In the era of increased antibiotic resistance, targeting enzymes involved in bacterial communication (quorum sensing) represents a new strategy to fight bacterial infections. LsrK is a kinase responsible for the phosphorylation of autoinducer-2, a signaling molecule involved in quorum sensing. Inhibiting LsrK would lead to quorum sensing inactivation and interfere with the pathogenesis. In this study, we built the first LsrK 3D model and performed virtual screening of a locally available database. Selected compounds were tested against LsrK, and the analogue search conducted based on the positive hits led to the identification of low-micromolar LsrK inhibitors. These results prove the utility of the model and provide the first class of LsrK inhibitors to be further optimized as antivirulence agents.

Antibiotic resistance is posing a continuous threat to global public health and represents a huge burden for society as a whole. In the last decade, the interference with bacterial Quorum Sensing (QS) (i.e., cell-cell communication) mechanisms has extensively been investigated as a valid therapeutic approach in the pursuit of a next generation of antimicrobials. (S)-4,5-dihydroxy-2,3pentanedione, commonly known as (S)-DPD, a small signaling molecule that modulates QS in both Gram-negative and Gram-positive bacteria, is phosphorylated by LsrK and the resulting phospho-DPD activates QS. We designed and prepared a small library of DPD derivatives, characterized by five different scaffolds and evaluated their LsrK inhibition in the context of QS interference. SAR studies highlighted the pyrazole moiety as an essential structural element for LsrK inhibition. Particularly, four compounds were found to be micromolar LsrK inhibitors (IC50 ranging between 100 µM and 500 µM) encouraging further exploration of novel analogues as potential new antimicrobials. imination reagent and then deprotected (Scheme 1). Briefly, alkyne 1 was dissolved in DMF and then Pd(OAc)2, PPh3, KOAc and the appropriate o-bromoaldehyde were added. The reaction mixtures were irradiated at 80 °C for 1-2 hours, ammonium acetate was added and the resulting mixtures were irradiated again at 150 °C, thus affording the desired protected products 3a-g (Scheme 1). Heteroaromatic o-bromoaldehydes (i.e., 3-bromofuran-2-carbaldehyde 2e, 3-bromothiophene-2carbaldehyde 2f and 5-bromo-2-methyl-4-thiazolecarboxaldehyde 2g) led to isolation of the corresponding furopyridine, thienopyridine and thiazolopyridine derivatives 3e-f. Final acidic treatment yielded the monosubstituted annulated pyridines-Het-DPD derivatives 4a-g (Scheme 1). Scheme 1: Synthesis of monosubstituted annulated pyridines-Het-DPD derivatives 4a-g. Reagents and conditions: (a) 2a-g (0.9 eq), Pd(OAc)2 (1.8% mol), PPh3 (3.6% mol), KOAc (1.8 eq), DMF, mw, 80 °C, 1h-2h; (a') NH4OAc (1.8 eq), MW, 150 °C, 2h-3h; (b) 12M HCl (cat.), 1,4-dioxane, 0 °C to rt, 1h-3h Sonogashira coupling 43 of terminal alkyne 1 with acyl chlorides, followed by addition of amidinium salts to the corresponding ynones and final acidic removal of the acetal protecting group provided rapid access to 2,4,6-trisubstituted pyrimidines-Het-DPD derivatives (Figure 2, series B). Benzoyl chloride 5a was selected to screen three different conditions for the synthesis of ynone 6a. Surprisingly, copper-, ligand-and solvent-free acylation 51 as well as the use of a mixture of PdCl2(CH3CN)2, Sphos and Cs2CO3 26 did not furnish the desired product. Good results were obtained with the copper-and palladium-catalyzed system proposed by Karpov et al. 50 which allowed us to obtain compound 6a with a yield of 87%. 52 Three different ynones (6a-c) were reacted with six different amidinium salts and the resulting products 7a-f treated under acidic conditions (i.e., Scheme 2, conditions c) to afford the 2,4,6trisubstituted pyrimidines-Het-DPD derivat...

Pseudomonas aeruginosa is an opportunistic human pathogen recognized as a critical threat by the World Health Organization because of the dwindling number of effective therapies available to treat infections. Over the past decade, it has become apparent that the glyoxylate shunt plays a vital role in sustaining P. aeruginosa during infection scenarios. The glyoxylate shunt comprises two enzymes: isocitrate lyase and malate synthase isoform G. Inactivation of these enzymes has been reported to abolish the ability of P. aeruginosa to establish infection in a mammalian model system, yet we still lack the structural information to support drug design efforts. In this work, we describe the first X-ray crystal structure of P. aeruginosa malate synthase G in the apo form at 1.62 Å resolution. The enzyme is a monomer composed of four domains and is highly conserved with homologues found in other clinically relevant microorganisms. It is also dependent on Mg 2+ for catalysis. Metal ion binding led to a change in the intrinsic fluorescence of the protein, allowing us to quantitate its affinity for Mg 2+ . We also identified putative drug binding sites in malate synthase G using computational analysis and, because of the high resolution of the experimental data, were further able to characterize its hydration properties. Our data reveal two promising binding pockets in malate synthase G that may be exploited for drug design.

Quorum sensing is being investigated as an alternative therapeutic strategy in antibacterial drug discovery programs aimed at combatting bacterial resistance. LsrK is an autoinducer-2 kinase (belongs to the sugar kinase family), playing a key role in the phosphorylation of the autoinducer-2 (AI-2) signaling molecules involved in quorum sensing. Inhibiting LsrK could result in reduced pathogenicity by interfering with quorum sensing signaling. Previously, we have generated homology models to employ in structure-based virtual screening and successfully identified the first class of LsrK inhibitors. While conducting these studies, the crystal structure of LsrK was released, providing us with an opportunity to evaluate the reliability and quality of our models. A comparative structural analysis of the crystal structure and homology models revealed consistencies among them in the overall structural fold and binding site. Furthermore, the binding characteristics and conformational changes of LsrK have been investigated using molecular dynamics to inspect whether LsrK undergoes similar conformational changes as that of sugar kinases. These studies revealed the flexibility of the LsrK C-terminal domain (Domain II) attributing to the conformational changes in LsrK resulting in open and closed states during the phosphorylation. Further, simulations provided us with insights into the flexibility of a loop in Domain I that can influence the ligand accessibility to the LsrK binding site.

The Cover Feature shows the bacterial quorum sensing (QS) signaling and role of LsrK kinase enzyme in the phosphorylation of signaling molecules involved in QS. The LsrK protein structure was modelled and utilized to identify new inhibitors of LsrK kinase. The structure‐based drug design studies and following experimental efforts revealed the potency of anthranilic acid derivatives as LsrK kinase inhibitors. These are the first class of LsrK kinase inhibitors reported to date. More information can be found in the Full Paper by Antti Poso et al. on page 2400 in Issue 22, 2018 (DOI: 10.1002/cmdc.201800548).