Structural studies on molecular mechanisms of Nelfinavir resistance caused by non-active site mutation V77I in HIV-1 protease

Abstract

BackgroundThe human immunodeficiency virus (HIV-1) is a retrovirus causing acquired immunodeficiency syndrome (AIDS), which has become a serious problem across the world and has no cure reported to date. Human immunodeficiency virus (HIV-1) protease is an attractive target for antiviral treatment and a number of therapeutically useful inhibitors have been designed against it. The emergence of drug resistant mutants of HIV-1 poses a serious problem for conventional therapies that have been used so far. Until now, thirteen protease inhibitors (PIs), major mutation sites and many secondary mutations have been listed in the HIV Drug Resistance Database. In this study, we have studied the effect of the V77I mutation in HIV-PR along with the co-occurring mutations L33F and K20T through multi-nanosecond molecular dynamics simulations. V77I is known to cause Nelfinavir (NFV) resistance in the subtype B population of HIV-1 protease. We have for the first time reported the effect of this clinically relevant mutation on the binding of Nelfinavir and the conformational flexibility of the protease.ResultsTwo HIV-PR mutants have been considered in this study - the Double Mutant Protease (DBM) V77I-L33F and Triple Mutant Protease (TPM) V77I-K20T-L33F. The molecular dynamics simulation studies were carried out and the RMSD trajectories of the unliganded wild type and mutated protease were found to be stable. The binding affinity of NFV with wild type HIV-PR was very high with a Glide XP docking score of -9.3 Kcal/mol. NFV showed decreased affinity towards DBM with a docking score of -8.0 Kcal/mol, whereas its affinity increased towards TPM (Glide XP score: -10.3). Prime/MM-GBSA binding free energy of the wild type, DBM and TPM HIV-PR docked structures were calculated as -38.9, -11.1 and -42.6 Kcal/mol respectively. The binding site cavity volumes of wild type, DBM and TPM protease were 1186.1, 1375.5 and 1042.5 Å3 respectively.ConclusionIn this study, we have studied the structural roles of the two HIV-PR mutations by conducting molecular dynamics simulation studies of the wild type and mutant HIV-1 PRs. The present study proposes that DBM protease showed greater flexibility and the flap separation was greater with respect to the wild type protease. The cavity size of the MD-stabilized DBM was also found to be increased, which may be responsible for the decreased interaction of Nelfinavir with the cavity residues, thus explaining the decreased binding affinity. On the other hand, the binding affinity of NFV for TPM was found to be enhanced, accounted for by the decrease in cavity size of the mutant which facilitated strong interactions with the flap residues. The flap separation of TPM was less than the wild type protease and the decreased cavity size may be responsible for its lower resistance, and hence, may be the reason for its lower clinical relevance.

Structural analyses of 2015-updated drug-resistant mutations in HIV-1 protease: an implication of protease inhibitor cross-resistance

“…Current studies of protease structures bearing different resistance mutations [7, 18–20, 27–33] are generally focussed on the protease flaps located above the protease active site and how the flaps mediate PI accessibility. Mutations in this area naturally affect the flap motions, reducing PI accessibility and binding.…”

HIV-1 drug resistance mutations emerging on darunavir therapy in PI-naive and -experienced patients in the UK

“…An in silico structural study has shown that HIV-1 proteases containing the V77I substitution impact the cavity size of the protease active site and its binding affinity for nelfinavir. 22 Phenotypic experiments may be warranted to explore whether codon 77 is involved in darunavir susceptibility. It is surprising that the present analysis did not identify more positively selected darunavir resistance-associated sites, but this could reflect the rarity of emergent resistance to this agent.…”

Role of Conformational Motions in Enzyme Function: Selected Methodologies and Case Studies

“…Studies using the DEER method concluded that the semi-open conformation is the most populated state, independent of the subtype [22,47], while NMR residual dipolar coupling (RDC) measurements suggested that the closed conformation was predominant [90]. However, all studies agree that the populations of these conformers change upon inhibitor binding, with the most potent inhibitors favoring the “closed” conformation [47,91–93]. In all common subtypes of WT HIV-1 PR (subtypes B, C, and CRF_01 A/E), the residence time of inhibitors in the active-site pocket, and thus the inhibitory strength, is correlated with the degree to which said inhibitor induces flap closure.…”

“…For example, 13 out of the 19 hydrophobic core residues are associated with drug resistance acquisition, despite the absence of direct contacts with inhibitors [94]. Importantly, the accumulation of mutations in HIV-1 PR does not necessarily lead to increased drug resistance [48,93,99]. Indeed, alterations in inhibitor-protein interactions caused by secondary mutations correlate only partly with K i variations.…”

“…Indeed, alterations in inhibitor-protein interactions caused by secondary mutations correlate only partly with K i variations. The explanation most often put forward is that these secondary mutations alter the dynamic ensemble of the protein, which propagates through the entire structure (Figure 3b), sometimes even affecting the dynamically constrained catalytic Asp25 residue [90,92,93,98]. Thus, if a secondary mutation does not alter protein dynamics, it will have no effect on inhibitor binding.…”