Phenethylthiazolethiourea (PETT) compounds, a new class of HIV-1 reverse transcriptase inhibitors. 1. Synthesis and basic structure-activity relationship studies of PETT analogs.

A novel series of potent specific HIV-1 inhibitory compounds is described. The lead compound in the series, N-(2-phenethyl)-N'-(2-thiazolyl)thiourea (1), inhibits HIV-1 RT using rCdG as the template with an IC50 of 0.9 microM. In MT-4 cells, compound 1 inhibits HIV-1 with an ED50 of 1.3 microM. The 50% cytotoxic dose in cell culture is > 380 microM. The chemical structure-activity relationship (SAR) was developed by notionally dividing the lead compound in four quadrants. The SAR strategy had two phases. The first phase involved optimization of antiviral activity through independent variation of quadrants 1-4. The second phase involved the preparation of hybrid structures combining the best of these substituents. Further SAR studies and pharmacokinetic considerations led to the identification of N-(2-pyridyl)-N'-(5-bromo-2-pyridyl)-thiourea (62; LY300046.HCl) as a candidate for clinical evaluation. LY300046.HCl inhibits HIV-1 RT with an IC50 of 15 nM and in cell culture has an ED50 of 20 nM.

Phenethylthiazolylthiourea (PETT) compounds as a new class of HIV-1 reverse transcriptase inhibitors. 2. Synthesis and further structure-activity relationship studies of PETT analogs.

Phenylethylthiazolylthiourea (PETT) derivatives have been identified as a new series of non-nucleoside inhibitors of HIV-1 RT. Structure-activity relationship studies of this class of compounds resulted in the identification of N-[2-(2-pyridyl)ethyl]-N'-[2-(5-bromopyridyl)]-thiourea hydrochloride (trovirdine; LY300046.HCl) as a highly potent anti-HIV-1 agent. Trovirdine is currently in phase one clinical trials for potential use in the treatment of AIDS. Extension of these structure-activity relationship studies to identify additional compounds in this series with improved properties is ongoing. A part of this work is described here. Replacement of the two aromatic moieties of the PETT compounds by various substituted or unsubstituted heteroaromatic rings was investigated. In addition, the effects of multiple substitution in the phenyl ring were also studied. The antiviral activities were determined on wild-type and constructed mutants of HIV-1 RT and on wild-type HIV-1 and mutant viruses derived thereof, Ile100 and Cys181, in cell culture assays. Some selected compounds were determined on double-mutant viruses, HIV-1 (Ile 100/Asn103) and HIV-1 (Ile100/Cys181). A number of highly potent analogs were synthesized. These compounds displayed IC50's against wild-type RT between 0.6 and 5 nM. In cell culture, these agents inhibited wild-type HIV-1 with ED50's between 1 and 5 nM in MT-4 cells. In addition, these derivatives inhibited mutant HIV-1 RT (Ile 100) with IC50's between 20 and 50 nM and mutant HIV-1 RT (Cys 181) with IC50's between 4 and 10 nM, and in cell culture they inhibited mutant HIV-1 (Ile100) with ED50's between 9 and 100 nM and mutant HIV-1 (Cys181) with ED50's between 3 and 20 nM.

Induction of VanA vancomycin resistance genes in Enterococcus faecalis: use of a promoter fusion to evaluate glycopeptide and nonglycopeptide induction signals.

To characterize induction of VanA resistance a plasmid was constructed in which the gene for firefly luciferase lucA was placed under the control of the promoter for the VanA resistance genes, the vanH promoter. This system afforded convenient quantitative measurement of induction of the VanA genes. Glycopeptide antibiotics and antibiotics representing 19 different mechanisms of action were evaluated for their ability to induce. Antibiotics that acted as inducers were all inhibitors of late steps of peptidoglycan synthesis. These included moenomycin, bacitracin, tunicamycin, ramoplanin and glycopeptides, but not penicillin or other beta-lactam antibiotics. Glycopeptide antibiotics were the most potent inducers. Both glycopeptides with little or no antimicrobial activity and semisynthetic glycopeptides active against VanA resistant enterococci were inducers. Overall, results suggest that an induction response may involve both an internal signal, such as precursor accumulation, and the glycopeptide molecule itself as a signal. The system may be useful as a screen for new antimicrobial agents.

Cationic liposome-mediated uptake of human immunodeficiency virus type 1 Tat protein into cells.

The human immunodeficiency virus type 1 (HIV-1) Tat protein strongly transactivates gene expression from the viral long terminal repeat (LTR) and is required for virus efficient replication. Previous studies have shown that cells scrape-loaded in the presence of purified recombinant Tat can absorb the protein in a receptor-independent fashion. Using recombinant Tat in which cysteine residues were blocked by sulfitolysis to prevent disulfide aggregation (S-Tat) we were unable to observe this phenomenon, possibly because of improper protein folding. In this study we report that the block to cellular uptake could be overcome by mixing S-Tat with a cationic liposome, Lipofectin. When mixed with Lipofectin, S-Tat effected a specific, concentration-dependent transactivation of HIV-1 LTR-directed reporter gene activity in Hela Cells. Cellular uptake was confirmed by Western blot analysis with an anti-Tat antibody. The method described utilizes cells plated in a 96-well format, requires only nanogram quantities of S-Tat protein and is much less labor-intensive than assays involving scrape-loading, making it suitable for use as a high-throughput screen for detecting Tat inhibitors. The method may have applications for the analysis of other recombinant proteins that require uptake into intact cells for determination of functionality and presents a general technique for introducing exogenous proteins into cells.

Detection of specific human immunodeficiency virus type 1 Tat-TAR complexes in the presence of mild denaturing conditions.

Gene expression from the human immunodeficiency virus 1 (HIV-1) is greatly enhanced by binding of the virally encoded Tat protein to a 59-base RNA stem-loop structure, the Transactivation Responsive Element (TAR), located at the 5'-termini of all viral transcripts. This interaction was investigated in vitro using 32P-labelled TAR and highly purified Tat in which cysteine residues were blocked by sulpitolysis (S-Tat). It is shown that specific complex formation between S-Tat and TAR can occur in the presence of urea, with urea concentrations between 5 and 6 M causing an approximately two-fold increase in the level of binding. Two conditions favoring RNA secondary structure, low temperature (0 degree C) and the presence of divalent cations (Mg2+), diminished the level of specific binding. These observations suggest that the presence of mild denaturants promoted macromolecular refolding or rearrangement in a manner that increased the number of molecules available for binding, and present a general method for studying protein/RNA interactions where analysis has been obstructed by improper protein or RNA conformation.

Isolation of E. coli mutants containing multiple transpositions of IS sequences.

The characterization of three E. coli mutants that appeared to have unselected IS1 insertions on the chromosome are described. One had a single new IS1 sequence. The second had three new IS1 sequences. The third had two new IS1 sequences and one of the IS1 sequences in the parent was missing. These mutants were found in a collection on strains that contained IS insertions in the spc operon. The frequency of finding mutants with unselected IS1 transpositions was at least 100 times greater than expected. The results suggest several transposition events may frequently occur in the same cell.

Organization of ribosomal protein genes of Escherichia coli as analyzed by polar insertion mutations.

Several mutants of lambdaspc1 and lambdafus3 have been isolated carrying DNA insertion elements that were selected for their ability to reduce the expression of the spc gene. The sizes and locations of the insertions on the phage genomes were determined by heteroduplex analysis. They were found to be located at different positions in the Spc transcription unit. The effect of these insertions on the expression of the ribosomal protein genes carried by these phages in ultraviolet light-irradiated bacteria was investigated. The insertions at intermediate positions in the transcription unit reduced the expression of some of the genes in the unit but not others. Assuming that the genes whose expressions were reduced are distal to the insertion, it was possible to determine the relative position of most of the genes in the unit. The results indicate the order of genes in the Spc transcription unit is: promoter, L14, L24, L5, S14, S8, L6, L18, (S5, L15, L30).

Effect of fialuridine on replication of mitochondrial DNA in CEM cells and in human hepatoblastoma cells in culture.

Fialuridine (FIAU) is a nucleoside analog with potent activity against hepatitis B virus in vitro and in vivo. In this report, the effect of FIAU on mitochondrial DNA (mtDNA) replication in vitro was investigated. CEM cells, a cell line derived from human T cells, were incubated for 6 days in up to 20 microM FIAU. Total cellular DNA was isolated, normalized for the number of cells, and slot hybridized to a probe specific for mtDNA sequences. Treatment of CEM cells with FIAU did not result in a dose-dependent decrease in the amount of mtDNA. In contrast, dideoxycytidine (ddC) inhibited mtDNA replication by 50% at a concentration of approximately 0.1 microM. After 6 days of incubation, both compounds displayed a 50% toxic dose at a concentration of approximately 2 microM in CEM cells and approximately 34 microM in human hepatoblastoma cells (HepG2). In further experiments, CEM cells were incubated for 15 days in up to 2.5 microM FIAU, and again, no inhibition of mtDNA was observed. Over a 6-day incubation, FIAU, at concentrations of up to 200 microM, also failed to inhibit mtDNA replication in either HepG2 or HepG2 cells which constitutively replicate duck hepatitis B virus. In contrast, ddC inhibited mtDNA replication in these cells with a 50% inhibitory concentration of approximately 0.2 microM over a 6-day incubation. Treatment of cells with either FIAU or ddC resulted in a dose-dependent increase in lactate levels in the cell medium, indicating that any effect of FIAU on mitochondrial function may not be related to inhibition of mtDNA replication on the basis of the in vitro data. Alternative explanations for mitochondrial toxicity are considered.

Specialized transducing phages for ribosomal protein genes of Escherichia coli.

Specialized lambda transducing phages have been isolated carrying approximately half the ribosomal protein genes of E. coli. These phages carry regions of the bacterial chromosome between aroE and fus. The ribosomal protein genes on these phages have been identified by the stimulation of ribosomal protein synthesis in ultraviolet-irradiated bacteria following infection by the transducing phage, and by the in vitro synthesis of ribosomal proteins in a DNA-dependent protein synthesizing system. The results indicate lambdadspcl probably carries at least 22 ribosomal protein genes and lambdadspc2 at least 26 genes. All these genes are clustered between trkA and strA. At least 13 of them have not been previously mapped.

Identification of a gene for the alpha-subunit of RNA polymerase at the str-spc region of the Escherichia coli chromosome.

A structural gene for the alpha-subunit of RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase; EC 2.7.7.6) has been identified and mapped between spcA and trkA, near 64 min on the E. coli chromosome. It appears to be coordinately expressed and possibly cotranscribed with the genes for ribosomal proteins S11, S4, and L17.

Identification of two copies of the gene for the elongation factor EF-Tu in E. coli.

Two copies of the structural gene for the elongation factor EF-Tu have been identified in Escherichia coli: one near rif and the other near str. The latter seems to belong to a single transcriptional unit together with the genes for ribosomal protein S7, S12 (str) and the elongation factor EF-G (fus).

Fate of donor insertion sequence IS1 during transposition.

The number of insertion sequence IS1 segments in Escherichia coli K-12 and 20 mutants in which an ISI had been inserted at a new site was measured by Southern blot hybridization analysis. The parent strain appeared to contain seven IS1 segments. Each of the mutants contained these seven IS1 and one additional IS1 corresponding to the new IS1 insertion. These results suggest that a copy of a donor ISI is inserted at the new site. A model for transposition is prevented that postulates that a reactive intermediate is formed by a tandem duplication of a transposable sequence.

Role of short regions of homology in intermolecular illegitimate recombination events.

The structures of three recombinants between bacteriophage lambda DNA and plasmid pBR322 that were generated in a recA derivative of Escherichia coli are described. Each resulted from two illegitimate recombination events that resulted in the substitution of part of the lambda genome by part of the plasmid genome. The nucleotide sequences at the six lambda-plasmid junctions were determined and compared with the sequences of the lambda and plasmid genomes before recombination. Each recombination occurred at a short region of homology in the two genomes, and other short regions of homology were found near some of the junctions. The structures of these junctions are similar to those resulting from illegitimate recombination in animal cells. A model to explain how these multiple illegitimate recombination events could result from a cascade of DNA gyrase-catalyzed recombinations is discussed.

Identification and organization of ribosomal protein genes of Escherichia coli carried by lambdafus2 transducing phage.

We describe the isolation of lambdafus2, which carries bacterial DNA from the str-spc region of the Escherichia coli chromosome. Genes for 27 ribosomal proteins were found on the genome of this transducing phage by identifying the ribosomal proteins whose synthesis was stimulated after infection of UV-irradiated bacteria. These were genes for S3, S4, S5, S7, S8, S10, S11, S12, S13, S14, S17, S19, L2, L3, L4, L5, L6, L14, L15, L16, L17, L18, L22 L23, L24, L29, and L30. Subsets of these genes were identified on the genomes of the phages lambdaspc1, lambdaspc2-delta 9, and lambdaspc2-delta16, all of which carry parts of the bacterial DNA present on lambdafus2. From the known structures of these phage genomes, it has been possible to determine the relative order of many of these genes on the lambdafus2 genome and thus on the E. coli chromosome. Our evidence also suggests that the genes for S3, S17, S19, L2, L4, L16, L22, L23, and L29 are part of a single transcription unit. These results, along with the observations described in the previous and accompanying papers, indicate that the ribosomal protein genes on lambdafus2 are organized into at least four transcription units.

IS1 and IS2 mutations in the ribosomal protein genes of E. coli K-12.

The insertion mutations I15 and I16 in the ribosomal protein gene cluster at 64 min in Escherichia coli are identified as IS1 and IS2 using electron microscope heteroduplex analysis.

Cluster of genes in Escherichia coli for ribosomal proteins, ribosomal RNA, and RNA polymerase subunits.

The transducing phage lambdarifd18 isolated by Kirschbaum and Konrad [(1973 J. Bacteriol. 116, 517-526] was found to carry structural genes for several 50S ribosomal proteins and 16S and 23S rRNA. It has previously been demonstrated [Kirschbaum & Scaife (1974) Mol. Gen. Genet. 132, 193-201] that this phage carries genes for the DNA-dependent RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase; EC 2.7.7.6) subunits beta and beta'. Thus, the region of the E. coli chromosome carried by lambdarifd18 contains a cluster of genes essential for transcription and translation.