terized [19-25], understanding the subtle balance of molecular recognition events that confer drug resistance in HIV-1 is crucial to the development of second generation drugs in the treatment of HIV-1 infection. Summary HIV protease is the aspartyl protease that processes the Gag and Pol polyproteins and allows for the matura-The homodimeric HIV-1 protease is the target of some tion of the immature HIV virion, thus allowing the spread of the most effective antiviral AIDS therapy, as it facili-of the virus. Remarkably, the precise physical parame-tates viral maturation by cleaving ten asymmetric and ters that govern how HIV-1 protease binds to its ten nonhomologous sequences in the Gag and Pol poly-natural, nonhomologous substrates [26-31] (Table 1) re-proteins. Since the specificity of this enzyme is not main poorly understood. The active site of the homodi-easily determined from the sequences of these cleav-meric protease is at the dimer interface [18, 32]. Despite age sites alone, we solved the crystal structures of the symmetry conferred on its active site because it is complexes of an inactive variant (D25N) of HIV-1 prote-a homodimer, the enzyme recognizes asymmetric sub-ase with six peptides that correspond to the natural strate sites within the Gag and Pol polyproteins. The substrate cleavage sites. When the protease binds to amino acid sequences of these substrates are asymmet-its substrate and buries nearly 1000 A ˚ 2 of surface area, ric around the cleavage sites in both size and charge the symmetry of the protease is broken, yet most inter-distribution. In addition, these sites share little sequence nal hydrogen bonds and waters are conserved. How-homology. How then does the protease recognize a ever, no substrate side chain hydrogen bond is con-particular peptide sequence as being a substrate? There served. Specificity of HIV-1 protease appears to be must be a breakdown in the symmetry within the individ-determined by an asymmetric shape rather than a par-ual protease dimer when it binds to its substrates. This ticular amino acid sequence. breakdown has often been difficult to characterize, however , since many of the complexes of HIV protease Introduction bound to asymmetric ligands do not uniquely orient the protease dimer in the crystal cell. This lack of unique As the worldwide AIDS epidemic continues into its third orientation resulted in protease-substrate structures decade, a cure for HIV-1 still eludes the medical commu-with 50% of the ligand oriented in one direction and nity [1]. In the absence of a cure for HIV-1 pathogenesis, 50% in the other, thus averaging out the asymmetry suppressing viral replication and maintaining it at low within the protease. To elucidate how HIV-1 protease to undetectable levels have become critical goals in the recognizes its substrates, we determined the crystal field of HIV-1 research [2-5]. To this end, highly active structures of six complexes of HIV-1 protease with de-antiretroviral therapy (HAART) has become a successful cameric peptides that correspond ...

TMC114, a newly designed human immunodeficiency virus type 1 (HIV-1) protease inhibitor, is extremely potent against both wild-type (wt) and multidrug-resistant (MDR) viruses in vitro as well as in vivo. Although chemically similar to amprenavir (APV), the potency of TMC114 is substantially greater. To examine the basis for this potency, we solved crystal structures of TMC114 complexed with wt HIV-1 protease and TMC114 and APV complexed with an MDR (L63P, V82T, and I84V) protease variant. In addition, we determined the corresponding binding thermodynamics by isothermal titration calorimetry. TMC114 binds approximately 2 orders of magnitude more tightly to the wt enzyme (K d ‫؍‬ 4.5 ؋ 10 ؊12 M) than APV (K d ‫؍‬ 3.9 ؋ 10 ؊10 M). Our X-ray data (resolution ranging from 2.2 to 1.2 Å) reveal strong interactions between the bis-tetrahydrofuranyl urethane moiety of TMC114 and main-chain atoms of D29 and D30. These interactions appear largely responsible for TMC114's very favorable binding enthalpy to the wt protease (؊12.1 kcal/mol). However, TMC114 binding to the MDR HIV-1 protease is reduced by a factor of 13.3, whereas the APV binding constant is reduced only by a factor of 5.1. However, even with the reduction in binding affinity to the MDR HIV protease, TMC114 still binds with an affinity that is more than 1.5 orders of magnitude tighter than the first-generation inhibitors. Both APV and TMC114 fit predominantly within the substrate envelope, a property that may be associated with decreased susceptibility to drug-resistant mutations relative to that of first-generation inhibitors. Overall, TMC114's potency against MDR viruses is likely a combination of its extremely high affinity and close fit within the substrate envelope.

The screening of known HIV-1 protease inhibitors against a panel of multi-drug-resistant viruses revealed the potent activity of TMC126 on drug-resistant mutants. In comparison to amprenavir, the improved affinity of TMC126 is largely the result of one extra hydrogen bond to the backbone of the protein in the P2 pocket. Modification of the substitution pattern on the phenylsulfonamide P2' substituent of TMC126 created an interesting SAR, with the close analogue TMC114 being found to have a similar antiviral activity against the mutant and the wild-type viruses. X-ray and thermodynamic studies on both wild-type and mutant enzymes showed an extremely high enthalpy driven affinity of TMC114 for HIV-1 protease. In vitro selection of mutants resistant to TMC114 starting from wild-type virus proved to be extremely difficult; this was not the case for other close analogues. Therefore, the extra H-bond to the backbone in the P2 pocket cannot be the only explanation for the interesting antiviral profile of TMC114. Absorption studies in animals indicated that TMC114 has pharmacokinetic properties comparable to currently approved HIV-1 protease inhibitors.

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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 wildtype 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.