Thermodynamic dissection of the binding energetics of KNI-272, a potent HIV-1 protease inhibitor

KNI-272 is a powerful HIV-1 protease inhibitor with a reported inhibition constant in the picomolar range. In this paper, a complete experimental dissection of the thermodynamic forces that define the binding affinity of this inhibitor to the wild-type and drug-resistant mutant V82F/184V is presented. Unlike other protease inhibitors, KNI-272 binds to the protease with a favorable binding enthalpy. The origin of the favorable binding enthalpy has been traced to the coupling of the binding reaction to the burial of six water molecules. These bound water molecules, previously identified by NMR studies, optimize the atomic packing at the inhibitor/protein interface enhancing van der Waals and other favorable interactions. These interactions offset the unfavorable enthalpy usually associated with the binding of hydrophobic molecules. The association constant to the drug resistant mutant is 100-500 times weaker. The decrease in binding affinity corresponds to an increase in the Gibbs energy of binding of 3-3.5 kcal/mol, which originates from less favorable enthalpy (1.7 kcal/mol more positive) and entropy changes. Calorimetric binding experiments performed as a function of pH and utilizing buffers with different ionization enthalpies have permitted the dissection of proton linkage effects. According to these experiments, the binding of the inhibitor is linked to the protonation/deprotonation of two groups. In the uncomplexed form these groups have pKs of 6.0 and 4.8, and become 6.6 and 2.9 in the complex. These groups have been identified as one of the aspartates in the catalytic aspartyl dyad in the protease and the isoquinoline nitrogen in the inhibitor molecule. The binding affinity is maximal between pH 5 and pH 6. At those pH values the affinity is close to 6 x 10(10) M(-1) (Kd = 16 pM). Global analysis of the data yield a buffer- and pH-independent binding enthalpy of -6.3 kcal/mol. Under conditions in which the exchange of protons is zero, the Gibbs energy of binding is -14.7 kcal/mol from which a binding entropy of 28 cal/K mol is obtained. Thus, the binding of KNI-272 is both enthalpically and entropically favorable. The structure-based thermodynamic analysis indicates that the allophenylnorstatine nucleus of KNI-272 provides an important scaffold for the design of inhibitors that are less susceptible to resistant mutations.     

Design of a folding inhibitor of the HIV-1 protease

A novel way to inhibit HIV-1 protease by destabilizing its native state is discussed. A simplified protein model is used together with Monte Carlo simulations, to assess the destabilizing effect of peptides displaying the same sequence as specific fragments of the protein which are essential for its stability. Model calculations also show that it is unlikely that the protein can escape the inhibitory peptide by point mutations.     

Structure-based phenotyping predicts HIV-1 protease inhibitor resistance

Mutations in HIV-1 drug targets lead to resistance and consequent therapeutic failure of antiretroviral drugs. Phenotypic resistance assays are time-consuming and costly, and genotypic rules-based interpretations may fail to predict the effects of multiple mutations. We have developed a computational procedure that rapidly evaluates changes in the binding energy of inhibitors to mutant HIV-1 PR variants. Models of WT complexes were produced from crystal structures. Mutant complexes were built by amino acid substitutions in the WT complexes with subsequent energy minimization of the ligand and PR binding site residues. Accuracy of the models was confirmed by comparison with available crystal structures and by prediction of known resistance-related mutations. PR variants from clinical isolates were modeled in complex with six FDA-approved PIs, and changes in the binding energy (DeltaE(bind)) of mutant versus WT complexes were correlated with the ratios of phenotypic 50% inhibitory concentration (IC(50)) values. The calculated DeltaE(bind) of five PIs showed significant correlations (R(2) = 0.7-0.8) with IC(50) ratios from the Virco Antivirogram assay, and the DeltaE(bind) of six PIs showed good correlation (R(2) = 0.76-0.85) with IC(50) ratios from the Virologic PhenoSense assay. DeltaE(bind) cutoffs corresponding to a four-fold increase in IC(50) were used to define the structure-based phenotype as susceptible, resistant, or equivocal. Blind predictions for 78 PR variants gave overall agreement of 92% (kappa = 0.756) and 86% (kappa = 0.666) with PhenoSense and Antivirogram phenotypes, respectively. The structural phenotyping predicted drug resistance of clinical HIV-1 PR variants with an accuracy approaching that of frequently used cell-based phenotypic assays.     

Molecular dynamics studies on HIV-1 protease drug resistance and folding pathways

Drug resistance to HIV-1 protease involves the accumulation of multiple mutations in the protein. We investigate the role of these mutations by using molecular dynamics simulations that exploit the influence of the native-state topology in the folding process. Our calculations show that sites contributing to phenotypic resistance of FDA-approved drugs are among the most sensitive positions for the stability of partially folded states and should play a relevant role in the folding process. Furthermore, associations between amino acid sites mutating under drug treatment are shown to be statistically correlated. The striking correlation between clinical data and our calculations suggest a novel approach to the design of drugs tailored to bind regions crucial not only for protein function, but for folding as well.     

A poke in the eye: inhibiting HIV-1 protease through its flap-recognition pocket

A novel mechanism of inhibiting HIV-1 protease (HIVp) is presented. Using computational solvent mapping to identify complementary interactions and the Multiple Protein Structure method to incorporate protein flexibility, we generated a receptor-based pharmacophore model of the flexible flap region of the semiopen, apo state of HIVp. Complementary interactions were consistently observed at the base of the flap, only within a cleft with a specific structural role. In the closed, bound state of HIVp, each flap tip docks against the opposite monomer, occupying this cleft. This flap-recognition site is filled by the protein and cannot be identified using traditional approaches based on bound, closed structures. Virtual screening and dynamics simulations show how small molecules can be identified to complement this cleft. Subsequent experimental testing confirms inhibitory activity of this new class of inhibitor. This may be the first new inhibitor class for HIVp since dimerization inhibitors were introduced 17 years ago.     

Translation‐coupled protein folding assay using a protease to monitor the folding status

Protein folding is an essential prerequisite for proteins to execute nearly all cellular functions. There is a growing demand for a simple and robust method to investigate protein folding on a large‐scale under the same conditions. We previously developed a global folding assay system, in which proteins translated using an Escherichia coli‐based cell‐free translation system are centrifuged to quantitate the supernatant fractions. Although the assay is based on the assumption that the supernatants contain the folded native states, the supernatants also include nonnative unstructured proteins. In general, proteases recognize and degrade unstructured proteins, and thus we used a protease to digest the unstructured regions to monitor the folding status. The addition of Lon protease during the translation of proteins unmasked subfractions, not only in the soluble fractions but also in the aggregation‐prone fractions. We translated ～90 E. coli proteins in the protease‐inclusion assay, in the absence and presence of chaperones. The folding assay, which sheds light on the molecular mechanisms underlying the aggregate formation and the chaperone effects, can be applied to a large‐scale analysis.     

Nonpeptidic HIV protease inhibitors possessing excellent antiviral activities and therapeutic indices. PD 178390: a lead HIV protease inhibitor

With the insight generated by the availability of X-ray crystal structures of various 5,6-dihydropyran-2-ones bound to HIV PR, inhibitors possessing various alkyl groups at the 6-position of 5,6-dihydropyran-2-one ring were synthesized. The inhibitors possessing a 6-alkyl group exhibited superior antiviral activities when compared to 6-phenyl analogues. Antiviral efficacies were further improved upon introduction of a polar group (hydroxyl or amino) on the 4-position of the phenethyl moiety as well as the polar group (hydroxymethyl) on the 3-(tert-butyl-5-methyl-phenylthio) moiety. The polar substitution is also advantageous for decreasing toxicity, providing inhibitors with higher therapeutic indices. The best inhibitor among this series, (S)-6-[2-(4-aminophenyl)-ethyl]-(3-(2-tert-butyl-5-methyl-phenylsulfanyl)-4-hydroxy-6-isopropyl-5,6-dihydro-pyran-2-one (34S), exhibited an EC₅₀ of 200 nM with a therapeutic index of >1000. More importantly, these non-peptidic inhibitors, 16S and 34S, appear to offer little cross-resistance to the currently marketed peptidomimetic PR inhibitors. The selected inhibitors tested in vitro against mutant HIV PR showed a very small increase in binding affinities relative to wild-type HIV PR. C_max and absolute bioavailability of 34S were higher and half-life and time above EC₉₅ were longer compared to 16S. Thus 34S, also known as PD 178390, which displays good antiviral efficacy, promising pharmacokinetic characteristics and favorable activity against mutant enzymes and CYP3A4, has been chosen for further preclinical evaluation.     

Disabling the folding catalyst is the last critical step in alpha-lytic protease folding

Alpha-Lytic protease (alphaLP) is an extracellular bacterial pro-protease marked by extraordinary conformational rigidity and a highly cooperative barrier to unfolding. Although these properties successfully limit its proteolytic destruction, thereby extending the functional lifetime of the protease, they come at the expense of foldability (t(1/2) = 1800 yr) and thermodynamic stability (native alphaLP is less stable than the unfolded species). Efficient folding has required the coevolution of a large N-terminal pro region (Pro) that rapidly catalyzes alphaLP folding (t(1/2) = 23 sec) and shifts the thermodynamic equilibrium in favor of folded protease through tight native-state binding. Release of active alphaLP from this stabilizing, but strongly inhibitory, complex requires the proteolytic destruction of Pro. alphaLP is capable of initiating Pro degradation via cleavage of a flexible loop within the Pro C-terminal domain. This single cleavage event abolishes Pro catalysis while maintaining strong native-state binding. Thus, the loop acts as an Achilles' heel by which the Pro foldase machinery can be safely dismantled, preventing Pro-catalyzed unfolding, without compromising alphaLP native-state stability. Once the loop is cleaved, Pro is rapidly degraded, releasing active alphaLP.     

Computational proteomics analysis of HIV-1 protease interactome

HIV-1 protease is a small homodimeric enzyme that ensures maturation of HIV virions by cleaving the viral precursor Gag and Gag-Pol polyproteins into structural and functional elements. The cleavage sites in the viral polyproteins share neither sequence homology nor binding motif and the specificity of the HIV-1 protease is therefore only partially understood. Using an extensive data set collected from 16 years of HIV proteome research we have here created a general and predictive rule-based model for HIV-1 protease specificity based on rough sets. We demonstrate that HIV-1 protease specificity is much more complex than previously anticipated, which cannot be defined based solely on the amino acids at the substrate's scissile bond or by any other single substrate amino acid position only. Our results show that the combination of at least three particular amino acids is needed in the substrate for a cleavage event to occur. Only by combining and analyzing massive amounts of HIV proteome data it was possible to discover these novel and general patterns of physico-chemical substrate cleavage determinants. Our study is an example how computational biology methods can advance the understanding of the viral interactomes.     

Design of HIV-1-PR inhibitors that do not create resistance: blocking the folding of single monomers

The main problems found in designing drugs are those of optimizing the drug-target interaction and of avoiding the insurgence of resistance. We suggest a scheme for the design of inhibitors that can be used as leads for the development of a drug and that do not face either of these problems, and then apply it to the case of HIV-1-PR. It is based on the knowledge that the folding of single-domain proteins, such as each of the monomers forming the HIV-1-PR homodimer, is controlled by local elementary structures (LES), stabilized by local contacts among hydrophobic, strongly interacting, and highly conserved amino acids that play a central role in the folding process. Because LES have evolved over many generations to recognize and strongly interact with each other so as to make the protein fold fast and avoid aggregation with other proteins, highly specific (and thus little toxic) as well as effective folding-inhibitor molecules suggest themselves: short peptides (or eventually their mimetic molecules) displaying the same amino acid sequence of that of LES (p-LES). Aside from being specific and efficient, these inhibitors are expected not to induce resistance; in fact, mutations in HIV-1-PR that successfully avoid the action of p-LES imply the destabilization of one or more LES and thus should lead to protein denaturation. Making use of Monte Carlo simulations, we first identify the LES of the HIV-1-PR and then show that the corresponding p-LES peptides act as effective inhibitors of the folding of the protease.     

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.     

X-ray structure of HIV-1 protease in situ product complex

HIV-1 protease is an effective target for design of different types of drugs against AIDS. HIV-1 protease is also one of the few enzymes that can cleave substrates containing both proline and nonproline residues at the cleavage site. We report here the first structure of HIV-1 protease complexed with the product peptides SQNY and PIV derived by in situ cleavage of the oligopeptide substrate SQNYPIV, within the crystals. In the structure, refined against 2.0-A resolution synchrotron data, a carboxyl oxygen of SQNY is hydrogen-bonded with the N-terminal nitrogen atom of PIV. At the same time, this proline nitrogen atom does not form any hydrogen bond with catalytic aspartates. These two observations suggest that the protonation of scissile nitrogen, during peptide bond cleavage, is by a gem-hydroxyl of the tetrahedral intermediate rather than by a catalytic aspartic acid.     

Critical differences in HIV-1 and HIV-2 protease specificity for clinical inhibitors

Clinical inhibitor amprenavir (APV) is less effective on HIV‐2 protease (PR₂) than on HIV‐1 protease (PR₁). We solved the crystal structure of PR₂ with APV at 1.5 Å resolution to identify structural changes associated with the lowered inhibition. Furthermore, we analyzed the PR₁ mutant (PR_1M) with substitutions V32I, I47V, and V82I that mimic the inhibitor binding site of PR₂. PR_1M more closely resembled PR₂ than PR₁ in catalytic efficiency on four substrate peptides and inhibition by APV, whereas few differences were seen for two other substrates and inhibition by saquinavir (SQV) and darunavir (DRV). High resolution crystal structures of PR_1M with APV, DRV, and SQV were compared with available PR₁ and PR₂ complexes. Val/Ile32 and Ile/Val47 showed compensating interactions with SQV in PR_1M and PR₁, however, Ile82 interacted with a second SQV bound in an extension of the active site cavity of PR_1M. Residues 32 and 82 maintained similar interactions with DRV and APV in all the enzymes, whereas Val47 and Ile47 had opposing effects in the two subunits. Significantly diminished interactions were seen for the aniline of APV bound in PR_1M and PR₂ relative to the strong hydrogen bonds observed in PR₁, consistent with 15‐ and 19‐fold weaker inhibition, respectively. Overall, PR_1M partially replicates the specificity of PR₂ and gives insight into drug resistant mutations at residues 32, 47, and 82. Moreover, this analysis provides a structural explanation for the weaker antiviral effects of APV on HIV‐2.     

Lack of synergy for inhibitors targeting a multi-drug-resistant HIV-1 protease

The three-dimensional structures of indinavir and three newly synthesized indinavir analogs in complex with a multi-drug-resistant variant (L63P, V82T, I84V) of HIV-1 protease were determined to approximately 2.2 A resolution. Two of the three analogs have only a single modification of indinavir, and their binding affinities to the variant HIV-1 protease are enhanced over that of indinavir. However, when both modifications were combined into a single compound, the binding affinity to the protease variant was reduced. On close examination, the structural rearrangements in the protease that occur in the tightest binding inhibitor complex are mutually exclusive with the structural rearrangements seen in the second tightest inhibitor complex. This occurs as adaptations in the S1 pocket of one monomer propagate through the dimer and affect the conformation of the S1 loop near P81 of the other monomer. Therefore, structural rearrangements that occur within the protease when it binds to an inhibitor with a single modification must be accounted for in the design of inhibitors with multiple modifications. This consideration is necessary to develop inhibitors that bind sufficiently tightly to drug-resistant variants of HIV-1 protease to potentially become the next generation of therapeutic agents.     

Single point mutation induced alterations in the equilibrium structural transitions on the folding landscape of HIV-1 protease

Equilibrium folding–unfolding transitions are hard to study in HIV-1 protease (PR) because of its autolytic properties. Further, the protease exhibits many tolerant point mutations some of which also impart drug resistance to the protein. It is conceivable that the mutations affect protein's function by altering its folding characteristics; these would clearly depend on the nature of the mutations themselves. In this background, we report here NMR studies on the effects of D25?N mutation, which removes one negative charge from the protein at the active site, on the equilibrium folding behaviour of PR starting from its acetic acid denatured state. It is observed that in PR_D25N two slowly exchanging conformations are present at the N-terminal. One of them is similar to that of PR. Though the conformational and dynamics preferences of PR and PR_D25N are fairly similar in 9?M acetic acid, they seem to undergo different folding transitions when acetic acid concentration is reduced. The differences are seen in the active site, in the flap, and in the hinge of the flap regions. The present study suggests that such differences, though different in detail, would occur for other mutations as well, and also for different initial denatured states. These would have significant regulatory implications for the efficacy of protease function.     

Purification of HIV-1 wild-type protease and characterization of proteolytically inactive HIV-1 protease mutants by pepstatin A affinity chromatography

Recombinant wild-type protease of human immunodeficiency virus, type [(HIV-1) expressed in E. coli was purified by pepstatin A affinity chromatography. An 88-fold purification was achieved giving a protease preparation with a specific enzymatic activity of approximately 3700 pmol/min/μg. Two proteolytically inactive HIV-1 mutant proteases (Arg-87 → Lys; Asn-88 → Glu) were found to bind to pepstatin A agarose, and they were purified as the wild-type protease. A third mutant protease (Arg-87 → Glu) was apparently unable to bind to pepstatin A under similar conditions. Binding to pepstatin A indicates the binding ability of the substrate binding site and the ability to form dimers. These features may be used to purify and to characterize other mutated HIV-1 proteases.     

Organization of focal adhesion plaques is disrupted by action of the HIV-1 protease

Focal adhesion plaques were severely affected in human embryonic fibroblasts permeabilized with digitonin and incubated in buffer containing the human immunodeficiency virus type 1 protease (HIV-1 PR). A mutant HIV-1 PR (3271 HIV-1 PR) had no effect on focal adhesion plaques. Similar effects were seen with cells microinjected with either HIV-1 PR or 3271 HIV-1 PR. Immunoblots of the human embryonic fibroblasts demonstrated that a number of focal adhesion plaque proteins were specifically cleaved by HIV-1 PR. These included fimbrin, focal adhesion plaque kinase (FAK), talin, and, to a lesser extent, filamin, spectrin and fibronectin. Proteins detected by antibodies to beta 4 integrin and alpha 3 integrin were also cleaved by the HIV-1 PR. Control experiments demonstrated that the effect and protein cleavages described are due to action of the HIV-1 PR and not to the action of endogenous host cell proteases.     

Protease pro region required for folding is a potent inhibitor of the mature enzyme.

alpha-Lytic protease, an extracellular bacterial serine protease, is synthesized with a large pro region that is required in vivo for the proper folding of the protease domain. To allow detailed mechanistic study, we have reconstituted pro region-dependent folding in vitro. The pro region promotes folding of the protease domain in the absence of other protein factors or exogenous energy sources. Surprisingly, we find that the pro region is a high affinity inhibitor of the mature protease. The pro region also inhibits the closely related Streptomyces griseus protease B, but not the more distantly related, yet structurally similar protease, elastase. Based on these data, we suggest a mechanism in which pro region binding reduces the free energy of a late folding transition state having native-like structure.