4-(Arylthio)-pyridin-2(1H)-ones variously substituted in their 3-, 5-, and 6-positions have been synthesized as a new series of 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT)-pyridinone hybrid molecules. Biological studies revealed that some of them show potent HIV-1 specific reverse transcriptase inhibitory properties. Compounds 16 and 7c, the most active ones, inhibit the replication of HIV-1 at 3 and 6 nM, respectively.

Removal of 3-azido-3deoxythymidine (AZT) 3-azido-3-deoxythymidine 5-monophosphate (AZTMP) from the terminated primer mediated by the human HIV-1 reverse transcriptase (RT) has been proposed as a relevant mechanism for the resistance of HIV to AZT. Here we compared wild type and AZT-resistant (D67N/K70R/ T215Y/K219Q) RTs for their ability to unblock the AZTMP-terminated primer by phosphorolysis in the presence of physiological concentrations of pyrophosphate or ATP. The AZT-resistant enzyme, as it has been previously described, showed an increased ability to unblock the AZTMP-terminated primer by an ATP-dependent mechanism. We found that only mutations in the p66 subunit were responsible for this ability. We also found that three structurally divergent non-nucleoside reverse transcriptase inhibitor (NNRTI), nevirapine, TIBO, and a 4-arylmethylpyridinone derivative, were able to inhibit the phosphorolytic activity of the enzyme, rendering the AZT-resistant RT sensitive to AZTTP. The 4-arylmethylpyridinone derivative proved to be about 1000-fold more potent in inhibiting phosphorolysis than nevirapine or TIBO. Moreover, combinations of AZTTP with NNRTIs exhibited an exceptionally high degree of synergy in the inhibition of AZT-resistant enzyme only when ATP or PP i were present, indicating that inhibition of phosphorolysis was responsible for the synergy found in the combination. Our results not only demonstrate the importance of phosphorolysis concerning HIV-1 RT resistance to AZT but also point to the implication of this activity in the strong synergy found in some combinations of NNRTIs with AZT.Human immunodeficiency virus, type 1 (HIV-1) 1 reverse transcriptase (RT) is responsible for the conversion of singlestranded viral RNA into double-stranded DNA prior to integration into the genome of the human host. Numerous compounds that inhibit the DNA polymerase activity of RT have been described. They can be divided into two broad classes. The first group, that of nucleoside analogs, includes dideoxynucleoside compounds, such as 2Ј,3Ј-dideoxycytidine and AZT, that inhibit viral replication by acting as chain terminators of DNA synthesis (1). The second group, the non-nucleoside reverse transcriptase inhibitors (NNRTI), includes a large number of structurally dissimilar hydrophobic compounds that bind to a site on the RT palm subdomain adjacent to but distinct from the polymerase active site (2).The FDA-approved HIV-1 therapies involve drugs that inhibit two viral enzymes, reverse transcriptase and protease. AZT was the first drug approved against HIV-1 and is still widely used in combination with other antiretroviral drugs. The prolonged clinical use of this nucleoside analog in monotherapy gives rise to highly resistant viruses containing mutations in the RT enzyme, D67N, K70R, T215F/Y, K219E/Q (3), and, in some cases, M41L and L210W. Viruses carrying at least four mutations are more than 100-fold less sensitive to AZT than wild type virus in cell culture. Although the genotype for AZT resistance is well character...

To test the concept that HIV reverse transcriptase could be effectively inhibited by "mixed site inhibitors", a series of seven conjugates containing both a nucleoside analogue component (AZT 1, ddC 2) and a nonnucleoside type inhibitor (HEPT analogue 12, pyridinone 27) were synthesized and evaluated for their ability to block HIV replication. The (N-3 and C-5)AZT-HEPT conjugates 15, 22, and 23 displayed 2-5 microM anti-HIV activity, but they had no effect on the replication of HIV-2 or the HIV-1 strain with the Y181C mutation. The (C-5)AZT-pyridinone conjugates 34-37 were found to be inactive. In marked contrast, the ddC-HEPT molecule 26 displayed the same potency (EC(50) = 0.45 microM) against HIV-1 (wild type and the Y181C nevirapine-resistant strain) and HIV-2 in cell culture. No synergistic effect was observed for these bis-substrate inhibitors, suggesting that the two individual inhibitor components in these molecules do not bind simultaneously in their respective sites. Interestingly, however, the results indicate that the AZT-HEPT conjugates and the ddC-HEPT derivative 26 inhibit reverse transcriptase (RT) in an opposite manner. One explanation for this difference is that the former compounds interact preferentially with the hydrophobic pocket in RT, whereas 26 (after supposed triphosphorylation) inhibits RT through binding in the catalytic site.