Preclinical evaluation of 1H-benzylindole derivatives as novel human immunodeficiency virus integrase strand transfer inhibitors

Abstract

We have identified 1H-benzylindole analogues as a novel series of human immunodeficiency virus (HIV) integrase inhibitors with antiretroviral activities against different strains of HIV type 1 (HIV-1), HIV-2, and simian immunodeficiency virus strain MAC(251) [SIV(MAC(251))]. Molecular modeling and structure-activity relationship-based optimization resulted in the identification of CHI/1043 as the most potent congener. CHI/1043 inhibited the replication of HIV-1(III(B)) in MT-4 cells at a 50% effective concentration (EC(50)) of 0.60 microM, 70-fold below its cytotoxic concentration. Equal activities against HIV-1(NL4.3), HIV-2(ROD), HIV-2(EHO), and SIV(MAC(251)) were observed. CHI/1043 was equally active against virus strains resistant against inhibitors of reverse transcriptase or protease. Replication of both X4 and R5 strains in peripheral blood mononuclear cells was sensitive to the inhibitory effect of CHI/1043 (EC(50), 0.30 to 0.38 microM). CHI/1043 inhibited integrase strand transfer activity in oligonucleotide-based enzymatic assays at low micromolar concentrations. Time-of-addition experiments confirmed CHI/1043 to interfere with the viral replication cycle at the time of retroviral integration. Quantitative Alu PCR corroborated that the anti-HIV activity is based upon the inhibition of proviral DNA integration. An HIV-1 strain selected for 70 passages in the presence of CHI/1043 was evaluated genotypically and phenotypically. The mutations T66I and Q146K were present in integrase. Cross-resistance to other integrase strand transfer inhibitors, such as L-708,906, the naphthyridine analogue L-870,810, and the clinical drugs GS/9137 and MK-0518, was observed. In adsorption, distribution, metabolism, excretion, and toxicity studies, antiviral activity was strongly reduced by protein binding, and metabolization in human liver microsomes was observed. Transport studies with Caco cells suggest a low oral bioavailability.

FIG. 1.

Structures of INSTIs.

FIG. 2.

Time-of-addition assay. MT-4 cells were infected with HIV-1(III_B) at an MOI of 0.5, and the test compounds were added at different times postinfection. Viral p24 antigen production was determined at 31 h postinfection. Compounds used were DS (20 μM), AZT (1.9 μM), ritonavir (2.8 μM), MK-0518 (0.05 μM), and CHI/1043 (27 μM). The results are from a representative experiment that was repeated at least once.

FIG. 3.

Kinetics of VSV G protein-pseudotyped HIV-1 vector transduction in the presence of CHI/1043. 293T cells (∼1.5 × 10⁵ cells) were transduced with VSV G protein-pseudotyped HIV-1 vector at an MOI of 10 in the absence (⋄) or presence (▴) of 12.2 μM CHI/1043. DNA extracts were made from samples taken at different time points, and formations of total HIV-1 DNA (A), integrated proviral DNA (B) and 2-LTR circles (C) were quantified using Q-PCR. Kinetics of HIV-1 infection in the presence of CHI/1043. MT-4 cells (1.5 × 10⁶ cells) were infected with HIV-1(NL4.3) (150 ng p24) in the absence or presence of 12.2 μM CHI/1043. DNA extracts were made from samples taken at different time points, and formations of total HIV-1 DNA (D), integrated proviral DNA (E), and 2-LTR circles (F) were quantified using Q-PCR. All data represent mean values ± standard deviations from at least two separate experiments.