Small-molecule inhibitor binding to an N -acyl-homoserine lactone synthase

Abstract

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...

Frontier studies on highly selective bio-regulators useful for environmentally benign agricultural production

“…74) Analog 79 showed 91% and 51% inhibitory activities at concentrations of 100 and 10 μM, respectively. A kinetic analysis indicated that 79 acted as a competitive inhibitor of TofI activity with a K i value of 4.8 μM.…”

“…This is the first study to show that flavonoids exuded from roots inhibited the function of strigolactones. Alpinumisoflavone (74), which was one of the most potent inhibitors of germ tube growth, did not inhibit the formation of new branching hyphae at 0.31, 0.63, or 1.25 μg/disk. Unexpectedly, high-order branches up to sixth or seventh hyphae were formed by the treatment with 74 at the three tested concentrations in the presence of 68.…”

“…4). The other four compounds were identified as the pyranoisoflavones, licoisoflavone B (72), sophoraisoflavone A (73), alpinumisoflavone (74), and 3′-hydroxy-4′-O-methylalupinumisoflavone (75) (Fig. 4).…”

Exploiting Quorum Sensing To Confuse Bacterial Pathogens

“…In contrast, E9C-3oxoC6 was found to competitively inhibit C8HSL binding to the AHL receptor TofR. Structural studies of TofI in its apo form and in complex with J8-C8 revealed that the inhibitor binds in a pocket with high specificity for the octanoyl chain, suggesting that J8-C8 is a competitive inhibitor of the acyl-ACP carrier utilized for C8HSL synthesis, and thus it has been postulated that modulation of acyl chain length in J8-C8 mimics may be a means by which to rationally design enzyme-specific LuxI family inhibitors (119). Separately, holo-ACP, the SAM analogues S-adenosylhomocysteine, S-adenosylcysteine, and sinefungin, the reaction product methylthioadenosine (MTA), and the possible reaction intermediate butyryl-SAM were found to inhibit C4HSL production by RhlI in vitro (27), demonstrating that LuxI-type synthase activity could also potentially be inhibited by end products, reaction intermediates, and/or substrate analogues.…”

“…A screen of 55 "hits" from a previous quorum-sensing screen in P. aeruginosa (118) yielded the identification of two strong inhibitors of QS in B. glumae (119). Although both compounds inhibited C8HSL production, they were found to do so in different ways; whereas both E9C-3oxoC6 and J8-C8 (Table 3) suppressed toxoflavin production in WT cells, only E9C-3oxo-C6 had an effect in a tofI mutant strain, suggesting that the two compounds functioned by different mechanisms and that the molecular target of J8-C8 was the TofI enzyme (119). Direct inhibition of TofI by J8-C8 was subsequently confirmed in vitro.…”