Novel Postentry Inhibitor of Human Immunodeficiency Virus Type 1 Replication Screened by Yeast Membrane-Associated Two-Hybrid System

Abstract

Human immunodeficiency virus (HIV)Gag protein targets to the plasma membrane and assembles into viral particles. In the next round of infection, the mature Gag capsids disassemble during viral entry. Thus, Gag plays a central role in the HIV life cycle. Using a yeast membrane-associated two-hybrid assay based on the SOS-RAS signaling system, we developed a system to measure the Gag-Gag interaction and isolated 6 candidates for Gag assembly inhibitors from a chemical library composed of 20,000 small molecules. When tested in the human MT-4 cell line and primary peripheral blood mononuclear cells, one of the candidates, 2-(benzothiazol-2-ylmethylthio)-4-methylpyrimidine (BMMP), displayed an inhibitory effect on HIV replication, although a considerably high dose was required. Unexpectedly, neither particle production nor maturation was inhibited by BMMP. Confocal microscopy confirmed that BMMP did not block Gag plasma membrane targeting. Single-round infection assays with envelope-pseudotyped and luciferase-expressing viruses revealed that BMMP inhibited HIV replication postentry but not simian immunodeficiency virus (SIV) or murine leukemia virus infection. Studies with HIV/SIV Gag chimeras indicated that the Gag capsid (CA) domain was responsible for the BMMP-mediated HIV postentry block. In vitro studies indicated that BMMP accelerated disassembly of HIV cores and, conversely, inhibited assembly of purified CA protein in a dosedependent manner. Collectively, our data suggest that BMMP primarily targets the HIV CA domain and disrupts viral infection postentry, possibly through inducing premature disassembly of HIV cores. We suggest that BMMP is a potential lead compound to develop antiretroviral drugs bearing novel mechanisms of action.Over 2 decades, research has developed antiretroviral therapy (ART) with a combination of antiretroviral drugs for human immunodeficiency virus type 1 (HIV-1) infection (10). ART has dramatically improved the survival of HIV-1-infected individuals. Current ART involves a combination of inhibitors of HIV-specific enzymes, such as protease (PR), reverse transcriptase (RT), and integrase (IN). In some cases, inhibitors of HIV-1 entry are also used. However, the emergence of HIV-1 variants resistant to antiretroviral drugs during ART stresses the need for novel HIV-1 inhibitors against distinct targets.Multiple screening approaches have been employed for HIV-1 drug discovery (37) and have successfully discovered HIV-1 inhibitors that are currently available: nucleoside analogue RT inhibitors were discovered by HIV replication assays (23) and PR inhibitors were produced by structure-based drug design (25). In general, cell-free assays allow discovery of compounds with a relatively low 50% effective dose (ED 50 ) in vitro. However, many such compounds often fail to inhibit HIV-1 replication in in vivo assays, because they may not penetrate the cell membrane or may easily be catalyzed in metabolic environments. Also, possible toxic effects of the compounds must be tested in a subsequent c...

A Triazinone Derivative Inhibits HIV‐1 Replication by Interfering with Reverse Transcriptase Activity

“…The early steps of reverse transcription were attenuated by compound 1 , as well as by EFV, an NNRTI (Figure C). Compound 1 had no inhibitory activity against HIV‐1 RT‐associated RNase H or multimerization of Gag …”

“…Primary isolates of HIV‐1 were obtained from the Henry M. Jackson Foundation . HIV‐1 replication was monitored by measuring viral p24 capsid antigen in culture supernatant using an ELISA kit, as described previously3 . The molecular clone of simian immunodeficiency virus (SIV), the lentiviral vector, and the murine leukemia virus vector were described previously .…”

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Novel Role of HSP40/DNAJ in the Regulation of HIV-1 Replication

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Cyclophilin A-Dependent Restriction to Capsid N121K Mutant Human Immunodeficiency Virus Type 1 in a Broad Range of Cell Lines

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