Dihydroxythiophenes are novel potent inhibitors of human immunodeficiency virus integrase with a diketo acid-like pharmacophore

Abstract

We have identified dihydroxythiophenes (DHT) as a novel series of human immunodeficiency virus type 1 (HIV-1) integrase inhibitors with broad antiviral activities against different HIV isolates in vitro. DHT were discovered in a biochemical integrase high-throughput screen searching for inhibitors of the strand transfer reaction of HIV-1 integrase. DHT are selective inhibitors of integrase that do not interfere with virus entry, as shown by the inhibition of a vesicular stomatitis virus G-pseudotyped retroviral system. Moreover, in quantitative real-time PCR experiments, no effect on the synthesis of viral cDNA could be detected but rather an increase in the accumulation of 2-long-terminal-repeat cycles was detected. This suggests that the integration of viral cDNA is blocked. Molecular modeling and the structure activity relationship of DHT demonstrate that our compound fits into a two-metal-binding motif that has been suggested as the essential pharmacophore for diketo acid (DKA)-like strand transfer inhibitors (Grobler et al., Proc. Natl. Acad. Sci. USA 99:6661-6666, 2002.). This notion is supported by the profiling of DHT on retroviral vectors carrying published resistance mutations for DKA-like inhibitors where DHT showed partial cross-resistance. This suggests that DHT bind to a common site in the catalytic center of integrase, albeit with an altered binding mode. Taken together, our findings indicate that DHT are novel selective strand transfer inhibitors of integrase with a pharmacophore homologous to DKA-like inhibitors.

FIG. 1.

Time-of-addition experiment. MT-4 cells were infected with HIV-1 (D117/II) at an MOI of >1, and test compounds were added at different time points after infection (0, 1, 3, 8, or 27 h). The production of viral p24 Ag was assessed 36 h after infection. Concentrations of compounds were as follows: T20, 5 μM; DKA, 5 μM; EFV, 0.25 μM; SQV, 0.5 μM; and DHT-2, 15 μM. OD₄₅₀, optical density at 450 nm; OD₆₂₀, optical density at 620 nm.

FIG. 2.

Effect of DHT-2 on production of HIV-1 by persistently infected U1 cells. U1 cells were treated with PMA to induce HIV-1 production in the absence or presence of AZT, SQV, or DHT-2 and incubated for 48 h. The amount of p24 in the supernatants is expressed as the percentage of untreated control. Note that the upper concentration range (a) is given for AZT and SQV, while the lower range (b) is given for DHT-2.

FIG. 3.

Effect of DHT on HIV DNA during HIV vector transduction. 293T cells were transduced with HIV-1 vectors at a high MOI in the absence or presence of inhibitors (AZT, DHT-2, S-1360, L-870.810, and dimethyl sulfoxide [DMSO]). Cells were harvested, and DNA was extracted at different time points after infection. Samples were analyzed by quantitative PCR for late reverse transcripts (A), 2-LTR circles (B), and integrated proviral DNA (C).

FIG. 4.

Three-dimensional-structure of DHT and alignment to reference INSTI. (A) Three-dimensional model of DHT-1. Values for distances d1 and d2 and the angle theta are given. (B) Overlay of DHT-1 (in gray), S-1360 (in orange), and L-870,810 (in green). Values for distances d1 and d2 and angle theta shown in panel A are listed in the table. Circles mark residues putatively involved in chelating active site metal ions.