Adam B. Shapiro

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.

Correction for interference by test samples in high-throughput assays.

In high-throughput biochemical assays performed in multiwell plates, the effect of test samples on the activity of the biochemical system is usually measured by optical means such as absorbance, fluorescence, luminescence, or scintillation counting. The test sample often causes detection interference when it remains in the well during the measurement. Interference may be due to light absorption, fluorescence quenching, sample fluorescence, chemical interaction of the sample with a detection reagent, or depression of the meniscus. A simple method is described that corrects for such interference well by well. The interference is measured in a separate artifact assay plate. An appropriate arithmetic correction is then applied to the measurement in the corresponding well of the activity assay plate. The correction procedure can be used for single-point screening or potency measurements on serial dilutions of test samples.

Inhibitors of the acetyltransferase domain of N-acetylglucosamine-1-phosphate-uridylyltransferase/glucosamine-1-phosphate-acetyltransferase (GlmU). Part 2: Optimization of physical properties leading to antibacterial aryl sulfonamides

A previously described aryl sulfonamide series, originally found through HTS, targets GlmU, a bifunctional essential enzyme involved in bacterial cell wall synthesis. Using structure-guided design, the potency of enzyme inhibition was increased in multiple isozymes from different bacterial species. Unsuitable physical properties (low LogD and high molecular weight) of those compounds prevented them from entering the cytoplasm of bacteria and inhibiting cell growth. Further modifications described herein led to compounds that possessed antibacterial activity, which was shown to occur through inhibition of GlmU. The left-hand side amide and the right-hand side sulfonamides were modified such that enzyme inhibitory activity was maintained (IC(50) <0.1 μM against GlmU isozymes from Gram-negative organisms), and the lipophilicity was increased giving compounds with LogD -1 to 3. Antibacterial activity in an efflux-pump deficient mutant of Haemophilus influenzae resulted for compounds such as 13.

Molecular Mechanisms of Sulbactam Antibacterial Activity and Resistance Determinants in Acinetobacter baumannii

ABSTRACT Sulbactam is a class A β-lactamase inhibitor with intrinsic whole-cell activity against certain bacterial species, including Acinetobacter baumannii. The clinical use of sulbactam for A. baumannii infections is of interest due to increasing multidrug resistance in this pathogen. However, the molecular drivers of its antibacterial activity and resistance determinants have yet to be precisely defined. Here we show that the antibacterial activities of sulbactam vary widely across contemporary A. baumannii clinical isolates and are mediated through inhibition of the penicillin-binding proteins (PBPs) PBP1 and PBP3, with very low frequency of resistance; the rare pbp3 mutants with high levels of resistance to sulbactam are attenuated in fitness. These results support further investigation of the potential clinical utility of sulbactam.

A Homogeneous, High-Throughput Fluorescence Anisotropy-Based DNA Supercoiling Assay

The degree of supercoiling of DNA is vital for cellular processes, such as replication and transcription. DNA topology is controlled by the action of DNA topoisomerase enzymes. Topoisomerases, because of their importance in cellular replication, are the targets of several anticancer and antibacterial drugs. In the search for new drugs targeting topoisomerases, a biochemical assay compatible with automated high-throughput screening (HTS) would be valuable. Gel electrophoresis is the standard method for measuring changes in the extent of supercoiling of plasmid DNA when acted upon by topoisomerases, but this is a low-throughput and laborious method. A medium-throughput method was described previously that quantitatively distinguishes relaxed and supercoiled plasmids by the difference in their abilities to form triplex structures with an immobilized oligonucleotide. In this article, the authors describe a homogeneous supercoiling assay based on triplex formation in which the oligonucleotide strand is labeled with a fluorescent dye and the readout is fluorescence anisotropy. The new assay requires no immobilization, filtration, or plate washing steps and is therefore well suited to HTS for inhibitors of topoisomerases. The utility of this assay is demonstrated with relaxation of supercoiled plasmid by Escherichia coli topoisomerase I, supercoiling of relaxed plasmid by E. coli DNA gyrase, and inhibition of gyrase by fluoroquinolones and nalidixic acid.

A High-Throughput, Homogeneous, Fluorescence Resonance Energy Transfer-Based Assay for Phospho-N-acetylmuramoyl-pentapeptide Translocase (MraY)

Peptidoglycan biosynthesis is an essential process in bacteria and is therefore a suitable target for the discovery of new antibacterial drugs. One of the last cytoplasmic steps of peptidoglycan biosynthesis is catalyzed by the integral membrane protein MraY, which attaches soluble UDP-N-acetylmuramoyl-pentapeptide to the membrane-bound acceptor undecaprenyl phosphate. Although several natural product–derived inhibitors of MraY are known, none have the properties necessary to be of clinical use as antibacterial drugs. Here we describe a novel, homogeneous, fluorescence resonance energy transfer–based MraY assay that is suitable for high-throughput screening for novel MraY inhibitors. The assay allows for continuous measurement, or it can be quenched prior to measurement.

Continuous fluorescence anisotropy-based assay of BOCILLIN FL penicillin reaction with penicillin binding protein 3

We report a simple, rapid, and reproducible fluorescence anisotropy-based method for measuring rate constants for acylation and deacylation of soluble penicillin binding protein (PBP) constructs by compounds in microtiter plates by means of competition with time-dependent acylation by BOCILLIN FL. The method is demonstrated by measuring the acylation rate constants of the PBP3 periplasmic domains from Pseudomonas aeruginosa and Acinetobacter baumannii by BOCILLIN FL, aztreonam, meropenem, and ceftazidime. The new method requires very little protein and can be completed in approximately 1h per compound. A set of BOCILLIN FL acylation progress curves collected over a range of competitor concentrations is fit globally to a kinetic model by numerical integration. First-order deacylation rate constants could also be measured, as demonstrated with a catalytically impaired mutant OXA-10 β-lactamase.

Inhibitors of Acetyltransferase Domain of N-Acetylglucosamine-1-Phosphate-Uridyltransferase/ Glucosamine-1-Phosphate-Acetyltransferase (Glmu). Part 1: Hit to Lead Evaluation of a Novel Arylsulfonamide Series.

A novel arylsulfonamide-containing series of compounds represented by 1, discovered by highthroughput screening, inhibit the acetyltransferase domain of N-acetylglucosamine-1-phosphate-uridyltransferase/glucosamine-1-phosphate-acetyltransferase (GlmU). X-ray structure determination confirmed that inhibitor binds at the site occupied by acetyl-CoA, indicating that series is competitive with this substrate. This letter documents our early hit-to-lead evaluation of the chemical series and some of the findings that led to improvement in in-vitro potency against Gram-negative and Gram-positive bacterial isozymes, exemplified by compound 40.

Allosteric inhibition of the DNA-dependent ATPase activity of Escherichia coli DNA gyrase by a representative of a novel class of inhibitors

A novel class of bacterial DNA gyrase inhibitors has been shown previously to form a ternary complex with DNA and gyrase in a site distinct from the fluoroquinolone and ATP binding sites and does not cause double-strand-cleaved complex stabilization like fluoroquinolones. We show that, unlike fluoroquinolones, a representative compound inhibits DNA-dependent ATP hydrolysis by Escherichia coli gyrase and also blocks cleaved complex stabilization by ciprofloxacin. Conversely, ciprofloxacin blocks ATPase inhibition by the novel compound. We conclude that the compound acts allosterically to inhibit ATP binding or hydrolysis and interferes with the gyrase catalytic cycle at a different point than ciprofloxacin.

A high-throughput fluorescence anisotropy-based assay for human topoisomerase II β-catalyzed ATP-dependent supercoiled DNA relaxation

Because of their essentiality for DNA replication, transcription, and repair, type II topoisomerases are targets for antibacterial and anticancer drugs. There are two type II topoisomerases in humans, topoisomerase IIα (TOP2A) and topoisomerase IIβ (TOP2B), and two in bacteria, gyrase and topoisomerase IV. Inhibition of one or both of the human type II topoisomerases by antibacterial compounds targeting their bacterial counterparts could result in toxicity. In addition, side effects of anticancer drugs targeting TOP2A could result from inhibition of TOP2B. A simple and rapid biochemical assay for the activity of TOP2A and TOP2B would be advantageous for screening for novel inhibitors, testing them for selectivity for one enzyme over the other, and testing for potential toxicity of antibacterial type II topoisomerases mediated by human topoisomerase II inhibition. In this paper, we show that a previously reported high-throughput, fluorescence anisotropy-based assay for ATP-dependent relaxation of supercoiled DNA by human TOP2A can also be used under identical conditions for human TOP2B. We used this assay to compare the potencies versus both enzymes of 19 compounds reported in the literature to inhibit human and/or bacterial type II topoisomerases. We also used the assay to investigate the effect of ATP concentration on inhibitor potencies.