Mode Of Inhibition of a novel DNA gyrase inhibitor with activity against Neisseria gonorrhoeae Inhibition of Neisseria gonorrhoeae type II Topoisomerases by the Novel Spiropyrimidinetrione AZD 0914 * #

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