br Acknowledgements This work was supported by a
Acknowledgements This work was supported by a Grant for the Program for the Strategic Research Foundation at Private Universities S1101017 organized by the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), Japan and JSPS KAKENHI Grant Numbers JP22560209 and JP5K05842.
There is an increased need for new death associated protein kinase because many bacterial pathogens are developing resistance to existing drugs due to overuse and overprescription of these drugs. The problem is magnified by the lack of new antibacterial agents and the reduced interest of the pharmaceutical industry to invest in their discovery and development. There is a more significant need for treatments in the Gram-negative arena as there are less options for treatment and target inhibition is more difficult. One strategy for overcoming resistance to existing antibacterial drugs is the development of drugs that inhibit new or novel targets. Bacterial NAD-dependent DNA ligase (LigA) was selected as a new target because of its essentiality for growth of a broad spectrum of Gram-positive and Gram-negative pathogens and limited homology to the human DNA ligases, which use ATP as a substrate instead of NAD. DNA ligases are critical in DNA repair, replication, and recombination. LigA inhibitors have been reported by several groups. Bayer reported a series of potent pyridochromanone inhibitors of LigA. AstraZeneca reported a series of potent adenosine based inhibitors. There have also been reports of inhibitors from a number of classes including: arylamino acids, tetracyclic indoles, glycosyl ureides and glycosylamines, pyrimidopyrimidines, aminoalkoxypyrimidines, thienopyridines, and anilines., In an effort to develop a novel broad spectrum LigA inhibitor, we used previously disclosed structure–activity relationships (SAR) for LigA inhibitors; results from in-house high-throughput screens (HTS), including a high-concentration fragment screen and X-ray crystal structures of LigA with inhibitors bound., , In the design and build we also included that would provide desirable physical properties that may influence target access and in vivo stability in the process. Two earlier scaffolds that were used for mapping the active site and that suggested specific interactions that could be accessed via fragments are shown in . Compound potently inhibits LigA activity in enzyme inhibition assays. The compound () also efficiently inhibits bacterial growth in vitro (cellular assay to evaluate in vitro potency), but is weak (⩾32μg/ml) against the other species evaluated and has low solubility. In the crystal structure, , the amino and carboxamide groups hydrogen bond to the backbone carbonyl and amide of Leu117. The amino group hydrogen bonds with Glu114. The carbonyl group is directed toward the ribose binding site and hydrogen bonds with a network of water molecules. The ring nitrogen and oxygen are directed toward Lys291. Lastly the aromatic core π-stacks with Tyr226, behind inhibitors, but not highlighted in . The adenosine based inhibitor, compound also potently inhibits LigA in biochemical enzyme assays but weakly inhibits bacterial growth in cells. The compound has good solubility and low plasma protein binding. In the crystal structure, , the Lys291 is pushed out of the way and Glu114 interacts with the exocyclic amine. The nitrogen at position-9 of the adenine ring hydrogen bonds with the Leu117 backbone amide. The adenine ring π-stacks with Tyr226 and the thioether fills a hydrophobic pocket which was found to be key for binding affinity in the adenosine series. The ribose 2′-OH also hydrogen bonds with the Glu174 side chain and the 5′-OH interacts with Leu82. An overlay of compounds and obtained from the LigA X-ray structures is shown in with several key active site residues highlighted. Compounds and are fragments from an earlier LigA NMR screen for which ICs and a crystal structure were determined. The X-ray crystal structure of the complex of LigA with compound is shown in . Two hydrogen bonds are made with the quinoline nitrogen (3.24Å) and the phenol OH (2.96Å) by Lys291. Additionally the carboxamide makes two hydrogen bonds with the backbone of Leu117. The methyl group partially fills the hydrophobic pocket and the aromatic core π-stacks with Tyr226.