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Some therapeutics for parasitic, cardiac and neurological diseases activate apoptosis. Therefore, the study of apoptotic proteins in pathogenic organisms is relevant. However, the molecular mechanism of apoptosis in unicellular organisms remain elusive, despite morphological evidence of its occurrence. In Trypanosoma cruzi, the causative agent of Chagas disease, metacaspase 3 (TcMCA3), seems to have a key role in parasite apoptosis. Accordingly, this work provides data concerning TcMCA3 regulation through its interaction with procaspase-activating compound 1 (PAC-1), a procaspase 3 activator. Indeed, PAC-1 reduced T. cruzi epimastigote viability with an IC of 14.12 µM and induced loss of mitochondrial potential and exposure of phosphatidylserine, features of the apoptotic process. Notwithstanding, those PAC-1-inducible effects were not conserved in metacyclic trypomastigotes. Moreover, PAC-1 reduced the viability of mammalian cells with a greater IC (25.70 µM) compared to T. cruzi epimastigotes, indicating distinct modes of binding between caspases and metacaspases. To shed light on the selectivity of metacaspases and caspases, we determined the structural features related to the PAC-1 binding sites in both types of proteins. These data are important for improving the understanding of the apoptosis pathway in T. cruzi so that TcMCA3 could be better targeted with future pharmaceuticals. [ABSTRACT FROM AUTHOR]

A sensitive and simple HPLC method for simultaneous determination of PAC-1 (first procaspase-activating compound), phenol red, and permeability markers (carbamazepine and furosemide) in perfusion samples was developed and validated to assess intestinal absorption of PAC-1 using single-pass intestinal perfusion technique (SPIP) in rats. The chromatographic separation was carried out on a Kromasil C column (150 mm × 4.6 mm, 5 μm) with acetonitrile-methanol-30 mmol L phosphate buffer (pH 3.0, 25:10:65, v/v/v) as mobile phase at a flow rate of 1.0 mL min, and the wavelength of the UV detector was set at 281 nm. The calibration curves were linear in the ranges of 2.40-48.0 μg mL for PAC-1; 3.60-72.0 μg mL for carbamazepine; 3.20-64.0 μg mL for furosemide, and 4.80-96.0 μg mL for phenol red ( r > 0.999). Both the intra- and inter-day precisions (RSD%) of all analytes were less than 6.8 % at three concentration levels, while accuracy ranged from 95.4 to 104.5 %. Data obtained in all method validation studies indicated that the method was suitable for the intended purpose. The effective permeability values ( P ) considering water flux with the help of non-permeable marker phenol red was calculated to be 0.42 × 10, 0.62 × 10, 0.32 × 10 cm s for PAC-1; 0.72 × 10, 0.77 × 10, 0.52 × 10 cm s for carbamazepine; 0.20 × 10, 0.16 × 10, 0.12 × 10 cm s for furosemide in duodenum, jejunum and ileum, respectively. The P value can be increased by co-perfusion with verapamil, indicating that absorption of PAC-1 is efficiently transported by P-glycoprotein (P-gp) in the gut wall. [ABSTRACT FROM AUTHOR]

Purpose: To develop more potent procaspase-3 activator, 7 novel derivatives of PAC-1 were synthesized and evaluated. Among them, SM-1 stood out for its promising activity and good pharmacokinetics properties. The purpose of this study is to elucidate the pharmacological mechanism of SM-1 and evaluate its efficacy and toxicity in-depth.Methods: To reveal the effects of SM-1 on caspase-3 activity, both in vitro activation assay and in cells fluorometric assay were tested. The protein levels and distributions of procaspase-3 and cleaved caspase-3 were also measured by western blot and immunostaining. MTT assay, apoptosis assay and mouse xenograft model were applied to evaluate the efficacy of SM-1. Preliminary safety assessments also tested the acute toxicity and tissue distribution of SM-1.Results: Compared to PAC-1, SM-1 showed higher cytotoxicity in cancer cells. Further investigation demonstrated that SM-1 relieved zinc-mediated inhibition of procaspase-3 and activated the caspase-3 activity both in tube test and in cells. Efficacy evaluation showed SM-1-induced cell apoptosis mainly via activation of caspase-3 and reduced tumor size in mouse xenograft model. Its apoptosis induction efficacy was higher than PAC-1. The preliminary safety assessment demonstrated that the overall LD50 of SM-1 lied between 500 and 1000 mg/kg and the distribution of SM-1 in brain was low.Conclusions: We identified SM-1 as a promising antitumor candidate, which displayed enhanced procaspase-3 activating activity and potent cytotoxicity for cancer cells but low toxicity for normal cells. [ABSTRACT FROM AUTHOR]

Flow cytometric evaluation of platelet function extends our understanding of platelets’ role in various clinical conditions associated with either bleeding disorders, thrombosis, or monitoring of antiplatelet therapy. The use of suboptimal concentrations of various agonists may allow assessing the “activatability” of platelets. We determined platelet responsiveness to thrombin-receptor-activating peptide-6, arachidonic acid, adenosine 5c-diphosphate (ADP), epinephrine, collagen, and ristocetin at suboptimal concentrations by determination of P-selectin expression and binding of PAC-1 in 26 healthy male individuals. The response varied considerably from one individual to the next. However, within individuals, responses to all agonists except collagen correlated strongly ( p<0.05), suggesting a global variability of platelet responses. Moreover, P-selectin expression and PAC-1 binding were strongly correlated ( p<0.05). Interestingly, with epinephrine, PAC-1 positive events outnumbered P-selectin positive events, while this was not seen with the other agonists. Thus, epinephrine may specifically affect the conformational switch mechanism and receptor clustering. Our data indicate that the in vitro response to suboptimal concentrations of agonists varies, but individuals with selective platelet defects may still be identified based on data obtained with the various agonists. [ABSTRACT FROM AUTHOR]

Pituitary adenylate cyclase-activating polypeptide (PACAP) modulates glutamatergic neurotransmission and induces the expression of glutamate transporters EAAT1 and EAAT2 in newborn mouse astroglial cell cultures. Since nanomolar concentrations of PACAP exert this effect, signal transduction via the high affinity PACAP-type I-receptor PAC1 was assumed. To test this hypothesis and to assess the importance of PAC1-signallingin vivo, we analyzed glutamate transporter expression in mice with a PAC1 knockout.EAAT1 and EAAT2 expression was investigated in the hippocampus and the cerebral cortex of PAC1 mutant mice and wildtype littermates by semiquantitativein-situ-hybridization.PAC1-knockout mice show a subtle but significant reduction of EAAT1 expression in the dentate gyrus. In contrast, reduced expression levels of EAAT1 in the cerebral cortex did not reach statistical significance and EAAT2 expression was unchanged in CA3 and cerebral cortex of PAC1 mutant mice.Our data confirm the previously reportedin-vitro-regulation of EAAT1 in the adult nervous systemin vivo. EAAT2 expression, however, is unchanged in PAC1 knockout mice, most likely due to counterbalancing factors. [ABSTRACT FROM AUTHOR]