Heterocyclic Compounds That Inhibit Rev-RRE Function and Human Immunodeficiency Virus Type 1 Replication

Abstract

A cell-based screening assay was performed to identify compounds that inhibited the postintegration stage of the human immunodeficiency virus (HIV) life cycle. This assay utilized a cell line that contains the HIV gag and pol genes expressed in a Rev-dependent fashion. The cell line produces about 10 to 15 ng of p24 per milliliter of medium over a 24-h period in the form of viruslike particles. Any compound that inhibits a postintegration step in the HIV life cycle scores in this assay by decreasing particle production. Forty thousand compounds were screened, and 192 compounds were selected from the original screen because they showed more than 50% inhibition at a 10 M concentration. The cumulative evidence presented in this study strongly suggests that 2 of the 192 compounds work as inhibitors of HIV Rev function. This was determined by a variety of cell-based assays, although the compounds do not interfere with Rev-RRE (Rev response element) binding in vitro. Both compounds inhibit replication of the lab isolate NL4-3 as well as an HIV primary isolate from Brazil (93BR021) and thus are promising leads as therapeutic candidates that target HIV replication through inhibition of Rev function.Most of the current drugs in use for the treatment of AIDS work by targeting the enzymatic activities of the human immunodeficiency virus (HIV) reverse transcriptase or protease, although entry (7) and integrase (13) inhibitors are starting to be used, and presently there is also promising development of other novel targets (51, 59). However, because of the emergence of drug-resistant virus that commonly occurs as the result of treatment, there remains a great need to continue the search for alternative therapies that target other essential viral activities.The Rev protein is absolutely essential for HIV replication (for a review see reference 49). Proviral clones lacking a functional rev gene have no replicative ability, even in established tissue culture cell lines or peripheral blood mononuclear cells (PBMCs). In the absence of Rev, genomic RNA and several other HIV mRNAs cannot exit the nucleus (22,30,42). Thus, viral structural proteins are not made and the infectious cycle cannot continue. It is thus clear that modalities inhibiting the function of Rev could form the basis for therapy against HIV infection and AIDS.Although the Rev/RRE (Rev response element) export pathway is still not fully understood, several important steps have been identified (see reference 49). The pathway starts with the import of Rev into the nucleus (34, 58). Rev then binds specifically to RNA containing the RRE (17,28,30,42,53) and multimerizes on the RRE in a process believed to involve protein-protein as well as protein-RNA interactions (12,14,16,32,36,43,67). The Rev-RRE complex is then recognized by Crm1 (exportin 1; official gene symbol, XPO1) and RAN-GTP (1), which initiates the export process and eventually targets the complex to the nuclear pore, where it interacts with nucleoporins (1,4,25,70). This results in translocation of the com...

Single-Nucleotide Changes in the HIV Rev-Response Element Mediate Resistance to Compounds That Inhibit Rev Function

“…Previous experiments suggested that the two compounds used in this study do not directly affect Rev-RRE binding since they do not inhibit binding in vitro (46). Nevertheless, cells treated with the compounds clearly constitute an environment that compromises Rev function.…”

“…In a previous study, we described two compounds, 3-amino-5-ethyl-4,6-dimethylthieno [2,3-b]pyridine-2-carboxamide (compound 103833) and 4-amino-6-methoxy-2-(trifluoromethyl)-3-quinolinecarbonitrile (compound 104366), which were identified from a screen of 40,000, as inhibitors of Rev-RRE function and viral replication (46). In that study, using a series of reporter assays, we showed that Rev-dependent gene expression was preferentially inhibited by the compounds in cell culture, relative to control genes that did not require Rev.…”

“…pCMV Gag-Pol-RRE (where CMV is the simian promoter cytomegalovirus; the construct expresses HIV Gag-Pol proteins in a Rev-dependent manner) was used in transient transfections of 293T cells as previously described (25,35,46). SupT1 or Rev-CEM cells were infected as previously described (27), with the specific modifications given in each figure legend.…”

Synthesis of some new Thieno[2,3-b]pyridines, Pyrimidino[4',5':4,5]thieno[2,3-b]pyridine and Pyridines Incorporating 5-Bromobenzofuran-2-yl Moiety

“…Beige crystals, Yield: 84%, melting point: 160-162 °C (acetic acid). IR (KBr, cm (12). Yellow crystals, Yield: 90%, melting point: 220-222 °C (dioxane).…”

“…The thieno [2,3-b]pyridine derivatives occupy special place and have attracted considerable attention because of their broad pharmacological activities, including anticancer [1][2][3][4][5][6][7][8][9], antiviral [10][11][12][13], anti-inflammatory [14][15][16][17], antimicrobial [18,19], antidiabetic [20][21][22][23], antihypertensive [24][25][26] and osteogenic [27,28] activities, in addition to treatment of CNS disorders [29][30][31]. Also, pyridine derivatives of different heterocyclic nucleus have shown potent pharmacological properties like antifungal [32,33], antitubercular [34], antibacterial [35], antimicrobial [36], insecticida [37].…”