Consistent Prediction of Mutation Effect on Drug Binding in HIV-1 Protease Using Alchemical Calculations

Abstract

Despite a large number of antiretroviral drugs targeting HIV-1 protease for inhibition, mutations in this protein during the course of patient treatment can render them inefficient. This emerging resistance inspired numerous computational studies of the HIV-1 protease aimed at predicting the effect of mutations on drug binding in terms of free binding energy ΔG, as well as in mechanistic terms. In this study, we analyze ten different protease-inhibitor complexes carrying major resistance-associated mutations (RAMs) G48V, I50V, and L90M using molecular dynamics simulations. We demonstrate that alchemical free energy calculations can consistently predict the effect of mutations on drug binding. By explicitly probing different protonation states of the catalytic aspartic dyad, we reveal the importance of the correct choice of protonation state for the accuracy of the result. We also provide insight into how different mutations affect drug binding in their specific ways, with the unifying theme of how all of them affect the crucial drug binding regions of the protease. ■ INTRODUCTIONHIV-1 (human immunodeficiency virus-1, further denoted as HIV) has caused a global epidemic that affects approximately 37 million people worldwide.1 There is no vaccine or cure available against HIV, but antiretroviral therapy (ART) is recommended for every infected individual 1 to suppress the virus. Success of ART has led to a near-normal life expectancy of HIV infected patients.2 Nevertheless, resistance toward drugs remains a major issue, making it necessary to switch therapy during the course of treatment of a single patient. 1One of the main targets of ART is the HIV protease, a protein responsible for cleaving HIV polyproteins during virus maturation ( Figure 1a). HIV protease is a homodimer with two 99 residues long subunits. The major structural components of the binding pocket of this protein are the active site at its bottom, which is followed in sequence by a loop and a short β-sheet (residues 26−32) that constitute the side of the pocket together with the so-called 80s loop (residues 79−84) ( Figure 1a). On the top the pocket is covered by the so-called flap region. In the framework of ART, HIV protease can be inhibited by protease inhibitors (PIs) (Figure 1b), a class of competitive inhibitors which are currently recommended as second-and third-line ART treatment.1 Specific substitutions in 13 different positions in the protease are considered to be major resistanceassociated mutations (RAMs). 3I50V is a RAM toward protease inhibitors Lopinavir (LPV) 4,5 and Darunavir (DRV) 6,7 and has been associated with particularly strong resistance to Amprenavir (APV) 8−10 and correspondingly to its prodrug Fosamprenavir (FPV). It also has been suggested to cause resistance toward Indinavir (IDV), 9,11 and at the same time, there is evidence pointing that this mutation has been associated with sensitivity toward Atazanavir (ATV) 12,13 and Tipranavir (TPV).14 I50 is located in the protease flap region (Figure 1a). This flap is...

Non-Active Site Mutants of HIV-1 Protease Influence Resistance and Sensitisation Towards Protease Inhibitors

“…For this purpose, we performed MD simulations for the protease mutants and wildtype in inhibitor-bound and unbound state, where in the bound state the simulations were performed in both alternative protonation states of the catalytic residues of the active site, D25 and D25 , to account for asymmetry of this complex. This allows us to identify which protonation state is more likely for both wildtype and mutant complexes, as well as to increase the accuracy of the ∆∆G estimation, as we reported previously [17]. The resulting ∆∆G calculations (Tables 2 and S2) overall indicated a good agreement in discriminating resistant and sensitising effects of mutations on the protein-ligand binding, including the opposite effects of N88S towards IDV and APV.…”

“…Numerous studies have addressed the molecular effects of RAMs. Some studies analyse the effects of selected major RAMs on binding of different inhibitors [10,11,12,13,14,15,16,17] and others the effect of different RAMs on binding of the same inhibitor [18,19,20,21,22,23,24,25,26,27]. Most of the studies are however focused on a single mutation-inhibitor combination, particularly for major RAMs outside of the binding pocket, and thus offer only a limited perspective on molecular mechanisms of the protease resistance.…”

Large scale relative protein ligand binding affinities using non-equilibrium alchemy

“…In turn, application of FEP to a vast range of protein-ligand systems revealed that the method can indeed deliver accurate relative binding affinity predictions with an error of <1 kcal mol À1 with respect to experiment. [23][24][25][26][27][28][29][30][31][32][33][34][35][36] However, the application of FEP using most MD soware remains challenging, preventing its widescale uptake.…”

“…57 Notwithstanding the lack of consensus in the scientic community on this matter, our non-equilibrium protocols 58,59 have already provided high-accuracy predictions in a number of applications involving amino acid and nucleotide mutations. 27,29,34,35,59,60 Here, we use pmx to calculate the difference in binding free energy for 482 ligand perturbations across 13 different ligandprotein activity datasets in two contemporary force elds. The calculated free energy differences were combined into a consensus estimate from the results of both force elds providing further increase in accuracy.…”

Exploring molecular mechanism of allosteric inhibitor to relieve drug resistance of multiple mutations in HIV‐1 protease by enhanced conformational sampling

“…Despite the initial success of these drugs in clinical treatments of HIV‐infected patients, unfortunately, PR has evolved into mutants to weaken the clinical efficiency of PIs, leading to multidrug resistance (MDR) toward the existing drugs . Except for the experimental studies, different simulation methods were also employed to partially probe drug resistance induced by the mutated PR . It is worth noting that Schiffer's group used crystallography and simulations to study bindings of inhibitors to PR, and their results clarified drug‐resistant mechanism of mutations in PR and binding modes of inhibitors to PR .…”