The mechanistic modelling of HIV-1 protease and its natural substrates: a theoretical perspective.
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Date
2020
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Abstract
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.
Description
Doctoral Degree. University of KwaZulu-Natal, Durban.