?Fig.2B2B also show that JPL-32 inhibited HIV-2 production to the same extent that this known transactivation inhibitor K-37 did. in acutely HIV-1-infected cells but also in chronically HIV-infected cells. A dose-dependent inhibition of computer virus particle release and viral protein expression was observed gamma-secretase modulator 1 upon exposure to the pyridine oxide derivatives. Finally, inhibition of HIV-1 long terminal repeat-mediated green fluorescence protein expression in quantitative transactivation bioassays indicated that the additional target of action of the pyridine oxide derivatives may be located at the level of HIV gene expression. During the last decade, numerous compounds have been reported to inhibit the replication of human immunodeficiency computer virus (HIV) in cell culture. Among these, a variety of HIV type 1 (HIV-1) reverse transcriptase (RT) and protease inhibitors and one fusion inhibitor have been formally licensed for clinical use in the treatment of HIV-1 infections (8, 9). Treatment of HIV-infected individuals at present is based on combination therapy with HIV RT and/or protease inhibitors. Since these compounds interact with virus-specific enzymes, the emergence of drug-resistant viruses may not be avoidable during long-term drug treatment. Indeed, multidrug-resistant computer virus strains have progressively been reported in patients receiving highly active antiretroviral therapy (19). Therefore, the discovery of new antiviral targets or the development of novel antiviral strategies for anti-HIV treatment is necessary. Several targets in the HIV-1 replication cycle other than RT, protease, and computer virus entry have been identified as possible intervention sites for antiviral chemotherapy. Among these, viral gene regulation (processes) seems to be very attractive, as it would open the possibility to control HIV-1 replication not only in acutely infected cells but also in chronically infected cells. In this way, inhibitors of HIV gene regulation may have great potential in anti-HIV drug combination therapy because they can force the computer virus to slow down its replication rate or even shut off replication and establish a dormant state. Furthermore, it may be argued that the use of antivirals targeted at HIV gene expression would result in the development of a lower incidence of drug resistance, as HIV gene regulation requires the interplay of both viral and cellular components (2, 6). Recently, we have reported gamma-secretase modulator 1 the identification and structure-antiviral activity relationship of a new class of anti-HIV compounds that may have multiple mechanisms of antiviral action (4). Furthermore, it has recently been shown that this HIV-1-specific inhibitors gamma-secretase modulator 1 within the class of pyridine oxide derivatives were endowed with properties characteristic of those of nonnucleoside RT inhibitors (NNRTIs) (18). Here, we provide convincing evidence that, in addition to HIV-1 RT inhibition, the pyridine oxide derivatives also interfere with viral gene expression RPTOR after the proviral DNA has been incorporated into the genomes of the target cells. MATERIALS AND METHODS Compounds. Pyridine oxide derivatives (Fig. ?(Fig.1)1) were synthesized at and supplied by Crompton Corporation (Middlebury, Conn., and Guelph, Ontario, Canada). The thiocarboxanilide NNRTI UC-781 was obtained from W. Brouwer (Crompton Corporation, Guelph, Ontario, Canada). Tenofovir [(expression and, thus, the appearance of blue-stained syncytia after contamination with HIV-1(IIIB). The pyridine oxide derivatives JPL-32, JPL-58, and JPL-88 inhibited both HIV-1(IIIB)- and HIV-2(ROD)-directed expression, with IC50s of 0.5, 4, and 6 g/ml, gamma-secretase modulator 1 respectively, for HIV-1 and 2, 12, and 20 g/ml, respectively, for HIV-2. The fluoroquinoline K-37, known as an HIV transactivation inhibitor (13), prevented the appearance of blue-stained syncytium formation, with an IC50 as low as 0.02 g/ml. Next, a time-of-addition experiment with HeLa-CD4-LTR–galactosidase cells was performed to determine the possible step(s) in the HIV replication cycle that is inhibited by the pyridine oxide compounds (Fig. 2A and B). Dextran sulfate was found to interact at a very early step.