized. Several distances, different angles between certain residues were all taken into consideration. Our findings do show that apart from binding free-energy calculations, not one single factor but several factors contribute to the binding affinity of protease inhibitors. It is clear from these results that in the development of new HIV-1 drugs, more emphasis should be made in the design of drugs with, not only better binding in the active site but also with better interaction with other regions of the enzyme. Another interesting emphasis drawn from this study is that there is still need for drug development targeting HIV-1 PR C-SA as the currently available drugs were modelled around the inhibition of HIV-1 subtype B

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

The mechanistic modelling of HIV-1 protease and its natural substrates: a theoretical perspective.

Doctoral Degree. University of KwaZulu-Natal, Durban.An epidemic that has had profound impact on humanity both culturally and health-wise in recent decades is the Acquired immunodeficiency syndrome (AIDS) triggered by the Human immunodeficiency virus (HIV). The developments of drugs, impeding specific enzymes essential for the replication of the HIV-1 virus, has been a breakthrough in the treatment of the virus. These enzymes include the HIV-1 protease (PR), which is a significant degrading enzyme necessary for the proteolytic cleavage of the Gag and Gag-Pol polyproteins, needed for the maturation of viral protein. The catalytic mechanism of the HIV-1 PR of these polyproteins is a major subject of investigation over the past decades. Most research on this topic explores the HIV-1 PR mechanism of action on its target as a stepwise general acid-base mechanism with little or no attention to the concerted process. Among the limitations of the stepwise reaction model is the presence of more than two transition state (TS) steps and this led to different views on the precise rate-determining step of the reaction as well as the protonation state of the catalytic aspartate in the active site of the HIV-1 PR. Likewise, consensus on the exact recognition mechanism of the natural substrates by HIV-1 PR is not forthcoming. The present study investigates the recognition approach and mechanism of reaction of the HIV-1 PR with its natural substrate by a means of computational models. It is intended to explain the cleavage mechanism of the reaction as a concerted process through the application of in-silico techniques. This is achieved by computing the activation energies and elucidating the quantum chemical properties of the enzyme-substrate system. An improved understanding of the mechanism will assist in the design of new HIV-1 PR inhibitors. The molecular dynamics (MD) technique with hybrid quantum mechanics and molecular mechanics (QM/MM) method that includes the density functional theory (DFT) and Amber model were utilized to investigate the concerted hydrolysis process. Based on previous studies in our group involving concerted TS modeling, a six-membered ring TS pathway was first considered. This was achieved by employing a small model system and QM methods (Hartree-Fock and DFT) for the enzymatic mechanism of the HIV-1 PR. A general-acid base (GA/GB) model where both catalytic aspartate (Asp) groups are involved in the mechanism, and the water molecule at the active site attacks the natural substrate synchronously, was utilized. A new perspective arose from the study where an acyclic concerted computational model offered activation energies closer to experiment observations than the six-membered ring model. Hence, the proposed concerted acyclic mechanism of the HIV-1 natural substrate within the entire protease was investigated using both multi-layered QM/MM “Our own N-layered Integrated molecular Orbital and molecular Mechanics” (ONIOM) theory and QM/MM MD umbrella sampling method. A comprehensive review about experimental and theoretical results for the interactions between HIV PR and its natural substrates was presented. An important output in the present study is that the acyclic TS model barrier with one water molecule at the HIV-1 PR active site (DFT study), provides marginally, the most accurate activation energies. Similarly, the computational model demonstrated that optimum recognition specificity of the enzyme depends on structural details of the substrates as well as the number of amino acids in the substrate sequence (minimum P5-P5ʹ required). By modelling the entire enzyme—substrate system using a hybrid ONIOM QM/MM method, it was observed that although both subtype B and C-SA HIV-1 PR recognize and cleave at the scissile and non-scissile regions of the natural substrate sequence, the scissile region has a lower activation free energy. In all cases we found activation free energies that are in good agreement with experimental results. Also, the free energy profiles obtained from the umbrella sampling model were in absolute agreement with experimental in vitro HIV-1 PR hydrolysis data. The outcome of this investigations offers a plausible theoretical yardstick for the concerted enzymatic mechanism of the HIV-1 PRs that is pragmatic to related aspartate proteases and possibly other enzymatic processes. Future studies on the reaction mechanism of HIV-1 PR and its natural substrate should encompass the use of advanced theoretical techniques aimed at exploring more than the energetics of the system. The prospect of integrated computational algorithms that does not involve cropped/partitioning/constraining or restraining model systems of the enzyme—substrate mechanism to accurately elucidate the HIV-1 PR catalytic process on natural substrates/inhibitors will be undertaken in our group. Computational investigations on the enzymatic mechanism of the HIV-1 PR—natural substrate involves fine-tuning the scissile amide bond strength through steric and electronic factors. This may lead to the development of potential substrate-based inhibitors with better potency and reduced toxicity. ISIQEPHU Ubhubhane olube nomthelela omkhulu ebuntwini bobabili ngokwemvelo nangokuqonda kwezempilo emashumini eminyaka amuva nje yi-Acquired immunodeficiency syndrome (AIDS) okubangelwa yi-Human immunodeficiency virus (HIV). Ukuthuthuka kwezidakamizwa, okufaka amandla ama-enzyme athile abalulekile ekuphindaphindweni kwegciwane le-HIV-1, kube yimpumelelo ekwelashweni kwaleli gciwane. La ma-enzyme afaka i-HIV-1 proteinase (PR), okuyi-enzyme ebalulekile eyonakalisayo edingekayo ekuhlanzeni kwe-protein ye-Gag ne-GagPol, edingeka ekuvuthweni kweprotheni yegciwane. Indlela ebusayo ye-HIV-1 PR yalezi zipolyprotein iyinto enkulu ephenywayo emashumini eminyaka edlule. Ucwaningo oluningi ngalesi sihloko luhlola indlela esebenza ngayo ye-HIV-1 PR kulokho okukuhlosile njengenyathelo elisisekelo le-acid-base elisebenzayo ngaphandle kokunaka noma lengenayo inqubo ehlanganisiwe. Phakathi kokukhawulelwa kwemodeli yokusabela esezingeni eliphansi kukhona ubukhona bezinyathelo ezingaphezu kwezimbili zokuguqula isimo (TS) futhi lokhu kuholele ekubukweni okuhlukile esilinganisweni esinqunyiwe sokulinganisa sokuphendula kanye nesimo sokuhlasela sethonya elishukumisayo kulowo osebenzayo indawo ye-HIV-1 PR. Ngokunjalo, ukuvumelana mayelana nendlela ngqo yokuqashelwa kwezakhi zemvelo nge-HIV-1 PR akusondeli. Ucwaningo lwamanje luphenya indlela yokuqashelwa kanye nendlela yokusabela kwe-HIV-1 PR ngesakhiwo sayo esingokwemvelo ngezindlela zamamodeli wokuncintisana. Kuhloswe ukuchaza indlela ye-cleavage yokusabela njengenqubo ekhonjiwe ngokusebenzisa amasu we-in-silico. Lokhu kutholakala ngokuhlanganisa amandla we-activation amandla kanye nokucacisa izakhiwo zamakhemikhali we-quantum wohlelo lwangaphansi lwe-enzyme. Ukuqonda okungcono kwendlela ezokusiza ekwakhiweni kwama-inhibitors amasha we-HIV-1 PR. Indlela esetshenziswayo yama-molecule (i-MD) ene-hybrid quantum mechanics kanye nemolecule mechanics (QM / MM) efaka inqubo yokusizakala yokusebenza kwe-density theory (DFT) kanye ne-Amber model ukuphenya inqubo ekhonjiwe ye-hydrolysis. Ngokusekelwe kwizifundo zangaphambili eqenjini lethu ezibandakanya ukumodelwa kwe-TS ekhonjiwe, indlela eyindilinga eyisithupha yomgwaqo eyi-TS yaqala ukubhekwa. Lokhu kutholwe ngokusebenzisa uhlelo olusha lwemodeli nezindlela ze-QM (Hartree-Fock ne-DFT) ngomshini we-enzymatic we- HIV-1 PR. Imodeli ejwayelekile ye-acid-(GA / GB) lapho amaqembu womabili we-catalytic aspartate (Asp) abandakanyeka khona emshinini, futhi i-molecule lamanzi esakhiweni esisebenzayo lihlasela i-substrate yemvelo ngokuvumelanisa, lalisetshenziswa. Kuqhamuke umbono omusha ocwaningweni lapho imodeli ye-acyclic ekhonjiwe yokuhlinzekwa kwamandla inika amandla okusebenzisa eduze nokuhlolwa okubonwayo kunasekuqaleni kwendandatho eyindandatho eyisithupha. Ngakho-ke, indlela ehlongozwayo ekhonjwe ngendlela ekhanyayo yeHIV-1 substrate yemvelo kuyo yonke iprotease iphenyisisiwe kusetshenziswa ama-QM / MM amaningi ahlukaniswe ngama-Mechanics”(ONIOM) kanye ne-QM / MM MD isampula isambulela indlela. Ukubuyekezwa okuphelele mayelana nemiphumela yokulinga kanye nemibhalo theory yokuxhumana phakathi kwe-HIV PR nezakhi zayo zemvelo kwalethwa. Umphumela obalulekile ocwaningweni lwamanje ukuthi isithintelo se-acyclic TS imodeli nge-mocule eyodwa yamanzi kwisiza esisebenzayo se-HIV-1 PR (i-DFT), sinikela ngamandla, amandla anembe kakhulu okusebenza. Ngokufanayo, imodeli yokuhlanganisa ibonise ukuthi ukuqashelwa okuphelele kweenzyme kuncike kwimininingwane yokwakheka kwama-substrates kanye nenani lama-amino acid ngokulandelana kwe-substrate (ubuncane be-P5-P5'). Ngokumodela yonke i-enzyme — uhlelo olusebenzisa uhlelo lwe-hybrid ONIOM QM / MM, kwaqapheleka ukuthi yize zombili izifunda ezingaphansi kwe-B ne-C-SA ye-HIV-1 PR zibona futhi zinamathele ezindaweni ezibucayi nezingasontekile zendlela yokulandelana engokwemvelo. isifunda esinomswakama sinamandla aphansi we-activation mahhala. Kuzo zonke izimo sithole amandla we-activation mahhala avumelane kahle nemiphumela yokuhlolwa. Futhi, amaphrofayili wamandla wamahhala atholakala kusampuli yesampuli ye-umbrella ayesesivumelwaneni ngokuphelele nedatha yokuhlolwa kwe-vitro HIV-1 PR hydrolysis. Umphumela walolu phenyo uhlinzeka ngokungenaphutha kwethiyori eyingqophamlando ye-enzymatic mechanism ye-HIV-1 PRs edlulele kumaphrotheni ahlobene ne-aspartate kanye nezinye izinqubo ze-enzymatic. Izifundo zesikhathi esizayo mayelana nendlela yokusebenza kwe-HIV-1 PR kanye nengxenye yayo yemvelo kufanele ifake phakathi ukusetshenziswa kwamasu athuthukile we-theorytical okuhloswe ngawo ukuthola ngaphezu komfutho we-system. Ithemba lama-algorithms ahlanganisiwe wokubandakanya okungabandakanyanga okuhlanganisiwe / ukwahlukanisa / ukuphoqelela noma ukuvimba izindlela eziyimodeli ze-enzyme-inqubo engaphansi yokwengeza ukucacisa ngokunembile inqubo yokulwa ne-HIV-1 PR kuzakhi zangaphansi zemvelo / ezinqandweni kuzokwenziwa eqenjini lethu. Uphenyo lwe-computational mayelana ne-enzymatic mechanism ye-HIV-1 PR-substrate yemvelo ifaka phakathi ukulungisa kahle amandla e-bond ayisihlanganisi nge-steric ne-elekthronikhi. Lokhu kungaholela ekwakhiweni kwama-inhibitors angaphansi komhlaba angaphansi nge-potency engcono nokunciphisa ubuthi

Multidimensional computational modeling of Potent BACE1 (β-Secretase) inhibitors towards Alzheimer’s disease treatment.

Doctoral Degree. University of KwaZulu-Natal, Durban.Alzheimer’s disease (AD), as a progressive multifactorial neurodegenerative abnormality of the brain, is often connected with loss or death of neurons as its primary pathogenesis. Another kind of dementia is associated with memory loss and unstable and irrational behaviors, especially among the elderly above 60 years. In South Africa, there are over four million people above the age of 60 years, with an approximation of one hundred and eighty-seven thousand living with dementia. The two distinguishing features (hallmarks) of AD are neurofibrillary tangles and β-amyloid plaques. The β-amyloid plaques result when amyloid precursor protein (APP) is cleaved by β-amyloid precursor protein cleaving enzyme1 (BACE1), otherwise known as β-secretase. Since 1999 the first BACE1 was discovered, it has become a major interest in attempting to develop drugs for the inhibition or reduction of the β-amyloid aggregates in the brain. Reducing or inhibiting the accumulation of β-amyloid has long been the target in the design of drugs for AD treatment. Having a good knowledge of the characteristic properties (BACE1) would assist in the design of potent selective BACE1 inhibitors with fewer or no side effects. Hitherto, only five drugs have been approved by the Food and Drug Administration (FDA) for the remediation of Alzheimer’s disease, and none of the approved drugs targets BACE1. In about twenty years of its discovery, several past and ongoing studies have focused on BACE1 therapeutic roles as a target in managing AD. Several attempts have previously beenmade in designing some small drugmolecules capable of good BACE1 inhibition. Some of the initially discovered BACE1 inhibitors include verubecestat, lanabecestat, atabecestat, and umibecestat (CNP-520). Although these inhibitors significantly lowered β-amyloid plaques in persons having neurological Alzheimer’s at its clinical trials (phase 3), they were suddenly terminated for some health concerns. The termination contributed to the reasons why there are insufficient BACE-targeted drugs for AD treatment. Lately, a novel potent, orally effective, and highly selective AM-6494 BACE1 inhibitor was discovered. This novel BACE1 inhibitor exhibited no fur coloration and common skin alteration, as observed with some initial BACE1 inhibitors. AM-6494 with an IC50 value of 0.4 nM in vivo is presently selected and at the preclinical phase trials. Before this study, the inhibition properties of this novel BACE1 inhibitor at the atomistic and molecular level of BACE1 inhibition remained very unclear. The first manuscript (chapter two) is a literature review on Alzheimer's disease and β-secretase inhibition: An update focusing on computer-aided inhibitor design. We provide an introductory background of the subject with a brief discussion on Alzheimer’s pathology. The review features computational methods involved in designing BACE1 inhibitors including the discontinued drugs. Using the topical keywords BACE1, inhibitor design, and computational/theoretical study in theWeb of Science and Scopus database, we retrieved over 49 relevant articles. The search years are from 2010 and 2020, with analysis conducted from May 2020 to March 2021. Our second manuscript (chapter three) reviewed BACE1 exosite-binding antibody and allosteric inhibition as an alternative therapeutic development. We studied BACE1 biological functions, the pathogenesis of the associated diseases, and the enzymatic properties of the APP site cleavage. We suggested an extensive application of advanced computational simulations in the investigation of anti-BACE1 body and allosteric exosites. It is believed that this investigation will further help in reducing the associated challenges with designing BACE1 inhibitors while exploring the opportunities in the design of allosteric antibodies. The review also revealed that some molecules exhibited dual binding sites at the active site and allosteric site. As a result, we recommend an extensive investigation of the binding free energy beyond molecular docking (such as advanced molecular dynamic simulations) as this promises to reveal the actual binding site for the compounds under investigation. Chapter four contains the detailed computational science techniques which cover the application of the vitally essential methods of molecular mechanics (MM), quantum mechanics (QM), hybrid of QM/MM, basis sets, and other computational instruments employed in this study. In the third manuscript (chapter five), we carried out computational simulations of AM-6494 and CNP- 520.CNP520 was one of the earliest BACE1 drugs that were terminated, chosen in this study forcomparative reasons. This simulation was to elucidate and understand the binding affinities of these two inhibitors at the atomistic level. We explored the quantum mechanics (QM) density functional theory (DFT) and hybrid QM/MM of Our Own N-layered Integrated molecular Orbital and Molecular Mechanics (ONIOM) in these simulations. These computational approaches helped in predicting the electronic properties of AM-6494 and CNP-520, including their binding energies when in complex with BACE1. Considering the debates on which protonated forms of Asp 32 and Asp 288 gives a more favorable binding energy, we analysed the two forms which involved the protonation and un-protonation of Asp 32 and Asp 228.The ONIOM protonated model calculation gave binding free energy of -33.463 kcal/mol (CNP-520)and 62.849 kcal/mol (AM-6494) while the binding free energy of -59.758 kcal/mol was observed for the unprotonated AM-6494 model. These results show the protonated model as a more favourable binding free energy when compared with the un-protonation AM-6494 model. Further thermochemistry processes coupled with molecular interaction plots indicate that AM-6494 has better inhibition properties thanCNP-520.However, it was observed that the protonation and the un-protonation of Asp 32 and Asp 228 modelscould adequately illustrate the interatomic binding of the ligands-BACE1 complex. To further explicate the binding mechanism, conformational and structural dynamism of AM-6494 relative to CNP-520 in complex with BACE1, we carried out advanced computational simulations in the fourth manuscript (chapter six). The extensive application of accelerated molecular dynamics simulations, as well as principal component analysis, were involved. From the results, AM-6494 further exhibited higher binding affinity with van der Waals as the predominant contributing energy relative to CNP-520. Furthermore, conformational analysis of the β-hairpin (flap) within the BACE1 active site exhibited efficient closed flap conformations in complex withAM-6494 relative to CNP-520, whichmostly alternated between closed and semi-open conformational dynamics. These observations further elucidate that AM- 6494 shows higher inhibitory potential towards BACE1. The catalytic dyad (Asp32/228), Tyr14, Leu30, Tyr71, and Gly230 constitute essential residues in both AM-6494 potencies CNP-520 at the BACE1 binding interface. The results from these extensive computational simulations and analysis undoubtedly elucidate AM-6494 higher inhibition potentials that will further help develop new molecules with improved potency and selectivity for BACE1. Besides, grasping the comprehensive molecular mechanisms of the selected inhibitors would also help in fundamental pharmacophore investigation when designing BACE1 inhibitors. Finally, the implementation of computational techniques in the designing of BACE1 inhibitors has been quite interesting. Nevertheless, the designing of potent BACE1 inhibitors through the computational application of the QM method such as the density functional theory (DFT), MM, and a hybrid QM/MM method should be extensively explored. We highly recommend that experimentalists should always collaborate with computational chemists to save time and other resources. ISIZULU ABSTRACT Iqoqa Isifo se-Alzheimer (AD), njengoba siqhubeka siyinhlanganisela yezimbangela ze- neurodegenerative engajwayelekile ebuchosheni, isikhathi esiningi kuxhumana nokulahleka noma ukufa kwama-neurons njengongqaphambili we-pathogenesis. Kungolunye uhlobo lwedementia oluhambisana nokulahlekelwa ukukhumbula kanyenokuxenga kanye nokuphanjanelwa ingqondo, ikakhulukazi kubantu abadala esebeneminyaka engaphezulu kuka-60. ENingizimu Afrikha, kunabantu abangaphezulu kwezigidi ezine abangephezulu kweminyaka ewu-60, ngokuhlawumbisela nje abayinkulungwane namashumi ayisishayangolombili nesikhombisa baphila nedemetia. Zimbili izimpawu ezihlukanisekayo ze-AD ziba-ama-neurofibrillary tangles kanye ne-B-amyloid plaques. I-B-amyloid plaques ingumphumela ngesikhathi i-amyloid eyiprotheni egijimayo iqhwakele oketshezini i-enzyme1 (BACEI), ngale kwalokho yaziwanjenge B-secretase. Kusukela ngo 1999 i-BAC1 yatholakala, isiphenduke ungqaphambili emizamweni yokwakha isidakamizwa sokwehlisa i-B-amyloid ngokwezinga lengqondo. Ngokunciphisa ukwanda kwe-B-amyloid isiphenduke okuqondiwe mayelana nokuqopha isidakamizwa ukuze kwelashwe i-AD. Ukuba nolwazi oluhle oluthinta isici sezakhi ze-BACE1 kuzosiza ekubazeni amandla akhethiwe i-BACE1 ukuvimbela imiphumela engaqondiwe. Kuze kube manje mihlanu imithi esiphasisiwe ngabezokuphatha ukudla kanye nezidakamizwa (FDA) ukwelapha isifo se-Alzheimer kanye nokuthi azikho kulezi eziphasisiwe izidakamizwa ebhekana ngqo ne-BACE1. Emva kokuba selitholakele lapho nje eminyakeni engu 20, sekunezinye esikhathini esedlule kanye nezifundo ezisaqhubeka zigxile ngokubheka kakhulu iqhaza lokwelapha i-BAC1 njengokuqondiswe ekungameleni u-AD. Imizamo eminingana yenziwa esikhathini esedlule ukuqopha uketshezi lwezidakamizwa olukwazi ukuvimba kahle i-BACE1. i-B-amyloid plaques kumuntu one-neurological ye-Alzheimer’s kumzamo (isigaba 3), kwabuye kwanqanyulwa ngenxa yokukhathazeka ngokwezempilo. Ukunqanyulwa kwanikela kuzizathu zokusilele kwezidakamizwa okuqondene nokulashwa kwe-AD. Kamuva, i-novel enamandla, ngisho ngawo umlomo kanye neyakhethwa ngezinga eliphezulu i-AM-6494 BACE1 evikelayo yatholakala. Le noveli i-BACE1 evimbayo yabukisa hhayi ukushintsha kombala woboya kanye nokushintsha kwesikhumba okujwayelekile, njengoba kubukwa nezivimbo zokuqala ze-BACE1. I-AM-6494 ne-IC50 enobumqoka buka 0.4nM kuyo i-vivo ekhethwa ngokwamanje kanye nesigaba sembulambethe yemizamo. Ngaphambi kwalesi sifundo, izakhi zesivimbela zale noveli i-BACE1zivimba ngokwe-atomistic kanye neqophelo le-molecular ye-B ACE1evimbayo kusale nje kungacacile. Umqulu wokuqala (isahluko sesibili) ukubuyekezwa kwesifo se-Alzheimer’s kanye no-B-secretase ovimbayo: ezikhumbuzayo ezigxile ngokusizwa yikhompuyutha eyisivimbo ngokwakhiwa. Sethula isendlalelo sesifundo kanye nengxoxo kafushane nezimbangela nemiphumela ye-Alzheimer. Ukubukezwa kwezimpawu zendlela zobukhompuyutha kufaka ekuqopheni isivimbo se-BACE1 nokuqhutshekiswa kwesidakamizwa. Ngokusebenzisa ofeleba begama BACE1, kusho ukwakha isivimbo, kanye nesifundo senjulalwazi kulwembu lobuchwepheshe kanye ne-Scopus sesizindalwazi. Sathola amaphepha acwaningiwe anokuhlobana angaphezulu kuka 49. Unyaka wokuthungatha usukela ku2010 kuya ku2020, nohlaziyo lwenziwa kusukela kuNhlaba 2020 kuya kuNdasa 2021. Umqulu wethu wesibili (isahluko sesithathu) sabuyekeza i-BACE ehlanganisa i-exosite antibody kanye ne-allosteric yokuthuthukisa ukwelashwa. Sakufunda ukusebenza kwesayensi yokuphila ye-BACE1, i-pathogenesis ehambisana nezifo kanye nezakhi zama-enzymatic esizinda sokuhlukana se-APP. Saphakamisa ukufakwa okunzulu nokucokeme kokulinganisa ngobuchwepheshe bekhompuyutha ekuphenyeni ama-anti-BACE1 omzimba kanye ne-allosteric ye-exosites. Kuyakholeka ukuthi uphenyo luzoqhubeka nokusiza ekwehliseni izinselelo ezihambisana nokwakha isithiyo se-BACE1 ngesikhathi kuhlolwa amathuba okwakheka kwe-allosteric yama-antibodies. Ubuyekezo luphinde lwaveza uketshezi olubukisa isizinda sokuhlanganisa kabili kusizinda esikhuthele kanye nesizinda se-allosteric. Umphumela, kube ukwenza isincomo mayelana nocwaningo olunzulu oluzohlanganisa umfutho okhululekile odlulele ku-molecular docking (njengesicokeme se-molecular yokuhlukahlukana kokulinganisa) njengoba lokhu kuthembisa ukuveza isiza esibopha ngempela ama-compounds angaphansi Isahluko sesine siqukethe imininingwane ngamaqhinga e-computational sayensi efaka isicelo esibalulekile sezindlela ezibalulekile ze-molecular mechanics (MM), i-quantum mechanics (QM), i-hybrid ye-QM/MM, ngesisekelo samasethi kanye namanye amathuluzi ekhompuyutha akhethwa kulesi sifundo. Kumqulu wesithathu (isahluko sesihlanu), siqhube isilinganiso se-computational ye-AM-6494 kanye CNP-520.I-CNP-520 kwakungenye yezidakamizwa zokuqala zeBACE1 ezashatshalaliswa, zakhethwa kulesisifundo ngezizathu zokuqhathanisa. Ukulinganisa kwakuchaza kanye nokuqonda ukusondelana ngokuhlanganiswa kwezithiyo ezimbili kusigaba se-atomistic. Kwahlolwa i-quatum mechanics (QM) yesisindo yokusebenza kwenjulalwazi (DFT) kanye ne-hybrid QM/MM yokwethu okuno-N oluwugqinsi lwe-molecular Orbital kanye ne-Molecular Mechanics (ONIOM) kulolu linganiso. Lezi zindlelakwenza ze-computational zasiza ekuqageleni kwezakhiwo zama-electronic e-AM-6494 kanye CNP-520, kungena namandla okuhlanganisa ngesikhathi kuba lukhuni ne-BACE1. Ngokucabanga izinkulumo mpikiswano mayelana nokuma kwe-protonated ye-Asp32 kanye Asp288 kunika ukuvumelana namandla okuhlanganisa, nokuhlaziya izimo ezimbili ezifaka i-protonation kanye ne-unprotonation ye-Asp32 kanye Asp228. I-ONIOM ye-protonated yomfanekiso wokubala wanikeza amandla akhululekile okuhlanganisa -33,463kcal/mol (NP-520) kanye 62.849 kcal /mol kwavela i-unprotonate ye-AM6494. Imiphumela itshengisa ukuthi i-protonated iyisifanekiso njengoba kuyisona esivumela ukuhlanganiswa ngokukhululeka ngesikhathi lapho bekuqhathanisa ne-unprotonation yomfanekiso u-AM-649. Kuqhutshelwa phambili nemisebenzi ye-thermochemistry kuhlangana nokudlelana ne-molecular plots kutshengisa ukuthi i-AM-649 inezakhiwo ezinhle zokuvimba kune CNP-520. Yize kunjalo kwabonakala ukuthi i-protonation kanye ne-unprotonation ye-Asp32 kanye neyomfanekiso owu- Asp228 bekungatshengisa ngokwenele ukuhlanganisa ngokwe-interatomic yama-ligands EBACE1 ebilukhuni. be-BACE1 ngokwedlulele isilinganiso se-computational. Ukwenza ngokujulile kuphangiswa isilinganiso se-molecular ngokuhlukana, kwakakwa nohlaziyo olusemqoka lwezingxenyana. Imiphumela ye-AM-6494 yaqhubeka yatshengisa ukusondelana kokuhlanganiswayo no-van der Waals njengohamba phambili ekunikeleni amandla ahlobene ne-CNP-520. Ukuvuma kohlaziyo lwe-B-hairpin ngaphakathi ku-BACE1 kutshengiswa esizeni esiphilayo esivala ngendlela umnyakazo wokuvuma kobunkimbinkimbi be-AM-6494 ehlobene neCNP-520, ngokuvamile eshitshashintshayo phakathi kwevalekile kanye nezishaya sakuvuleka kokuvuma okunhlobonhlobo. Lokhu kuhlolwa kuqhubeke kwachazwa ngokuthi i-AM-6494 itshengisa ukuvimba okukhulu nokunethemba mayelana ne-BACE1. Isikhuthazizinguquko se-dyad (Asp32/228), Tyr14, Leu 30, Tyr 71, kanye ne-Gly230 kwakha izinsalela ezibalulekile nxazombili kuAM-6494ne-potencies yeCNP-520 kuBACE1 nesixhumanisi esihlanganisayo. Imiphumela ivela kulama-computational anzulu ayisilinganiso kanye nohlaziyo olucacisa ngokungangabazi i-AM-6494 enesivimbelo esiphakeme esingakwazi ukuqhubeka nokusiza intuthuko yama-molecules amasha anamandla athuthukile kanye nakhethelwe i-BACE1. Ngaphandle kwalokhu, ukucosha izinkambiso ezibanzi ze-moleculor mayelana nezivimbo ezikhethiwe kuzosiza mayelana nophenyo olubalulekile lwe- pharmacophore ngesikhathi kuqoshwa izivimbo se-BACE1. Ekugcineni, ukwenziwa kwe-computational ngokwamacebo ekubazeni izivimbo ze-BACE1 kube into ehlaba umxhwele. Nokho ukubaza izivimbo ezinamandla ze-BACE1 ngokusebenzisa i-computational yendlela ye-QM njengenjulalwazi yesisindo esisebenzayo (DFT), MM, kanye nendlela ye-hybrid QM/MM kufanele iphenywe kanzulu. Sincoma kakhulu ukuthi ongoti abenza izibonisi kufanele njalo bahlangane nama-computational chemists ukonga isikhathi kanye nezinye izinsiza