Analysis of the S3 and S3′ subsite specificities of feline immunodeficiency virus (FIV) protease: Development of a broad-based protease inhibitor efficacious against FIV, SIV, and HIV in vitro and ex vivo

Abstract

The S3 and S3 subsite binding specificities of HIV and feline immunodeficiency virus proteases (FIV) proteases (PRs) have been explored by using C 2 -symmetric competitive inhibitors. The inhibitors evaluated contained (1S, 2R, 3R, 4S)-1,4-diamino-1,4-dibenzyl-2,3-diol as P1 and P1 units, Val as P2 and P2 residues, and a variety of amino acids at the P3 and P3 positions. All inhibitors showed very high potency against HIV PR in vitro, and their K i values ranged between 1.1 and 2.6 nM. In contrast to the low restriction of P3 and P3 residues observed in HIV PR, FIV PR exhibited strong preference for small hydrophobic groups at the S3 and S3 subsites. Within this series, the most effective inhibitor against FIV PR contained Ala at P3 and P3. Its K i of 41 nM was 415-and 170-fold lower than those of the inhibitors without the P3 and P3 moieties or with the Phe at these positions, respectively. In addition, these compounds were tested against mutant FIV PRs, which contain amino acid substitutions corresponding to those in native HIV PR at homologous sites, and their efficacy of inhibition progressively increased up to 5-fold. The most potent FIV PR inhibitor was selected for examination of its effectiveness in tissue culture, and it was able to block nearly 100% of virus production in an acute infection at 1 g͞ml (1.1 M) against HIV, FIV, and simian immunodeficiency virus. Furthermore, it was not toxic to cells, and even after 2 months of culture there was no sign of resistance development by virus. The findings suggest that inhibitors with small P3 residue may be efficacious against a broad range of HIV variants as well as interspecies PRs.

Generation of Infectious Feline Immunodeficiency Virus (FIV) Encoding FIV/Human Immunodeficiency Virus Chimeric Protease

“…WT FIV showed sensitivity to TL-3 down to 200 nM and was completely inhibited at concentrations of 400 nM and higher (Fig. 7C), consistent with previous ex vivo results (31). The infectivity of mutant FIV carrying 6s-98H or 6s-98S PRs was tested in the absence and presence of DRV (50 nM, 100 nM, and 200 nM), LPV (50 nM, 100 nM, and 200 nM), and TL-3 (50 nM, 100 nM, and 150 nM).…”

“…The full-length chimeric PR genes, including 6s (I37V N55M V59I I98P Q99V P100N), 6s-98H, and 6s-98S, were PCR amplified and cloned into the pET21a(ϩ) expression vector (EMD Chemicals, Inc.) for protein expression, using unique NdeI and HindIII restriction sites. PR expression was induced with 1 mM isopropyl ␤-D-1-thiogalactoptranoside for 4 h at 37°C in the transformed Rosetta 2 strain of Escherichia coli (EMD Chemicals, Inc.), and PR was subsequently purified using ion-exchange chromatography and fast protein liquid chromatography (FPLC) from inclusion bodies as described previously (20,31).…”

“…The pCFIV expression plasmids were transfected into 293T cells for expression of FIV Gag and Gag-Pol polyprotein as described previously (38). HIV-1 PR inhibitors, including fosamprenavir (FPV), ATV, RTV, LPV, TPV, DRV, and TL-3 (31,32), and other compounds, including APV-1 (35) and AB-2 (8, 9), were used to evaluate the inhibitor sensitivity of PRs in the context of Gag-Pol polyprotein expression in 293T cells. The following reagents were obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: APV, ATV, LPV, TPV, and DRV (catalog no.…”

“…Comparisons of the 3 dimensional structures of the two PRs led to the rational design of TL-3, a broad-based PR inhibitor capable of blocking infection by FIV, simian immunodeficiency virus (SIV), and HIV-1, as well as many drug-resistant HIV-1 variants (10,12,21,23,31,32). Similar approaches comparing the structures of FIV and drug-resistant HIV-1 PRs to that of WT HIV-1 PR have led to the development of additional PR-inhibiting compounds with broadened efficacy (8,9,19,29,41,42).…”

Analysis of HIV Wild-Type and Mutant Structures via in Silico Docking against Diverse Ligand Libraries

“…A similar method has previously proven successful in developing an inhibitor effective against FIV, SIV, and HIV. 3 Due to the rapid evolution of drug resistance, such approaches are vital in the design of new inhibitors. Crossresistance involving current Food and Drug Adminsitration (FDA)-approved drugs is also a continuing problem.…”