HIV-1 protease molecular dynamics of a wild-type and of the V82F/I84V mutant: possible contributions to drug resistance and a potential new target site for drugs

The protease from type 1 human immunodeficiency virus (HIV-1) is a critical drug target against which many therapeutically useful inhibitors have been developed; however, the set of viral strains in the population has been shifting to become more drug-resistant. Because indirect effects are contributing to drug resistance, an examination of the dynamic structures of a wild-type and a mutant could be insightful. Consequently, this study examined structural properties sampled during 22 nsec, all atom molecular dynamics (MD) simulations (in explicit water) of both a wild-type and the drug-resistant V82F/I84V mutant of HIV-1 protease. The V82F/I84V mutation significantly decreases the binding affinity of all HIV-1 protease inhibitors currently used clinically. Simulations have shown that the curling of the tips of the active site flaps immediately results in flap opening. In the 22-nsec MD simulations presented here, more frequent and more rapid curling of the mutant's active site flap tips was observed. The mutant protease's flaps also opened farther than the wild-type's flaps did and displayed more flexibility. This suggests that the effect of the mutations on the equilibrium between the semiopen and closed conformations could be one aspect of the mechanism of drug resistance for this mutant. In addition, correlated fluctuations in the active site and periphery were noted that point to a possible binding site for allosteric inhibitors.     

Atomic resolution crystal structures of HIV-1 protease and mutants V82A and I84V with saquinavir

Due to the limited distance data available from the experiments, the structures determined by NMR Spectroscopy may not always be as accurate as desired. Further refinement of the structures is often required and sometimes critical. With the increase of high quality protein structures determined and deposited in PDB Data Bank, commonly shared protein conformational properties can be extracted based on the statistical distributions of the properties in the structural database and used to improve the outcomes of the NMR-determined structures. Here we examine the distributions of protein interatomic distances in known protein structures. We show that based on these distributions, a set of mean-force potentials can be defined for proteins and employed to refine the NMR-determined structures. We report the test results on 70 NMR-determined structures and compare the potential energy, the Ramachandran plot, and the ensemble RMSD of the structures refined with and without using the derived mean-force potentials.     

Ligand modifications to reduce the relative resistance of multi-drug resistant HIV-1 protease

Proper proteolytic processing of the HIV-1 Gag/Pol polyprotein is required for HIV infection and viral replication. This feature has made HIV-1 protease an attractive target for antiretroviral drug design for the treatment of HIV-1 infected patients. To examine the role of the P1 and P1′positions of the substrate in inhibitory efficacy of multi-drug resistant HIV-1 protease 769 (MDR 769), we performed a series of structure–function studies. Using the original CA/p2 cleavage site sequence, we generated heptapeptides containing one reduced peptide bond with an L to F and A to F double mutation at P1 and P1′ (F-r-F), and an A to F at P1′ (L-r-F) resulting in P1/P1′ modified ligands. Here, we present an analysis of co-crystal structures of CA/p2 F-r-F, and CA/p2 L-r-F in complex with MDR 769. To examine conformational changes in the complex structure, molecular dynamic (MD) simulations were performed with MDR769–ligand complexes. MD trajectories show the isobutyl group of both the lopinavir analog and the CA/p2 L-r-F substrate cause a conformational change of in the active site of MDR 769. IC₅₀ measurements suggest the non identical P1/P1′ ligands (CA/p2 L-r-F and lopinavir analog) are more effective against MDR proteases as opposed to identical P1/P1′ligands. Our results suggest that a non identical P1/P1′composition may be more favorable for the inhibition of MDR 769 as they induce conformational changes in the active site of the enzyme resulting in disruption of the two-fold symmetry of the protease, thus, stabilizing the inhibitor in the active site.     

Structural and kinetic analysis of pyrrolidine-based inhibitors of the drug-resistant Ile84Val mutant of HIV-1 protease

Human immunodeficiency virus (HIV) protease is a well-established drug target in HIV chemotherapy. However, continuously increasing resistance towards approved drugs inevitably requires the development of new inhibitors preferably showing no susceptibility against resistant HIV protease strains. Recently, symmetric pyrrolidine-3,4-bis-N-benzyl-sulfonamides have been developed as a new class of HIV-1 protease inhibitors. The most promising candidate exhibited a K_i of 74 nM towards a wild-type protease. Herein, we report the influence of the active-site mutations Ile50Val and Ile84Val on these inhibitors by structural and kinetic analysis. Although the Ile50Val mutation leads to a significant decrease in affinity for all compounds in this series, they retain or even show increased affinity towards the important Ile84Val mutation. By detailed analysis of the crystal structures of two representatives in complex with wild-type and mutant proteases, we were able to elucidate the structural basis of this phenomenon.     

Effects of the V82A and I54V mutations on the dynamics and ligand binding properties of HIV-1 protease

A major problem in the antiretroviral treatment of HIV-infections with protease-inhibitors is the emergence of resistance, resulting from the occurrence of distinct mutations within the protease molecule. In the present work we investigated the structural properties of a triple mutant (I54V-V82A-L90M) and a double mutant (V82A-L90M) that both confer strong resistance to ritonavir (RTV), but not to amprenavir (APV). For the unliganded double mutant protease molecular dynamics simulations revealed a contraction of the ligand binding pocket, which is enhanced by the I54V mutation. The observed displacement of backbone atoms of the 80s loops (residues 80–85 and 80’–85’ of the dimer) was found to primarily affect binding of the larger RTV molecule. The pocket contraction detected for the unbound protease upon mutation is also observed in the presence of APV, but not of RTV. As a consequence, the protein-ligand contacts lost upon the V82A mutation are restored by 80s loop motions for the APV-bound, but not for the RTV-bound form. RTV binding is therefore both hampered in the initial recognition step due to the poor fit of the bulky inhibitor into the small pocket of the mutant free protease and by the loss of protein-ligand interactions in the RTV-bound protease. The synergistic nature of both effects offers an explanation for the high level of resistance observed. These findings demonstrate that large inhibitors, which tightly bind to wild-type protease, may nevertheless be prone to the emergence of resistance in the presence of particular patterns of mutations. This information should be helpful for the design of novel and more effective drugs, e.g., by targeting different residues or by developing allosteric inhibitors that are capable of regulating protease dynamics.     

Evaluation of triazolamers as active site inhibitors of HIV-1 protease

Proteases typically recognize their peptide substrates in extended conformations. General approaches for designing protease inhibitors often consist of peptidomimetics that feature this conformation. Herein we discuss a combination of computational and experimental studies to evaluate the potential of triazole-linked β-strand mimetics as inhibitors of HIV-1 protease activity.     

Structural basis of HIV-1 and HIV-2 protease inhibition by a monoclonal antibody

BACKGROUND: Since the demonstration that the protease of the human immunodeficiency virus (HIV Pr) is essential in the viral life cycle, this enzyme has become one of the primary targets for antiviral drug design. The murine monoclonal antibody 1696 (mAb1696), produced by immunization with the HIV-1 protease, inhibits the catalytic activity of the enzyme of both the HIV-1 and HIV-2 isolates with inhibition constants in the low nanomolar range. The antibody cross-reacts with peptides that include the N terminus of the enzyme, a region that is highly conserved in sequence among different viral strains and that, furthermore, is crucial for homodimerization to the active enzymatic form. RESULTS: We report here the crystal structure at 2.7 A resolution of a recombinant single-chain Fv fragment of mAb1696 as a complex with a cross-reactive peptide of the HIV-1 protease. The antibody-antigen interactions observed in this complex provide a structural basis for understanding the origin of the broad reactivity of mAb-1696 for the HIV-1 and HIV-2 proteases and their respective N-terminal peptides. CONCLUSION: A possible mechanism of HIV-protease inhibition by mAb1696 is proposed that could help the design of inhibitors aimed at binding inactive monomeric species.     

Multidrug resistance to HIV-1 protease inhibition requires cooperative coupling between distal mutations

The appearance of viral strains that are resistant to protease inhibitors is one of the most serious problems in the chemotherapy of HIV-1/AIDS. The most pervasive drug-resistant mutants are those that affect all inhibitors in clinical use. In this paper, we have characterized a multiple-drug-resistant mutant of the HIV-1 protease that affects indinavir, nelfinavir, saquinavir, ritonavir, amprenavir, and lopinavir. This mutant (MDR-HM) contains six amino acid mutations (L10I/M46I/I54V/V82A/I84V/L90M) located within and outside the active site of the enzyme. Microcalorimetric and enzyme kinetic measurements indicate that this mutant lowers the affinity of all inhibitors by 2-3 orders of magnitude. By comparison, the multiiple-drug-resistant mutant only increased the K(m) of the substrate by a factor of 2, indicating that the substrate is able to adapt to the changes caused by the mutations and maintain its binding affinity. To understand the origin of resistance, three submutants containing mutations in specific regions were also studied, i.e., the active site (V82A/I84V), flap region (M46I/I54V), and dimerization region (L10I/L90M). None of these sets of mutations by themselves lowered the affinity of inhibitors by more than 1 order of magnitude, and additionally, the sum of the effects of each set of mutations did not add up to the overall effect, indicating the presence of cooperative effects. A mutant containing only the four active site mutations (V82A/I84V/M46I/I54V) only showed a small cooperative effect, suggesting that the mutations at the dimer interface (L10I/L90M) play a major role in eliciting a cooperative response. These studies demonstrate that cooperative interactions contribute an average of 1.2 +/- 0.7 kcal/mol to the overall resistance, most of the cooperative effect (0.8 +/- 0.7 kcal/mol) being mediated by the mutations at the dimerization interface. Not all inhibitors in clinical use are affected the same by long-range cooperative interactions between mutations. These interactions can amplify the effects of individual mutations by factors ranging between 2 and 40 depending on the inhibitor. Dissection of the energetics of drug resistance into enthalpic and entropic components provides a quantitative account of the inhibitor response and a set of thermodynamic guidelines for the design of inhibitors with a lower susceptibility to this type of mutations.     

Generation and characterization of a human immunodeficiency virus type 1 (HIV-1) mutant resistant to an HIV-1 protease inhibitor.

A synthetic peptide, RPI 312, that specifically inhibits the protease of the human immunodeficiency virus type 1 (HIV-1) showed a potent inhibition on virus production, maturation, and infectivity. Treatment with this agent prevented the cleavage of Gag protein at the site between p17 and p24 in HIV-1 chronically infected MOLT-4 cells as well as in the released virus. Passage of HIV-1 in the presence of gradually increasing concentrations of this protease inhibitor resulted in emergence of a variant that could evade the drug effects. In the resistant variant the maturation of Gag proteins appeared normal, but its infectivity was reduced compared with that of the parent virus. The nucleotides coding the amino acids at and around the cleavage site between Gag proteins p17 and p24 were not changed. One point mutation (A-->G) at site 2082 of the pol gene that resulted in one amino acid change at site 84 of the protease from isoleucine to valine (I-84-->V) could be detected in the resistant variant. An HIV-1 infectious DNA clone with the I-84-->V mutation also showed reduced sensitivity to this protease inhibitor. The findings that the resistant variant had lower infectivity and was still affected by higher doses of the drug support the speculation that resistance to protease inhibitors may not be as problematic as other drug resistance.     

Thermodynamic linkage between the binding of protons and inhibitors to HIV-1 protease

The aspartyl dyad of free HIV-1 protease has apparent pK(a)s of approximately 3 and approximately 6, but recent NMR studies indicate that the aspartyl dyad is fixed in the doubly protonated form over a wide pH range when cyclic urea inhibitors are bound, and in the monoprotonated form when the inhibitor KNI-272 is bound. We present computations and measurements related to these changes in protonation and to the thermodynamic linkage between protonation and inhibition. The Poisson-Boltzmann model of electrostatics is used to compute the apparent pK(a)s of the aspartyl dyad in the free enzyme and in complexes with four different inhibitors. The calculations are done with two parameter sets. One assigns epsilon = 4 to the solute interior and uses a detailed model of ionization; the other uses epsilon = 20 for the solute interior and a simplified representation of ionization. For the free enzyme, both parameter sets agree well with previously measured apparent pK(a)s of approximately 3 and approximately 6. However, the calculations with an internal dielectric constant of 4 reproduce the large pKa shifts upon binding of inhibitors, but the calculations with an internal dielectric constant of 20 do not. This observation has implications for the accurate calculation of pK(a)s in complex protein environments. Because binding of a cyclic urea inhibitor shifts the pK(a)s of the aspartyl dyad, changing the pH is expected to change its apparent binding affinity. However, we find experimentally that the affinity is independent of pH from 5.5 to 7.0. Possible explanations for this discrepancy are discussed.     

Insights into a mutation-assisted lateral drug escape mechanism from the HIV-1 protease active site

We provide insight into the first stages of a kinetic mechanism of lateral drug expulsion from the active site of HIV-1 protease, by conducting all atom molecular dynamics simulations with explicit solvent over a time scale of 24 ns for saquinavir bound to the wildtype, G48V, L90M and G48V/L90M mutant proteases. We find a consistent escape mechanism associated with the G48V mutation. First, increased hydrophilic and hydrophobic flap coupling and water mediated disruption of catalytic dyad hydrogen bonding induce drug motion away from the dyad and promote protease flap transition to the semi-open form. Conversely, flap-inhibitor motion is decoupled in the wildtype. Second, the decrease of total interactions causes unidirectional lateral inhibitor translation by up to 4 A toward the P3 subsite exit of the active site, increased P3 subsite exposure to solvent and a complete loss of hydrophobic interactions with the opposite end of the active site. The P1 subsite moves beyond the hydrophobic active site side pocket, the only remaining steric barrier to complete expulsion being the "breathable" residue, P81. Significant inhibitor deviation is reported over 24 ns, and subsequent complete expulsion, implemented using steered molecular dynamics simulations, is shown to occur most easily for the G48V-containing mutants. Our simulations thus provide compelling support for lateral drug escape from a protease in a semi-open flap conformation. It is likely that some mutations take advantage of this escape mechanism to increase the rate of inhibitor dissociation from the protease. Finally, unidirectional translation may be countered by designing inhibitors with terminal subsites that provide sufficient anchoring to the flaps, thus increasing the steric barrier for translation in either direction.     

Structural basis for coevolution of a human immunodeficiency virus type 1 nucleocapsid-p1 cleavage site with a V82A drug-resistant mutation in viral protease

Maturation of human immunodeficiency virus (HIV) depends on the processing of Gag and Pol polyproteins by the viral protease, making this enzyme a prime target for anti-HIV therapy. Among the protease substrates, the nucleocapsid-p1 (NC-p1) sequence is the least homologous, and its cleavage is the rate-determining step in viral maturation. In the other substrates of HIV-1 protease, P1 is usually either a hydrophobic or an aromatic residue, and P2 is usually a branched residue. NC-p1, however, contains Asn at P1 and Ala at P2. In response to the V82A drug-resistant protease mutation, the P2 alanine of NC-p1 mutates to valine (AP2V). To provide a structural rationale for HIV-1 protease binding to the NC-p1 cleavage site, we solved the crystal structures of inactive (D25N) WT and V82A HIV-1 proteases in complex with their respective WT and AP2V mutant NC-p1 substrates. Overall, the WT NC-p1 peptide binds HIV-1 protease less optimally than the AP2V mutant, as indicated by the presence of fewer hydrogen bonds and fewer van der Waals contacts. AlaP2 does not fill the P2 pocket completely; PheP1' makes van der Waals interactions with Val82 that are lost with the V82A protease mutation. This loss is compensated by the AP2V mutation, which reorients the peptide to a conformation more similar to that observed in other substrate-protease complexes. Thus, the mutant substrate not only binds the mutant protease more optimally but also reveals the interdependency between the P1' and P2 substrate sites. This structural interdependency results from coevolution of the substrate with the viral protease.     

Atomistic simulations of the HIV-1 protease folding inhibition

Biochemical experiments have recently revealed that the p-S8 peptide, with an amino-acid sequence identical to the conserved fragment 83-93 (S8) of the HIV-1 protease, can inhibit catalytic activity of the enzyme by interfering with protease folding and dimerization. In this study, we introduce a hierarchical modeling approach for understanding the molecular basis of the HIV-1 protease folding inhibition. Coarse-grained molecular docking simulations of the flexible p-S8 peptide with the ensembles of HIV-1 protease monomers have revealed structurally different complexes of the p-S8 peptide, which can be formed by targeting the conserved segment 24-34 (S2) of the folding nucleus (folding inhibition) and by interacting with the antiparallel termini β-sheet region (dimerization inhibition). All-atom molecular dynamics simulations of the inhibitor complexes with the HIV-1 PR monomer have been independently carried out for the predicted folding and dimerization binding modes of the p-S8 peptide, confirming the thermodynamic stability of these complexes. Binding free-energy calculations of the p-S8 peptide and its active analogs are then performed using molecular dynamics trajectories of the peptide complexes with the HIV-1 PR monomers. The results of this study have provided a plausible molecular model for the inhibitor intervention with the HIV-1 PR folding and dimerization and have accurately reproduced the experimental inhibition profiles of the active folding inhibitors.     

Evaluating the potency of HIV-1 protease drugs to combat resistance

HIV-1 protease has been an important drug target for the antiretroviral treatment of HIV infection. The efficacy of protease drugs is impaired by the rapid emergence of resistant virus strains. Understanding the molecular basis and evaluating the potency of an inhibitor to combat resistance are no doubt important in AIDS therapy. In this study, we first identified residues that have significant contributions to binding with six substrates using molecular dynamics simulations and Molecular Mechanics Generalized Born Surface Area calculations. Among the critical residues, Asp25, Gly27, Ala28, Asp29, and Gly49 are well conserved, with which the potent drugs should form strong interactions. We then calculated the contribution of each residue to binding with eight FDA approved drugs. We analyzed the conservation of each protease residue and also compared the interaction between the HIV protease and individual residues of the drugs and substrates. Our analyses showed that resistant mutations usually occur at less conserved residues forming more favorable interactions with drugs than with substrates. To quantitatively integrate the binding free energy and conservation information, we defined an empirical parameter called free energy/variability (FV) value, which is the product of the contribution of a single residue to the binding free energy and the sequence variability at that position. As a validation, the FV value was shown to identify single resistant mutations with an accuracy of 88%. Finally, we evaluated the potency of a newly approved drug, darunavir, to combat resistance and predicted that darunavir is more potent than amprenavir but may be susceptible to mutations on Val32 and Ile84.     

Structural and kinetic analysis of drug resistant mutants of HIV-1 protease.

Mutants of HIV-1 protease that are commonly selected on exposure to different drugs, V82S, G48V, N88D and L90M, showed reduced catalytic activity compared to the wild-type protease on cleavage site peptides, CA-p2, p6pol-PR and PR-RT, critical for viral maturation. Mutant V82S is the least active (2-20% of wild-type protease), mutants N88D, R8Q, and L90M exhibit activities ranging from 20 to 40% and G48V from 50 to 80% of the wild-type activity. In contrast, D30N is variable in its activity on different substrates (10-110% of wild-type), with the PR-RT site being the most affected. Mutants K45I and M46L, usually selected in combination with other mutations, showed activities that are similar to (60-110%) or greater than (110-530%) wild-type, respectively. No direct relationship was observed between catalytic activity, inhibition, and structural stability. The mutants D30N and V82S were similar to wild-type protease in their stability toward urea denaturation, while R8Q, G48V, and L90M showed 1.5 to 2.7-fold decreased stability, and N88D and K45I showed 1.6 to 1.7-fold increased stability. The crystal structures of R8Q, K45I and L90M mutants complexed with a CA-p2 analog inhibitor were determined at 2.0, 1.55 and 1.88 A resolution, respectively, and compared to the wild-type structure. The intersubunit hydrophobic contacts observed in the crystal structures are in good agreement with the relative structural stability of the mutant proteases. All these results suggest that viral resistance does not arise by a single mechanism.     

Solution 1H NMR study of the active site structure for the double mutant H64Q/V68F cyanide complex from mouse neuroglobin

Solution (1)H NMR spectroscopy has been carried out to investigate the molecular and electronic structures of the active site in H64Q/V68F double mutant mouse neuroglobin in the cyanomet form. Two heme orientations resulting from a 180 degrees rotation about the alpha-gamma-meso axis were observed with a population ratio about 1:1, and the clearly distinguished B isomer was used to perform the study. Based on the analysis of the dipolar shifts and paramagnetic relaxation constants, the distal Gln(64)(E7) side chain is obtained to adopt an orientation that may produce hydrogen bond between the N(epsilon)H(1) and the Fe-bound cyanide. The side chain of Phe(68)(E11) is oriented out of the heme pocket just like that in triple mutant of cyanide complex of sperm whale myoglobin. A 15 degrees rotation of the imidazole ring in axial His(96) is observed, which is close to the varphi angle determined from the crystal structure of NgbCO. The quantitative determinations of the orientation and anisotropies of the paramagnetic susceptibility tensor reveal that cyanide is tilted by 8 degrees from the heme normal which allows for contact to the Gln(64)(E7) N(epsilon)H(1). The E7 and E11 residues appear to control the direction and the extent of tilt of the bound ligand. Furthermore, the tilt of the ligand has no obvious influence on the heme heterogeneity of cyanide ligation for isomer A/B of the wild type and mutant protein, indicating that factors other than steric effects, such as polarity of heme pocket, impacts on ligand binding affinity.     

Selection and analysis of HIV-1 integrase strand transfer inhibitor resistant mutant viruses

This report describes methods for the selection and analysis of antiretroviral resistance to HIV integrase strand transfer inhibitors (InSTIs) in cell culture. The method involves the serial passage of HIV-1 in the presence of increasing concentrations of test inhibitors, followed by the cloning and sequencing of the integrase coding region from the selected viruses. The identified mutations are subsequently re-engineered into a reference wild-type molecular clone, and the resulting replication capacity and level of drug resistance are determined relative to the wild-type virus. Here we describe examples of selection and analysis of InSTI-resistant viruses using four integrase inhibitors from three structurally distinct chemical classes; a diketo acid, two naphthyridines, and a pyrimidinecarboxamide. Each inhibitor selected an independent route to resistance. Interestingly, the shift in the IC₅₀ required to suppress the re-engineered resistant mutant viruses closely matched the concentration of compound used during the selection of drug resistance.     

Structural implications of drug-resistant mutants of HIV-1 protease: high-resolution crystal structures of the mutant protease/substrate analogue complexes

Emergence of drug-resistant mutants of HIV-1 protease is an ongoing problem in the fight against AIDS. The mechanisms governing resistance are both complex and varied. We have determined crystal structures of HIV-1 protease mutants, D30N, K45I, N88D, and L90M complexed with peptide inhibitor analogues of CA-p2 and p2-NC cleavage sites in the Gag-pol precursor in order to study the structural mechanisms underlying resistance. The structures were determined at 1.55-1.9-A resolution and compared with the wild-type structure. The conformational disorder seen for most of the hydrophobic side-chains around the inhibitor binding site indicates flexibility of binding. Eight water molecules are conserved in all 9 structures; their location suggests that they are important for catalysis as well as structural stability. Structural differences among the mutants were analyzed in relation to the observed changes in protease activity and stability. Mutant L90M shows steric contacts with the catalytic Asp25 that could destabilize the catalytic loop at the dimer interface, leading to its observed decreased dimer stability and activity. Mutant K45I reduces the mobility of the flap and the inhibitor and contributes to an enhancement in structural stability and activity. The side-chain variations at residue 30 relative to wild-type are the largest in D30N and the changes are consistent with the altered activity observed with peptide substrates. Polar interactions in D30N are maintained, in agreement with the observed urea sensitivity. The side-chains of D30N and N88D are linked through a water molecule suggesting correlated changes at the two sites, as seen with clinical inhibitors. Structural changes seen in N88D are small; however, water molecules that mediate interactions between Asn88 and Thr74/Thr31/Asp30 in other complexes are missing in N88D.