Quorum sensing (QS) controls certain behaviors of bacteria in response to population density. In Gram-negative bacteria, QS is often mediated by N-acyl-L-homoserine lactones (acyl-HSLs). Because QS influences the virulence of many pathogenic bacteria, synthetic inhibitors of acyl-HSL synthases might be useful therapeutically for controlling pathogens. However, rational design of a potent QS antagonist has been thwarted by the lack of information concerning the binding interactions between acyl-HSL synthases and their ligands. In the Gram-negative bacterium Burkholderia glumae, QS controls virulence, motility, and protein secretion and is mediated by the binding of N-octanoyl-L-HSL (C8-HSL) to its cognate receptor, TofR. C8-HSL is synthesized by the acyl-HSL synthase TofI. In this study, we characterized two previously unknown QS inhibitors identified in a focused library of acyl-HSL analogs. Our functional and X-ray crystal structure analyses show that the first inhibitor, J8-C8, binds to TofI, occupying the binding site for the acyl chain of the TofI cognate substrate, acylated acyl-carrier protein.Moreover, the reaction byproduct, 5′-methylthioadenosine, independently binds to the binding site for a second substrate, Sadenosyl-L-methionine. Closer inspection of the mode of J8-C8 binding to TofI provides a likely molecular basis for the various substrate specificities of acyl-HSL synthases. The second inhibitor, E9C-3oxoC6, competitively inhibits C8-HSL binding to TofR. Our analysis of the binding of an inhibitor and a reaction byproduct to an acyl-HSL synthase may facilitate the design of a new class of QS-inhibiting therapeutic agents. Q uorum sensing (QS) is an intercellular signaling process that mediates certain behaviors of bacteria (including bioluminescence, biofilm formation, motility, and virulence factor production) in response to the bacterial cell population density (1-3). In Gram-negative bacteria, QS is often mediated by Nacyl-L-homoserine lactones (acyl-HSLs), which are synthesized by the LuxI family of acyl-HSL synthases from S-adenosyl-Lmethionine (SAM) and acylated acyl-carrier protein (acyl-ACP), with the release of holo-ACP and 5′-methylthioadenosine (MTA) as byproducts (SI Appendix, Fig. S1A) (4, 5). Compounds of the acyl-HSL class share a homoserine lactone ring moiety, but the acyl chains conjugated to the ring via an amide bond vary in length, oxidation state at C3, and amount of saturation (SI Appendix, Fig. S1A). The recent finding that p-coumarate is an alternative substrate for acyl-ACP has extended the known range of possible acyl-HSL substrates (6). On the other hand, the acyl-HSL receptor is a transcriptional regulator that controls the expression of target genes in response to acyl-HSL binding (1-3).Among the hundreds of genes regulated by QS, the most widely studied genes are those related to virulence; these genes are of particular interest because QS disruption is being investigated as a strategy for controlling virulent pathogens (7-9). QS inhibitors can act by suppressing a...

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Kynurenine 3-monooxygenase (KMO) inhibitors have been developed for the treatment of neurodegenerative disorders. The mechanisms of flavin reduction and hydrogen peroxide production by KMO inhibitors are unknown. Herein, we report the structure of human KMO and crystal structures of Saccharomyces cerevisiae (sc) and Pseudomonas fluorescens (pf) KMO with Ro 61-8048. Proton transfer in the hydrogen bond network triggers flavin reduction in p-hydroxybenzoate hydroxylase, but the mechanism triggering flavin reduction in KMO is different. Conformational changes via π-π interactions between the loop above the flavin and substrate or non-substrate effectors lead to disorder of the C-terminal α helix in scKMO and shifts of domain III in pfKMO, stimulating flavin reduction. Interestingly, Ro 61-8048 has two different binding modes. It acts as a competitive inhibitor in scKMO and as a non-substrate effector in pfKMO. These findings provide understanding of the catalytic cycle of KMO and insight for structure-based drug design of KMO inhibitors.

Intestinal epithelial cells are adapted in mucosal hypoxia and hypoxia-inducible factors in these cells can fortify barrier integrity to support mucosal tissue healing. Here we investigated whether hypoxia-related pathways could be proposed as potential therapeutic targets for inflammatory bowel disease. We developed a novel hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitor, CG-598 which stabilized HIF-1α in the gut tissue. Treatment of CG-598 did not affect extra-intestinal organs or cause any significant adverse effects such as erythropoiesis. In the experimental murine colitis model, CG-598 ameliorated intestinal inflammation with reduction of inflammatory lesions and pro-inflammatory cytokines. CG-598 treatment fortified barrier function by increasing the expression of intestinal trefoil factor, CD73, E-cadherin and mucin. Also, IL-10 and IL-22 were induced from lamina propria CD4+ T-cells. The effectiveness of CG-598 was comparable to other immunosuppressive therapeutics such as TNF-blockers or JAK inhibitors. These results suggest that CG-598 could be a promising therapeutic candidate to treat inflammatory bowel disease.

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