Improved cyclic urea inhibitors of the HIV-1 protease: synthesis, potency, resistance profile, human pharmacokinetics and X-ray crystal structure of DMP 450.

BACKGROUND: Effective HIV protease inhibitors must combine potency towards wild-type and mutant variants of HIV with oral bioavailability such that drug levels in relevant tissues continuously exceed that required for inhibition of virus replication. Computer-aided design led to the discovery of cyclic urea inhibitors of the HIV protease. We set out to improve the physical properties and oral bioavailability of these compounds. RESULTS: We have synthesized DMP 450 (bis-methanesulfonic acid salt), a water-soluble cyclic urea compound and a potent inhibitor of HIV replication in cell culture that also inhibits variants of HIV with single amino acid substitutions in the protease. DMP 450 is highly selective for HIV protease, consistent with displacement of the retrovirus-specific structural water molecule. Single doses of 10 mg kg-1 DMP 450 result in plasma levels in man in excess of that required to inhibit wild-type and several mutant HIVs. A plasmid-based, in vivo assay model suggests that maintenance of plasma levels of DMP 450 near the antiviral IC90 suppresses HIV protease activity in the animal. We did identify mutants that are resistant to DMP 450, however; multiple mutations within the protease gene caused a significant reduction in the antiviral response. CONCLUSIONS: DMP 450 is a significant advance within the cyclic urea class of HIV protease inhibitors due to its exceptional oral bioavailability. The data presented here suggest that an optimal cyclic urea will provide clinical benefit in treating AIDS if it combines favorable pharmacokinetics with potent activity against not only single mutants of HIV, but also multiply-mutant variants.

In vitro susceptibility of clinical isolates of HIV-1 to XM323, a non-peptidyl HIV protease inhibitor.

OBJECTIVE: To determine the in vitro susceptibility of primary clinical isolates and laboratory strains of HIV-1 to XM323. METHODS: The AIDS Clinical Trials Group/US Department of Defense p24 antigen-based consensus assay was used to determine in vitro susceptibility of 57 primary clinical isolates and three laboratory strains of HIV-1 to XM323, zidovudine, zalcitabine (ddC), and didanosine (ddI). RESULTS: The concentrations of compound required to inhibit viral p24 antigen production by 50% [median inhibitory concentration (IC50)] for nucleosides were as follows: zidovudine, 0.001-->5 microM; ddC, < 0.01-0.23 microM; ddI, 0.2-->25 microM). Against both nucleoside susceptible and resistant isolates XM323 exhibited potent inhibition with IC50 values of < 0.02-0.27 microM and IC90 values of 0.03-1.17 microM. CONCLUSIONS: XM323 is a potent inhibitor of diverse clinical isolates of HIV-1 in vitro and represents a novel class of non-peptidyl inhibitors of HIV-1 protease.

Selection conditions affect the evolution of specific mutations in the reverse transcriptase gene associated with resistance to DMP 266.

OBJECTIVE: To monitor the appearance of HIV-1 variants resistant to inhibition by DMP 266, a benzoxazinone non-nucleoside reverse transcriptase inhibitor using two different protocols for applying drug selective pressure in tissue culture. To compare the phenotype and genotype of viral isolates selected by each method. METHODS: MT-2 cells and fresh donor peripheral blood mononuclear cells (PBMC) were infected with HIV-1 strain RF. The MT-2 cells were infected in the presence of a 50% inhibitory concentration (IC50) of DMP 266 and the concentration was slowly increased during the selection period. The PBMC were infected for 1 week in the absence of inhibitor and then a single concentration was maintained throughout the selection period. Both cultures were passaged for approximately 4 months. Virus and cell pellets were harvested over this in vitro selection period, the RT genes amplified by polymerase chain reaction from the cell pellets, and the proviral DNAs sequenced. Isolated virus was tested for DMP 266 susceptibility in either the AIDS Clinical Trials Group/Department of Defense consensus assay or MT-2 yield reduction assay. RESULTS: Passage in MT-2 cells resulted in accumulation of three substitutions in RT (V179D, L1001, Y181C) after 24 passages associated with 1000-fold reduced susceptibility to DMP 266. In PBMC cultures treated with 0.96 microM DMP 266, virus replication was completely suppressed after 2 weeks; no regrowth occurred in the presence of compound after 10 weeks or in the absence of compound for 3 additional weeks. The 0.096 microM treated cultures had an initial 2.5-log reduction in infectious virus titre followed by rapid regrowth. Virus obtained at week 6 displayed a 28-fold reduction in susceptibility with an L1001 substitution in RT, and by week 11 displayed a 1000-fold reduction in susceptibility with an additional V1081 substitution. CONCLUSIONS: High-level resistance to DMP 266 may develop by at least two pathways and experimental conditions influence the genotype selected. The continued absence of detectable virus in the PBMC cultures grown at 0.96 microM is supportive evidence that maintaining trough plasma levels of DMP 266 which result in sustained antiviral activity in vivo may delay emergence of highly resistant viral variants. Confirmation of this hypothesis will require clinical trials.

Cyclic HIV protease inhibitors: synthesis, conformational analysis, P2/P2' structure-activity relationship, and molecular recognition of cyclic ureas.

High-resolution X-ray structures of the complexes of HIV-1 protease (HIV-1PR) with peptidomimetic inhibitors reveal the presence of a structural water molecule which is hydrogen bonded to both the mobile flaps of the enzyme and the two carbonyls flanking the transition-state mimic of the inhibitors. Using the structure-activity relationships of C2-symmetric diol inhibitors, computed-aided drug design tools, and first principles, we designed and synthesized a novel class of cyclic ureas that incorporates this structural water and preorganizes the side chain residues into optimum binding conformations. Conformational analysis suggested a preference for pseudodiaxial benzylic and pseudodiequatorial hydroxyl substituents and an enantiomeric preference for the RSSR stereochemistry. The X-ray and solution NMR structure of the complex of HIV-1PR and one such cyclic urea, DMP323, confirmed the displacement of the structural water. Additionally, the bound and "unbound" (small-molecule X-ray) ligands have similar conformations. The high degree of preorganization, the complementarity, and the entropic gain of water displacement are proposed to explain the high affinity of these small molecules for the enzyme. The small size probably contributes to the observed good oral bioavailability in animals. Extensive structure-based optimization of the side chains that fill the S2 and S2' pockets of the enzyme resulted in DMP323, which was studied in phase I clinical trials but found to suffer from variable pharmacokinetics in man. This report details the synthesis, conformational analysis, structure-activity relationships, and molecular recognition of this series of C2-symmetry HIV-1PR inhibitors. An initial series of cyclic ureas containing nonsymmetric P2/P2' is also discussed.

Limited sequence diversity of the HIV type 1 protease gene from clinical isolates and in vitro susceptibility to HIV protease inhibitors.

Proviral DNAs from 3 laboratory strains and 21 clinical isolates of HIV-1 were extracted from infected cells after proteinase K digestion and the protease gene was PCR amplified and sequenced directly by the Sanger method. In vitro susceptibilities of the virus isolates to protease inhibitors were determined by the ACTG/DoD consensus assay. Four different HIV protease inhibitors were tested including P9941, a C2 symmetrical diol (Du Pont-Merck); A80987, an asymmetric mono-ol (Abbott); XM323, a cyclic urea (Du Pont-Merck); and Ro31-8959, an asymmetric hydroxyethylene isostere (Roche). Maximum sequence variation was 10% at both the nucleic and amino acid levels. Purine-purine substitutions were most common. Five noncontiguous regions were conserved across all isolates and corresponded to amino acids 1-9 (amino terminal), 21-32 (catalytic site), 47-56 ("flap" region), 78-88 (substrate-binding region), and 94-99 (carboxy terminal). All clinical isolates demonstrated in vitro susceptibility to the protease inhibitors. There was no significant difference between the susceptibility of the reference strains and the clinical isolates. These data suggest that the variable regions of protease do not contain sites that are important for interactions with the inhibitors tested.

Identification of a clinical isolate of HIV-1 with an isoleucine at position 82 of the protease which retains susceptibility to protease inhibitors.

The HIV-1 protease (PR) is essential for the production of mature virions. As such, it has become a target for the development of anti-HIV chemotherapeutics. Multiple passages of virus in cell culture in the presence of PR inhibitors have resulted in the selection of variants with decreased sensitivity to inhibitors of the PR. The most common alteration observed is a single amino acid change at position 82. This particular position has been well characterized by several laboratories as being important for the susceptibility of the virus to inhibitors of PR function. Mutations which result in the substitution of the wild-type valine with alanine, phenylalanine, threonine or isoleucine at position 82 of the PR have been associated with decreased sensitivity to several PR inhibitors. We describe here a clinical strain of HIV-1 that contains an isoleucine at position 82 of the PR instead of the usual valine. This strain is unique in that it was isolated from a patient that was anti-retroviral naive, and in the past, variants at position 82 of the PR have only been found after treatment of patients or cell culture with PR inhibitors. Moreover, this virus remains sensitive to PR inhibitors of the cyclic urea and C-2 symmetrical diol classes.

Wound infection and septic shock due to Vibrio vulnificus.

Vibrio vulnificus is an organism commonly found in the marine environment whose pathogenic potential for humans has been recently recognized. Two patients are described who developed rapidly progressing wound infections and bacteremia due to this halophilic, lactose-positive vibrio. The clinical manifestations of the resulting infection differed from patterns reported by other authors.

In vitro activities of trimethoprim and sulfamethoxazole against Listeria monocytogenes.

The in vitro activities of trimethoprim and sulfamethoxyazole against clinical isolates of Listeria monocytogenes were examined separately and in combination with a microtiter broth dilution system. Sulfamethoxazole demonstrated variable activity and was generally bacteriostatic. Trimethoprim alone was bactericidal against 96% of isolates at less than 0.5 microgram/ml. The bactericidal action of trimethoprim against L. monocytogenes was generally potentiated by sulfamethoxyazole even when isolates were relatively resistant to sulfamethoxyazole alone.