The discovery and optimization of a new class of bacterial topoisomerase (DNA gyrase and topoisomerase IV) inhibitors binding in the ATP domain are described. A fragment molecule, 1-ethyl-3-(2-pyridyl)urea, provided sufficiently potent enzyme inhibition (32 μM) to prompt further analogue work. Acids and acid isosteres were incorporated at the 5-pyridyl position of this fragment, bridging to a key asparagine residue, improving enzyme inhibition, and leading to measurable antibacterial activity. A CF3-thiazole substituent at the 4-pyridyl position improved inhibitory potency due to a favorable lipophilic interaction. Promising antibacterial activity was seen versus the Gram-positive pathogens Staphylococcus aureus and Streptococcus pneumoniae and the Gram-negative pathogens Haemophilus influenzae and Moraxella catarrhalis . Precursor metabolite incorporation and mutant analysis studies support the mode-of-action, blockage of DNA synthesis by dual target topoisomerase inhibition. Compound 35 was efficacious in a mouse S. aureus disease model, where a 4.5-log reduction in colony forming units versus control was demonstrated.

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 ␤-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.

A main challenge in the development of new agents for the treatment of Pseudomonas aeruginosa infections is the identification of chemotypes that efficiently penetrate the cell envelope and are not susceptible to established resistance mechanisms. Siderophore-conjugated monocarbams are attractive because of their ability to hijack the bacteria's iron uptake machinery for transport into the periplasm and their inherent stability to metallo-β-lactamases. Through development of the SAR we identified a number of modifications to the scaffold that afforded active anti-P. aeruginosa agents with good physicochemical properties. Through crystallographic efforts we gained a better understanding into how these compounds bind to the target penicillin binding protein PBP3 and factors to consider for future design. KEYWORDS: Pseudomonas aeruginosa, β-lactam, penicillin binding protein, siderophore, monocarbam, structure-guided design O ne of the largest challenges in the discovery of new Gram-negative antibacterial agents is the identification of chemotypes that can penetrate the cell envelope. 1 The pathogens, such as Pseudomonas aeruginosa (P. aeruginosa), have evolved complex cell architectures including an outer membrane composed of charged lipopolysaccharides, a thin peptidogylcan layer, and an inner membrane made up of phospholipids. 2 Identifying molecules with the necessary features to enable permeation has been a challenge.β-Lactams are one chemotype that have been successful in the treatment of P. aeruginosa. 3 Since the discovery of penicillin, β-lactams have been used in the clinic and are still widely prescribed. One of the most effective classes of β-lactams for the treatment of P. aeruginosa infections are the carbapenems (e.g., meropenem, 1, Figure 1). 4 Their effectiveness stems from their ability to acylate their penicillin-binding protein (PBP) targets, aided by high permeability into P. aeruginosa through the outer membrane porins. 5 However, they are currently being challenged in the clinic by the increasing emergence of broadspectrum serine β-lactamases and metallo-β-lactamases which hydrolyze most β-lactams and render them ineffective. 6 There is one class of β-lactams that is stable to the metallo-β-lactamases: the monocyclic β-lactams represented by aztreonam (2). Aztreonam however has limited effectiveness against P. aeruginosa presumably due to its poor permeation of the outer membrane, β-lactamase susceptibility, and high propensity for efflux (P. aeruginosa MIC 90 ≥ 1024 μg/mL, n = 20 panel). 3,7 To address the poor activity of 2, we focused on improving permeation by introducing siderophore mimics to promote uptake. 8−10 Siderophores are small Fe-chelating molecules synthesized and secreted by bacteria to scavenge iron from the host. 9,11 The iron-complexed siderophores are then brought into the cell via the iron uptake machinery, enabling the bacteria to access the much needed nutrient. It has been shown that introduction of iron-chelating groups to a monocyclic β-lactam scaffold ca...