Hoan Huynh

Novel Bacterial NAD+-Dependent DNA Ligase Inhibitors with Broad-Spectrum Activity and Antibacterial Efficacy In Vivo

ABSTRACT DNA ligases are indispensable enzymes playing a critical role in DNA replication, recombination, and repair in all living organisms. Bacterial NAD+-dependent DNA ligase (LigA) was evaluated for its potential as a broad-spectrum antibacterial target. A novel class of substituted adenosine analogs was discovered by target-based high-throughput screening (HTS), and these compounds were optimized to render them more effective and selective inhibitors of LigA. The adenosine analogs inhibited the LigA activities of Escherichia coli, Haemophilus influenzae, Mycoplasma pneumoniae, Streptococcus pneumoniae, and Staphylococcus aureus, with inhibitory activities in the nanomolar range. They were selective for bacterial NAD+-dependent DNA ligases, showing no inhibitory activity against ATP-dependent human DNA ligase 1 or bacteriophage T4 ligase. Enzyme kinetic measurements demonstrated that the compounds bind competitively with NAD+. X-ray crystallography demonstrated that the adenosine analogs bind in the AMP-binding pocket of the LigA adenylation domain. Antibacterial activity was observed against pathogenic Gram-positive and atypical bacteria, such as S. aureus, S. pneumoniae, Streptococcus pyogenes, and M. pneumoniae, as well as against Gram-negative pathogens, such as H. influenzae and Moraxella catarrhalis. The mode of action was verified using recombinant strains with altered LigA expression, an Okazaki fragment accumulation assay, and the isolation of resistant strains with ligA mutations. In vivo efficacy was demonstrated in a murine S. aureus thigh infection model and a murine S. pneumoniae lung infection model. Treatment with the adenosine analogs reduced the bacterial burden (expressed in CFU) in the corresponding infected organ tissue as much as 1,000-fold, thus validating LigA as a target for antibacterial therapy.

ETX2514 is a broad-spectrum β-lactamase inhibitor for the treatment of drug-resistant Gram-negative bacteria including Acinetobacter baumannii

Multidrug-resistant (MDR) bacterial infections are a serious threat to public health. Among the most alarming resistance trends is the rapid rise in the number and diversity of β-lactamases, enzymes that inactivate β-lactams, a class of antibiotics that has been a therapeutic mainstay for decades. Although several new β-lactamase inhibitors have been approved or are in clinical trials, their spectra of activity do not address MDR pathogens such as Acinetobacter baumannii. This report describes the rational design and characterization of expanded-spectrum serine β-lactamase inhibitors that potently inhibit clinically relevant class A, C and D β-lactamases and penicillin-binding proteins, resulting in intrinsic antibacterial activity against Enterobacteriaceae and restoration of β-lactam activity in a broad range of MDR Gram-negative pathogens. One of the most promising combinations is sulbactam–ETX2514, whose potent antibacterial activity, in vivo efficacy against MDR A. baumannii infections and promising preclinical safety demonstrate its potential to address this significant unmet medical need.

Discovery of inhibitors of 4'-phosphopantetheine adenylyltransferase (PPAT) to validate PPAT as a target for antibacterial therapy.

ABSTRACT Inhibitors of 4′-phosphopantetheine adenylyltransferase (PPAT) were identified through high-throughput screening of the AstraZeneca compound library. One series, cycloalkyl pyrimidines, showed inhibition of PPAT isozymes from several species, with the most potent inhibition of enzymes from Gram-positive species. Mode-of-inhibition studies with Streptococcus pneumoniae and Staphylococcus aureus PPAT demonstrated representatives of this series to be reversible inhibitors competitive with phosphopantetheine and uncompetitive with ATP, binding to the enzyme-ATP complex. The potency of this series was optimized using structure-based design, and inhibition of cell growth of Gram-positive species was achieved. Mode-of-action studies, using generation of resistant mutants with targeted sequencing as well as constructs that overexpress PPAT, demonstrated that growth suppression was due to inhibition of PPAT. An effect on bacterial burden was demonstrated in mouse lung and thigh infection models, but further optimization of dosing requirements and compound properties is needed before these compounds can be considered for progress into clinical development. These studies validated PPAT as a novel target for antibacterial therapy.

Discovery of bacterial NAD⁺-dependent DNA ligase inhibitors: improvements in clearance of adenosine series.

Optimization of clearance of adenosine inhibitors of bacterial NAD(+)-dependent DNA ligase is discussed. To reduce Cytochrome P-450-mediated metabolic clearance, many strategies were explored; however, most modifications resulted in compounds with reduced antibacterial activity and/or unchanged total clearance. The alkyl side chains of the 2-cycloalkoxyadenosines were fluorinated, and compounds with moderate antibacterial activity and favorable pharmacokinetic properties in rat and dog were identified.

Enantioselective Synthesis and Profiling of Two Novel Diazabicyclooctanone β-Lactamase Inhibitors

The enantioselective synthesis of two novel cyclopropane-fused diazabicyclooctanones is reported here. Starting from butadiene monoxide, the key enone intermediate 7 was prepared in six steps. Subsequent stereoselective introduction of the cyclopropane group and further transformation led to compounds 1a and 1b as their corresponding sodium salt. The great disparity regarding their hydrolytic stability was rationalized by the steric interaction between the cyclopropyl methylene and urea carbonyl. These two novel β-lactamase inhibitors were active against class A, C, and D enzymes.