Thermodynamic basis of resistance to HIV-1 protease inhibition: calorimetric analysis of the V82F/I84V active site resistant mutant

One of the most serious side effects associated with the therapy of HIV-1 infection is the appearance of viral strains that exhibit resistance to protease inhibitors. The active site mutant V82F/I84V has been shown to lower the binding affinity of protease inhibitors in clinical use. To identify the origin of this effect, we have investigated the binding thermodynamics of the protease inhibitors indinavir, ritonavir, saquinavir, and nelfinavir to the wild-type HIV-1 protease and to the V82F/I84V resistant mutant. The main driving force for the binding of all four inhibitors is a large positive entropy change originating from the burial of a significant hydrophobic surface upon binding. At 25 degrees C, the binding enthalpy is unfavorable for all inhibitors except ritonavir, for which it is slightly favorable (-2.3 kcal/mol). Since the inhibitors are preshaped to the geometry of the binding site, their conformational entropy loss upon binding is small, a property that contributes to their high binding affinity. The V82F/I84V active site mutation lowers the affinity of the inhibitors by making the binding enthalpy more positive and making the entropy change slightly less favorable. The effect on the enthalpy change is, however, the major one. The predominantly enthalpic effect of the V82F/I84V mutation is consistent with the idea that the introduction of the bulkier Phe side chain at position 82 and the Val side chain at position 84 distort the binding site and weaken van der Waals and other favorable interactions with inhibitors preshaped to the wild-type binding site. Another contribution of the V82F/I84V to binding affinity originates from an increase in the energy penalty associated with the conformational change of the protease upon binding. The V82F/I84V mutant is structurally more stable than the wild-type protease by about 1.4 kcal/mol. This effect, however, affects equally the binding affinity of substrate and inhibitors.     

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.     

X-ray crystal structures of human immunodeficiency virus type 1 protease mutants complexed with atazanavir

Atazanavir, which is marketed as REYATAZ, is the first human immunodeficiency virus type 1 (HIV-1) protease inhibitor approved for once-daily administration. As previously reported, atazanavir offers improved inhibitory profiles against several common variants of HIV-1 protease over those of the other peptidomimetic inhibitors currently on the market. This work describes the X-ray crystal structures of complexes of atazanavir with two HIV-1 protease variants, namely, (i) an enzyme optimized for resistance to autolysis and oxidation, referred to as the cleavage-resistant mutant (CRM); and (ii) the M46I/V82F/I84V/L90M mutant of the CRM enzyme, which is resistant to all approved HIV-1 protease inhibitors, referred to as the inhibitor-resistant mutant. In these two complexes, atazanavir adopts distinct bound conformations in response to the V82F substitution, which may explain why this substitution, at least in isolation, has yet to be selected in vitro or in the clinic. Because of its nearly symmetrical chemical structure, atazanavir is able to make several analogous contacts with each monomer of the biological dimer.     

Exploring the energy landscapes of molecular recognition by a genetic algorithm: Analysis of the requirements for robust docking of HIV-1 protease and FKBP-12 complexes

Energy landscapes of molecular recognition are explored by performing “semi-rigid” docking of FK-506 and rapamycin with the Fukisawa binding protein (FKBP-12), and flexible docking simulations of the Ro-31-8959 and AG-1284 inhibitors with HIV-1 protease by a genetic algorithm. The requirements of a molecular recognition model to meet thermodynamic and kinetic criteria of ligand-protein docking simultaneously are investigated using a family of simple molecular recognition energy functions. The critical factor that determines the success rate in predicting the structure of ligand-protein complexes is found to be the roughness of the binding energy landscape, in accordance with a minimal frustration principle. The results suggest that further progress in structure prediction of ligand-protein complexes can be achieved by designing molecular recognition energy functions that generate binding landscapes with reduced frustration. © 1996 Wiley-Liss, Inc.     

High resolution crystal structures of HIV-1 protease with a potent non-peptide inhibitor (UIC-94017) active against multi-drug-resistant clinical strains

The compound UIC-94017 (TMC-114) is a second-generation HIV protease inhibitor with improved pharmacokinetics that is chemically related to the clinical inhibitor amprenavir. UIC-94017 is a broad-spectrum potent inhibitor active against HIV-1 clinical isolates with minimal cytotoxicity. We have determined the high-resolution crystal structures of UIC-94017 in complexes with wild-type HIV-1 protease (PR) and mutant proteases PR(V82A) and PR(I84V) that are common in drug-resistant HIV. The structures were refined at resolutions of 1.10-1.53A. The crystal structures of PR and PR(I84V) with UIC-94017 ternary complexes show that the inhibitor binds to the protease in two overlapping positions, while the PR(V82A) complex had one ordered inhibitor. In all three structures, UIC-94017 forms hydrogen bonds with the conserved main-chain atoms of Asp29 and Asp30 of the protease. These interactions are proposed to be critical for the potency of this compound against HIV isolates that are resistant to multiple protease inhibitors. Other small differences were observed in the interactions of the mutants with UIC-94017 as compared to PR. PR(V82A) showed differences in the position of the main-chain atoms of residue 82 compared to PR structure that better accommodated the inhibitor. Finally, the 1.10A resolution structure of PR(V82A) with UIC-94017 showed an unusual distribution of electron density for the catalytic aspartate residues, which is discussed in relation to the reaction mechanism.     

Nine Crystal Structures Determine the Substrate Envelope of the MDR HIV-1 Protease

Under drug selection pressure, emerging mutations render HIV-1 protease drug resistant, leading to the therapy failure in anti-HIV treatment. It is known that nine substrate cleavage site peptides bind to wild type (WT) HIV-1 protease in a conserved pattern. However, how the multidrug-resistant (MDR) HIV-1 protease binds to the substrate cleavage site peptides is yet to be determined. MDR769 HIV-1 protease (resistant mutations at residues 10, 36, 46, 54, 62, 63, 71, 82, 84, and 90) was selected for present study to understand the binding to its natural substrates. MDR769 HIV-1 protease was co-crystallized with nine substrate cleavage site hepta-peptides. Crystallographic studies show that MDR769 HIV-1 protease has an expanded substrate envelope with wide open flaps. Furthermore, ligand binding energy calculations indicate weaker binding in MDR769 HIV-1 protease-substrate complexes. These results help in designing the next generation of HIV-1 protease inhibitors by targeting the MDR HIV-1 protease.     

Insights into the structural function of the complex of HIV-1 protease with TMC-126: molecular dynamics simulations and free-energy calculations

The binding properties of the protein–inhibitor complex of human immunodeficiency virus type 1 (HIV-1) protease with the inhibitor TMC-126 are investigated by combining computational alanine scanning (CAS) mutagenesis with binding free-energy decomposition (BFED). The calculated results demonstrate that the flap region (residues 38–58) and the active site region (residues 23–32) in HIV-1 protease contribute 63.72% of the protease to the binding of the inhibitor. In particular, the mechanisms for the interactions of key residues of these species are fully explored and analyzed. Interestingly, the regression analyses show that both CAS and BFED based on the generalized Born model yield similar results, with a correlation coefficient of 0.94. However, compared to CAS, BFED is faster and can decompose the per-residue binding free-energy contributions into backbone and side-chain contributions. The results obtained in this study are useful for studying the binding mechanism between receptor and ligand and for designing potent inhibitors that can combat diseases.     

Exploring the drug resistance mechanism of active site,non-active site mutations and their cooperative effects in CRF01_AE HIV-1 protease: molecular dynamics simulations and free energy calculations

Human immunodeficiency virus type 1 protease is essential for virus replication and maturation and has been considered as one of the important drug target for the antiretroviral treatment of HIV infection. The majority of HIV infections are caused due to non-B subtypes in developing countries. Subtype AE is spreading rapidly and infecting huge population worldwide. Understanding the interdependence of active and non-active site mutations in conferring drug resistance is crucial for the development effective inhibitors in subtype AE protease. In this work, we have investigated the mechanism of resistance against indinavir (IDV) due to therapy selected active site mutation V82F, non-active site mutations PF82V and their cooperative effects PV82F in subtype AE-protease using molecular dynamics simulations and binding free energy calculations. The simulations suggested all the three complexes lead to decrease in binding affinity of IDV, whereas the PF82V complex resulted in an enhanced binding affinity compared to V82F and PV82F complexes. Large positional deviation of IDV was observed in V82F complex. The preservation of hydrogen bonds of IDV with active site Asp25/Asp25′ and flap residue Ile50/50′ via a water molecule is crucial for effective binding. Owing to the close contact of 80s loop with Ile50′ and Asp25, the alteration between residues Thr80 and Val82, further induces conformational change thereby resulting in loss of interactions between IDV and the residues in the active site cavity, leading to drug resistance. Our present study shed light on the effect of active, non-active site mutations and their cooperative effects in AE protease.

Communicated by Ramaswamy H. Sarma     

A structural and thermodynamic escape mechanism from a drug resistant mutation of the HIV-1 protease

The efficacy of HIV-1 protease inhibition therapies is often compromised by the appearance of mutations in the protease molecule that lower the binding affinity of inhibitors while maintaining viable catalytic activity and substrate affinity. The V82F/I84V double mutation is located within the binding site cavity and affects all protease inhibitors in clinical use. KNI-764, a second-generation inhibitor currently under development, maintains significant potency against this mutation by entropically compensating for enthalpic losses, thus minimizing the loss in binding affinity. KNI-577 differs from KNI-764 by a single functional group critical to the inhibitor response to the protease mutation. This single difference changes the response of the two inhibitors to the mutation by one order of magnitude. Accordingly, a structural understanding of the inhibitor response will provide important guidelines for the design of inhibitors that are less susceptible to mutations conveying drug resistance. The structures of the two compounds bound to the wild type and V82F/I84V HIV-1 protease have been determined by X-ray crystallography at 2.0 A resolution. The presence of two asymmetric functional groups, linked by rotatable bonds to the inhibitor scaffold, allows KNI-764 to adapt to the mutated binding site cavity more readily than KNI-577, with a single asymmetric group. Both inhibitors lose about 2.5 kcal/mol in binding enthalpy when facing the drug-resistant mutant protease; however KNI-764 gains binding entropy while KNI-577 loses binding entropy. The gain in binding entropy by KNI-764 accounts for its low susceptibility to the drug-resistant mutation. The heat capacity change associated with binding becomes more negative when KNI-764 binds to the mutant protease, consistent with increased desolvation. With KNI-577, the opposite effect is observed. Structurally, the crystallographic B factors increase for KNI-764 when it is bound to the drug-resistant mutant. The opposite is observed for KNI-577. Consistent with these observations, it appears that KNI-764 is able to gain binding entropy by a two-fold mechanism: it gains solvation entropy by burying itself deeper within the binding pocket and gains conformational entropy by losing interaction with the protease.     

Adaptability and flexibility of HIV-1 protease.

Even though more than 200 three-dimensional structures of HIV-1 protease complexed to a variety of inhibitors are available in the Protein Data Bank; very few structures of unliganded protein have been determined. We have recently solved structures of unliganded HIV-1 protease tethered dimer mutants to resolutions of 1.9 A and 2.1 A, and have found that the flaps assume closed-flap conformation even in the absence of any bound ligand. We report comparison of the unliganded closed-flap structure with structures of HIV-1 protease inhibitor complexes with a view to accurately identifying structural changes that the ligand can induce on binding to HIV-1 protease in the crystal. These studies reveal that the least flexible region present in the active site of HIV-1 protease need not also be the least adaptable to external stress, thus highlighting the conceptual difference between flexibility and adaptability of proteins in general.     

Drug resistance mutations can effect dimer stability of HIV-1 protease at neutral pH.

The monomer-dimer equilibrium for the human immunodeficiency virus type 1 (HIV-1) protease has been investigated under physiological conditions. Dimer dissociation at pH 7.0 was correlated with a loss in beta-sheet structure and a lower degree of ANS binding. An autolysis-resistant mutant, Q7K/L33I/L63I, was used to facilitate sedimentation equilibrium studies at neutral pH where the wild-type enzyme is typically unstable in the absence of bound inhibitor. The dimer dissociation constant (KD) of the triple mutant was 5.8 microM at pH 7.0 and was below the limit of measurement (approximately 100 nM) at pH 4.5. Similar studies using the catalytically inactive D25N mutant yielded a KD value of 1.0 microM at pH 7.0. These values differ significantly from a previously reported value of 23 nM obtained indirectly from inhibitor binding measurements (Darke et al., 1994). We show that the discrepancy may result from the thermodynamic linkage between the monomer-dimer and inhibitor binding equilibria. Under conditions where a significant degree of monomer is present, both substrates and competitive inhibitors will shift the equilibrium toward the dimer, resulting in apparent increases in dimer stability and decreases in ligand binding affinity. Sedimentation equilibrium studies were also carried out on several drug-resistant HIV-1 protease mutants: V82F, V82F/I84V, V82T/I84V, and L90M. All four mutants exhibited reduced dimer stability relative to the autolysis-resistant mutant at pH 7.0. Our results indicate that reductions in drug affinity may be due to the combined effects of mutations on both dimer stability and inhibitor binding.     

Towards understanding the mechanisms of molecular recognition by computer simulations of ligand-protein interactions

The thermodynamic and kinetic aspects of molecular recognition for the methotrexate (MTX)-dihydrofolate reductase (DHFR) ligand-protein system are investigated by the binding energy landscape approach. The impact of 'hot' and 'cold' errors in ligand mutations on the thermodynamic stability of the native MTX-DHFR complex is analyzed, and relationships between the molecular recognition mechanism and the degree of ligand optimization are discussed. The nature and relative stability of intermediates and thermodynamic phases on the ligand-protein association pathway are studied, providing new insights into connections between protein folding and molecular recognition mechanisms, and cooperativity of ligand-protein binding. The results of kinetic docking simulations are rationalized based on the thermodynamic properties determined from equilibrium simulations and the shape of the underlying binding energy landscape. We show how evolutionary ligand selection for a receptor active site can produce well-optimized ligand-protein systems such as MTX-DHFR complex with the thermodynamically stable native structure and a direct transition mechanism of binding from unbound conformations to the unique native structure.     

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.     

Computational analysis of plasmepsin IV bound to an allophenylnorstatine inhibitor

The plasmepsin proteases from the malaria parasite Plasmodium falciparum are attracting attention as putative drug targets. A recently published crystal structure of Plasmodium malariae plasmepsin IV bound to an allophenylnorstatine inhibitor [Clemente, J.C. et al. (2006) Acta Crystallogr. D 62, 246-252] provides the first structural insights regarding interactions of this family of inhibitors with plasmepsins. The compounds in this class are potent inhibitors of HIV-1 protease, but also show nM binding affinities towards plasmepsin IV. Here, we utilize automated docking, molecular dynamics and binding free energy calculations with the linear interaction energy LIE method to investigate the binding of allophenylnorstatine inhibitors to plasmepsin IV from two different species. The calculations yield excellent agreement with experimental binding data and provide new information regarding protonation states of active site residues as well as conformational properties of the inhibitor complexes.     

Active site binding modes of curcumin in HIV-1 protease and integrase

Structure models for the interaction of curcumin with HIV-1 integrase (IN) and protease (PR) were investigated using computational docking. Curcumin was found to bind preferentially in similar ways to the active sites of both IN and PR. For IN, the binding site is formed by residues Asp64, His67, Thr66, Glu92, Thr93, Asp116, Ser119, Asn120, and Lys159. Docked curcumin contacts the catalytic residues adjacent to Asp116 and Asp64, and near the divalent metal (Mg2+). In the PR docking, the curcumin structure fitted well to the active site, interacting with residues Asp25, Asp29, Asp30, Gly27', Asp29', and Asp30'. The results suggest that o-hydroxyl and/or keto-enol structures are important for both IN and PR inhibitory actions. The symmetrical structure of curcumin seems to play an important role for binding to the PR protein, whereas the keto-enol and only one side of the terminal o-hydroxyl showed tight binding to the IN active site.     

Ligand-induced conformational changes: improved predictions of ligand binding conformations and affinities

A computational docking strategy using multiple conformations of the target protein is discussed and evaluated. A series of low molecular weight, competitive, nonpeptide protein tyrosine phosphatase inhibitors are considered for which the x-ray crystallographic structures in complex with protein tyrosine phosphatase 1B (PTP1B) are known. To obtain a quantitative measure of the impact of conformational changes induced by the inhibitors, these were docked to the active site region of various structures of PTP1B using the docking program FlexX. Firstly, the inhibitors were docked to a PTP1B crystal structure cocrystallized with a hexapeptide. The estimated binding energies for various docking modes as well as the RMS differences between the docked compounds and the crystallographic structure were calculated. In this scenario the estimated binding energies were not predictive inasmuch as docking modes with low estimated binding energies corresponded to relatively large RMS differences when aligned with the corresponding crystal structure. Secondly, the inhibitors were docked to their parent protein structures in which they were cocrystallized. In this case, there was a good correlation between low predicted binding energy and a correct docking mode. Thirdly, to improve the predictability of the docking procedure in the general case, where only a single target protein structure is known, we evaluate an approach which takes possible protein side-chain conformational changes into account. Here, side chains exposed to the active site were considered in their allowed rotamer conformations and protein models containing all possible combinations of side-chain rotamers were generated. To evaluate which of these modeled active sites is the most likely binding site conformation for a certain inhibitor, the inhibitors were docked against all active site models. The receptor rotamer model corresponding to the lowest estimated binding energy is taken as the top candidate. Using this protocol, correct inhibitor binding modes could successfully be discriminated from proposed incorrect binding modes. Moreover, the ranking of the estimated ligand binding energies was in good agreement with experimentally observed binding affinities.     

Experimental and computational studies indicate the mutation of Glu12 to increase the thermostability of oligomeric protease from <Emphasis Type="Italic">Pyrococcus horikoshii</Emphasis>

The intracellular protease from Pyrococcus horikoshii (PhpI) is a member of the DJ-1/ThiJ/PfpI superfamily, which is suggested to be involved in cellular protection against environmental stresses. In this study, flexible docking approach was employed to dock the ligand into the active site of PhpI. By analyzing the results, active site architecture and certain key residues responsible for substrate specificity were identified on the enzyme. Our docking result indicates that Glu12 plays an important role in substrate binding. The kinetic experiment conducted by Zhan shows that the E12T mutant is more stable than that of the wild-type. We also predict that Glu15, Lys43, and Tyr46 may be important in the catalytic efficiency and thermostability of enzyme. The new structural and mechanistic insights obtained from computational study should be valuable for detailed structures and mechanisms of the member of the DJ-1 superfamily.     

Insights into saquinavir resistance in the G48V HIV-1 protease: quantum calculations and molecular dynamic simulations

The spread of acquired immune deficiency syndrome has increasingly become a great concern owing largely to the failure of chemotherapies. The G48V is considered the key signature residue mutation of HIV-1 protease developing with saquinavir therapy. Molecular dynamics simulations of the wild-type and the G48V HIV-1 protease complexed with saquinavir were carried out to explore structure and interactions of the drug resistance. The molecular dynamics results combined with the quantum-based and molecular mechanics Poisson-Boltzmann surface area calculations indicated a monoprotonation took place on D25, one of the triad active site residues. The inhibitor binding of the triad residues and its interaction energy in the mutant were similar to those in the wild-type. The overall structure of both complexes is almost identical. However, the steric conflict of the substituted valine results in the conformational change of the P2 subsite and the disruption of hydrogen bonding between the -NH of the P2 subsite and the backbone -CO of the mutated residue. The magnitude of interaction energy changes was comparable to the experimental K(i) data. The designing for a new drug should consider a reduction of steric repulsion on P2 to enhance the activity toward this mutant strain.     

Analysis of HIV-1 CRF_01 A/E protease inhibitor resistance: structural determinants for maintaining sensitivity and developing resistance to atazanavir

A series of HIV-1 protease mutants has been designed in an effort to analyze the contribution to drug resistance provided by natural polymorphisms as well as therapy-selective (active and non-active site) mutations in the HIV-1 CRF_01 A/E (AE) protease when compared to that of the subtype B (B) protease. Kinetic analysis of these variants using chromogenic substrates showed differences in substrate specificity between pretherapy B and AE proteases. Inhibition analysis with ritonavir, indinavir, nelfinavir, amprenavir, saquinavir, lopinavir, and atazanavir revealed that the natural polymorphisms found in A/E can influence inhibitor resistance. It was also apparent that a high level of resistance in the A/E protease, as with B protease, is due to it aquiring a combination of active site and non-active site mutations. Structural analysis of atazanavir bound to a pretherapy B protease showed that the ability of atazanavir to maintain its binding affinity for variants containing some resistance mutations is due to its unique interactions with flap residues. This structure also explains why the I50L and I84V mutations are important in decreasing the binding affinity of atazanavir.     

Towards Ligand Docking Including Explicit Interface Water Molecules

Small molecule docking predicts the interaction of a small molecule ligand with a protein at atomic-detail accuracy including position and conformation the ligand but also conformational changes of the protein upon ligand binding. While successful in the majority of cases, docking algorithms including RosettaLigand fail in some cases to predict the correct protein/ligand complex structure. In this study we show that simultaneous docking of explicit interface water molecules greatly improves Rosetta’s ability to distinguish correct from incorrect ligand poses. This result holds true for both protein-centric water docking wherein waters are located relative to the protein binding site and ligand-centric water docking wherein waters move with the ligand during docking. Protein-centric docking is used to model 99 HIV-1 protease/protease inhibitor structures. We find protease inhibitor placement improving at a ratio of 9∶1 when one critical interface water molecule is included in the docking simulation. Ligand-centric docking is applied to 341 structures from the CSAR benchmark of diverse protein/ligand complexes [1]. Across this diverse dataset we see up to 56% recovery of failed docking studies, when waters are included in the docking simulation.