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 PR1 (M) 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.

Potent new antiviral compound shows similar inhibition and structural interactions with drug resistant mutants and wild type HIV-1 protease.

The potent new antiviral inhibitor GRL-98065 (1) of HIV-1 protease (PR) has been studied with PR variants containing the single mutations D30N, I50V, V82A, and I84V that provide resistance to the major clinical inhibitors. Compound 1 had inhibition constants of 17-fold, 8-fold, 3-fold, and 3-fold, respectively, for PR(D30N), PR(I50V), PR(V82A), and PR(I84V) relative to wild type PR. The chemically related darunavir had similar relative inhibition, except for PR(D30N), where inhibitor 1 was approximately 3-fold less potent. The high resolution (1.11-1.60 Angstrom) crystal structures of PR mutant complexes with inhibitor 1 showed small changes relative to the wild type enzyme. PR(D30N) and PR(V82A) showed compensating interactions with inhibitor 1 relative to those of PR, while reduced hydrophobic contacts were observed with PR(I50V) and PR(I84V). Importantly, inhibitor 1 complexes showed fewer changes relative to wild type enzyme than reported for darunavir complexes. Therefore, inhibitor 1 is a valuable addition to the antiviral inhibitors with high potency against resistant strains of HIV.

HIV-1 protease dimer interface mutations that compensate for viral reverse transcriptase instability in infectious virions.

Mature enzymes encoded within the human immunodeficiency virus type 1 (HIV-1) genome (protease (PR), reverse transcriptase (RT) and integrase (IN)) derive from proteolytic processing of a large polyprotein (Gag-Pol). Gag-Pol processing is catalyzed by the viral PR, which is active as a homodimer. The HIV-1 RT functions as a heterodimer (p66/p51) composed of subunits of 560 and 440 amino acid residues, respectively. Both subunits have identical amino acid sequence, but p51 lacks 120 residues that are removed by the HIV-1 PR during viral maturation. While p66 is the catalytic subunit, p51 has a primarily structural role. Amino acid substitutions affecting the stability of p66/p51 (i.e. F130W) have a deleterious effect on viral fitness. Previously, we showed that the effects of F130W are mediated by p51 and can be compensated by mutation T58S. While studying the dynamics of emergence of the compensatory mutation, we observed that mutations in the viral PR-coding region were selected in HIV clones containing the RT substitution F130W, before the imposition of T58S/F130W mutations. The PR mutations identified (G94S and T96S) improved the replication capacity of the F130W mutant virus. By using a trans-complementation assay, we demonstrate that the loss of p66/p51 heterodimer stability caused by Trp130 can be attributed to an increased susceptibility of RT to viral PR degradation. Recombinant HIV-1 PRs bearing mutations G94S or T96S showed decreased dimer stability and reduced catalytic efficiency. These results were consistent with crystallographic data showing the location of both residues in the PR dimerization interface.

A novel bis-tetrahydrofuranylurethane-containing nonpeptidic protease inhibitor (PI), GRL-98065, is potent against multiple-PI-resistant human immunodeficiency virus in vitro.

We designed, synthesized, and identified GRL-98065, a novel nonpeptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitor (PI) containing the structure-based designed privileged cyclic ether-derived nonpeptide P2 ligand, 3(R),3a(S),6a(R)-bis-tetrahydrofuranylurethane (bis-THF), and a sulfonamide isostere, which is highly potent against laboratory HIV-1 strains and primary clinical isolates (50% effective concentration [EC(50)], 0.0002 to 0.0005 microM) with minimal cytotoxicity (50% cytotoxicity, 35.7 microM in CD4(+) MT-2 cells). GRL-98065 blocked the infectivity and replication of each of the HIV-1(NL4-3) variants exposed to and selected by up to a 5 microM concentration of saquinavir, indinavir, nelfinavir, or ritonavir and a 1 microM concentration of lopinavir or atazanavir (EC(50), 0.0015 to 0.0075 microM), although it was less active against HIV-1(NL4-3) selected by amprenavir (EC(50), 0.032 microM). GRL-98065 was also potent against multiple-PI-resistant clinical HIV-1 variants isolated from patients who had no response to existing antiviral regimens after having received a variety of antiviral agents, HIV-1 isolates of various subtypes, and HIV-2 isolates examined. Structural analyses revealed that the close contact of GRL-98065 with the main chain of the protease active-site amino acids (Asp29 and Asp30) is important for its potency and wide-spectrum activity against multiple-PI-resistant HIV-1 variants. The present data demonstrate that the privileged nonpeptide P2 ligand, bis-THF, is critical for the binding of GRL-98065 to the HIV protease substrate binding site and that this scaffold can confer highly potent antiviral activity against a wide spectrum of HIV isolates.

Structural and kinetic analysis of caspase-3 reveals role for s5 binding site in substrate recognition.

The molecular basis for the substrate specificity of human caspase-3 has been investigated using peptide analog inhibitors and substrates that vary at the P2, P3, and P5 positions. Crystal structures were determined of caspase-3 complexes with the substrate analogs at resolutions of 1.7 A to 2.3 A. Differences in the interactions of caspase-3 with the analogs are consistent with the Ki values of 1.3 nM, 6.5 nM, and 12.4 nM for Ac-DEVD-Cho, Ac-VDVAD-Cho and Ac-DMQD-Cho, respectively, and relative kcat/Km values of 100%, 37% and 17% for the corresponding peptide substrates. The bound peptide analogs show very similar interactions for the main-chain atoms and the conserved P1 Asp and P4 Asp, while interactions vary for P2 and P3. P2 lies in a hydrophobic S2 groove, consistent with the weaker inhibition of Ac-DMQD-Cho with polar P2 Gln. S3 is a surface hydrophilic site with favorable polar interactions with P3 Glu in Ac-DEVD-Cho. Ac-DMQD-Cho and Ac-VDVAD-Cho have hydrophobic P3 residues that are not optimal in the polar S3 site, consistent with their weaker inhibition. A hydrophobic S5 site was identified for caspase-3, where the side-chains of Phe250 and Phe252 interact with P5 Val of Ac-VDVAD-Cho, and enclose the substrate-binding site by conformational change. The kinetic importance of hydrophobic P5 residues was confirmed by more efficient hydrolysis of caspase-3 substrates Ac-VDVAD-pNA and Ac-LDVAD-pNA compared with Ac-DVAD-pNA. In contrast, caspase-7 showed less efficient hydrolysis of the substrates with P5 Val or Leu compared with Ac-DVAD-pNA. Caspase-3 and caspase-2 share similar hydrophobic S5 sites, while caspases 1, 7, 8 and 9 do not have structurally equivalent hydrophobic residues; these caspases are likely to differ in their selectivity for the P5 position of substrates. The distinct selectivity for P5 will help define the particular substrates and signaling pathways associated with each caspase.

Mechanism of drug resistance revealed by the crystal structure of the unliganded HIV-1 protease with F53L mutation.

Mutations in HIV-1 protease (PR) that produce resistance to antiviral PR inhibitors are a major problem in AIDS therapy. The mutation F53L arising from antiretroviral therapy was introduced into the flexible flap region of the wild-type PR to study its effect and potential role in developing drug resistance. Compared to wild-type PR, PR(F53L) showed lower (15%) catalytic efficiency, 20-fold weaker inhibition by the clinical drug indinavir, and reduced dimer stability, while the inhibition constants of two peptide analog inhibitors were slightly lower than those for PR. The crystal structure of PR(F53L) was determined in the unliganded form at 1.35 Angstrom resolution in space group P4(1)2(1)2. The tips of the flaps in PR(F53L) had a wider separation than in unliganded wild-type PR, probably due to the absence of hydrophobic interactions of the side-chains of Phe53 and Ile50'. The changes in interactions between the flaps agreed with the reduced stability of PR(F53L) relative to wild-type PR. The altered flap interactions in the unliganded form of PR(F53L) suggest a distinct mechanism for drug resistance, which has not been observed in other common drug-resistant mutants.

Effectiveness of nonpeptide clinical inhibitor TMC-114 on HIV-1 protease with highly drug resistant mutations D30N, I50V, and L90M.

The potent new antiviral inhibitor TMC-114 (UIC-94017) of HIV-1 protease (PR) has been studied with three PR variants containing single mutations D30N, I50V, and L90M, which provide resistance to the major clinical inhibitors. The inhibition constants (K(i)) of TMC-114 for mutants PR(D30N), PR(I50V), and PR(L90M) were 30-, 9-, and 0.14-fold, respectively, relative to wild-type PR. The molecular basis for the inhibition was analyzed using high-resolution (1.22-1.45 A) crystal structures of PR mutant complexes with TMC-114. In PR(D30N), the inhibitor has a water-mediated interaction with the side chain of Asn30 rather than the direct interaction observed in PR, which is consistent with the relative inhibition. Similarly, in PR(I50V) the inhibitor loses favorable hydrophobic interactions with the side chain of Val50. TMC-114 has additional van der Waals contacts in PR(L90M) structure compared to the PR structure, leading to a tighter binding of the inhibitor. The observed changes in PR structure and activity are discussed in relation to the potential for development of resistant mutants on exposure to TMC-114.

Kinetic, stability, and structural changes in high-resolution crystal structures of HIV-1 protease with drug-resistant mutations L24I, I50V, and G73S.

The crystal structures, dimer stabilities, and kinetics have been analyzed for wild-type human immunodeficiency virus type 1 (HIV-1) protease (PR) and resistant mutants PR(L24I), PR(I50V), and PR(G73S) to gain insight into the molecular basis of drug resistance. The mutations lie in different structural regions. Mutation I50V alters a residue in the flexible flap that interacts with the inhibitor, L24I alters a residue adjacent to the catalytic Asp25, and G73S lies at the protein surface far from the inhibitor-binding site. PR(L24I) and PR(I50V), showed a 4% and 18% lower k(cat)/K(m), respectively, relative to PR. The relative k(cat)/K(m) of PR(G73S) varied from 14% to 400% when assayed using different substrates. Inhibition constants (K(i)) of the antiviral drug indinavir for the reaction catalyzed by the mutant enzymes were about threefold and 50-fold higher for PR(L24I) and PR(I50V), respectively, relative to PR and PR(G73S). The dimer dissociation constant (K(d)) was estimated to be approximately 20 nM for both PR(L24I) and PR(I50V), and below 5 nM for PR(G73S) and PR. Crystal structures of the mutants PR(L24I), PR(I50V) and PR(G73S) were determined in complexes with indinavir, or the p2/NC substrate analog at resolutions of 1.10-1.50 Angstrom. Each mutant revealed distinct structural changes relative to PR. The mutated residues in PR(L24I) and PR(I50V) had reduced intersubunit contacts, consistent with the increased K(d) for dimer dissociation. Relative to PR, PR(I50V) had fewer interactions of Val50 with inhibitors, in agreement with the dramatically increased K(i). The distal mutation G73S introduced new hydrogen bond interactions that can transmit changes to the substrate-binding site and alter catalytic activity. Therefore, the structural alterations observed for drug-resistant mutations were in agreement with kinetic and stability changes.

Molecular basis for substrate recognition and drug resistance from 1.1 to 1.6 angstroms resolution crystal structures of HIV-1 protease mutants with substrate analogs.

HIV-1 protease (PR) and two drug-resistant variants--PR with the V82A mutation (PR(V82A)) and PR with the I84V mutation (PR(I84V))--were studied using reduced peptide analogs of five natural cleavage sites (CA-p2, p2-NC, p6pol-PR, p1-p6 and NC-p1) to understand the structural and kinetic changes. The common drug-resistant mutations V82A and I84V alter residues forming the substrate-binding site. Eight crystal structures were refined at resolutions of 1.10-1.60 A. Differences in the PR-analog interactions depended on the peptide sequence and were consistent with the relative inhibition. Analog p6(pol)-PR formed more hydrogen bonds of P2 Asn with PR and fewer van der Waals contacts at P1' Pro compared with those formed by CA-p2 or p2-NC in PR complexes. The P3 Gly in p1-p6 provided fewer van der Waals contacts and hydrogen bonds at P2-P3 and more water-mediated interactions. PR(I84V) showed reduced van der Waals interactions with inhibitor compared with PR, which was consistent with kinetic data. The structures suggest that the binding affinity for mutants is modulated by the conformational flexibility of the substrate analogs. The complexes of PR(V82A) showed smaller shifts of the main chain atoms of Ala82 relative to PR, but more movement of the peptide analog, compared to complexes with clinical inhibitors. PR(V82A) was able to compensate for the loss of interaction with inhibitor caused by mutation, in agreement with kinetic data, but substrate analogs have more flexibility than the drugs to accommodate the structural changes caused by mutation. Hence, these structures help to explain how HIV can develop drug resistance while retaining the ability of PR to hydrolyze natural substrates.

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.

Crystal structures of HIV protease V82A and L90M mutants reveal changes in the indinavir-binding site.

The crystal structures of the wild-type HIV-1 protease (PR) and the two resistant variants, PR(V82A) and PR(L90M), have been determined in complex with the antiviral drug, indinavir, to gain insight into the molecular basis of drug resistance. V82A and L90M correspond to an active site mutation and nonactive site mutation, respectively. The inhibition (K(i)) of PR(V82A) and PR(L90M) was 3.3- and 0.16-fold, respectively, relative to the value for PR. They showed only a modest decrease, of 10-15%, in their k(cat)/K(m) values relative to PR. The crystal structures were refined to resolutions of 1.25-1.4 A to reveal critical features associated with inhibitor resistance. PR(V82A) showed local changes in residues 81-82 at the site of the mutation, while PR(L90M) showed local changes near Met90 and an additional interaction with indinavir. These structural differences concur with the kinetic data.