ABSTRACT

Bisheteroarylpiperazines are potent inhibitors of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). We describe a novel bisheteroarylpiperazine, U-90152 {1-(5-methanesulfonamido-IH-indol-2-yI-carbonyl)-4-[3-(1-methylethyl-amino)pyridinyllpiperazine}, which inhibited recombinant HIV-1 RT at a 50%Y inhibitory concentration (IC.) of 0.26 FLM (compared with IC50s of >440 ,uM for DNA polymerases at and B). U-90152 blocked the replication in peripheral blood lymphocytes of 25 primary HWV-i isolates, including variants that were highly resistant to 3'-azido-2',3'-dideoxythymidine (AZT) or 2',3'-dideoxyinosine, with a mean 50%o effective dose of 0.066 + 0.137 pM. U-90152 had low cellular cytotoxicity, causing less than 8% reduction in peripheral blood lymphocyte viability at 100 ,uM. In experiments assessing inhibition of the spread of HIV-MB in cell cultures, U-90152 was much more effective than AZT. When approximately 500 HIV-1l1m-infected MT-4 cells were mixed 1:1,000 with uninfected cells, 3 ,uM AZT delayed the evidence of rapid viral growth for 7 days. In contrast, 3 ,uM U-90152 totally prevented the spread of HIV-i, and death and/or dilution of the original inoculum of infected cells prevented renewed viral growth after U-90152 was removed at day 24. The combination of U-90152 and AZT, each at 0.5 ,uM, also totally prevented viral spread. Finally, although the RT amino acid substitutions K103N (lysine 103 to asparagine) and Y181C (tyrosine 181 to cysteine), which confer cross-resistance to several nonnucleoside inhibitors, also decrease the potency of U-90152, this drug retains significant activity against these mutant RTs in vitro (IC50s, approximately 8 ,uM).

PNU-140690 is a member of a new class of nonpeptidic human immunodeficiency virus (HIV) protease inhibitors (sulfonamide-containing 5,6-dihydro-4-hydroxy-2-pyrones) discovered by structure-based design. PNU-140690 has excellent potency against a variety of HIV type 1 (HIV-1) laboratory strains and clinical isolates, including those resistant to the reverse transcriptase inhibitors zidovudine or delavirdine. When combined with either zidovudine or delavirdine, PNU-140690 contributes to synergistic antiviral activity. PNU-140690 is also highly active against HIV-1 variants resistant to peptidomimetic protease inhibitors, underscoring the structural distinctions between PNU-140690 and substrate analog protease inhibitors. PNU-140690 retains good antiviral activity in vitro in the presence of human plasma proteins, and preclinical pharmacokinetic studies revealed good oral bioavailability. Accordingly, PNU-140690 is a candidate for clinical evaluation.

The cytochrome d complex is a two-subunit, membrane-bound terminal oxidase in the aerobic respiratory chain of Escherichia coli. The enzyme catalyzes the two-electron oxidation of ubiquinol and the four-electron reduction of oxygen to water. Previous work demonstrated that the site for ubiquinol oxidation was selectively inactivated by limited proteolysis by trypsin, which cleaves at a locus within subunit I. This work is extended to show that a similar phenomenon is observed with limited chymotrypsin proteolysis of the complex. The cleavage patterns are similar whether one uses the purified oxidase in nondenaturing detergent or reconstituted in proteoliposomes or uses spheroplasts of E. coli as the substrate for the proteolysis. Hence, the protease-sensitive locus is periplasmic in the cell. Fragments resulting from proteolysis were characterized by N-terminal sequencing and by immunoblotting with the use of a monoclonal antibody of known epitope within subunit I. The data indicate that inactivation of the ubiquinol oxidase activity results from cleavage at specific residues with a hydrophilic region previously defined as the Q loop. This domain has been already implicated in ubiquinol oxidation by the use of inhibitory monoclonal antibodies. Electrochemical and HPLC analysis of the protease-cleaved oxidase suggests no global changes in either the quaternary or tertiary structure of the enzyme. It is likely that the Q loop is directly involved in forming a portion of the ubiquinol binding site near the periplasmic surface of the membrane.

The cytochrome d complex is one of the two terminal oxidases in the aerobic respiratory system of Escherichia coli. This enzyme is not present in cells grown with high levels of dissolved oxygen in the culture medium but accumulates after mid-exponential growth, reaching high levels in stationary-phase cells. In this study, the transcriptional activity of the cyd operon, encoding the two subunits of the enzyme, was examined under a variety of growth conditions. This was accomplished by the use of a chromosomal operon fusion, cyd-lacZ, generated in vivo by a A plac-Mu hopper bacteriophage and also by the use of a cyd-lacZ protein fusion created in vitro on a plasmid, transferred onto a lambda transducing phage, and examined as a single-copy lysogen. Transcription of the gene fusions was monitored by determination of 0-galactosidase activity. The data clearly show that cyd is transcriptionally regulated and that induction is observed when the culture reaches a sufficient cell density so as to substantially reduce the steady-state levels of dissolved oxygen. The transcriptional activity is also regulated by other growth conditions, including the carbon source. The turn-on of cyd under semianaerobic conditions does not require thefnr gene product, cyclic AMP, or the cyclic AMP-binding protein.The aerobic respiratory chain of Escherichia coli contains two terminal oxidases, the cytrochrome o complex and the cytochrome d complex (1, 12). These membrane-bound enzymes each catalyze the oxidation of ubiquinol-8 and the reduction of oxygen to water. Under laboratory growth conditions, these enzymes are redundant. Mutants lacking either enzyme grow normally (2, 8). The two oxidases are encoded by the cyo and cyd operons, which have been mapped to min 10.2 and 16.5, respectively, on the genetic linkage map of E. coli (2, 8). Simultaneous mutations in both cyo and cyd result in the inability of the cells to grow aerobically on nonfermentable substrates such as DL-lactate or succinate (2).Although the two enzymes appear redundant under the conditions examined to date, their genetic regulation is quite different. The cytochrome o complex is predominant when the cells are grown with high oxygen levels (23). At low oxygen tension, the cytochrome d complex accumulates (15,23 obvious need for the enzyme, except perhaps as an oxygen scavenger. The regulation of gene expression by oxygen is being investigated in a number of laboratories, both in procaryotes (e.g., see references 13, 20, and 26) and in yeast cells (17,27). Several, although not all, of the genes encoding electron transport enzymes which are required for anaerobic respiration are positively regulated by the fnr gene product in E. coli (21) (oxrA in Salmonella typhimurium [13]). In the present report, it is shown that the fnr gene product is not required for the transcriptional activation of cyd. MATERIALS AND METHODSBacteria, bacteriophages, and genetic procedures. The sources and properties of the various bacterial strains and phages used in this work are listed in T...