Group A Streptococcus Secreted Esterase Hydrolyzes Platelet-Activating Factor to Impede Neutrophil Recruitment and Facilitate Innate Immune Evasion

The innate immune system is the first line of host defense against invading organisms. Thus, pathogens have developed virulence mechanisms to evade the innate immune system. Here, we report a novel means for inhibition of neutrophil recruitment by Group A Streptococcus (GAS). Deletion of the secreted esterase gene (designated sse) in M1T1 GAS strains with (MGAS5005) and without (MGAS2221) a null covS mutation enhances neutrophil ingress to infection sites in the skin of mice. In trans expression of SsE in MGAS2221 reduces neutrophil recruitment and enhances skin invasion. The sse deletion mutant of MGAS5005 (ΔsseMGAS5005) is more efficiently cleared from skin than the parent strain. SsE hydrolyzes the sn-2 ester bond of platelet-activating factor (PAF), converting biologically active PAF into inactive lyso-PAF. KM and kcat of SsE for hydrolysis of 2-thio-PAF were similar to those of the human plasma PAF acetylhydrolase. Treatment of PAF with SsE abolishes the capacity of PAF to induce activation and chemotaxis of human neutrophils. More importantly, PAF receptor-deficient mice significantly reduce neutrophil infiltration to the site of ΔsseMGAS5005 infection. These findings identify the first secreted PAF acetylhydrolase of bacterial pathogens and support a novel GAS evasion mechanism that reduces phagocyte recruitment to sites of infection by inactivating PAF, providing a new paradigm for bacterial evasion of neutrophil responses

3-(1H-Indol-3-yl)-2-[3-(4-nitrophenyl)ureido]propanamide Derivatives are Agonists of Human Formyl Peptide Receptor 2

N-formyl peptide receptor (FPR1) and FPR2 are G protein-coupledreceptors (GPCR) involved in host defense and sensing cellular dysfunction. Previously we found that antagonists of gastrin-releasing peptide/neuromedin B receptors (BB1/BB2), PD168368 and PD176252, were potent mixed FPR1/FPR2 agonists [Mol. Pharm. (2011) 79: 77-90]. In the present studies, we screened 13 structural derivatives of PD176252 for their ability to activate human neutrophils and HL-60 cells transfected with human FPR1 or FPR2. While none of the compounds had BB2 antagonist activity, five of the compounds stimulated Ca2+ flux in HL-60 cells expressing FPR2, but not in HL-60 cells expressing FPR1, suggesting they were selective for FPR2. The most potent compounds EMY-96 [(R)3-(1H-indol-3-yl)-2-(3-(4-nitrophenyl)ureido)-N-((1-(pyridin-2-yl)cyclohexyl) methyl)propanamide] and its S-isomer ML-16 induced Ca2+ flux with EC50 values in the low micromolar range. Furthermore, pretreatment of FPR2/HL-60 cells with specific the FPR2 antagonist WRW4 prevented Ca2+ flux activated by EMY-96 and ML-16. Interestingly, neither EMY-96 nor ML-16 was able to induce Ca2+ flux in human neutrophils; however, EMY-96 desensitized human neutrophils and FPR2/HL-60 cells to subsequent activation by the hexapeptide WKYMVM. In addition, all five active compounds dose-dependently stimulated human neutrophil chemotaxis. Lastly, these compounds induced β-arrestin binding to FPR2. Thus, these 3-(1H-Indol-3-yl)-2-[3-(4-nitrophenyl)ureido] propanamide derivatives represent unique FPR2 agonists and exhibit novel properties in human neutrophils and FPR-transfected HL-60 cells. This work was supported in part by National Institutes of Health grant P20 RR-020185, National Institutes of Health contract HHSN266200400009C, an equipment grant from the M.J. Murdock Charitable Trust, and the Montana State University Agricultural Experimental Station

3-(1H-indol-3-yl)-2-[3-(4-nitrophenyl)ureido]propanamide enantiomers with human formyl-peptide receptor agonist activity: Molecular modeling of chiral recognition by FPR2.

N-formyl peptide receptors (FPRs) are G protein-coupled receptors (GPCRs) that play critical roles in inflammatory reactions, and FPR-specific interactions can possibly be used to facilitate the resolution of pathological inflammatory reactions. Recent studies indicated that FPRs have stereo-selective preference for chiral ligands. Here, we investigated the structure-activity relationship of 24 chiral ureidopropanamides, including previously reported compounds PD168368/PD176252 and their close analogs, and used molecular modeling to define chiral recognition by FPR2. Unlike previously reported 6-methyl-2,4-disubstituted pyridazin-3(2H)-ones, whose R-forms preferentially activated FPR1/FPR2, we found that four S-enantiomers in the seven ureidopropanamide pairs tested preferentially activated intracellular Ca(2+) flux in FPR2-transfected cells, while the R-counterpart was more active in two enantiomer pairs. Thus, active enantiomers of FPR2 agonists can be in either R- or S-configurations, depending on the molecular scaffold and specific substituents at the chiral center. Using molecular modeling approaches, including field point methodology, homology modeling, and docking studies, we propose a model that can explain stereoselective activity of chiral FPR2 agonists. Importantly, our docking studies of FPR2 chiral agonists correlated well with the FPR2 pharmacophore model derived previously. We conclude that the ability of FPR2 to discriminate between the enantiomers is the consequence of the arrangement of the three asymmetric hydrophobic subpockets at the main orthosteric FPR2 binding site with specific orientation of charged regions in the subpockets

Novel 3-(1H-indol-3-yl)-2-[3-(4-methoxyphenyl)ureido]propanamides as selective agonists of human formyl-peptide receptor 2.

N-formyl peptide receptors (FPRs) are G protein-coupled receptors (GPCRs) that play critical roles in inflammatory reactions, and FPR-specific interactions can possibly be used to facilitate the resolution of pathological inflammatory reactions. We here report the synthesis and biological evaluation of six pairs of chiral ureidopropanamido derivatives as potent and selective formyl peptide receptor-2 (FPR2) agonists, that were designed starting from our lead agonist (S)-3-(1H-indol-3-yl)-2-[3-(4-methoxyphenyl)ureido]-N-[[1-(5-methoxy-2-pyridinyl)cyclohexyl]methyl]propanamide ((S)-9a). The new compounds were obtained in overall yields considerably higher than (S)-9a. Various of the new compounds showed agonist properties comparable to that of (S)-9a along with higher selectivity over FPR1. Molecular modeling was used to define chiral recognition by FPR2. In vitro metabolic stability of selected compounds was also assessed to obtain preliminary insight on drug-like properties of this class of compounds

Rehabilitating drug-induced long-QT promoters: In-silico design of hERG-neutral cisapride analogues with retained pharmacological activity

BACKGROUND: The human ether-a-go-go related gene 1 (hERG1), which codes for a potassium ion channel, is a key element in the cardiac delayed rectified potassium current, I(Kr), and plays an important role in the normal repolarization of the heart’s action potential. Many approved drugs have been withdrawn from the market due to their prolongation of the QT interval. Most of these drugs have high potencies for their principal targets and are often irreplaceable, thus “rehabilitation” studies for decreasing their high hERG1 blocking affinities, while keeping them active at the binding sites of their targets, have been proposed to enable these drugs to re-enter the market. METHODS: In this proof-of-principle study, we focus on cisapride, a gastroprokinetic agent withdrawn from the market due to its high hERG1 blocking affinity. Here we tested an a priori strategy to predict a compound’s cardiotoxicity using de novo drug design with molecular docking and Molecular Dynamics (MD) simulations to generate a strategy for the rehabilitation of cisapride. RESULTS: We focused on two key receptors, a target interaction with the (adenosine) receptor and an off-target interaction with hERG1 channels. An analysis of the fragment interactions of cisapride at human A(2A) adenosine receptors and hERG1 central cavities helped us to identify the key chemical groups responsible for the drug activity and hERG1 blockade. A set of cisapride derivatives with reduced cardiotoxicity was then proposed using an in-silico two-tier approach. This set was compared against a large dataset of commercially available cisapride analogs and derivatives. CONCLUSIONS: An interaction decomposition of cisapride and cisapride derivatives allowed for the identification of key active scaffolds and functional groups that may be responsible for the unwanted blockade of hERG1