RESUMEN
Cation channel of sperm (CatSper), the main sperm-specific Ca2+ channel, plays a key role in mammalian fertilization, and it is essential for male fertility, becoming an attractive target for contraception. Based on this, in the present work, we investigated the effects of CatSper inactivation on in vitro and in vivo sperm fertilizing ability and the mechanisms underlying such effects. Exposure of cauda epididymal mouse sperm to different concentrations (1-20 µM) of the potent CatSper inhibitor HC-056456 (HC) during in vitro capacitation showed no effects on sperm viability but significantly affected Ca2+ entry into the cells, progressive motility, protein tyrosine phosphorylation, induced acrosome reaction, and hyperactivation, as well as the sperm's ability to in vitro fertilize cumulus oocyte complexes and zona-free eggs. Whereas the presence of HC during gamete coincubation did not affect in vitro fertilization, exposure of either non-capacitating or already capacitated sperm to HC prior to gamete coincubation severely reduced fertilization, indicating that sperm function is affected by HC when the cells are incubated with the drug before sperm-egg interaction. Of note, insemination of HC-treated sperm into the uterus significantly or completely reduced the percentage of oviductal fertilized eggs showing, for the first time, the effects of a CatSper inhibitor on in vivo fertilization. These observations, together with the finding that HC affects sperm fertilizing ability independently of the sperm capacitation status, provide further insights on how CatSper regulates sperm function and represent a solid proof of concept for developing a male/female non-hormonal contraceptive based on the pharmacological blockage of CatSper activity.
RESUMEN
Ribose 5-phosphate isomerase type B (RPI-B) is a key enzyme of the pentose phosphate pathway that catalyzes the isomerization of ribose 5-phosphate (R5P) and ribulose 5-phosphate (Ru5P). Trypanosoma cruzi RPI-B (TcRPI-B) appears to be a suitable drug-target mainly due to: (i) its essentiality (as previously shown in other trypanosomatids), (ii) it does not present a homologue in mammalian genomes sequenced thus far, and (iii) it participates in the production of NADPH and nucleotide/nucleic acid synthesis that are critical for parasite cell survival. In this survey, we report on the competitive inhibition of TcRPI-B by a substrate - analogue inhibitor, Compound B (Ki = 5.5 ± 0.1 µM), by the Dixon method. This compound has an iodoacetamide moiety that is susceptible to nucleophilic attack, particularly by the cysteine thiol group. Compound B was conceived to specifically target Cys-69, an important active site residue. By incubating TcRPI-B with Compound B, a trypsin digestion LC-MS/MS analysis revealed the identification of Compound B covalently bound to Cys-69. This inhibitor also exhibited notable in vitro trypanocidal activity against T. cruzi infective life-stages co-cultured in NIH-3T3 murine host cells (IC50 = 17.40 ± 1.055 µM). The study of Compound B served as a proof-of-concept so that next generation inhibitors can potentially be developed with a focus on using a prodrug group in replacement of the iodoacetamide moiety, thus representing an attractive starting point for the future treatment of Chagas' disease.
Asunto(s)
Isomerasas Aldosa-Cetosa/antagonistas & inhibidores , Diseño de Fármacos , Inhibidores Enzimáticos/química , Proteínas Protozoarias/antagonistas & inhibidores , Tripanocidas/síntesis química , Trypanosoma cruzi/enzimología , Células 3T3 , Isomerasas Aldosa-Cetosa/metabolismo , Animales , Sitios de Unión , Dominio Catalítico , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Cinética , Ratones , Simulación de Dinámica Molecular , Proteínas Protozoarias/metabolismo , Especificidad por Sustrato , Tripanocidas/metabolismo , Tripanocidas/farmacología , Trypanosoma cruzi/efectos de los fármacosRESUMEN
The enzyme of the pentose phosphate pathway (PPP) ribulose-5-phosphate-epimerase (RPE) is encoded by two genes present in the genome of Trypanosoma cruzi CL Brener clone: TcRPE1 and TcRPE2. Despite high sequence similarity at the amino acid residue level, the recombinant isoenzymes show a strikingly different kinetics. Whereas TcRPE2 follows a typical michaelian behavior, TcRPE1 shows a complex kinetic pattern, displaying a biphasic curve, suggesting the coexistence of -at least- two kinetically different molecular forms. Regarding the subcellular localization in epimastigotes, whereas TcRPE1 is a cytosolic enzyme, TcRPE2 is localized in glycosomes. To our knowledge, TcRPE2 is the first PPP isoenzyme that is exclusively localized in glycosomes. Over-expression of TcRPE1, but not of TcRPE2, significantly reduces the parasite doubling time in vitro, as compared with wild type epimastigotes. Both TcRPEs represent single domain proteins exhibiting the classical α/ß TIM-barrel fold, as expected for enzymes with this activity. With regard to the architecture of the active site, all the important amino acid residues for catalysis -with the exception of M58- are also present in both TcRPEs models. The superimposition of the binding pocket of both isoenzyme models shows that they adopt essentially identical positions in the active site with a residue specific RMSD < 2Å, with the sole exception of S12, which displays a large deviation (residue specific RMSD: 11.07 Å). Studies on the quaternary arrangement of these isoenzymes reveal that both are present in a mixture of various oligomeric species made up of an even number of molecules, probably pointing to the dimer as their minimal functional unit. This multiplicity of oligomeric species has not been reported for any of the other RPEs studied so far and it might bear implications for the regulation of TcRPEs activity, although further investigation will be necessary to unravel the physiological significance of these structural findings.