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New Entry Inhibitor in Development
  "A small molecule HIV-1 inhibitor that targets the HIV-1 envelope and inhibits CD4 receptor binding"
Pin-Fang Lin, Bristol Myers Squibb. Proceedings of National Academy of Sciences (PNAS) journal. August 20, 2003.
This study indicates that BMS-378806, a newly synthesized small molecule HIV-1 inhibitor, blocks the first step of the infection process - the binding of the HIV-1 envelope glycoprotein gp120 to its cell surface receptor, CD4; shows good bioavailability in animals; is effective against HIV strains resistant to existing HIV-1 protease and reverse transcriptase inhibitors; and appears to be the first compound that blocks gp120-CD4 binding.
Abstract: BMS-378806 is a recently discovered small molecule HIV-1 inhibitor that blocks viral entrance to cells. The compound exhibits potent inhibitory activity against a panel of R5- (virus using the CCR5 coreceptor), X4- (virus using the CXCR4 coreceptor), and R5yX4 HIV-1 laboratory and clinical isolates of the B subtype (median EC50 of 0.04 mM) in culture assays.
BMS-378806 is selective for HIV-1 and inactive against HIV-2, SIV and a panel of other viruses, and exhibits no significant cytotoxicity in the 14 cell types tested (concentration for 50% reduction of cell growth, >225 mM). Mechanism of action studies demonstrated that BMS-378806 binds to gp120 and inhibits the interactions of the HIV-1 envelope protein to cellular CD4 receptors.
Further confirmation that BMS-378806 targets the envelope in infected cells was obtained through the isolation of resistant variants and the mapping of resistance substitutions to the HIV-1 envelope. In particular, two substitutions, M426L and M475I, are situated in the CD4 binding pocket of gp120. Recombinant HIV-1 carrying these two substitutions demonstrated significantly reduced susceptibility to compound inhibition.
BMS-378806 displays many favorable pharmacological traits, such as low protein binding, minimal human serum effect on anti-HIV-1 potency, good oral bioavailability in animal species, and a clean safety profile in initial animal toxicology studies.
Together, the data show that BMS-378806 is a representative of a new class of HIV inhibitors that has the potential to become a valued addition to our current armamentarium of antiretroviral drugs.
Pharmacokinetic and Safety Profiles Whether a wide spectrum of susceptibilities will be problematic for attachment inhibitors re-mains to be seen. Efficacy will be determined to a great extent by the drug exposure levels achievable in man. Because the efficacy of antiretroviral agents can be impacted significantly by serum protein binding, the effect of human serum on the anti-HIV activity of BMS-378806 was assessed by infecting either HIV-1LAI or HIV-1NL4- 3 strain in MT-2 cells in the presence of 40% human serum. Results showed that the presence of human serum only marginally increased the EC50 of BMS-378806 by 1.2-fold (data not shown). In agreement, the extent of protein binding in human plasma was determined to be relatively low at 73%. Together, these results suggested that human serum will likely exert a minimal effect on the anti-HIV-1 activity of BMS-378806.
Oral bioavailability of BMS-378806 was assessed in rats, dogs, and cynomolgus monkeys, and determined to be 19%, 77%, and 24%, respectively. Species differences in oral absorption likely contributed to different bioavailabilities across species. The steady-state volume of distribution ranged from 0.4 to 0.6 liters/kg, suggesting that the compound is distributed outside plasma.
BMS-378806 did not cross the blood–brain barrier to any appreciable extent. The total body clearance relative to the hepatic blood f low was intermediate in rats and low in dogs and monkeys. The terminal half-life after i.v. administration was 0.3, 1.2, and 0.9 h in rats, dogs, and monkeys, respectively. However, the apparent terminal half-life after oral administration was significantly longer in the rat and monkey (2.1 and 6.5 h, respectively) than that after i.v. dosing, suggesting prolonged oral absorption. In addition, in vitro metabolism studies demonstrated that the compound was metabolized by multiple cytochrome P450 enzymes and had low potential for inhibiting these major human drug-metabolizing enzymes. In a 2-week exploratory drug safety study in rats, BMS-378806 was well tolerated when given orally to rats for 2 weeks at daily doses of up to 100 mgykg. In addition, BMS-378806 was not mutagenic in an Ames reverse-substitution assay (data not shown).
Early efforts to block HIV-1 entry to cells focused on using sCD4 to inhibit virus infection. Although sCD4 demonstrated efficacy against many laboratory strains, it exhibited poor activity against primary isolates, which may have contributed to disappointing results in clinical trials. PRO-542 (a CD4-IgG fusion protein) was shown to be effective in neutralizing many clinical HIV-1 strains in culture and is efficacious in the clinics. However, this recombinant fusion protein requires i.v. administration, which may limit its clinical application. Recently, a 27-aa CD4 mimic, CD4M33, was shown to bind to gp120 and inhibit HIV-1 infection in vitro.
The clinical development potential of this peptide awaits future studies. BMS-378806 is a small molecule inhibitor that blocks the HIV entry process. The compound specifically inhibits gp120 binding to cellular CD4 receptors, functioning independently of viral coreceptors. The pharmacokinetic and pharmaceutic characteristics of BMS-378806 support an oral formulation in man, and initial toxicology studies raised no safety concerns. As such, this class of attachment inhibitors appears suitable for advancement into clinical development.
HIV-1 envelope is a heterogeneous protein with variable se-quences exposed on the outer surface of virions. Therefore, the finding of a specific and effective small molecule inhibitor of gp120 was somewhat unexpected. However, cumulative results from bio-chemical and genetic studies described here strongly suggest that
BMS-378806 indeed targets gp120 and interferes with CD4 binding. Further studies using a f luorescence quenching method showed that BMS-378806 binds directly to gp120 at a 1:1 stoichiometry (unpublished data). These studies establish the feasibility of targeting the HIV-1 gp120 envelope with a small molecule compound and support the envelope protein as a viable target for a new drug class. Of particular importance is that drugs targeting the envelope are effective against strains resistant to existing classes of drugs.
Because of the diversity of envelope glycoproteins, it is notsurprising that considerable variability was observed in the susceptibility of virus subtypes to BMS-378806. The most susceptible HIV-1 strains observed belong to subtype B (with a median EC50 of 0.04 mM). This finding is probably due to our utilization of a subtype B strain (HIV-1JRFL) in the initial screening process.
BMS-378806 does inhibit other subtypes, but at a lower potency. Enfuvirtide and coreceptor inhibitors also exhibit a range of susceptibilities when tested against various viruses. New structural information on the precise gp120 binding site of BMS-378806 and a knowledge of the structure-activity relationship of this series of compounds will assist in the understanding of the basis of anti-HIV spectrum. The information should also allow the design of additional attachment inhibitors with enhanced potency and an expanded spectrum.
The HIV entry process depends on multiple conformational changes of the envelope protein and, together with the rapid immune evasion by HIV-1, suggests that the viral envelope is a f lexible and adaptable protein. Consequently, resistance develop-ment may be an issue for compounds that target the envelope. The initial in vitro drug selection studies showed that BMS-378806 developed resistance in '20 days under our experimental condi-tions. This rate of resistance development was similar to that of the reverse transcriptase inhibitors, nevirapine and lamivudine, ob-served from parallel experiments (data not shown). Because HIV-1 envelope is a heterogeneous protein, it may in part explain the relatively rapid rate of resistance selection by BMS-378806. Genetic analysis of the drug selected viruses revealed that amino acid substitutions acquired during selection were mapped to regions spanning the entire HIV envelope. No changes were identified in the overall HIV-1 sequences outside the envelope region of the drug selected viruses after sequencing one entire BMS-378806-resistant HIV-1 strain (data not shown). Most significantly, M426L and M475I substitutions, located at or near gp120yCD4 contact sites, conferred high levels of resistance to the in vitro mutagenized HIV-1 variants, suggesting that the CD4 binding pocket of gp120 is the antiviral target. The M434I and other secondary changes (V68A and I595F) also affected drug susceptibility of recombinant viruses, presumably by influencing the gp120 conformation. Moreover, two of the selected substitutions, I595F and K655E, were situated at the gp41 region. It is interesting to note that the same I595 residue has been implicated in the development of sCD4 sensitivity. The reasons why these gp41 substitutions were selected by BMS-378806 and the impact these changes have on envelope conformation await further studies. We also noted that the selected gp120 substitutions varied depending on the viral background. This phenomenon was observed for HIV-1 protease inhibitors as well. Namely, M475I was the major change occurring in HIV-1NL4-3, and M434I was the major change in HIV-1LAI. It is likely that the collective interactions of these selected substitutions with other envelope residues determine the overall drug susceptibility. However, it is important to note that the available crystal structures are post-CD4 binding, and we do not know the actual conformation of gp120 targeted by BMS-378806. Finally, results from resistance studies, in conjunction with the [ 3 H]BMS-378806ygp120 binding data, support the envelope as the target site of the compound. The mapping of the BMS-378806 resistance to the viral envelope also demonstrated that BMS-378806 functions as an inhibitor of the envelope in HIV-1-infected cells.
BMS-378806 is effective against variants resistant to HIV-1 protease and reverse transcriptase inhibitors (unpublished data). In reciprocal experiments, BMS-378806 resistant HIV strains remain susceptible to these two classes of drugs as well. Therefore, BMS-378806 may represent an important therapeutic option, particularly for patients who harbor viruses resistant to existing antiretroviral agents.
In summary, results described here indicate that small molecule inhibitors of HIV-1 envelope could potentially be developed into a new class of antiretroviral drugs. The HIV-1 gp120 envelope represents a recently discovered phase of the viral life cycle that can be effectively targeted with small molecule compounds. Further studies with this class of inhibitors may also provide us with a greater understanding of the HIV-1 envelope structure and the HIV entry process.
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