In vitro and in vivo antibacterial activities of omadacycline, a novel aminomethylcycline.

Omadacycline is the first intravenous and oral 9-aminomethylcycline in clinical development for use against multiple infectious diseases including acute bacterial skin and skin structure infections (ABSSSI), community-acquired bacterial pneumonia (CABP), and urinary tract infections (UTI). The comparative in vitro activity of omadacycline was determined against a broad panel of Gram-positive clinical isolates, including methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE), Lancefield groups A and B beta-hemolytic streptococci, penicillin-resistant Streptococcus pneumoniae (PRSP), and Haemophilus influenzae (H. influenzae). The omadacycline MIC90s for MRSA, VRE, and beta-hemolytic streptococci were 1.0 µg/ml, 0.25 µg/ml, and 0.5 µg/ml, respectively, and the omadacycline MIC90s for PRSP and H. influenzae were 0.25 µg/ml and 2.0 µg/ml, respectively. Omadacycline was active against organisms demonstrating the two major mechanisms of resistance, ribosomal protection and active tetracycline efflux. In vivo efficacy of omadacycline was demonstrated using an intraperitoneal infection model in mice. A single intravenous dose of omadacycline exhibited efficacy against Streptococcus pneumoniae, Escherichia coli, and Staphylococcus aureus, including tet(M) and tet(K) efflux-containing strains and MRSA strains. The 50% effective doses (ED50s) for Streptococcus pneumoniae obtained ranged from 0.45 mg/kg to 3.39 mg/kg, the ED50s for Staphylococcus aureus obtained ranged from 0.30 mg/kg to 1.74 mg/kg, and the ED50 for Escherichia coli was 2.02 mg/kg. These results demonstrate potent in vivo efficacy including activity against strains containing common resistance determinants. Omadacycline demonstrated in vitro activity against a broad range of Gram-positive and select Gram-negative pathogens, including resistance determinant-containing strains, and this activity translated to potent efficacy in vivo.

Sequence analysis and functional characterization of the violacein biosynthetic pathway from Chromobacterium violaceum.

Violacein is a purple-colored, broad-spectrum antibacterial pigment that has a dimeric structure composed of 5-hydroxyindole, oxindole and 2-pyyrolidone subunits formed by the condensation of two modified tryptophan molecules. The violacein biosynthetic gene cluster from Chromobacterium violaceum was characterized by DNA sequencing, transposon mutagenesis, and chemical analysis of the pathway intermediates produced heterologously in Escherichia. coli. The violacein biosynthetic gene cluster spans eight kilobases and is comprised of the four genes, vioABCD, that are necessary for violacein production. Sequence analysis suggests that the products of vioA, vioC and vioD are nucleotide-dependent monooxygenases. Disruption of vioA or vioB completely abrogates the biosynthesis of violacein intermediates, while disruption of the vioC or vioD genes results in the production of violacein precursors.

Emergence of different mechanisms of resistance in the evolution of multidrug resistance in murine erythroleukemia cell lines.

We examined the genetic and biochemical bases for drug resistance and the order of appearance of different mechanisms underlying the increasingly more resistant murine erythroleukemia cell lines established in Adriamycin (ADR). In the first-step low-level resistant cell line PC4-A5 (able to grow in 5 ng/mL ADR), there was a 2-fold reduction in topoisomerase IIalpha and topoisomerase IIbeta mRNA levels, as well as topoisomerase IIalpha protein and activity levels as compared with the parental cell line. The topoisomerase IIalpha activity levels remained reduced as the cells became increasingly more resistant. In contrast, the topoisomerase II mRNA and protein levels returned to approximately the parental levels in resistant cells growing in higher drug concentrations (40-160 ng/mL). Parental cells expressed the multidrug resistance protein (MRP), but beginning with PC4-A5 MRP expression decreased and remained reduced in increasingly resistant cell lines. At high levels of ADR resistance, the cells expressed the mdr3 gene concomitant with the appearance of vincristine resistance and energy-dependent daunomycin and vincristine efflux. Glutathione levels, internal pH, and expression of the major vault protein (MVP) remained unchanged in all cell lines. Fluorescence microscopy revealed no alterations in daunomycin distribution or vesicle numbers between the parental and resistant cell lines. Different resistance mechanisms emerge sequentially as cells become more resistant to ADR; the mechanisms are retained during the development of multidrug resistance (MDR). In intermediate-level MDR cell lines (PC4-A10 and PC4-A20), resistance involves an as yet undetermined mechanism(s).

DBF2, a cell cycle-regulated protein kinase, is physically and functionally associated with the CCR4 transcriptional regulatory complex.

CCR4, a general transcriptional regulator affecting the expression of a number of genes in yeast, forms a multi-subunit complex in vivo. Using the yeast two-hybrid screen, we have identified DBF2, a cell cycle-regulated protein kinase, as a CCR4-associated protein. DBF2 is required for cell cycle progression at the telophase to G1 cell cycle transition. DBF2 co-immunoprecipitated with CCR4 and CAF1/POP2, a CCR4-associated factor, and co-purified with the CCR4 complex. Moreover, a dbf2 disruption resulted in phenotypes and transcriptional defects similar to those observed in strains deficient for CCR4 or CAF1. ccr4 and caf1 mutations, on the other hand, were found to affect cell cycle progression in a manner similar to that observed for dbf2 defects. These data indicate that DBF2 is involved in the control of gene expression and suggest that the CCR4 complex regulates transcription during the late mitotic part of the cell cycle.

Differential changes in genome structure and expression of the mdr gene family in multidrug-resistant murine erythroleukemia cell lines.

The genome structure and expression of mdr genes were examined in multidrug-resistant sublines of two different murine (DBA/2J) Friend erythroleukemia cell lines, PC4 and C7D, derived by stepwise exposure to increasing concentrations of adriamycin beginning with 5 ng/ml. The PC4 cell lines selected in higher drug concentrations (80-1280 ng/ml) demonstrated amplification of all three mdr genes with preferential amplification of mdr3. Overexpression of the mdr2 and mdr3 genes accompanied their genomic amplification; however, expression of mdr1 was not seen despite amplification. In the C7D cell lines selected with higher drug concentrations (40-160 ng/ ml), amplification and overexpression of mdr1 and mdr2 without mdr3 was observed. Increased expression of mdr1 occurred prior to gene amplification. The distribution of mdr-specific genes in micrococcal nuclease-generated chromatin fractions differing in transcriptionally active sequences and proteins was different between the parent and drug-resistant sublines. An enrichment (two- to threefold) of mdr3 genes in the H1-depleted mononucleosome fraction enriched for actively transcribed genes (e.g., globin) was detected by Southern analysis of chromatin fractions in PC4-80 cells (selected in 80 ng/ml of adriamycin and overexpressing mdr3), compared to the parental cells. mdr3 enrichment was also detected using a new PCR-based method, which examined mdr3 genes and repetitive sequences. Of note, the H1-depleted chromatin fraction from PC4-20 showed enrichment of the mdr3 gene, although mdr3 expression was not detected in the cell line. These studies showed a different pattern of gene amplification and overexpression in genetically related erythroleukemia cell lines selected for resistance to the same chemotherapeutic agent. A change in chromatin organization of mdr genes preceded overexpression and amplification of the mdr3 gene.

Active efflux of the free acid form of the fluorescent dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein in multidrug-resistance-protein-overexpressing murine and human leukemia cells.

Murine and human cell lines overexpressing the multidrug-resistance protein (MRP) showed a marked decreased accumulation of the fluorescent dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). In contrast, less altered accumulation was seen in the P-glycoprotein(P-gp)-overexpressing cell lines. The decreased drug accumulation was reversed by the energy inhibitors sodium azide/2-deoxyglucose and by the vinca alkaloid, vincristine, but not by the chemotherapeutic agents, etoposide and adriamycin. Decreased accumulation was linked to active efflux of the hydrophilic free acid form of BCECF from the MRP-overexpressing cell lines, indicating that dye extrusion occurs after the dye ester has been converted to the free acid form in the cytoplasm. The finding suggests that MRP mediates removal of substrates from a cytoplasmic location. Buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis, decreased the vincristine and etoposide resistance displayed by the MRP-expressing murine cell lines, but did not affect the accumulation of BCECF. Thus, while glutathione may be involved in MRP-mediated resistance to some chemotherapeutic agents, it is not necessary for effiux of substrates such as BCECF.

Indomethacin-mediated reversal of multidrug resistance and drug efflux in human and murine cell lines overexpressing MRP, but not P-glycoprotein.

Decreased accumulation of the fluorescent dye BCECF [2', 7'-bis-(2-carboxyethyl)-5-(6)- carboxyfluorescein] characterized murine and human multidrug-resistant cell lines overexpressing the multidrug resistance protein (MRP). Indomethacin (10 microM), a known cyclo-oxygenase and glutathione-S-transferase inhibitor as well as a modulator of anion transport, increased accumulation and blocked efflux of BCECF in MRP-expressing murine and human cells. The drug did not affect P-glycoprotein (P-gp)-mediated export of rhodamine 123. The indomethacin effect on BCECF efflux was not reversed by the addition of exogenous prostaglandins, suggesting that the drug acts by a mechanism other than decreasing prostaglandin synthesis. Indomethacin also increased multidrug susceptibility of both murine and human cell lines overexpressing MRP, but not those displaying P-gp-associated resistance. In addition, indomethacin modulated the decreased vincristine accumulation in cells expressing MRP, but not in those expressing P-gp. These data suggest that indomethacin is a specific inhibitor of MRP, possibly functioning by inhibition of glutathione-S-transferase or, alternatively, by direct competition with the drug at the transport site.

Identification of a mouse protein whose homolog in Saccharomyces cerevisiae is a component of the CCR4 transcriptional regulatory complex.

The CCR4 protein from Saccharomyces cerevisiae is a component of a multisubunit complex that is required for the regulation of a number of genes in yeast cells. We report here the identification of a mouse protein (mCAF1 [mouse CCR4-associated factor 1]) which is capable of interacting with and binding to the yeast CCR4 protein. The mCAF1 protein was shown to have significant similarity to proteins from humans, Caenorhabditis elegans, Arabidopsis thaliana, and S. cerevisiae. The yeast gene (yCAF1) had been previously cloned as the POP2 gene, which is required for expression of several genes. Both yCAF1 (POP2) and the C. elegans homolog of CAF1 were shown to genetically interact with CCR4 in vivo, and yCAF1 (POP2) physically associated with CCR4. Disruption of the CAF1 (POP2) gene in yeast cells gave phenotypes and defects in transcription similar to those observed with disruptions of CCR4, including the ability to suppress spt10-enhanced ADH2 expression. In addition, yCAF1 (POP2) when fused to LexA was capable of activating transcription. mCAF1 could also activate transcription when fused to LexA and could functionally substitute for yCAF1 in allowing ADH2 expression in an spt10 mutant background. These data imply that CAF1 is a component of the CCR4 protein complex and that this complex has retained evolutionarily conserved functions important to eukaryotic transcription.

The yeast CCR4 protein is neither regulated by nor associated with the SPT6 and SPT10 proteins and forms a functionally distinct complex from that of the SNF/SWI transcription factors.

The CCR4 protein is specifically required for the increased transcription at the ADH2 locus resulting from mutations in the SPT10 (CRE1) and SPT6 (CRE2) genes and is also required for the expression of ADH2 and other genes under non-fermentative growth conditions. The mechanism by which mutations in CCR4 suppress defects in SPT10 and SPT6 was examined. The SPT10 and SPT6 genes were shown not to control CCR4 mRNA or protein expression nor did SPT10 and SPT6 proteins co-immuneprecipitate with CCR4. CCR4 association with two other proteins, 195 and 185 kDa in size, was unaffected by either spt10 or spt6 mutations. Also, the ability of CCR4 to activate transcription when fused to the LexA DNA binding domain was not specifically enhanced by defects in either SPT10 or SPT6. These results suggest that SPT10 and SPT6, in negatively regulating transcription at ADH2, act through a factor that requires CCR4 function, but do not regulate CCR4 expression, control its activity, physically interact with it, or affect its binding to other factors. The relationship of CCR4 to the group of general transcription factors, SNF2, SNF5, SNF6 and SWI1 and SWI3, which comprise a multisubunit complex required for ADH2 and other genes' expression, was also examined. CCR4 protein expression was not controlled by these factors nor did they co-immuneprecipitate or associate with CCR4. In addition, a ccr4 mutation had little effect on an ADH2 promoter alteration in contrast to the large effects displayed by mutations in SNF2 and SNF5. These data suggest that CCR4 acts by a separate mechanism from that used by the SNF/SWI general transcription factors in affecting gene expression.

CCR4 is a glucose-regulated transcription factor whose leucine-rich repeat binds several proteins important for placing CCR4 in its proper promoter context.

The yeast CCR4 protein is required for the expression of a number of genes involved in nonfermentative growth, including glucose-repressible ADH2, and is the only known suppressor of mutations in the SPT6 and SPT10 genes, two genes which are believed to be involved in chromatin maintenance. We show here that although CCR4 did not bind DNA under the conditions tested, it was able to activate transcription when fused to a heterologous DNA-binding domain. The transcriptional activation ability of CCR4, in contrast to that of many other activators, was glucose regulated. Two activation domains one of which was glucose responsive and encompassed a glutamine-proline-rich region similar to that found in other eukaryotic transcriptional factors were identified. The two transactivation regions, when separated from the leucine-rich repeat and the C terminus of CCR4, were unable to complement a defective ccr4 allele, suggesting that the leucine-rich repeat and the C terminus make contacts that link the activation regions to the proper gene context. Native immunoprecipitation of CCR4 revealed that CCR4 was complexed with at least four other proteins. The leucine-rich repeat of CCR4 was both necessary and sufficient for interaction with at least two of these factors. We propose that the leucine-rich repeat links CCR4 through its associated factors to its promoter context at ADH2 and other loci where it is required for proper transcriptional regulation.