A series of dipyridodiazepinones have been shown to be potent inhibitors of human immunodeficiency virus-1 (HIV-1) reverse transcriptase (RT). One compound, BI-RG-587, had a Ki of 200 nanomolar for inhibition of HIV-1 RT that was noncompetitive with respect to deoxyguanosine triphosphate. BI-RG-587 was specific for HIV-1 RT, having no effect on feline and simian RT or any mammalian DNA polymerases. BI-RG-587 inhibited HIV-1 replication in vitro as demonstrated by in situ hybridization, inhibition of protein p24 production, and the lack of syncytia formation in cultured human T cell lines and freshly isolated human peripheral blood lymphocytes. Cytotoxicity studies of BI-RG-587 on human cells showed a high therapeutic index (greater than 8000) in culture.

We have recently described a nonnuceoside compound that specifcally inhibits the reverse transcriptase of human immunodeficiency virus type 1 (HIV-1), the causative agent of AIDS. This compound, nevirapine (BI-RG-587), interacts with highly conserved tyrosine residues at positions 181 and 188 in the reverse t iptase to inhibit the recombinant enzyme and virus replication in cell culture with 50% inhibitory concentrations in the 40 nM range. HIV-1 variants resistant to nevirapine emerged with passage in cell culture in the presence of drug. This resistant phenotype was stable with continued passage in the absence of drug. These mutants had a substitution of cysteine for the tyrosine at position 181.

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), an important therapeutic target in the treatment of AIDS, is effectively inhibited by a class of nonnucleoside analog compounds that includes nevirapine (BI-RG-587) and tetrahydroimidazo[4,5,1-jk]-[1,4]benzodiazepin-2(1H)-one and -thione. We show that both tyrosine residues at positions 181 and 188 flanking the putative catalytic site of HIV-1 RT are required for sensitivity of the enzyme to these compounds. HIV-2 RT, which does not have tyrosines at these positions, is resistant to these nonnucleoside analog inhibitors. Substitution of the HIV-2 RT amino acid residues at position 181 or 188 into HIV-1 RT results in an enzyme that is resistant to these compounds while retaining sensitivity to 3'-azido-2',3'-dideoxythymidine triphosphate. HIV-2 RT substituted with amino acids 176-190 from HIV-1 RT acquires sensitivity to these nonnucleoside analog inhibitors.

We have determined the nucleotide sequence of the 2.2-kilobase-pair region upstream of the chicken c-myc coding exons. Using RNA blot analysis, we have localized a noncoding exon to a region that is separated from the c-myc coding sequences by an intron of 700-800 base pairs. In most avian leukosis virus-induced lymphomas proviral integration has occurred within, or downstream of, the first exon, thus presumably displacing the regulatory sequences that normally control c-myc expression. More than 70% of the integration sites were clustered in a 250-base-pair region in the first intron, immediately preceding the coding sequences. Sequences from the upstream noncoding exon were absent from the myc transcripts in these lymphomas; RNA transcripts from the normal c-myc allele were not expressed at detectable levels.

The antipsychotic drug trifluoperazine has been long considered a calmodulin inhibitor from in vitro studies but may function in vivo as a more general inhibitor by disturbing ion fluxes and altering the membrane potential. Resistance to trifluoperazine can arise in Saccharomyces cerevisiae cells by alterations in at least three distinct genetic loci. One locus, defined by a spontaneous dominant trifluoperazine resistance mutation (TFP1 408), was isolated and sequenced. The mechanism of action of the phenothiazine tranquilizers has been the subject of numerous studies (42). Studies reported in the pharmacological literature suggest that the phenothiazines inhibit one of the two types of dopamine receptors (24, 48), a hypothesis consistent with the antipsychotic and Parkinson's disease-like effects. Reports in the biochemical literature suggest that the phenothiazines may function as inhibitors of calcium-binding proteins, especially calmodulin (50) and protein kinase C (25), and may affect calcium-or calmodulin-regulated processes in cells (47). This alternative model is consistent with the effect of these drugs in vivo, since neurons and muscle cells are two types of cells in the body that are dependent on calcium gradients or currents for function.The medical effectiveness of the phenothiazines is directly related to the hydrophobicity of the molecule and varies with the side-chain modifications to the planar ring (50). Trifluoperazine (TFP) is the most hydrophobic of the medically effective phenothiazines. The hydrophobic character of the phenothiazine tranquilizers confers two properties to the drugs: lipid solubility and membrane association. Therefore these drugs can cross the blood-brain barrier readily, can get into any cell type, and can associate with or intercalate into any membranous structure.Since TFP is a medically important drug that affects cells that are dependent upon calcium gradients for function in vivo and inhibits calcium-binding proteins with hydrophobic domains in vitro, we isolated spontaneous TFP-resistant mutants of the simple eucaryote Saccharomyces cerevisiae in an attempt to find mutations in genes coding for proteins involved in calcium transport or regulated pathways. Calcium-sensitive or -dependent mutants of S. cerevisiae have been isolated before (30)(31)(32), and calcium-related biochemical activities have been documented in S. cerevisiae (8, 10-* Corresponding author.12, 29, 38). TFP-resistant pseudorevertants of a calciumdependent mutation have been isolated and are recessive to the wild type for TFP resistance and show TFP-dependent growth at 37°C (30). In this report we describe the isolation and characterization of a spontaneous mutation to dominant TFP resistance and the sequence of the gene encoding it.MATERIALS AND METHODS Strains and plasmids. S. cerevisiae strains were of the S288c background. The wild-type haploids used in this work were NF134 (Mata his4-539 lys2-801 ura3-52) and NF147 (MATTa ade2-101 ura3-52) (43). NF408 was a spontaneous mutation of NF147 to dom...