Pharmacodynamic Profiling of a Siderophore-Conjugated Monocarbam in Pseudomonas aeruginosa: Assessing the Risk for Resistance and Attenuated Efficacy.

The objective of this study was to investigate the risk of attenuated efficacy due to adaptive resistance for the siderophore-conjugated monocarbam SMC-3176 in Pseudomonas aeruginosa by using a pharmacokinetic/pharmacodynamic (PK/PD) approach. MICs were determined in cation-adjusted Mueller-Hinton broth (MHB) and in Chelex-treated, dialyzed MHB (CDMHB). Spontaneous resistance was assessed at 2× to 16× the MIC and the resulting mutants sequenced. Efficacy was evaluated in a neutropenic mouse thigh model at 3.13 to 400 mg/kg of body weight every 3 h for 24 h and analyzed for association with free time above the MIC (fT>MIC). To closer emulate the conditions of the in vivo model, we developed a novel assay testing activity mouse whole blood (WB). All mutations were found in genes related to iron uptake: piuA, piuC, pirR, fecI, and pvdS. Against four P. aeruginosa isolates, SMC-3176 displayed predictable efficacy corresponding to the fT>MIC using the MIC in CDMHB (R(2) = 0.968 to 0.985), with stasis to 2-log kill achieved at 59.4 to 81.1%. Efficacy did not translate for P. aeruginosa isolate JJ 4-36, as the in vivo responses were inconsistent with fT>MIC exposures and implied a threshold concentration that was greater than the MIC. The results of the mouse WB assay indicated that efficacy was not predictable using the MIC for JJ 4-36 and four additional isolates, against which in vivo failures of another siderophore-conjugated ß-lactam were previously reported. SMC-3176 carries a risk of attenuated efficacy in P. aeruginosa due to rapid adaptive resistance preventing entry via the siderophore-mediated iron uptake systems. Substantial in vivo testing is warranted for compounds using the siderophore approach to thoroughly screen for this in vitro-in vivo disconnect in P. aeruginosa.

Toward the rational design of carbapenem uptake in Pseudomonas aeruginosa.

Understanding how compound penetration occurs across the complex cell walls of Gram-negative bacteria is one of the greatest challenges in discovering new drugs to treat the infections they cause. A combination of next-generation transposon sequencing, computational metadynamics simulations (CMDS), and medicinal chemistry was used to define genetic and structural elements involved in facilitated carbapenem entry into Pseudomonas aeruginosa. Here we show for the first time that these compounds are taken up not only by the major outer membrane channel OccD1 (also called OprD or PA0958) but also by a closely related channel OccD3 (OpdP or PA4501). Transport-mediating molecular interactions predicted by CMDS for these channels were first confirmed genetically, then used to guide the design of carbapenem analogs with altered uptake properties. These results bring us closer to the rational design of channel transmissibility and may ultimately lead to improved permeability of compounds across bacterial outer membranes.

Novel antibacterial targets and compounds revealed by a high-throughput cell wall reporter assay.

UNLABELLED: A high-throughput phenotypic screen based on a Citrobacter freundii AmpC reporter expressed in Escherichia coli was executed to discover novel inhibitors of bacterial cell wall synthesis, an attractive, well-validated target for antibiotic intervention. Here we describe the discovery and characterization of sulfonyl piperazine and pyrazole compounds, each with novel mechanisms of action. E. coli mutants resistant to these compounds display no cross-resistance to antibiotics of other classes. Resistance to the sulfonyl piperazine maps to LpxH, which catalyzes the fourth step in the synthesis of lipid A, the outer membrane anchor of lipopolysaccharide (LPS). To our knowledge, this compound is the first reported inhibitor of LpxH. Resistance to the pyrazole compound mapped to mutations in either LolC or LolE, components of the essential LolCDE transporter complex, which is required for trafficking of lipoproteins to the outer membrane. Biochemical experiments with E. coli spheroplasts showed that the pyrazole compound is capable of inhibiting the release of lipoproteins from the inner membrane. Both of these compounds have significant promise as chemical probes to further interrogate the potential of these novel cell wall components for antimicrobial therapy. IMPORTANCE: The prevalence of antibacterial resistance, particularly among Gram-negative organisms, signals a need for novel antibacterial agents. A phenotypic screen using AmpC as a sensor for compounds that inhibit processes involved in Gram-negative envelope biogenesis led to the identification of two novel inhibitors with unique mechanisms of action targeting Escherichia coli outer membrane biogenesis. One compound inhibits the transport system for lipoprotein transport to the outer membrane, while the other compound inhibits synthesis of lipopolysaccharide. These results indicate that it is still possible to uncover new compounds with intrinsic antibacterial activity that inhibit novel targets related to the cell envelope, suggesting that the Gram-negative cell envelope still has untapped potential for therapeutic intervention.

Thinking outside the "bug": a unique assay to measure intracellular drug penetration in gram-negative bacteria.

Significant challenges are present in antibiotic drug discovery and development. One of these is the number of efficient approaches Gram-negative bacteria have developed to avoid intracellular accumulation of drugs and other cell-toxic species. In order to better understand these processes and correlate in vitro enzyme inhibition to whole cell activity, a better assay to evaluate a key factor, intracellular accumulation of the drug, is urgently needed. Here, we describe a unique liquid chromatography (LC)-mass spectrometry (MS) approach to measure the amount of cellular uptake of antibiotics by Gram-negative bacteria. This method, which measures the change of extracellular drug concentration, was evaluated by comparing the relative uptake of linezolid by Escherichia coli wild-type versus an efflux pump deficient strain. A higher dosage of the drug showed a higher accumulation in these bacteria in a dosing range of 5-50 ng/mL. The Escherichia coli efflux pump deficient strain had a higher accumulation of the drug than the wild-type strain as predicted. The approach was further validated by determining the relative meropenem uptake by Pseudomonas aeruginosa wild-type versus a mutant strain lacking multiple porins. These studies show great promise of being applied within antibiotic drug discovery, as a universal tool to aid in the search for compounds that can easily penetrate bacterial cells.

Target-based resistance in Pseudomonas aeruginosa and Escherichia coli to NBTI 5463, a novel bacterial type II topoisomerase inhibitor.

In a previous report (T. J. Dougherty, A. Nayar, J. V. Newman, S. Hopkins, G. G. Stone, M. Johnstone, A. B. Shapiro, M. Cronin, F. Reck, and D. E. Ehmann, Antimicrob Agents Chemother 58:2657-2664, 2014), a novel bacterial type II topoisomerase inhibitor, NBTI 5463, with activity against Gram-negative pathogens was described. First-step resistance mutations in Pseudomonas aeruginosa arose exclusively in the nfxB gene, a regulator of the MexCD-OprJ efflux pump system. The present report describes further resistance studies with NBTI 5463 in both Pseudomonas aeruginosa and Escherichia coli. Second-step mutations in P. aeruginosa arose at aspartate 82 of the gyrase A subunit and led to 4- to 8-fold increases in the MIC over those seen in the parental strain with a first-step nfxB efflux mutation. A third-step mutant showed additional GyrA changes, with no changes in topoisomerase IV. Despite repeated efforts, resistance mutations could not be selected in E. coli. Genetic introduction of the Asp82 mutations observed in P. aeruginosa did not significantly increase the NBTI MIC in E. coli. However, with the aspartate 82 mutation present, it was possible to select second-step mutations in topoisomerase IV that did lead to MIC increases of 16- and 128-fold. As with the gyrase aspartate 82 mutation, the mutations in topoisomerase IV did not by themselves raise the NBTI MIC in E. coli. Only the presence of mutations in both targets of E. coli led to an increase in NBTI MIC values. This represents a demonstration of the value of balanced dual-target activity in mitigating resistance development.

NBTI 5463 is a novel bacterial type II topoisomerase inhibitor with activity against gram-negative bacteria and in vivo efficacy.

The need for new antibiotics that address serious Gram-negative infections is well recognized. Our efforts with a series of novel bacterial type II topoisomerase inhibitors (NBTIs) led to the discovery of NBTI 5463, an agent with improved activity over other NBTIs against Gram-negative bacteria, in particular against Pseudomonas aeruginosa (F. Reck, D. E. Ehmann, T. J. Dougherty, J. V. Newman, S. Hopkins, G. Stone, N. Agrawal, P. Ciaccio, J. McNulty, H. Barthlow, J. O'Donnell, K. Goteti, J. Breen, J. Comita-Prevoir, M. Cornebise, M. Cronin, C. J. Eyermann, B. Geng, G. R. Carr, L. Pandarinathan, X. Tang, A. Cottone, L. Zhao, N. Bezdenejnih-Snyder, submitted for publication). In the present work, NBTI 5463 demonstrated promising activity against a broad range of Gram-negative pathogens. In contrast to fluoroquinolones, the compound did not form a double-strand DNA cleavable complex with Escherichia coli DNA gyrase and DNA, but it was a potent inhibitor of both DNA gyrase and E. coli topoisomerase IV catalytic activities. In studies with P. aeruginosa, NBTI 5463 was bactericidal. Resistant mutants arose at a low rate, and the mutations were found exclusively in the nfxB gene, a regulator of the MexCD-OprJ efflux system. Levofloxacin-selected resistance mutations in GyrA did not result in decreased susceptibility to NBTI 5463. Animal infection studies demonstrated that NBTI 5463 was efficacious in mouse models of lung, thigh, and ascending urinary tract infections.

FraG is necessary for filament integrity and heterocyst maturation in the cyanobacterium Anabaena sp. strain PCC 7120.

Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium that differentiates nitrogen-fixing heterocysts when fixed nitrogen becomes growth limiting in the medium. The gene alr2338 (designated fraG herein), located immediately upstream of the master regulator of differentiation hetR, was identified in a genetic screen for mutants unable to grow diazotrophically. Filaments with a mutation in fraG were unable to fix nitrogen or synthesize heterocyst-specific glycolipids, and they fragmented initially to approximately nine cells in length at 24 h after induction of heterocyst development and eventually became unicellular. The fragmentation phenotype could be duplicated in the presence of fixed nitrogen when differentiation of heterocysts was elicited by overexpression of hetR, suggesting that a defect in differentiation, and not a lack of fixed nitrogen in the medium, was the more direct cause of fragmentation. An intact fraG gene was necessary for differentiation of mature heterocysts, but was not required for proper pattern formation, as indicated by a normal pattern of expression of hetR in a fraG mutant. A transcriptional GFP reporter fusion indicated that the level of expression of fraG was low in vegetative cells in both nitrogen-replete and nitrogen-free media, and was induced in heterocysts. fraG appears to play a role in filament integrity and differentiation of proheterocysts into mature heterocysts.