Narrow substrate specificity and sensitivity toward ligand-binding site mutations of human T-cell Leukemia virus type 1 protease

Human T-cell leukemia virus type 1 (HTLV-1) is associated with a number of human diseases; therefore, its protease is a potential target for chemotherapy. To compare the specificity of HTLV-1 protease with that of human immunodeficiency virus type 1 (HIV-1) protease, oligopeptides representing naturally occurring cleavage sites in various retroviruses were tested. The number of hydrolyzed peptides as well as the specificity constants suggested a substantially broader specificity of the HIV protease. Amino acid residues of HTLV-1 protease substrate-binding sites were replaced by equivalent ones of HIV-1 protease. Most of the single and multiple mutants had altered specificity and a dramatically reduced folding and catalytic capability, suggesting that mutations are not well tolerated in HTLV-1 protease. The catalytically most efficient mutant was that with the flap residues of HIV-1 protease. The inhibition profile of the mutants was also determined for five inhibitors used in clinical practice and inhibitor analogs of HTLV-1 cleavage sites. Except for indinavir, the HIV-1 protease inhibitors did not inhibit wild type and most of the mutant HTLV-1 proteases. The wild type HTLV-1 protease was inhibited by the reduced peptide bond-containing substrate analogs, whereas the mutants showed various degrees of weakened binding capability. Most interesting, the enzyme with HIV-1-like residues in the flap region was the most sensitive to the HIV-1 protease inhibitors and least sensitive to the HTLV-1 protease inhibitors, indicating that the flap plays an important role in defining the specificity differences of retroviral proteases.     

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.     

A major role for a set of non-active site mutations in the development of HIV-1 protease drug resistance

A major problem in the chemotherapy of HIV-1 infection is the appearance of drug resistance. In the case of HIV-1 protease inhibitors, resistance originates from mutations in the protease molecule that lower the affinity of inhibitors while still maintaining a viable enzymatic profile. Drug resistance mutations can be classified as active site or non-active site mutations depending on their location within the protease molecule. Active site mutations directly affect drug/target interactions, and their action can be readily understood in structural terms. Non-active site mutations influence binding from distal locations, and their mechanism of action is not immediately apparent. In this paper, we have characterized a mutant form of the HIV-1 protease, ANAM-11, identified in clinical isolates from HIV-1 infected patients treated with protease inhibitors. This mutant protease contains 11 mutations, 10 of which are located outside the active site (L10I/M36I/S37D/M46I/R57K/L63P/A71V/G73S/L90M/I93L) and 1 within the active site (I84V). ANAM-11 lowers the binding affinity of indinavir, nelfinavir, saquinavir, and ritonavir by factors of 4000, 3300, 5800, and 80000, respectively. Surprisingly, most of the loss in inhibitor affinity is due to the non-active site mutations as demonstrated by additional experiments performed with a protease containing only the 10 non-active site mutations (NAM-10) and another containing only the active site mutation (A-1). Kinetic analysis with two different substrates yielded comparable catalytic efficiencies for A-1, ANAM-11, NAM-10, and the wild-type protease. These studies demonstrate that non-active site mutations can be the primary source of resistance and that their role is not necessarily limited to compensate deleterious effects of active site mutations. Analysis of the structural stability of the proteases by differential scanning calorimetry reveals that ANAM-11 and NAM-10 are structurally more stable than the wild-type protease while A-1 is less stable. Together, the binding and structural thermodynamic results suggest that the non-active site mutants affect inhibitor binding by altering the geometry of the binding site cavity through the accumulation of mutations within the core of the protease molecule.     

The binding energetics of first- and second-generation HIV-1 protease inhibitors: implications for drug design

KNI-764 is a powerful HIV-1 protease inhibitor with a reported low susceptibility to the effects of protease mutations commonly associated with drug resistance. In this paper the binding thermodynamics of KNI-764 to the wild-type and drug-resistant mutant V82F/I84V are presented and the results compared to those obtained with existing clinical inhibitors. KNI-764 binds to the wild-type HIV-1 protease with very high affinity (3.1 x 10(10) M(-1) or 32 pM) in a process strongly favored by both enthalpic and entropic contributions to the Gibbs energy of binding (Delta G = -RTlnK(a)). When compared to existing clinical inhibitors, the binding affinity of KNI-764 is about 100 fold higher than that of indinavir, saquinavir, and nelfinavir, but comparable to that of ritonavir. Unlike the existing clinical inhibitors, which bind to the protease with unfavorable or only slightly favorable enthalpy changes, the binding of KNI-764 is strongly exothermic (-7.6 kcal/mol). The resistant mutation V82F/I84V lowers the binding affinity of KNI-764 26-fold, which can be accounted almost entirely by a less favorable binding enthalpy to the mutant. Since KNI-764 binds to the wild type with extremely high affinity, even after a 26-fold decrease, it still binds to the resistant mutant with an affinity comparable to that of other inhibitors against the wild type. These results indicate that the effectiveness of this inhibitor against the resistant mutant is related to two factors: extremely high affinity against the wild type achieved by combining favorable enthalpic and entropic interactions, and a mild effect of the protease mutation due to the presence of flexible structural elements at critical locations in the inhibitor molecule. The conclusions derived from the HIV-1 protease provide important thermodynamic guidelines that can be implemented in general drug design strategies.     

Crystal structures of a multidrug-resistant human immunodeficiency virus type 1 protease reveal an expanded active-site cavity

The goal of this study was to use X-ray crystallography to investigate the structural basis of resistance to human immunodeficiency virus type 1 (HIV-1) protease inhibitors. We overexpressed, purified, and crystallized a multidrug-resistant (MDR) HIV-1 protease enzyme derived from a patient failing on several protease inhibitor-containing regimens. This HIV-1 variant contained codon mutations at positions 10, 36, 46, 54, 63, 71, 82, 84, and 90 that confer drug resistance to protease inhibitors. The 1.8-angstrom (A) crystal structure of this MDR patient isolate reveals an expanded active-site cavity. The active-site expansion includes position 82 and 84 mutations due to the alterations in the amino acid side chains from longer to shorter (e.g., V82A and I84V). The MDR isolate 769 protease "flaps" stay open wider, and the difference in the flap tip distances in the MDR 769 variant is 12 A. The MDR 769 protease crystal complexes with lopinavir and DMP450 reveal completely different binding modes. The network of interactions between the ligands and the MDR 769 protease is completely different from that seen with the wild-type protease-ligand complexes. The water molecule-forming hydrogen bonds bridging between the two flaps and either the substrate or the peptide-based inhibitor are lacking in the MDR 769 clinical isolate. The S1, S1', S3, and S3' pockets show expansion and conformational change. Surface plasmon resonance measurements with the MDR 769 protease indicate higher k(off) rates, resulting in a change of binding affinity. Surface plasmon resonance measurements provide k(on) and k(off) data (K(d) = k(off)/k(on)) to measure binding of the multidrug-resistant protease to various ligands. This MDR 769 protease represents a new antiviral target, presenting the possibility of designing novel inhibitors with activity against the open and expanded protease forms.     

Viability of a drug-resistant human immunodeficiency virus type 1 protease variant: structural insights for better antiviral therapy

Under the selective pressure of protease inhibitor therapy, patients infected with human immunodeficiency virus (HIV) often develop drug-resistant HIV strains. One of the first drug-resistant mutations to arise in the protease, particularly in patients receiving indinavir or ritonavir treatment, is V82A, which compromises the binding of these and other inhibitors but allows the virus to remain viable. To probe this drug resistance, we solved the crystal structures of three natural substrates and two commercial drugs in complex with an inactive drug-resistant mutant (D25N/V82A) HIV-1 protease. Through structural analysis and comparison of the protein-ligand interactions, we found that Val82 interacts more closely with the drugs than with the natural substrate peptides. The V82A mutation compromises these interactions with the drugs while not greatly affecting the substrate interactions, which is consistent with previously published kinetic data. Coupled with our earlier observations, these findings suggest that future inhibitor design may reduce the probability of the appearance of drug-resistant mutations by targeting residues that are essential for substrate recognition.     

Crystal structure of an in vivo HIV-1 protease mutant in complex with saquinavir: insights into the mechanisms of drug resistance

Saquinavir is a widely used HIV-1 protease inhibitor drug for AIDS therapy. Its effectiveness, however, has been hindered by the emergence of resistant mutations, a common problem for inhibitor drugs that target HIV-1 viral enzymes. Three HIV-1 protease mutant species, G48V, L90M, and G48V/L90M double mutant, are associated in vivo with saquinavir resistance by the enzyme (Jacobsen et al., 1996). Kinetic studies on these mutants demonstrate a 13.5-, 3-, and 419-fold increase in Ki values, respectively, compared to the wild-type enzyme (Ermolieff J, Lin X, Tang J, 1997, Biochemistry 36:12364-12370). To gain an understanding of how these mutations modulate inhibitor binding, we have solved the HIV-1 protease crystal structure of the G48V/L90M double mutant in complex with saquinavir at 2.6 A resolution. This mutant complex is compared with that of the wild-type enzyme bound to the same inhibitor (Krohn A, Redshaw S, Richie JC, Graves BJ, Hatada MH, 1991, J Med Chem 34:3340-3342). Our analysis shows that to accommodate a valine side chain at position 48, the inhibitor moves away from the protease, resulting in the formation of larger gaps between the inhibitor P3 subsite and the flap region of the enzyme. Other subsites also demonstrate reduced inhibitor interaction due to an overall change of inhibitor conformation. The new methionine side chain at position 90 has van der Waals interactions with main-chain atoms of the active site residues resulting in a decrease in the volume and the structural flexibility of S1/S1' substrate binding pockets. Indirect interactions between the mutant methionine side chain and the substrate scissile bond or the isostere part of the inhibitor may differ from those of the wild-type enzyme and therefore may facilitate catalysis by the resistant mutant.     

Secondary mutations M36I and A71V in the human immunodeficiency virus type 1 protease can provide an advantage for the emergence of the primary mutation D30N

Development of resistance mutations in enzymatic targets of human immunodeficiency virus 1 (HIV-1) hampers the ability to provide adequate therapy. Of special interest is the effect mutations outside the active site of HIV-1 protease have on inhibitor binding and virus viability. We engineered protease mutants containing the active site mutation D30N alone and with the nonactive site polymorphisms M36I and/or A71V. We determined the K(i) values for the inhibitors nelfinavir, ritonavir, indinavir, KNI272, and AG1776 as well as the catalytic efficiency of the mutants. Single and double mutation combinations exhibited a decrease in catalytic efficiency, while the triple mutant displayed catalytic efficiency greater than that of the wild type. Variants containing M36I or A71V alone did not display a significant change in binding affinities to the inhibitors tested. The variant containing mutation D30N displayed a 2-6-fold increase in K(i) for all inhibitors tested, with nelfinavir showing the greatest increase. The double mutants containing a combination of mutations D30N, M36I, and A71V displayed -0.5-fold to +6-fold changes in the K(i) of all inhibitors tested, with ritonavir and nelfinavir most affected. Only the triple mutant showed a significant increase (>10-fold) in K(i) for inhibitor nelfinavir, ritonavir, or AG-1776 displaying 22-, 19-, or 15-fold increases, respectively. Our study shows that the M36I and A71V mutations provide a greater level of inhibitor cross-resistance combined with active site mutation D30N. M36I and A71V, when present as natural polymorphisms, could aid the virus in developing active site mutations to escape inhibitor binding while maintaining catalytic efficiency.     

Efficiency of a second-generation HIV-1 protease inhibitor studied by molecular dynamics and absolute binding free energy calculations

A subnanomolar inhibitor of human immunodeficiency virus type 1 (HIV-1) protease, designated QF34, potently inhibits the wild-type and drug-resistant enzyme. To explain its broad activity, the binding of QF34 to the wild-type HIV-1 protease is investigated by molecular dynamics simulations and compared to the binding of two inhibitors that are used clinically, saquinavir (SQV) and indinavir (IDV). Analysis of the flexibility of protease residues and inhibitor segments in the complex reveals that segments of QF34 were more mobile during the dynamics studies than the segments of SQV and IDV. The dynamics of hydrogen bonding show that QF34 forms a larger number of stable hydrogen bonds than the two inhibitors that are used clinically. Absolute binding free energies were calculated with molecular mechanics-generalized Born surface area (MM-GBSA) methodology using three protocols. The most consistent results were obtained using the single-trajectory approach, due to cancellation of errors and inadequate sampling in the separate-trajectory protocols. For all three inhibitors, energy components in favor of binding include van der Waals and electrostatic terms, whereas polar solvation and entropy terms oppose binding. Decomposition of binding energies reveals that more protease residues contribute significantly to the binding of QF34 than to the binding of SQV and IDV. Moreover, contributions from protease main chains and side chains are balanced in the case of QF34 (52:48 ratio, respectively), whereas side chain contributions prevail in both SQV and IDV (main-chain:side-chain ratios of 41:59 and 45:55, respectively). The presented results help explain the ability of QF34 to inhibit multiple resistant mutants and should be considered in the design of broad-specificity second-generation HIV-1 protease inhibitors.     

Molecular dynamics simulations of 14 HIV protease mutants in complexes with indinavir

The molecular mechanisms of HIV drug resistance were studied using molecular dynamics simulations of HIV-1 protease complexes with the clinical inhibitor indinavir. One nanosecond molecular dynamics simulations were run for solvated complexes of indinavir with wild type protease, a control variant and 12 drug resistant mutants. The quality of the simulations was assessed by comparison with crystallographic and inhibition data. Molecular mechanisms that contribute to drug resistance include structural stability and affinity for inhibitor. The mutants showed a range of structural variation from 70 to 140% of the wild type protease. The protease affinity for indinavir was estimated by calculating the averaged molecular mechanics interaction energy. A correlation coefficient of 0.96 was obtained with observed inhibition constants for wild type and four mutants. Based on this good agreement, the trends in binding were predicted for the other mutants and discussed in relation to the clinical data for indinavir resistance. Figure Poincare map representation for WT protease-indinavir complex. The side chain of Tyr 59 showing the positions of hydrogen atoms.This revised version was published online in October 2004 with corrections to the Graphical Abstract.     

Comprehensive mutagenesis of HIV-1 protease: a computational geometry approach

A computational geometry technique based on Delaunay tessellation of protein structure, represented by C(alpha) atoms, is used to study effects of single residue mutations on sequence-structure compatibility in HIV-1 protease. Profiles of residue scores derived from the four-body statistical potential are constructed for all 1881 mutants of the HIV-1 protease monomer and compared with the profile of the wild-type protein. The profiles for an isolated monomer of HIV-1 protease and the identical monomer in a dimeric state with an inhibitor are analyzed to elucidate changes to structural stability. Protease residues shown to undergo the greatest impact are those forming the dimer interface and flap region, as well as those known to be involved in inhibitor binding.     

Structural insights into the mechanisms of drug resistance in HIV-1 protease NL4-3

The development of resistance to anti-retroviral drugs targeted against HIV is an increasing clinical problem in the treatment of HIV-1-infected individuals. Many patients develop drug-resistant strains of the virus after treatment with inhibitor cocktails (HAART therapy), which include multiple protease inhibitors. Therefore, it is imperative that we understand the mechanisms by which the viral proteins, in particular HIV-1 protease, develop resistance. We have determined the three-dimensional structure of HIV-1 protease NL4-3 in complex with the potent protease inhibitor TL-3 at 2.0 A resolution. We have also obtained the crystal structures of three mutant forms of NL4-3 protease containing one (V82A), three (V82A, M46I, F53L) and six (V82A, M46I, F53L, V77I, L24I, L63P) point mutations in complex with TL-3. The three protease mutants arose sequentially under ex vivo selective pressure in the presence of TL-3, and exhibit fourfold, 11-fold, and 30-fold resistance to TL-3, respectively. This series of protease crystal structures offers insights into the biochemical and structural mechanisms by which the enzyme can overcome inhibition by TL-3 while recovering some of its native catalytic activity.     

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.     

Amplification of the effects of drug resistance mutations by background polymorphisms in HIV-1 protease from African subtypes

The vast majority of HIV-1 infections worldwide are caused by the C and A viral subtypes rather than the B subtype prevalent in the United States and Western Europe. Genomic differences between subtypes give rise to sequence variations in the encoded proteins, including those identified as targets for antiretroviral therapies. In the case of the HIV-1 protease, we reported earlier [Velazquez-Campoy et al. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 6062-6067] that proteases from the C and A subtypes exhibit a higher biochemical fitness in the presence of widely prescribed protease inhibitors. In this paper we present a complete thermodynamic dissection of the differences between proteases from different subtypes and the effects of the V82F/I84V drug-resistant mutation within the framework of the B, C, and A subtypes. These studies involved four inhibitors in clinical use (indinavir, saquinavir, ritonavir, and nelfinavir) and a second-generation protease inhibitor (KNI-764). Naturally occurring amino acid polymorphisms found in proteases from the C and A subtypes lower the binding affinities of existing clinical inhibitors by factors ranging between 2 and 7.5 which by themselves are not enough to cause drug resistance. The preexisting lower affinity in the C and A subtypes, however, significantly amplifies the effects of the drug-resistant mutation. Relative to the wild-type B subtype protease, the V82F/I84V drug-resistant mutation within the C and A subtypes lowers the binding affinity of inhibitors by factors ranging between 40 and 3000. When the enzyme kinetic properties (k(cat) and K(m)) are included in the analysis, the biochemical fitness of the C and A subtype drug-resistant mutants can be up to 1000-fold higher than that of the wild-type B subtype protease in the presence of the studied inhibitors. From a thermodynamic standpoint, the combined effects of the drug-resistant mutations and the natural amino acid polymorphisms on the Gibbs energy are additive and involve significant alterations in the enthalpy and entropy changes associated with inhibitor binding. At the biochemical level, the combined effects of naturally existing polymorphisms and drug-resistant mutations might have important consequences on the long-term viability of current HIV-1 protease inhibitors.     

Active-site mutations in the South african human immunodeficiency virus type 1 subtype C protease have a significant impact on clinical inhibitor binding: kinetic and thermodynamic study

Human immunodeficiency virus (HIV) infections in sub-Saharan Africa represent about 56% of global infections. Study of active-site mutations (the V82A single mutation and the V82F I84V double mutation) in the less-studied South African HIV type 1 subtype C (C-SA) protease indicated that neither mutation had a significant impact on the proteolytic functioning of the protease. However, the binding affinities of, and inhibition by, saquinavir, ritonavir, indinavir, and nelfinavir were weaker for each variant than for the wild-type protease, with the double mutant exhibiting the most dramatic change. Therefore, our results show that the C-SA V82F I84V double mutation decreased the binding affinities of protease inhibitors to levels significantly lower than that required for effective inhibition.     

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.     

Non-active site changes elicit broad-based cross-resistance of the HIV-1 protease to inhibitors.

Three high level, cross-resistant variants of the HIV-1 protease have been analyzed for their ability to bind four protease inhibitors approved by the Food and Drug Administration (saquinavir, ritonavir, indinavir, and nelfinavir) as AIDS therapeutics. The loss in binding energy (DeltaDeltaG(b)) going from the wild-type enzyme to mutant enzymes ranges from 2.5 to 4.4 kcal/mol, 40-65% of which is attributed to amino acid substitutions away from the active site of the protease and not in direct contact with the inhibitor. The data suggest that non-active site changes are collectively a major contributor toward engendering resistance against the protease inhibitor and cannot be ignored when considering cross-resistance issues of drugs against the HIV-1 protease.     

Molecular mechanisms of resistance: free energy calculations of mutation effects on inhibitor binding to HIV-1 protease

The changes in the inhibitor binding constants due to the mutation of isoleucine to valine at position 84 of HIV-1 protease are calculated using molecular dynamics simulations. The calculations are done for three potent inhibitors--KNI-272, L-735,524 (indinavir or MK-639), and Ro 31-8959 (saquinavir). The calculations agree with the experimental data both in terms of an overall trend and in the magnitude of the resulting free energy change. HIV-1 protease is a homodimer, so each mutation causes two changes in the enzyme. The decrease in the binding free energy from each mutated side chain differs among the three inhibitors and correlates well with the size of the cavities induced in the protein interior near the mutated residue. The cavities are created as a result of a mutation to a smaller side chain, but the cavities are less than would be predicted from the wild-type structures, indicating that there is significant relaxation to partially fill the cavities.     

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.     

Extreme Entropy-Enthalpy Compensation in a Drug-Resistant Variant of HIV-1 Protease

The development of HIV-1 protease inhibitors has been the historic paradigm of rational structure-based drug design, where structural and thermodynamic analyses have assisted in the discovery of novel inhibitors. While the total enthalpy and entropy change upon binding determine the affinity, often the thermodynamics are considered in terms of inhibitor properties only. In the current study, profound changes are observed in the binding thermodynamics of a drug-resistant variant compared to wild-type HIV-1 protease, irrespective of the inhibitor bound. This variant (Flap+) has a combination of flap and active site mutations and exhibits extremely large entropy-enthalpy compensation compared to wild-type protease, 5-15 kcal/mol, while losing only 1-3 kcal/mol in total binding free energy for any of six FDA-approved inhibitors. Although entropy-enthalpy compensation has been previously observed for a variety of systems, never have changes of this magnitude been reported. The co-crystal structures of Flap+ protease with four of the inhibitors were determined and compared with complexes of both the wild-type protease and another drug-resistant variant that does not exhibit this energetic compensation. Structural changes conserved across the Flap+ complexes, which are more pronounced for the flaps covering the active site, likely contribute to the thermodynamic compensation. The finding that drug-resistant mutations can profoundly modulate the relative thermodynamic properties of a therapeutic target independent of the inhibitor presents a new challenge for rational drug design.