Pyrrolamide DNA gyrase inhibitors: fragment-based nuclear magnetic resonance screening to identify antibacterial agents.

DNA gyrase is an essential enzyme in bacteria, and its inhibition results in the disruption of DNA synthesis and, subsequently, cell death. The pyrrolamides are a novel class of antibacterial agents targeting DNA gyrase. These compounds were identified by a fragment-based lead generation (FBLG) approach using nuclear magnetic resonance (NMR) screening to identify low-molecular-weight compounds that bind to the ATP pocket of DNA gyrase. A pyrrole hit with a binding constant of 1 mM formed the basis of the design and synthesis of a focused library of compounds that resulted in the rapid identification of a lead compound that inhibited DNA gyrase with a 50% inhibitory concentration (IC(50)) of 3 µM. The potency of the lead compound was further optimized by utilizing iterative X-ray crystallography to yield DNA gyrase inhibitors that also displayed antibacterial activity. Spontaneous mutants were isolated in Staphylococcus aureus by plating on agar plates containing pyrrolamide 4 at the MIC. The resistant variants displayed 4- to 8-fold-increased MIC values relative to the parent strain. DNA sequencing revealed two independent point mutations in the pyrrolamide binding region of the gyrB genes from these variants, supporting the hypothesis that the mode of action of these compounds was inhibition of DNA gyrase. Efficacy of a representative pyrrolamide was demonstrated against Streptococcus pneumoniae in a mouse lung infection model. These data demonstrate that the pyrrolamides are a novel class of DNA gyrase inhibitors with the potential to deliver future antibacterial agents targeting multiple clinical indications.

Discovery of a novel azaindole class of antibacterial agents targeting the ATPase domains of DNA gyrase and Topoisomerase IV.

We present the discovery and optimization of a novel series of bacterial topoisomerase inhibitors. Starting from a virtual screening hit, activity was optimized through a combination of structure-based design and physical property optimization. Synthesis of fewer than a dozen compounds was required to achieve inhibition of the growth of methicillin-resistant Staphyloccus aureus (MRSA) at compound concentrations of 1.56 µM. These compounds simultaneously inhibit DNA gyrase and Topoisomerase IV at similar nanomolar concentrations, reducing the likelihood of the spontaneous occurrence of target-based mutations resulting in antibiotic resistance, an increasing threat in the treatment of serious infections.

Pyrrolamide DNA gyrase inhibitors: optimization of antibacterial activity and efficacy.

The pyrrolamides are a new class of antibacterial agents targeting DNA gyrase, an essential enzyme across bacterial species and inhibition results in the disruption of DNA synthesis and subsequently, cell death. The optimization of biochemical activity and other drug-like properties through substitutions to the pyrrole, piperidine, and heterocycle portions of the molecule resulted in pyrrolamides with improved cellular activity and in vivo efficacy.

The CARB-X Portfolio of Nontraditional Antibacterial Products.

The growing prevalence of antibiotic-resistant bacterial pathogens and the lack of new medicines to treat the infections they cause remain a significant global threat. In recent years, this ongoing unmet need has encouraged more research groups to focus on the discovery and development of nontraditional antibacterial agents, ranging from anti-virulence strategies to bacteriophage and ways to modulate the microbiome. The Combating Antibiotic-Resistant Bacteria Biopharmaceutical Accelerator (CARB-X) is a global nonprofit public-private partnership dedicated to accelerating antibacterial-related research. Importantly, the CARB-X portfolio supports a wide variety of novel and innovative nontraditional programs to help the global antibacterial research ecosystem understand the potential that these modalities can play in the management or prevention of serious infections. We describe here the breadth of the CARB-X pipeline of novel nontraditional products.

Genetic invalidation of Lp-PLA2 as a therapeutic target: Large-scale study of five functional Lp-PLA2-lowering alleles.

Aims Darapladib, a potent inhibitor of lipoprotein-associated phospholipase A2 (Lp-PLA2), has not reduced risk of cardiovascular disease outcomes in recent randomized trials. We aimed to test whether Lp-PLA2 enzyme activity is causally relevant to coronary heart disease. Methods In 72,657 patients with coronary heart disease and 110,218 controls in 23 epidemiological studies, we genotyped five functional variants: four rare loss-of-function mutations (c.109+2T > C (rs142974898), Arg82His (rs144983904), Val279Phe (rs76863441), Gln287Ter (rs140020965)) and one common modest-impact variant (Val379Ala (rs1051931)) in PLA2G7, the gene encoding Lp-PLA2. We supplemented de-novo genotyping with information on a further 45,823 coronary heart disease patients and 88,680 controls in publicly available databases and other previous studies. We conducted a systematic review of randomized trials to compare effects of darapladib treatment on soluble Lp-PLA2 activity, conventional cardiovascular risk factors, and coronary heart disease risk with corresponding effects of Lp-PLA2-lowering alleles. Results Lp-PLA2 activity was decreased by 64% ( p = 2.4 × 10-25) with carriage of any of the four loss-of-function variants, by 45% ( p < 10-300) for every allele inherited at Val279Phe, and by 2.7% ( p = 1.9 × 10-12) for every allele inherited at Val379Ala. Darapladib 160 mg once-daily reduced Lp-PLA2 activity by 65% ( p < 10-300). Causal risk ratios for coronary heart disease per 65% lower Lp-PLA2 activity were: 0.95 (0.88-1.03) with Val279Phe; 0.92 (0.74-1.16) with carriage of any loss-of-function variant; 1.01 (0.68-1.51) with Val379Ala; and 0.95 (0.89-1.02) with darapladib treatment. Conclusions In a large-scale human genetic study, none of a series of Lp-PLA2-lowering alleles was related to coronary heart disease risk, suggesting that Lp-PLA2 is unlikely to be a causal risk factor.

Discovery of efficacious Pseudomonas aeruginosa-targeted siderophore-conjugated monocarbams by application of a semi-mechanistic pharmacokinetic/pharmacodynamic model.

To identify new agents for the treatment of multi-drug-resistant Pseudomonas aeruginosa, we focused on siderophore-conjugated monocarbams. This class of monocyclic ß-lactams are stable to metallo-ß-lactamases and have excellent P. aeruginosa activities due to their ability to exploit the iron uptake machinery of Gram-negative bacteria. Our medicinal chemistry plan focused on identifying a molecule with optimal potency and physical properties and activity for in vivo efficacy. Modifications to the monocarbam linker, siderophore, and oxime portion of the molecules were examined. Through these efforts, a series of pyrrolidinone-based monocarbams with good P. aeruginosa cellular activity (P. aeruginosa MIC90 = 2 µg/mL), free fraction levels (>20% free), and hydrolytic stability (t1/2 ≥ 100 h) were identified. To differentiate the lead compounds and enable prioritization for in vivo studies, we applied a semi-mechanistic pharmacokinetic/pharmacodynamic model to enable prediction of in vivo efficacy from in vitro data.

Novel structures derived from 2-[[(2-pyridyl)methyl]thio]-1H-benzimidazole as anti-Helicobacter pylori agents, Part 2.

A parallel chemistry expansion of the 2-([3-[(1H-benzimidazol-2-ylsulfanyl)methyl]-phenyl]sulfanyl)-1-ethanol scaffold (2) successfully provided a set of 2-([3-[(1H-benzimidazol-2-ylsulfanyl)methyl]-2-methylphenyl]sulfanyl)ethyl carbamates with the generic structure 12, which displayed potent and selective activities against the gastric pathogen Helicobacter pylori. A prototype carbamate 12a was studied further and found to meet several significant in vitro microbiological criteria required for a novel anti-H. pylori agent. The compound displayed low minimal inhibition concentration (MIC) values against a panel of 27 different clinically relevant H. pylori strains (MIC(90) = 0.25 microg/mL), including strains resistant to either metronidazole or clarithromycin or both. Additionally, 12a was almost inactive against a wide range of commensal or pathogenic microorganisms comprising panels of 25 aerobic bacterial strains including two strains of methicillin resistant Staphylococcus aureus (MIC(90) = >64 microg/mL) and 18 anaerobic bacterial strains (MIC(90) = >64 microg/mL). The measured rate of resistance development against 12a was found to be less than 10(-9), a clinically acceptable level, and pharmacokinetic studies revealed in vivo exposure levels comparable with those established for antimicrobials currently used in H. pylori triple regimen.

Optimization of pyrrolamide topoisomerase II inhibitors toward identification of an antibacterial clinical candidate (AZD5099).

AZD5099 (compound 63) is an antibacterial agent that entered phase 1 clinical trials targeting infections caused by Gram-positive and fastidious Gram-negative bacteria. It was derived from previously reported pyrrolamide antibacterials and a fragment-based approach targeting the ATP binding site of bacterial type II topoisomerases. The program described herein varied a 3-piperidine substituent and incorporated 4-thiazole substituents that form a seven-membered ring intramolecular hydrogen bond with a 5-position carboxylic acid. Improved antibacterial activity and lower in vivo clearances were achieved. The lower clearances were attributed, in part, to reduced recognition by the multidrug resistant transporter Mrp2. Compound 63 showed notable efficacy in a mouse neutropenic Staphylococcus aureus infection model. Resistance frequency versus the drug was low, and reports of clinical resistance due to alteration of the target are few. Hence, 63 could offer a novel treatment for serious issues of resistance to currently used antibacterials.

Novel DNA gyrase inhibitors: microbiological characterisation of pyrrolamides.

Pyrrolamides are a novel class of antibacterial agents that target DNA gyrase, resulting in inhibition of DNA synthesis and bacterial cell death. In these studies, advanced compounds were shown to have potent in vitro activity against selected Gram-positive and Gram-negative pathogens, including meticillin-resistant Staphylococcus aureus, meticillin- and quinolone-resistant S. aureus, vancomycin-resistant enterococci, penicillin-resistant Streptococcus pneumoniae and ß-lactamase-producing Haemophilus influenzae and Moraxella catarrhalis. Representatives of the class were demonstrated to be bactericidal, with frequencies of spontaneous resistance ≤1×10(-7) when plated at concentrations equivalent to their minimum inhibitory concentration. Mode of action studies suggested that the activity of these compounds is due to inhibition of the GyrB subunit of DNA gyrase in key pathogens. The antibacterial activity, spectrum and mode of action of these compounds underscore the promise of the pyrrolamide series as attractive candidates for the treatment of several clinical indications, including respiratory and soft tissue infections.

Novel topoisomerase inhibitors: microbiological characterisation and in vivo efficacy of pyrimidines.

Pyrimidine compounds were identified as inhibitors of DNA topoisomerase IV through high-throughput screening. This study was designed to exemplify the in vitro activity of the pyrimidines against Gram-positive and Gram-negative microorganisms, to reveal the mode of action of these compounds and to demonstrate their in vivo efficacy. Frequencies of resistance to pyrimidines among Staphylococcus aureus and Streptococcus pneumoniae were <10(-10) at four times their minimum inhibitory concentrations (MICs). These compounds exhibited a dual mode of action through inhibition of the ParE subunit of DNA topoisomerase IV as well as the GyrB subunit of DNA gyrase, a homologue of DNA topoisomerase IV. Pyrimidines were shown to have MIC(90) values (MIC that inhibited 90% of the strains tested) of ≤2 mg/L against Gram-positive pathogens, including meticillin-resistant S. aureus, quinolone- and meticillin-resistant S. aureus, vancomycin-resistant enterococci, penicillin-non-susceptible S. pneumoniae and Streptococcus pyogenes, and MIC(90) values of 2- to >16 mg/L and ≤0.5 mg/L against the Gram-negative pathogens Haemophilus influenzae and Moraxella catarrhalis, respectively. The pyrimidines were bactericidal and exhibited a ca. 1000-fold reduction of the bacterial counts at 300 mg/kg in a S. pneumoniae lung infection model. The microbiological properties and in vivo efficacy of pyrimidines underscore their potential as candidates for the treatment of soft-tissue infections and hospital-acquired pneumonia.

Fragment-to-hit-to-lead discovery of a novel pyridylurea scaffold of ATP competitive dual targeting type II topoisomerase inhibiting antibacterial agents.

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.