Design, Synthesis, and Biological Evaluation of 3‑(1-Aryl‑1<i>H</i>‑indol-5-yl)propanoic Acids as New Indole-Based Cytosolic Phospholipase A₂α Inhibitors

This article describes the design, synthesis, and biological evaluation of new indole-based cytosolic phospholipase A₂α (cPLA₂α, a group IVA phospholipase A₂) inhibitors. A screening-hit compound from our library, (<i>E</i>)-3-{4-[(4-chlorophenyl)­thio]-3-nitrophenyl}­acrylic acid (<b>5</b>), was used to design a class of 3-(1-aryl-1<i>H</i>-indol-5-yl)­propanoic acids as new small molecule inhibitors. The resultant structure–activity relationships studied using the isolated enzyme and by cell-based assays revealed that the 1-(<i>p</i>-<i>O</i>-substituted)­phenyl, 3-phenylethyl, and 5-propanoic acid groups on the indole core are essential for good inhibitory activity against cPLA₂α. Optimization of the <i>p</i>-substituents on the N1 phenyl group led to the discovery of <b>56n</b> (ASB14780), which was shown to be a potent inhibitor of cPLA₂α via enzyme assay, cell-based assay, and guinea pig and human whole-blood assays. It displayed oral efficacy toward mice tetradecanoyl phorbol acetate-induced ear edema and guinea pig ovalbumin-induced asthma models

Inflammation and Resolution Are Associated with Upregulation of Fatty Acid β-Oxidation in Zymosan-Induced Peritonitis

<div><p>Inflammation is a fundamental defensive response to harmful stimuli. However, it can cause damage if it does not subside. To avoid such damage, organisms have developed a mechanism called resolution of inflammation. Here we applied an untargeted metabolomics approach to a sterile and self-resolving animal model of acute inflammation, namely zymosan-induced peritonitis in mice, to examine the effect of inflammation and resolution on the metabolomic profiles. Significant and time-dependent changes in metabolite profiles after zymosan administration were observed in both peritoneal wash fluid (PWF) and plasma. These metabolomic changes correlated well with inflammatory chemokine or cytokine production. In PWF, most of metabolites that could detected increased in zymosan-treated mice, which is suggestive of inflammation, oxidative stress and increased energy demands. In plasma, most metabolites in the central metabolic pathway (glycolysis and TCA cycle) were significantly downregulated after zymosan administration. The concentration of the ketone body 3-hydroxybutyric acid (3-HB) in plasma and PWF increased in zymosan-injected animals indicating upregulation of fatty acid β-oxidation. Increased 3-HB level was observed in the cells that infiltrated into the peritoneal cavity and these infiltrated cells might contribute, at least in part, to the production of 3-HB in the peritoneal cavity.</p></div

Time course of principle component analysis (PCA) score plot of PWF metabolomics data.

<p>Black symbols: control animals, blue: zymosan 1 mg administered animals, red: zymosan 10 mg administered animals, respectively. Black eclipse in the score plot illustrates the 95% confidence regions.</p

Concentration time courses of IL-1β, TNF-α, IFN-γ and MIP-1α in peritoneal wash fluid (PWF) after an intraperitoneal administration of zymosan or vehicle control in mice.

<p>Cytokine and chemokine levels are expressed as mean±s.e.m. (n = 5). LLOQ means lower limit of quantification.</p

Metabolite Profiles Correlate Closely with Neurobehavioral Function in Experimental Spinal Cord Injury in Rats

<div><p>Traumatic spinal cord injury (SCI) results in direct physical damage and the generation of local factors contributing to secondary pathogenesis. Untargeted metabolomic profiling was used to uncover metabolic changes and to identify relationships between metabolites and neurobehavioral functions in the spinal cord after injury in rats. In the early metabolic phase, neuronal signaling, stress, and inflammation-associated metabolites were strongly altered. A dynamic inflammatory response consisting of elevated levels of prostaglandin E2 and palmitoyl ethanolamide as well as pro- and anti-inflammatory polyunsaturated fatty acids was observed. <em>N-</em>acetyl-aspartyl-glutamate (NAAG) and <em>N-</em>acetyl-aspartate (NAA) were significantly decreased possibly reflecting neuronal cell death. A second metabolic phase was also seen, consistent with membrane remodeling and antioxidant defense response. These metabolomic changes were consistent with the pathology and progression of SCI. Several metabolites, including NAA, NAAG, and the ω-3 fatty acids docosapentaenoate and docosahexaenoate correlated greatly with the established Basso, Beattie and Bresnahan locomotive score (BBB score). Our findings suggest the possibility of a biochemical basis for BBB score and illustrate that metabolites may correlate with neurobehavior. In particular the NAA level in the spinal cord might provide a meaningful biomarker that could help to determine the degree of injury severity and prognosticate neurologic recovery.</p> </div

Correlation plots comparing BBB scores with metabolite levels at days 11 (circle) and 30 (square).

<p>The eight named metabolites with the highest |R| score are displayed (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043152#pone.0043152.s002" target="_blank">Table S2</a> for complete list). Sham animals (day 11 and 30) received full BBB score ( = 21), consistent with normal movement. SCI at day 11 and 30 are depicted as •(n = 7) and ▪(n = 8). Sham at day 11 and 30 are displayed as ○(n = 8) and □(n = 8). All data points of Sham animals and SCI animals were used to calculate the R values.</p

Time courses of leukocytes infiltrated into peritoneal cavity after an intraperitoneal administration of zymosan (1 or 10 mg).

<p>(A) Total cell number, (B) cell number of PMNs as determined by CD115⁻ Gr-1⁺ cells, (C) cell number of macrophages as determined by CD115⁺Gr-1⁻ cells, (D) cell number of eosinophils as determined by CCR3⁺Siglec-F⁺ cells. The asterisks indicate significant differences (*<i>P</i><.05 and **<i>P</i><.01) compared to the 0 h (control). Since the numbers of eosinophils were not counted at 0 h (normal) or 6 h after administration of zymosan, statistical analysis of eosinophil numbers could not be performed. (n = 3 per group mean±s.e.m.)</p

Summary of altered metabolites in peritoneal wash fluid (PWF) after zymosan intraperitoneal (i.p.) administration.

<p>Statistically significant changes are in bold (<i>P</i><0.05, compared with vehicle control). GPC refers to glycerophosphocholine and GPE refers to glycerophosphoethanolamine.</p

Table 3. Concentrations of 3-HB in the cells that infiltrated into the peritoneal cavity, PWF, and plasma in control and zymosan-treated (10 mg) animals.

<p>Concentrations are expressed as mean ± s.e.m (n = 5). The asterisks indicate significant differences (** <i>P</i><.01 and * <i>P</i><.05) compared to the vehicle control. Since the 3-HB concentrations of PWF in vehicle control animals were not detectable (ND, detection limit; 0.001 mM), statistical analysis could not be performed.</p

Correlation between absolute NAA concentration in the spinal cord and the corresponding BBB scores 32–34 days after SCI.

<p>SCI was induced by 100, 150 or 200-kdyn impact force. Sham animals received full BBB score ( = 21), consistent with normal movement.</p