A Computational perspective on the concerted cleavage mechanism of the natural targets of HIV-1 protease.
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Date
2018
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Abstract
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.
Description
Doctoral Degree. University of KwaZulu-Natal, Durban.