We characterized the inhibition of Neisseria gonorrhoeae type II topoisomerases gyrase and topoisomerase IV by AZD0914 (Figure 1), a novel spiropyrimidinetrione antibacterial compound that is currently in clinical trials for treatment of drug-resistant gonorrhea. AZD0914 has potent bactericidal activity against Neisseria gonorrhoeae, including multidrug-resistant strains; key Gram-positive; fastidious Gram-negative; atypical and anaerobic bacterial species (Huband, M.D., Bradford, P.A., Otterson, L.G., Basrab, G.S., Giacobe, R.A., Patey, S.A., Kutschke, A.C., Johnstone, M.R., Potter, M.E., Miller, P.F., and Mueller, J.P. (2014) In Vitro Antibacterial Activity of AZD0914: A New Spiropyrimidinetrione DNA Gyrase/Topoisomerase Inhibitor with Potent Activity against Gram-Positive, Fastidious Gram-Negative, and Atypical Bacteria. Antimicrob. Agents Chemother.. 59:467-474.). AZD0914 inhibited DNA biosynthesis preferentially to other macromolecules in Escherichia coli, and induced the SOS response to DNA damage in E. coli. AZD0914 stabilized the enzyme-DNA cleaved-complex for N. gonorrhoeae gyrase and topoisomeraseIV. The potency of AZD0914 for inhibition of supercoiling and the stabilization of cleaved complex by N. gonorrhoeae gyrase increased in a fluoroquinolone-resistant mutant enzyme. When a mutation, conferring mild resistance to AZD0914, was present in the fluoroquinolone-resistant mutant, the potency of ciprofloxacin for inhibition of supercoiling and stabilization of cleaved complex was increased greater than 20-fold. In contrast to ciprofloxacin, religation of the cleaved DNA did not occur in the presence of AZD0914 upon removal of magnesium from the DNAgyrase-inhibitor complex. AZD0914 had relatively low potency for inhibition of human type II topoisomerases α and β . In 2013 the Center of Disease Control (CDC) classified the threat level associated with the unmet medical need resulting from multi drug resistant N. gonorrhoeae as urgent (1) and it estimated that at least 800,000 cases of gonorrhea occur per year in the US alone (2). Fluoroquinolone antibacterial drugs previously offered an effective treatment option for gonorrhea. Over the last decade, however, development of resistance, first against fluoroquinolones and subsequently against all drugs used for first line treatment, such as cefixime and ceftriaxone (3,4) demanded the development of novel agents to combat highly resistant N. gonorrhoeae. Fluoroquinolones, one of the most successful classes of antibiotics on the market (5,6) target the homologous bacterial type II topoisomerases gyrase and topoisomerase IV (TopoIV). Both enzymes are conserved across most bacterial pathogens and are essential for cellular functions including DNA replication and decatenation. DNA gyrase, a heterotetramer of two subunits, GyrA2-GyrB2, introduces negative supercoils in DNA ahead of the replication fork, thereby relieving torsional strain during replication (6-8). TopoIV, a ParC2-ParE2 heterotetramer, catalyzes decatenation, which is essential for separating linked catenanes of two DNA molecules during replication. Type II topoisomerases modulate the topology of DNA in eukaryotes (6-11). The human nuclear type II topoisomerases TopoIIα and β are the targets of inhibitors that have clinical utility for the treatment of cancer (12,13). Sufficient selectivity by antibacterial drugs for inhibition of the bacterial over human topoisomerases at clinically relevant doses has been achieved, encouraging continued exploration of these enzymes as viable targets for novel antibacterial drugs. The molecular mechanism of type II topoisomerases is described by a functional model termed the two-gate mechanism (14-16). The catalytic cycle has several stages that can be blocked by inhibitors. Aminocoumarins, such as novobiocin, inhibit gyrase by competing with at C aeton U iv O C U L on July 9, 2015 hp://w w w .jb.org/ D ow nladed from Mode Of Inhibition of a novel DNA gyrase inhibitor with activity against Neisseria gonorrhoeae 3 ATP, thereby blocking the ATPase activity of the GyrB subunit. Fluoroquinolones, such as ciprofloxacin, stabilize the DNA-cleaved gyraseDNA complex by binding to an interface between DNA, GyrA and GyrB (17). Recently, a few novel classes of inhibitors have been reported that target bacterial topoisomerase II with modes of inhibition distinct from the fluoroquinolones and aminocoumarins (18,19). In this paper, we characterize the mechanism of inhibition of gyrase and TopoIV from N. gonorrhoeae by the novel spiropyrimidinetrione AZD0914 (Figure 1), which is currently in clinical trials as a treatment for drug-resistant N. gonorrhoeae infections. We examine the effects of ciprofloxacin and AZD0914 resistance mutations on inhibition of N. gonorrhoeae gyrase by these compounds, and measure inhibition of human TopoIIα and β by AZD0914. MATERIALS AND METHODS Materials – Buffers, salts, and routine biochemicals were sourced from Sigma-Aldrich (St. Louis, MO) and were of reagent grade or higher purity. Plasmid NTC0109711-U6shRNA, a derivative of pCR4-TOPO, was used in supercoiling, cleaved complex and religation assays. Relaxation of the supercoiled form was done as previously described (20). It was obtained in supercoiled form from Nature Technologies (Lincoln, NE). Kinetoplast DNA used in decatenation assays was obtained from Topogen, Inc. (Port Orange, FL). Ciprofloxacin HCl was from MP Biomedicals (Santa Ana, CA). Etoposide and ATP were from SigmaAldrich. Human TopoIIα was from Affymetrix (Santa Clara, CA). Human TopoIIβ was supplied by Prof. Caroline A. Austin, University of Newcastle-upon-Tyne. Chemistry — AZD0914 was synthesized as described by Basarab et al. (21). Inhibition of Macromolecule Biosynthesis — The procedure was performed according to Hilliard (10), with modifications published previously (22). E. coli was grown at room temperature in cation-adjusted Mueller Hinton Broth 1 (Sigma-Aldrich, St. Louis MO) in the presence of radiolabeled precursors. As positive controls, rifamycin blocked the incorporation of labeled uridine into RNA, erythromycin blocked labeled valine and leucine incorporation into protein, penicillin G blocked labeled Nacetylglucosamine incorporation into cell wall, triclosan blocked labeled acetic acid incorporation into fatty acids, and the aminocoumarin novobiocin as well as the fluoroquinolone norfloxacin blocked DNA synthesis. SOS induction assay — The SOS induction assay was performed as described (23). DNA manipulations and plasmid

Background: Inhibition of Neisseria gonorrhoeae type II topoisomerases gyrase and TopoIV by the antibacterial spiropyrimidinetrione AZD0914 was investigated. Results: AZD0914 stabilized the gyrase-DNA complex with double strand DNA cleavage, retaining potency in a fluoroquinolone-resistant mutant, with little inhibition of human type II topoisomerases. Conclusion: AZD0914 displays mechanistic differences from fluoroquinolones. Significance: AZD0914 has the potential to combat drug-resistant gonorrhea. We characterized the inhibition of Neisseria gonorrhoeae type II topoisomerases gyrase and topoisomerase IV by AZD0914 (AZD0914 will be henceforth known as ETX0914 (Entasis Therapeutics)), a novel spiropyrimidinetrione antibacterial compound that is currently in clinical trials for treatment of drug-resistant gonorrhea. AZD0914 has potent bactericidal activity against N. gonorrhoeae, including multidrug-resistant strains and key Gram-positive, fastidious Gram-negative, atypical, and anaerobic bacterial species (Huband, M. D., Bradford, P. A., Otterson, L. G., Basrab, G. S., Giacobe, R. A., Patey, S. A., Kutschke, A. C., Johnstone, M. R., Potter, M. E., Miller, P. F., and Mueller, J. P. (2014) In Vitro Antibacterial Activity of AZD0914: A New Spiropyrimidinetrione DNA Gyrase/Topoisomerase Inhibitor with Potent Activity against Gram-positive, Fastidious Gram-negative, and Atypical Bacteria. Antimicrob. Agents Chemother. 59, 467–474). AZD0914 inhibited DNA biosynthesis preferentially to other macromolecules in Escherichia coli and induced the SOS response to DNA damage in E. coli. AZD0914 stabilized the enzyme-DNA cleaved complex for N. gonorrhoeae gyrase and topoisomerase IV. The potency of AZD0914 for inhibition of supercoiling and the stabilization of cleaved complex by N. gonorrhoeae gyrase increased in a fluoroquinolone-resistant mutant enzyme. When a mutation, conferring mild resistance to AZD0914, was present in the fluoroquinolone-resistant mutant, the potency of ciprofloxacin for inhibition of supercoiling and stabilization of cleaved complex was increased greater than 20-fold. In contrast to ciprofloxacin, religation of the cleaved DNA did not occur in the presence of AZD0914 upon removal of magnesium from the DNA-gyrase-inhibitor complex. AZD0914 had relatively low potency for inhibition of human type II topoisomerases α and β.

Respiratory syncytial virus (RSV) drug discovery has been hindered by the lack of good chemistry starting points and would benefit from robust and convenient assays for high-throughput screening (HTS). In this paper, we present the development and optimization of a 384-well RSV replicon assay that enabled HTS for RSV replication inhibitors with a low bio-containment requirement. The established replicon assay was successfully implemented for high-throughput screening. A validation screen was performed which demonstrated high assay performance and reproducibility. Assay quality was further confirmed via demonstration of appropriate pharmacology for different classes of RSV replication tool inhibitors. RSV replicon and cytotoxicity assays were further developed into a multiplexed format that measured both inhibition of viral replication and cytotoxicity from the same well. This provided a time and cost efficient approach to support lead optimization. In summary, we have developed a robust RSV replicon assay to help expedite the discovery of novel RSV therapeutics.

ABSTRACT Bacterial biosensor strains can be useful tools for the discovery and characterization of antibacterial compounds. A plasmid-based reporter vector containing a transcriptional fusion between the recA promoter and green fluorescence protein gene was introduced into an Escherichia coli Δ tolC strain to create a biosensor strain that selectively senses inhibitors of DNA metabolism via the SOS response. The strain was used to develop a high-throughput assay to identify new inhibitors of DNA metabolism. Screening of the AstraZeneca compound library with this strain identified known inhibitors of DNA metabolism, as well as novel chemotypes. The cellular target of one novel series was elucidated as DNA gyrase through genetic characterization of laboratory-generated resistant mutants followed by 50% inhibitory concentration measurements in a DNA gyrase activity assay. These studies validated the use of this antibiotic biosensor strain to identify novel selective inhibitors of DNA metabolism by high-throughput screening.

The respiratory syncytial virus (RSV) L protein is a viral RNA-dependent RNA polymerase that contains multiple enzyme activities required for RSV replication. The RSV L inhibitors described in literature are limited by their cytotoxicity or the lack of RSV B subtype coverage. Here, we characterize a new RSV L inhibitor with strong antiviral activity against both RSV A and B subtypes and no detectable cytotoxicity. This compound, AZ-27, was equally active against RSV live viruses and subgenomic replicons and demonstrated advantages over other classes of RSV inhibitors in time-of-addition and cell line dependency studies. Resistance studies identified a dominant mutation in the putative capping enzyme domain of L protein, which conferred strong resistance to the AZ-27 series but not other classes of RSV inhibitors, supporting RSV L protein as the direct target for AZ-27. This novel and broad-spectrum RSV L polymerase inhibitor may pave the way toward an efficacious RSV therapeutic and provide a new tool for interrogation of the L protein function.

Targeting viral polymerases has been a proven and attractive strategy for antiviral drug discovery. Herein we describe our effort in improving the antiviral activity and physical properties of a series of benzothienoazepine compounds as respiratory syncytial virus (RSV) RNA polymerase inhibitors. The antiviral activity and spectrum of this class was significantly improved by exploring the amino substitution of the pyridine ring, resulting in the discovery of the most potent RSV A polymerase inhibitors reported to date.