Michael Draper

In Vitro and In Vivo Antibacterial Activities of Omadacycline, a Novel Aminomethylcycline

ABSTRACT 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.

Mechanism of Action of the Novel Aminomethylcycline Antibiotic Omadacycline

ABSTRACT Omadacycline is a novel first-in-class aminomethylcycline with potent activity against important skin and pneumonia pathogens, including community-acquired methicillin-resistant Staphylococcus aureus (MRSA), β-hemolytic streptococci, penicillin-resistant Streptococcus pneumoniae, Haemophilus influenzae, and Legionella. In this work, the mechanism of action for omadacycline was further elucidated using a variety of models. Functional assays demonstrated that omadacycline is active against strains expressing the two main forms of tetracycline resistance (efflux and ribosomal protection). Macromolecular synthesis experiments confirmed that the primary effect of omadacycline is on bacterial protein synthesis, inhibiting protein synthesis with a potency greater than that of tetracycline. Biophysical studies with isolated ribosomes confirmed that the binding site for omadacycline is similar to that for tetracycline. In addition, unlike tetracycline, omadacycline is active in vitro in the presence of the ribosomal protection protein Tet(O).

A Randomized, Evaluator-Blind, Phase 2 Study Comparing the Safety and Efficacy of Omadacycline to Those of Linezolid for Treatment of Complicated Skin and Skin Structure Infections

ABSTRACT A randomized, investigator-blind, multicenter phase 2 trial involving patients with complicated skin and skin structure infections (cSSSI) compared the safety and efficacy of omadacycline, a broad-spectrum agent with activity against methicillin-resistant Staphylococcus aureus (MRSA), to those of linezolid (with or without aztreonam). Patients were randomized 1:1 to omadacycline (100 mg intravenously [i.v.] once a day [QD] with an option to transition to 200 mg orally QD) or linezolid (600 mg i.v. twice daily [BID] with an option to transition to 600 mg orally BID) at 11 U.S. sites. Patients suspected or documented to have infections caused by Gram-negative bacteria were given aztreonam (2 g i.v. every 12 h [q12h]) if randomized to linezolid or matching placebo infusions if randomized to omadacycline. Adverse events were reported in 46 (41.4%) omadacycline-treated and 55 (50.9%) linezolid-treated patients. Adverse events related to treatment were assessed by investigators in 24 (21.6%) omadacycline-treated and 33 (30.6%) linezolid-treated patients. The gastrointestinal tract was most commonly involved, with adverse events reported in 21 (18.9%) patients exposed to omadacycline and 20 (18.5%) exposed to linezolid. Rates of successful clinical response in the intent-to-treat (ITT) and clinical evaluable (CE) populations favored omadacycline (ITT, 88.3% versus 75.9%; 95% confidence interval [CI], 1.9 to 22.9; CE, 98.0% versus 93.2%; 95% CI, −1.7 to 11.3). For microbiologically evaluable (ME) patients with S. aureus infections, the clinical success rates were 97.2% (70/72) in omadacycline-treated and 92.7% (51/55) in linezolid-treated patients. This phase 2 experience supports conclusions that omadacycline is well tolerated in cSSSI patients and that this aminomethylcycline has potential to be an effective treatment for serious skin infections.

N-Hydroxybenzimidazole inhibitors of ExsA MAR transcription factor in Pseudomonas aeruginosa: In vitro anti-virulence activity and metabolic stability.

ExsA is a multiple adaptational response (MAR) transcription factor, regulating the expression of a virulence determinant, the type III secretion system (T3SS) in Pseudomonas aeruginosa. Non-cytotoxic, non-antibacterial N-hydroxybenzimidazoles were identified as effective inhibitors of ExsA-DNA binding, and their potential utility as anti-virulence agents for P. aeruginosa was demonstrated in a whole cell assay. Select N-hydroxybenzimidazole inhibitors were stable in an in vitro human liver microsomal assay.

The marC gene of Escherichia coli is not involved in multiple antibiotic resistance.

The marC gene of Escherichia coli is divergently transcribed from the marRAB operon involved in resistance to multiple antibiotics (5, 8), oxidative stress agents (2), and organic solvents (3, 16). Previous data from our laboratory had suggested a role for marC in intrinsic multiple antibiotic resistance (4, 9, 16), and the gene has been so annotated in most databases. Because that earlier work had suggested that marC was regulated by the repressor MarR and induced by tetracycline, we sought the transcriptional start site for marC to see if the marC promoter might overlap the MarR binding sites within the marRAB promoter (5′ rapid amplification of cDNA end [RACE] system of Gibco/BRL Life Technologies, cells grown with 2 μg/ml tetracycline to increase the amount of mRNA). Transcription of marC started 30 nucleotides upstream from the putative ATG initiation codon of MarC (bp 1266 of Cohen et al. [4]). Therefore, the marC promoter does not contain the MarR binding sites. Moreover, Northern blot analysis of AG100 and its isogenic marR mutant AG112 (in which MarR is inactive [8, 12]) showed no differences in levels of marC mRNA between the two strains (data not shown), nor was expression of marC induced by salicylate, which inactivates the repressor MarR (1) (Fig. ​(Fig.1).1). We conclude that marC is not regulated by MarR. Since chloramphenicol does not bind to MarR (1), the apparent up-regulation by tetracycline and chloramphenicol (Fig. ​(Fig.1)1) (4) likely reflects stabilization of mRNA rather than true induction (11, 13, 14). FIG. 1. Northern blot analysis of marC from E. coli. RNA was isolated from mid-exponential-phase cells grown in LB broth at 30°C and treated for 1 h with the specified compounds. Separate cultures of E. coli AG100 following exposure to 5 mM salicylate ... We replaced the marC locus in E. coli AG100 with a kanamycin cassette as described previously (10) and looked for any increase in susceptibilities to antimicrobials. MICs were determined on LB agar by use of Etest strips (AB Biodisk, Solna, Sweden) with about three dozen different agents, including beta lactams, tetracyclines, fluoroquinolones, cephalosporins, imipenem, macrolides, aminoglycosides, chloramphenicol, fusidic acid, trimethoprim, and rifampin. Gradient plates (6) were used for oxidative stress agents (plumbagin, paraquat, phenylmethylsulfonate, dinitrophenol, and menadione) and for ethidium bromide. No differences in susceptibilities were seen for the marC::kan deletion mutant relative to the wild type. We then replaced the genes for the E. coli MarC paralogs YchE and YhgN in the marC::kan strain by use of spectinomycin and gentamicin cassettes, respectively, to create a triple knockout mutant, but again no differences in susceptibilities were seen. We also used three plasmid constructs designed to overexpress marC via the araBAD, T7, or native marC promoter. Plasmid pHA-1::marC (obtained from D. Daley) specifies membrane-bound MarC-PhoA regulated by Salmonella enterica serovar Typhimurium pBAD/AraC (7). We constructed pETmarC11 (T7 promoter/lac operator with lac repressor; specifying MarC-6H) and pACmarC1 (marC promoter starting 58 bp upstream of the transcriptional start site; specifying native MarC) by cloning PCR-amplified DNA into vectors pET21b (Novagen) and pACYC184, respectively. None of these three plasmids led to a change in susceptibility of cells to a variety of antibiotics and oxidative stress agents. We suggest that MarC no longer be classified as a multiple antibiotic resistance protein. However, we do not advise a name change until a function is found.

Gng12 is a novel negative regulator of LPS-induced inflammation in the microglial cell line BV-2

BackgroundInflammation plays a central role in many neurodegenerative diseases, including Parkinson’s, Alzheimer’s, multiple sclerosis, amyotrophic lateral sclerosis, and AIDS dementia. Microglia are the resident macrophages of the central nervous system and are the cells primarily responsible for the inflammatory component of these diseases.MethodsUsing gene expression profiling, we compared the profile of the neurospecific microglial cell line BV-2 after LPS stimulation to that of a macrophage cell line (J774A.1) stimulated with LPS.ResultsA set of 77 genes that were modulated only in microglial cells after LPS stimulation was identified. One gene of interest, Gng12, was investigated further to determine its ability to modify the inflammatory response. Specifically, Gng12 mRNA levels were transiently increased after LPS stimulation. In addition, overall levels of Gng12 mRNA after LPS stimulation were significantly higher in BV-2 cells as compared to macrophage cells.ConclusionModulating Gng12 mRNA levels using RNAi revealed a novel role for the factor in the negative regulation of the overall inflammatory response as based on effects on nitrite and TNFα levels. These data suggest that Gng12 is a negative regulator of the LPS response and may be an important factor in the overall inflammatory signaling cascade.

In Vitro and In Vivo Antimalarial Efficacies of Optimized Tetracyclines

ABSTRACT With increasing resistance to existing antimalarials, there is an urgent need to discover new drugs at affordable prices for countries in which malaria is endemic. One approach to the development of new antimalarial drugs is to improve upon existing antimalarial agents, such as the tetracyclines. Tetracyclines exhibit potent, albeit relatively slow, action against malaria parasites, and doxycycline is used for both treatment (with other agents) and prevention of malaria. We synthesized 18 novel 7-position modified tetracycline derivatives and screened them for activity against cultured malaria parasites. Compounds with potent in vitro activity and other favorable drug properties were further tested in a rodent malaria model. Ten compounds inhibited the development of cultured Plasmodium falciparum with a 50% inhibitory concentration (IC50) after 96 h of incubation of <30 nM, demonstrating activity markedly superior to that of doxycycline (IC50 at 96 h of 320 nM). Most compounds showed little mammalian cell cytotoxicity and no evidence of in vitro phototoxicity. In a murine Plasmodium berghei model, 13 compounds demonstrated improved activity relative to that of doxycycline. In summary, 7-position modified tetracyclines offer improved activity against malaria parasites compared to doxycycline. Optimized compounds may allow lower doses for treatment and chemoprophylaxis. If safety margins are adequate, dosing in children, the group at greatest risk for malaria in countries in which it is endemic, may be feasible.

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).