Identification and Optimization of Thienopyridine Carboxamides as Inhibitors of HIV Regulatory Complexes

Abstract

Viral regulatory complexes perform critical functions during virus replication and are important targets for therapeutic intervention. In HIV, the Tat and Rev proteins form complexes with multiple viral and cellular factors to direct transcription and export of the viral RNA. These complexes are composed of many proteins and are dynamic, making them difficult to fully recapitulate in vitro. Therefore, we developed a cell-based reporter assay to monitor the assembly of viral complexes for inhibitor screening. We screened a small-molecule library and identified multiple hits that inhibit the activity of the viral complexes. A subsequent chemistry effort was focused on a thieno[2,3-b]pyridine scaffold, examples of which inhibited HIV replication and the emergence from viral latency. Notable aspects of the effort to determine the structure-activity relationship (SAR) include migration to the regioisomeric thieno[2,3-c]pyridine ring system and the identification of analogs with single-digit nanomolar activity in both reporter and HIV infectivity assays, an improvement of Ͼ100-fold in potency over the original hits. These results validate the screening strategy employed and reveal a promising lead series for the development of a new class of HIV therapeutics.KEYWORDS antivirals, HIV, RNA, Rev R NA-protein complexes are essential to the assembly and activity of many viral regulatory systems and represent an important target class for antiviral drug discovery. In HIV, the Rev-Rev response element (RRE) protein-RNA complex is one such target due to its essential activity in mediating the export of unspliced and partially spliced RNAs from the nucleus to the cytoplasm (1, 2). Disrupting the Rev-RRE interaction prevents the expression of late viral proteins and the packaging of viral RNA, thus inhibiting virus replication.Rev is a 116-amino-acid RNA-binding protein that is expressed from fully spliced mRNAs early in the virus life cycle (3). Rev binds the RRE, a highly structured ϳ350-nucleotide (nt) RNA element encoded within the env gene (reviewed in references 2 and 4). Current models suggest that about six Rev molecules bind the RNA in order to properly position two of the nuclear export sequences on Rev for binding to a dimer of the Crm1-RanGTP export complex (5, 6). This complex is then exported through the nuclear pore, after which it disassembles in the cytoplasm to allow translation of the late HIV proteins and packaging of the viral genome (2, 7).The formation of the export complex is driven by several critical intermolecular interactions. Rev binds the RNA primarily through its arginine-rich motif (ARM), an ␣-helical domain that forms several important hydrogen bonds with the RNA (8, 9). The nuclear magnetic resonance (NMR) structures of the Rev peptide complexed to RRE IIB or to an RNA aptamer and a recent cocrystal structure of a Rev-RRE dimer (10) show that

Exploring the HIV-1 Rev Recognition Element (RRE)–Rev Inhibitory Capacity and Antiretroviral Action of Benfluron Analogs

“…The current antiretroviral therapy is not curative, making the discovery of new anti-HIV agents with alternative mechanisms of action a necessity [1]. Among the possible targets for direct action not used by presently marketed antiretrovirals, the complex formed between the virus-encoded protein Rev and the RRE of the viral RNA genome remains unexploited despite different efforts carried out to develop inhibitors (e.g., [2][3][4][5][6][7][8][9][10][11]). This ribonucleoprotein complex comprises several Rev protein monomers bound to the RRE RNA structure (Figure 1) and enables the nuclear export of unspliced or singly spliced HIV-1 transcripts, an essential step in the virus replication cycle [12].…”

Nucleic acid recognition and antiviral activity of 1,4-substituted terphenyl compounds mimicking all faces of the HIV-1 Rev protein positively-charged α-helix

“…Nevertheless, despite its importance in the viral replication cycle, the RRE-Rev ribonucleoprotein complex remains an unexploited target for HIV-1 chemotherapy. While a number of small-molecule compounds with substantial anti-HIV activity have been reported to block Rev function, most of them do not directly inhibit the formation of the RRE-Rev complex, or were found to bind to Rev partners in the host cell such as Crm1 or the cap-binding complex [15][16][17][18][19] .…”