Synthesis, Biological Evaluation, and Molecular Modeling Studies of New Thiadiazole Derivatives as Potent P2X7 Receptor Inhibitors

Twenty new 2-(1H-pyrazol-1-yl)-1,3,4-thiadiazole analogs were synthetized to develop P2X7 receptor (P2X7R) inhibitors. P2X7R inhibition in vitro was evaluated in mouse peritoneal macrophages, HEK-293 cells transfected with hP2X7R (dye uptake assay), and THP-1 cells (IL-1β release assay). The 1-(5-phenyl-1,3,4-thiadiazol-2-yl)-1H-pyrazol-5-amine derivatives 9b, 9c, and 9f, and 2-(3,5-dimethyl-1H-pyrazol-1-yl)-5-(4-fluorophenyl)-1,3,4-thiadiazole (11c) showed inhibitory effects with IC50 values ranging from 16 to 122 nM for reduced P2X7R-mediated dye uptake and 20 to 300 nM for IL-1β release. In addition, the in vitro ADMET profile of the four most potent derivatives was determined to be in acceptable ranges concerning metabolic stability and cytotoxicity. Molecular docking and molecular dynamics simulation studies of the molecular complexes human P2X7R/9f and murine P2X7R/9f indicated the putative intermolecular interactions. Compound 9f showed affinity mainly for the Arg268, Lys377, and Asn266 residues. These results suggest that 2-(1H-pyrazol-1-yl)-1,3,4-thiadiazole analogs may be promising novel P2X7R inhibitors with therapeutic potential

Structural and functional analysis of a platelet-activating lysophosphatidylcholine of Trypanosoma cruzi

BACKGROUND: Trypanosoma cruzi is the causative agent of the life-threatening Chagas disease, in which increased platelet aggregation related to myocarditis is observed. Platelet-activating factor (PAF) is a potent intercellular lipid mediator and second messenger that exerts its activity through a PAF-specific receptor (PAFR). Previous data from our group suggested that T. cruzi synthesizes a phospholipid with PAF-like activity. The structure of T. cruzi PAF-like molecule, however, remains elusive. METHODOLOGY/PRINCIPAL FINDINGS: Here, we have purified and structurally characterized the putative T. cruzi PAF-like molecule by electrospray ionization-tandem mass spectrometry (ESI-MS/MS). Our ESI-MS/MS data demonstrated that the T. cruzi PAF-like molecule is actually a lysophosphatidylcholine (LPC), namely sn-1 C18:1(delta 9)-LPC. Similar to PAF, the platelet-aggregating activity of C18:1-LPC was abrogated by the PAFR antagonist, WEB 2086. Other major LPC species, i.e., C16:0-, C18:0-, and C18:2-LPC, were also characterized in all T. cruzi stages. These LPC species, however, failed to induce platelet aggregation. Quantification of T. cruzi LPC species by ESI-MS revealed that intracellular amastigote and trypomastigote forms have much higher levels of C18:1-LPC than epimastigote and metacyclic trypomastigote forms. C18:1-LPC was also found to be secreted by the parasite in extracellular vesicles (EV) and an EV-free fraction. A three-dimensional model of PAFR was constructed and a molecular docking study was performed to predict the interactions between the PAFR model and PAF, and each LPC species. Molecular docking data suggested that, contrary to other LPC species analyzed, C18:1-LPC is predicted to interact with the PAFR model in a fashion similar to PAF. CONCLUSIONS/SIGNIFICANCE: Taken together, our data indicate that T. cruzi synthesizes a bioactive C18:1-LPC, which aggregates platelets via PAFR. We propose that C18:1-LPC might be an important lipid mediator in the progression of Chagas disease and its biosynthesis could eventually be exploited as a potential target for new therapeutic interventions

Activity of C16:0-PAF and different <i>T. cruzi</i> LPC species on the aggregation of rabbit platelets.

<p>Platelet aggregation assays were performed as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003077#s2" target="_blank">Materials and Methods</a>, using synthetic 16:0-PAF and C16:0-, C18:0-, C18:1-LPC, and purified C18:2-LPC. Control platelets or platelets pre-treated for 30 min with 10 µM WEB 2086 were assayed in the absence or presence of 1 µM C16:0-PAF or the LPC species at 10 µM (C16:0-LPC, C18:0-LPC, C18:1-LPC, and C18:2-LPC) and 100 µM (C18:1-LPC). Each lipid was tested in duplicate as indicated by black and blue curves in each graph.</p

Proposed molecular structures of the three major LPC species of <i>T. cruzi</i>.

<p>Fragment ions obtained from the MSⁿ analysis of <i>T. cruzi</i> ion species at <i>m/z</i> 530, 528, and 526 are indicated. The tandem fragmentation of C16:0-PAF standard is included as a reference.</p

The content of C18:2-, C18:1-, C18:0- and C16:0-LPC in different life-cycle stages of <i>T. cruzi</i>.

<p>^<i>a</i>The molar relative response factors (MRRF) of C10:0-LPC and LPC standards were used to calculate the amount of each LPC molecular species in Folch lower-phase fractions of <i>T. cruzi</i>.</p><p>^<i>b</i>The number of parasites was determined before lipid extraction by counting live parasites in a hemocytometer. Values are means of three determinations. The standard deviation in all cases was <15%.</p><p>^<i>c</i>Determined by multiplying the number of moles by the Avogadro's constant.</p><p>^<i>d</i>Estimated based on the parasite's length and diameter as determined by scanning electron microscopy, and assuming that each parasite is cylindrical, as previous described <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003077#pntd.0003077-PereiraChioccola1" target="_blank">[103]</a>.</p><p>^<i>e</i>Obtained by dividing the number of LPC molecules per cell by the surface area of each parasite form.</p><p>^<i>f</i>Obtained by dividing the amount of picomoles of LPC per 10⁶ cells of each parasite form (column 1) by the values obtained for Epi.</p><p>^<i>g</i>Not analyzed due to absence or trace amounts of the compound.</p