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 that provide resistance to the major clinical inhibitors. The inhibition constants (Ki) 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 Å) 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.

SUMMARYTMC114 (darunavir) is a promising clinical inhibitor of HIV-1 protease (PR) for treatment of drug resistant HIV/AIDS. We report the ultra-high 0.84Å resolution crystal structure of the TMC114 complex with PR containing the drug resistant mutation V32I (PR V32I ), and the 1.22 Å resolution structure of a complex with PR M46L . These structures show TMC114 bound at two distinct sites, one in the active-site cavity and the second on the surface of one of the flexible flaps in the PR dimer. Remarkably, TMC114 binds at these two sites simultaneously in two diastereomers related by inversion of the sulfonamide nitrogen. Moreover, the flap site is shaped to accommodate the diastereomer with the S-enantiomeric nitrogen rather than the one with the R-enantiomeric nitrogen. The existence of the second binding site and two diastereomers suggest a mechanism for the high effectiveness of TMC114 on drug resistant HIV and the potential design of new inhibitors. KeywordsHIV-1 protease; drug resistance; darunavir; allosteric binding site; ultra-high resolution crystal structure; enantiomer Two decades of research have yielded about twenty antiretroviral drugs and different drug regimens for treatment of HIV-1 infection. 1 Highly active antiretroviral therapy (HAART), in which a cocktail of drugs containing HIV-1 reverse transcriptase (RT) and protease (PR) inhibitors is administered to patients, 2,3 has dramatically improved the survival of infected people and transformed the previously deadly disease into a treatable medical condition in many cases.HIV-1 protease inhibitors (PIs) were developed successfully by structure-guided drug design. 4 They work by blocking the activity of PR to process the viral polypeptides Gag and Gag-Pol into the structural and enzymatic proteins during the final stages of viral particle maturation. The Food and Drug Administration (FDA) has approved eight PIs and ten PI combinations to be used in HAART. Nonetheless, the drug resistant strains develop quickly due to the infidelity * Correspondence should be addressed to Irene T. Weber. Department of Biology, Georgia State University, P.O. Box 4010, Atlanta, GA 30302-4010; USA; Phone: 404-651-0098, Fax: 404-651-2509, E-mail: iweber@gsu.edu We describe crystallographic analysis of the effects of TMC114 on mutants PR V32I and PR M46L . The crystal structures have been determined at 0.84Å and 1.22Å resolution for PR V32I -TMC114 and PR M46L -TMC114 complexes, respectively. The first ultra-high resolution structure of a PR-inhibitor complex showed two molecular species at 60% and 40% occupancy. The higher occupancy conformer has TMC114 bound at two distinct sites: the active site cavity and a second, new site on the surface of one of the flaps, while the lower occupancy conformer showed TMC114 only in the active site cavity. These results suggest an alternative mechanism for the effectiveness of TMC114 against many clinical drug resistant isolates of HIV-1 and may provide a distinct target for the design of novel inhibitors that bind to the second...

All aspartic proteases, including retroviral proteases, share the triplet DTG critical for the active site geometry and catalytic function. These residues interact closely in the active, dimeric structure of HIV-1 protease (PR). We have systematically assessed the effect of the D25N mutation on the structure and stability of the mature PR monomer and dimer. The D25N mutation (PR D25N ) increases the equilibrium dimer dissociation constant by a factor >100-fold (1.3 ؎ 0.09 M) relative to PR. In the absence of inhibitor, NMR studies reveal clear structural differences between PR and PR D25N in the relatively mobile P1 loop (residues 79 -83) and flap regions, and differential scanning calorimetric analyses show that the mutation lowers the stabilities of both the monomer and dimer folds by 5 and 7.3°C, respectively. Only minimal differences are observed in high resolution crystal structures of PR D25N complexed to darunavir (DRV), a potent clinical inhibitor, or a non-hydrolyzable substrate analogue, Ac-Thr-Ile-Nle-r-Nle-Gln-Arg-NH 2 (RPB), as compared with PR⅐DRV and PR⅐RPB complexes. Although complexation with RPB stabilizes both dimers, the effect on their T m is smaller for PR D25N (6.2°C) than for PR (8.7°C). The T m of PR D25N ⅐DRV increases by only 3°C relative to free PR D25N , as compared with a 22°C increase for PR⅐DRV, and the mutation increases the ligand dissociation constant of PR D25N ⅐DRV by a factor of ϳ10 6 relative to PR⅐DRV. These results suggest that interactions mediated by the catalytic Asp residues make a major contribution to the tight binding of DRV to PR.In HIV-1, 2 the protease is synthesized as part of a 165-kDa polyprotein (Gag-Pol). Gag-Pol comprises the matrix, capsid, P2, nucleocapsid, transframe, protease (PR), reverse transcriptase, and integrase domains (1). The protease mediates its own release and the processing of the viral polyproteins, Gag and Gag-Pol, into the necessary structural and functional proteins (1-3). This spatio-temporally regulated process is crucial for the maturation and propagation of HIV (4 -7). Because of this vital role, the mature protease dimer has proven to be a successful target for the development of antiviral agents. Structure-based design of drugs targeted against the mature protease has aided in the development of potent inhibitors that bind specifically to the active site (8, 9). Although several of these inhibitors are in clinical use and have curtailed the progression of the disease, the effectiveness of long term treatment has been limited due to naturally selected protease variants exhibiting lower affinity to the drugs than the wild-type enzyme, and this has been a challenge for the past decade (10). In recent years, a major emphasis in protease research has been to improve inhibitor design and treatment regimens, which include the highly active retroviral therapy, to overcome the problem of drug resistance and curb progress of the disease (11, 12).The HIV-1 protease is composed of 99 amino acids and is a member of the family of aspartic ac...

HIV-1 protease (PR) and its mutants are important antiviral drug targets. The PR flap region is critical for binding substrates or inhibitors and catalytic activity. Hence, mutations of flap residues frequently contribute to reduced susceptibility to PR inhibitors in drug resistant HIV. Structural and kinetic analyses were used to investigate the role of flap residues Gly48, Ile50 and Ile54 in the development of drug resistance. The crystal structures of flap mutants PRI50V, PRI54V, and PRI54M complexed with saquinavir (SQV) and PRG48V, PRI54V, and PRI54M complexed with darunavir (DRV) were determined at the resolutions of 1.05–1.40 Å. The PR mutants showed changes in flap conformation, interactions with adjacent residues, inhibitor binding and the conformation of the 80’s loop relative to the wild type PR. The PR contacts with darunavir were closer in PRG48V-DRV than in the wild type PR-DRV, while they were longer in PRI54M-DRV. The relative inhibition of PRs with mutations I54V and I54M was similar for saquinavir and darunavir. PRG48V was about 2-fold less susceptible to saquinavir than to darunavir, while the opposite was observed for PRI50V. The observed inhibition was in agreement with the association of G48V and I50V with clinical resistance to saquinavir and darunavir, respectively. This analysis of structural and kinetic effects of the mutants will assist in development of more effective inhibitors for drug resistant HIV.

The presence of pharmaceutical antibiotics in aquatic environments poses potential human health and ecological risks. We synthesized ordered micro- and mesoporous carbons, and further conducted batch experiments to systematically examine their adsorption properties toward three antibiotics, sulfamethoxazole, tetracycline, and tylosin, in aqueous solution. In comparison, nonporous graphite, single-walled carbon nanotubes, and two commercial microporous activated carbons were included as additional adsorbents. Adsorption of low-sized sulfamethoxazole was stronger on the activated carbons than on other carbonaceous adsorbents resulting from the pore-filling effect; in contrast, due to the size-exclusion effect adsorption of bulky tetracycline and tylosin was much lower on the activated carbons, especially for the more microporous one, than on the synthesized carbons. After normalizing for adsorbent surface area, adsorption of tetracycline and tylosin on the synthesized carbons was similar to that on nonporous graphite, reflecting complete accessibility of the adsorbent surface area in adsorption. Additionally, compared with other porous adsorbents the synthesized carbons showed faster adsorption kinetics of tetracycline and tylosin, which was attributed to their regular-shaped, open and interconnected three-dimensional pore structure. The findings indicate that template-synthesized micro- and mesoporous carbons are promising adsorbents for the removal of antibiotics, particularly, the bulky and flexible-structured compounds, from aqueous solution.

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 I50Valters 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 Å. 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.

HIV‐1 protease (PR) and two drug‐resistant variants – PR with the V82A mutation (PRV82A) and PR with the I84V mutation (PRI84V) – 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 Å. Differences in the PR–analog interactions depended on the peptide sequence and were consistent with the relative inhibition. Analog p6pol‐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. PRI84V 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 PRV82A 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. PRV82A 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.

Using a temperature-dependent synthetic approach, four distinct new silver(I) coordination polymers resulting from the different conformations adopted by the flexible ligand at different temperatures were obtained.