Browsing by Author "Sanusi, Zainab Kemi."
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Item Computational chemistry studies of subtypes B and South African C HIV proteases.(2016) Sanusi, Zainab Kemi.; Honarparvar, Bahareh.; Kruger, Hendrik Gerhardus.; Maguire, Glenn Eamonn Mitchel.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.Item The mechanistic modelling of HIV-1 protease and its natural substrates: a theoretical perspective.(2020) Sanusi, Zainab Kemi.; Maguire, Glenn Eamonn Mitchel.; Kruger, Hendrik Gerhardus.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.