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S-2720 [6-chloro-3,3-dimethyl-4-(isopropenyloxycarbonyl)-3,4- dihydroquinoxalin-2(1H)-thione], a quinoxaline derivative, was found to be a very potent inhibitor of both human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) activity and HIV-1 replication in tissue culture. Like other nonnucleoside RT inhibitors, S-2720 does not affect the HIV-2 RT. A S-2720-resistant virus was selected and shown to possess a mutation within the RT-coding region that has not previously been described. Notably, this mutation gives rise to a dramatic decrease in enzyme activity. S-2720, therefore, belongs to a new class of RT inhibitors that bind differently to the RT than other known nonnucleoside RT inhibitors. As no toxic effects were observed with S-2720 in mice, these quinoxaline derivatives deserve further evaluation to prove their potency as possible therapeutic agents for HIV-1 infection.

The quinoxaline nonnucleoside RT inhibitor (NNRTI) (S)-4-isopropoxycarbonyl-6-methoxy-3-(methylthiomethyl)-3,4-dihydroquinoxaline-2(1H)-thione (HBY 097) was used to select for drug-resistant HIV-1 variants in vitro. The viruses first developed mutations affecting the NNRTIbinding pocket, and five of six strains displayed the RT G190--E substitution, which is characteristic for HIV-1 resistance against quinoxalines. In one variant, a new mutant (G190->Q) most likely evolved from preexisting G190->E mutants. The negative charge introduced by the G190->E substitution was maintained at that site of the pocket by simultaneous selection for V179--D together with G190--Q. After continued exposure to the drug, mutations at positions so far known to be specific for resistance against nucleoside RT inhibitors (NRTIs) (L74-*V/I and V75->L/I) were consistently detected in all cultures. The inhibitory activities of the cellular conversion product of 2',3'-dideoxyinosine (ddl, didanosine), 2',3'-dideoxyadenosine (ddA) and of 2',3'-didehydro-3'-deoxythymidine (d4T, stavudine) against these late-passage viruses were shown to be enhanced with the L74->V/I RT mutant virus as compared with the wild-type (wt) HIV-1MN isolate. Clonal analysis proved linkage of the codon 74 and codon 75 mutations to the NNRTI-specific mutations in all RT gene fragments. The nonnucleoside-and nucleoside-resistance mutation sites are separated by approximately 35 A. We propose that the two sites "communicate" through the template-primer which is situated in the DNAbinding cleft between these two sites. Quinoxalines cause high selective pressure on HIV-1 replication in vitro; however, the implication of these findings for the treatment of infection has yet to be determined. Viral resistance against both nucleoside reverse transcriptase (RT) inhibitors (NRTIs) and nonnucleoside RT inhibitors (NNRTIs) of human immunodeficiency virus type 1 (HIV-1) replication develops in vitro (cell culture) and in vivo (patients). NRTIs select for RT amino acid substitutions at positions that can be allocated to different structural elements of the protein in the range of residue numbers 41-219 (1-5). A molecular mechanism for some alterations can be assumed on the basis of the RT crystal structure (6-8). In contrast, NNRTI-specific substitutions map exclusively to those protein secondary structure elements that together form a lipophilic pocket within the palm domain of the p66 RT subunit. All NNRTIs are believed to bind to this region, and mutations selected for by these drugs usuallyThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. occur in the segments composed of RT amino acids 98-108, 179-190, and 230-236 (9-16). Especially the Y181-*C RT mutant appears with many chemically distinct compounds, including different NNRTI combinations (12).6-Chloro-3,3-dimethyl-4-(isopropenyloxycarbonyl)-3,4-dihydroqu...

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In vitro resistance of HIV-1 against high levels of HBY 097 ((S)-4-isopropoxycarbonyl-6-methoxy-3-(methylthiomethyl)-3, 4-dihydro-quinoxaline-2(1H)-thione) and other quinoxaline nonnucleoside reverse transcriptase inhibitors (NNRTIs) is characterized by a specific amino acid substitution in the reverse transcriptase (RT), Gly 190Glu. This change results in decreased RT polymerase activity and in reduced growth properties of the corresponding viral variant. Here we show that the appearance of the crippling mutation at codon 190 can be prevented by lowering the selective pressure exerted by HBY 097. Under low selective pressure an accumulation of other NNRTI-specific mutations is observed. Up to five NNRTI-specific substitutions were detected in some of these virus lineages. In addition, we report novel RT amino acid changes which were not observed previously, including Val106lle, Val106Leu, and Gly190Thr. HBY 097 selects for different mutational patterns under high and low selective pressure conditions, respectively. Thus, the type of mutations which appear in HIV-infected patients undergoing therapy may be determined by the levels of the selecting drug.