Discovery and Optimization of Orally Bioavailable Phthalazone and Cinnolone Carboxylic Acid Derivatives as S1P2 Antagonists against Fibrotic Diseases.

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive lung disease. Current treatments only slow down disease progression, making new therapeutic strategies compelling. Increasing evidence suggests that S1P2 antagonists could be effective agents against fibrotic diseases. Our compound collection was mined for molecules possessing substructure features associated with S1P2 activity. The weakly potent indole hit 6 evolved into a potent phthalazone series, bearing a carboxylic acid, with the aid of a homology model. Suboptimal pharmacokinetics of a benzimidazole subseries were improved by modifications targeting potential interactions with transporters, based on concepts deriving from the extended clearance classification system (ECCS). Scaffold hopping, as a part of a chemical enablement strategy, permitted the rapid exploration of the position adjacent to the carboxylic acid. Compound 38, with good pharmacokinetics and in vitro potency, was efficacious at 10 mg/kg BID in three different in vivo mouse models of fibrotic diseases in a therapeutic setting.

Discovery of the S1P2 Antagonist GLPG2938 (1-[2-Ethoxy-6-(trifluoromethyl)-4-pyridyl]-3-[[5-methyl-6-[1-methyl-3-(trifluoromethyl)pyrazol-4-yl]pyridazin-3-yl]methyl]urea), a Preclinical Candidate for the Treatment of Idiopathic Pulmonary Fibrosis.

Mounting evidence from the literature suggests that blocking S1P2 receptor (S1PR2) signaling could be effective for the treatment of idiopathic pulmonary fibrosis (IPF). However, only a few antagonists have been so far disclosed. A chemical enablement strategy led to the discovery of a pyridine series with good antagonist activity. A pyridazine series with improved lipophilic efficiency and with no CYP inhibition liability was identified by scaffold hopping. Further optimization led to the discovery of 40 (GLPG2938), a compound with exquisite potency on a phenotypic IL8 release assay, good pharmacokinetics, and good activity in a bleomycin-induced model of pulmonary fibrosis.

Identification and Characterization of Novel CFTR Potentiators.

There is still a high unmet need for the treatment of most patients with cystic fibrosis (CF). The identification and development of new Cystic Fibrosis Transmembrane conductance Regulator (CFTR) modulators is necessary to achieve higher clinical benefit in patients. In this report we describe the characterization of novel potentiators. From a small screening campaign on F508del CFTR, hits were developed leading to the identification of pre-clinical candidates GLPG1837 and GLPG2451, each derived from a distinct chemical series. Both drug candidates enhance WT CFTR activity as well as low temperature or corrector rescued F508del CFTR, and are able to improve channel activity on a series of Class III, IV CFTR mutants. The observed activities in YFP halide assays translated well to primary cells derived from CF lungs when measured using Trans-epithelial clamp circuit (TECC). Both potentiators improve F508del CFTR channel opening in a similar manner, increasing the open time and reducing the closed time of the channel. When evaluating the potentiators in a chronic setting on corrected F508del CFTR, no reduction of channel activity in presence of potentiator was observed. The current work identifies and characterizes novel CFTR potentiators GLPG1837 and GLPG2451, which may offer new therapeutic options for CF patients.

2D proteome analysis initiates new insights on the Salmonella Typhimurium LuxS protein.

BACKGROUND: Quorum sensing is a term describing a bacterial communication system mediated by the production and recognition of small signaling molecules. The LuxS enzyme, catalyzing the synthesis of AI-2, is conserved in a wide diversity of bacteria. AI-2 has therefore been suggested as an interspecies quorum sensing signal. To investigate the role of endogenous AI-2 in protein expression of the Gram-negative pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium), we performed a 2D-DIGE proteomics experiment comparing total protein extract of wildtype S. Typhimurium with that of a luxS mutant, unable to produce AI-2. RESULTS: Differential proteome analysis of wildtype S. Typhimurium versus a luxS mutant revealed relatively few changes beyond the known effect on phase 2 flagellin. However, two highly differentially expressed protein spots with similar molecular weight but differing isoelectric point, were identified as LuxS whereas the S. Typhimurium genome contains only one luxS gene. This observation was further explored and we show that the S. Typhimurium LuxS protein can undergo posttranslational modification at a catalytic cysteine residue. Additionally, by constructing LuxS-betala and LuxS-PhoA fusion proteins, we demonstrate that S. Typhimurium LuxS can substitute the cognate signal peptide sequences of beta-lactamase and alkaline phosphatase for translocation across the cytoplasmic membrane in S. Typhimurium. This was further confirmed by fractionation of S. Typhimurium protein extracts, followed by Western blot analysis. CONCLUSION: 2D-DIGE analysis of a luxS mutant vs. wildtype Salmonella Typhimurium did not reveal new insights into the role of AI-2/LuxS in Salmonella as only a small amount of proteins were differentially expressed. However, subsequent in depth analysis of the LuxS protein itself revealed two interesting features: posttranslational modification and potential translocation across the cytoplasmic membrane. As the S. Typhimurium LuxS protein does not contain obvious signal motifs, it is speculated that LuxS is a new member of so called moonlighting proteins. These observations might have consequences in future studies on AI-2 quorum signaling in S. Typhimurium.

The proteome of Salmonella Typhimurium grown under in vivo-mimicking conditions.

To successfully infect a host, it is a prerequisite for enteric pathogens such as Salmonella enterica serovar Typhimurium to adapt to their environment, in casu the gastrointestinal tract. The adoption of an appropriate lifestyle is triggered by environmental signals such as the low oxygen availability and high osmolarity prevalent in the gut. In order to gain more insight in the changes that are induced when S. Typhimurium is adapting to these particular conditions, we used 2-D DIGE technology to investigate the combined effect of low oxygen tension and high osmolarity on the proteome of S. Typhimurium SL1344 compared to standard laboratory conditions. As a validation of the 2-D DIGE technique, preferential protein labeling by the Cy-dyes was assessed and proved to be negligible. The differentially expressed proteins identified reflect very well the applied culture conditions. Furthermore, reported transcriptional changes and observed changes at the translational level show overlap. Among the metabolic processes that are upregulated under in vivo-mimicking conditions are anaerobic fumarate respiration and the utilization of 1,2-propanediol. We also provide evidence that S. Typhimurium expresses an arginine deiminase pathway for the catabolism of L-arginine. The increased activity of this pathway was biochemically validated. Finally, also proteins involved in quorum sensing and virulence are differentially expressed under in vivo-mimicking conditions. These conditions offer possibilities as a simplified model system for the host environment given the high overlap of identifications in our study and reported genuine in vivo studies, respectively.

Let LuxS speak up in AI-2 signaling.

Quorum sensing is a process of bacterial cell-cell communication that uses small diffusible molecules to coordinate diverse behaviors in response to population density. The only quorum-sensing system shared by Gram-positive and Gram-negative bacteria involves the production of autoinducer-2 (AI-2). The AI-2 synthase LuxS is widely distributed among the Bacteria, which suggests that AI-2 is a language for interspecies communication. However, LuxS is also an integral component of the activated methyl cycle in bacteria. LuxS-based quorum sensing has been intensively studied in the past decade, mostly in relation to the AI-2 molecule and the downstream effects of luxS knockouts; few studies have focused on the gene and protein activity itself. Ongoing attempts to dissect the metabolic and signaling roles of LuxS leave little doubt that unraveling the regulation of luxS expression and cellular LuxS activity is the key to understanding LuxS-based quorum sensing.