Resolution of discordant HIV-1 protease resistance rankings using molecular dynamics simulations

The emergence of drug resistance is a major challenge for the effective treatment of HIV. In this article, we explore the application of atomistic molecular dynamics simulations to quantify the level of resistance of a patient-derived HIV-1 protease sequence to the inhibitor lopinavir. A comparative drug ranking methodology was developed to compare drug resistance rankings produced by the Stanford HIVdb, ANRS, and RegaDB clinical decision support systems. The methodology was used to identify a patient sequence for which the three rival online tools produced differing resistance rankings. Mutations at only three positions ( L10I , A71IV, and L90M ) influenced the resistance level assigned to the sequence. We use ensemble molecular dynamics simulations to elucidate the origin of these discrepancies and the mechanism of resistance. By simulating not only the full patient sequences but also systems containing the constituent mutations, we gain insight into why resistance estimates vary and the interactions between the various mutations. In the same way, we also gain valuable knowledge of the mechanistic causes of resistance. In particular, we identify changes in the relative conformation of the two beta sheets that form the protease dimer interface which suggest an explanation of the relative frequency of different amino acids observed in patients at residue 71.     

Analyzing the molecular basis of enzyme stability in ethanol/water mixtures using molecular dynamics simulations

One of the drawbacks of nonaqueous enzymology is the fact that enzymes tend to be less stable in organic solvents than in water. There are, however, some enzymes that display very high stabilities in nonaqueous media. In order to take full advantage of the use of nonaqueous solvents in enzyme catalysis, it is essential to elucidate the molecular basis of enzyme stability in these media. Toward this end, we performed μs-long molecular dynamics simulations using two homologous proteases, pseudolysin, and thermolysin, which are known to have considerably different stabilities in solutions containing ethanol. The analysis of the simulations indicates that pseudolysin is more stable than thermolysin in ethanol/water mixtures and that the disulfide bridge between C30 and C58 is important for the stability of the former enzyme, which is consistent with previous experimental observations. Our results indicate that thermolysin has a higher tendency to interact with ethanol molecules (especially through van der Waals contacts) than pseudolysin, which can lead to the disruption of intraprotein hydrophobic interactions and ultimately result in protein unfolding. In the absence of the C30-C58 disulfide bridge, pseudolysin undergoes larger conformational changes, becoming more open and more permeable to ethanol molecules which accumulate in its interior and form hydrophobic interactions with the enzyme, destroying its structure. Our observations are not only in good agreement with several previous experimental findings on the stability of the enzymes studied in ethanol/water mixtures but also give an insight on the molecular determinants of this stability. Our findings may, therefore, be useful in the rational development of enzymes with increased stability in these media.     

Absolute free energies of binding of peptide analogs to the HIV-1 protease from molecular dynamics simulations

The constants of binding of five peptide analogs to the active site of the HIV-1 aspartic-protease are calculated based on a novel sampling scheme that is efficient and does not introduce any approximations in addition to the energy function used to describe the system. The results agree with experiments. The squared correlation coefficient of the calculated vs. the measured values is 0.79. The sampling scheme consists of a series of molecular dynamics integrations with biases. The biases are selected based on an estimate of the probability density function of the system in a way to explore the conformational space and to reduce the statistical error in the calculated binding constants. The molecular dynamics integrations are done with a vacuum potential using a short cutoff scheme. To estimate the probability density of the simulated system, the results of the molecular dynamics integrations are combined using an extension of the weighted histogram analysis method (C. Bartels, Chem. Phys. Letters 331 (2000) 446-454). The probability density of the solvated ligand-protein system is obtained by applying a correction for the use of the short cutoffs in the simulations and by taking into account solvation with an electrostatic term and a hydrophobic term. The electrostatic part of the solvation is determined by finite difference Poisson-Boltzmann calculations; the hydrophobic part of the solvation is set proportional to the solvent accessible surface area. Setting the hydrophobic surface tension parameter equal to 8 mol(-1) K(-1) A(-2), absolute binding constants are in the muM to nM range. This is in agreement with experiments. The standard errors determined from eight repeated binding constant determinations are a factor of 14 to 411. A single determination of a binding constant is done with 499700 steps of molecular dynamics integration and 4500 finite difference Poisson-Boltzmann calculations. The simulations can be analyzed with respect to conformational changes of the active site of the HIV-1 protease or the ligands upon binding and provide information that complements experiments and can be used in the drug development process.     

Characterizing the dynamics and ligand-specific interactions in the human leukocyte elastase through molecular dynamics simulations

The human leukocyte elastase (HLE), a neutrophil serine protease of the chymotrypsin superfamily, is a major therapeutic target for a number of inflammatory diseases, such as chronic obstructive pulmonary disease (COPD). In this work, we present a comparative explicit water molecular dynamics (MD) study on the free and inhibitor-bound HLE. Knowledge of the flexibility and conformational changes induced by this irreversible inhibitor, whether in a prebound stage or covalently bound at the enzyme binding site, encases fundamental biological interest and is particularly relevant to ongoing structure-based drug design studies. Our results suggest that HLE operates by an induced-fit mechanism with direct intervention of a surface loop which is open toward the solvent in the free enzyme and closed while in the presence of the ligand. MM-PBSA free energy calculations furthermore elucidate the energetic contributions to the distinct conformations adopted by this loop. Additionally, a survey of the major contributions to the inhibitor binding free energies was attained. Our findings enforce the need to account for HLE flexibility, whether through the use of MD-generated ensembles of HLE conformations as targets for molecular docking or via sophisticated flexible-docking algorithms. We anticipate that inclusion of the observed HLE dynamic behavior into future drug design methodologies will have a relevant impact in the development of novel, more efficient, inhibitors.     

Understanding the basis of I50V‐induced affinity decrease in HIV‐1 protease via molecular dynamics simulations using polarized force field

Human immunodeficiency virus (HIV)‐1 protease is one of the most promising drug target commonly utilized to combat Acquired Immune Deficiency Syndrome (AIDS). However, with the emergence of drug resistance arising from mutations, the efficiency of protease inhibitors (PIs) as a viable treatment for AIDS has been greatly reduced. I50V mutation as one of the most significant mutations occurring in HIV‐1 protease will be investigated in this study. Molecular dynamics (MD) simulation was utilized to examine the effect of I50V mutation on the binding of two PIs namely indinavir and amprenavir to HIV‐1 protease. Prior to the simulations conducted, the electron density distributions of the PI and each residue in HIV‐1 protease are derived by combining quantum fragmentation approach molecular fractionation with conjugate caps and Poisson–Boltzmann solvation model based on polarized protein‐specific charge scheme. The atomic charges of the binding complex are subsequently fitted using delta restrained electrostatic potential (delta‐RESP) method to overcome the poor charge determination of buried atom. This way, both intraprotease polarization and the polarization between protease and the PI are incorporated into partial atomic charges. Through this study, the mutation‐induced affinity variations were calculated and significant agreement between experiments and MD simulations conducted was observed for both HIV‐1 protease‐drug complexes. In addition, the mechanism governing the decrease in the binding affinity of PI in the presence of I50V mutation was also explored to provide insights pertaining to the design of the next generation of anti‐HIV drugs. © 2015 Wiley Periodicals, Inc.     

Understanding the nonfouling mechanism of surfaces through molecular simulations of sugar-based self-assembled monolayers

This paper presents a molecular simulation study of the interactions of a protein (lysozyme) with self-assembled monolayers (SAMs) of mannitol and sorbitol terminated alkanethiols in the presence of explicit water molecules and ions. The all-atom simulations were performed to calculate the force generated on the protein as a function of its distance above the SAM surfaces. The structural and dynamic properties of water molecules both above the SAM surfaces and around the SAM head groups were analyzed to provide a better understanding of the nonfouling behavior of the sugar-based SAM surfaces. Results from this work suggest that both mannitol and sorbitol SAMs generate a tightly bound, structured water layer around the SAM chains. This hydration layer creates a repulsive force on the protein when it approaches the surface, resulting in a nonfouling surface despite the presence of hydrogen-bond donor groups. This work demonstrates the importance of strong surface-water interactions for surface resistance to nonspecific protein adsorption.     

Sommelet-hauser rearrangement of an ammonium ylide derived from the HIV-1 reverse transcriptase inhibitor nevirapine

Functionalization at the 3-position of the dipyridodiazepinone nevirapine ( 1 ) has been accomplished by Sommelet-Hauser rearrangement of an ylide derived from 1 . Treatment of N-cyanomethylpyrrolidinium salt 4 with potassium tert-butoxide in a mixture of dimethylsulfoxide and tetrahydrofuran at ?10°, followed by acid hydrolysis, afforded a mixture of compounds 5 and 6 in a ratio of 1:1.8. Upon treatment of 4 with sodium amide in liquid ammonia, 5 and 6 were obtained in a ratio of 1.5:1 and a combined yield of 83%. Compound 5 is the desired product resulting from Sommelet-Hauser rearrangement of 4 , whereas 6 derives from competing Stevens rearrangement and intramolecular cyclization of the aldehyde produced upon hydrolysis. Baeyer-Villiger oxidation of 5 afforded the 3-hydroxy derivative 2 , a recently identified metabolite of nevirapine.