Third target of rapamycin complex negatively regulates development of quiescence in Trypanosoma brucei.

African trypanosomes are protozoan parasites transmitted by a tsetse fly vector to a mammalian host. The life cycle includes highly proliferative forms and quiescent forms, the latter being adapted to host transmission. The signaling pathways controlling the developmental switch between the two forms remain unknown. Trypanosoma brucei contains two target of rapamycin (TOR) kinases, TbTOR1 and TbTOR2, and two TOR complexes, TbTORC1 and TbTORC2. Surprisingly, two additional TOR kinases are encoded in the T. brucei genome. We report that TbTOR4 associates with an Armadillo domain-containing protein (TbArmtor), a major vault protein, and LST8 to form a unique TOR complex, TbTORC4. Depletion of TbTOR4 caused irreversible differentiation of the parasite into the quiescent form. AMP and hydrolysable analogs of cAMP inhibited TbTOR4 expression and induced the stumpy quiescent form. Our results reveal unexpected complexity in TOR signaling and show that TbTORC4 negatively regulates differentiation of the proliferative form into the quiescent form.

A promising pipeline of preclinical drug candidates for leishmaniasis and chronic Chagas' disease.

The drug discovery pipeline for leishmaniasis and trypanosomiasis has been filling with novel chemical entities with known mechanisms of action. González et al. and Braillard et al. report a cytochrome bc1 complex inhibitor as another promising preclinical candidate for visceral leishmaniasis (VL) and, in combination with benznidazole, for chronic Chagas' disease (CCD).

Target of rapamycin (TOR) kinase in Trypanosoma brucei: an extended family.

The complex life cycle of Trypanosoma brucei provides an excellent model system to understand signalling pathways that regulate development. We described previously the classical functions of TOR (target of rapamycin) 1 and TOR2 in T. brucei. In a more recent study, we described a novel TOR kinase, named TOR4, which regulates differentiation from the proliferative infective form to the quiescent form. In contrast with TOR1 loss-of-function, down-regulation of TOR4 triggers an irreversible differentiation process through the development of the insect pre-adapted quiescent form. TOR4 governs a signalling pathway distinct from those controlled by the conventional TOR complexes TORC1 and TORC2. Depletion of TOR4 induces all well-known characteristics of the quiescent developmental stage in trypanosomes, including expression of the PAD (proteins associated with differentiation) surface proteins and transcriptional down-regulation of the VSG (variant surface glycoprotein) gene. TOR4 kinase forms a structurally and functionally distinct complex named TORC4. TOR4 associates with LST8 (lethal with sec-13 protein 8) and other factors including an armadillo-domain-containing protein and the major vault protein, which probably serves as a scaffold for this kinase. Research in T. brucei, a protozoan parasite that diverged from the eukaryotic tree early in evolution, may help to uncover new functions of TOR kinases.

Cyanotriazoles are selective topoisomerase II poisons that rapidly cure trypanosome infections.

Millions who live in Latin America and sub-Saharan Africa are at risk of trypanosomatid infections, which cause Chagas disease and human African trypanosomiasis (HAT). Improved HAT treatments are available, but Chagas disease therapies rely on two nitroheterocycles, which suffer from lengthy drug regimens and safety concerns that cause frequent treatment discontinuation. We performed phenotypic screening against trypanosomes and identified a class of cyanotriazoles (CTs) with potent trypanocidal activity both in vitro and in mouse models of Chagas disease and HAT. Cryo-electron microscopy approaches confirmed that CT compounds acted through selective, irreversible inhibition of trypanosomal topoisomerase II by stabilizing double-stranded DNA:enzyme cleavage complexes. These findings suggest a potential approach toward successful therapeutics for the treatment of Chagas disease.

The DNA methyltransferase inhibitors zebularine and decitabine induce mitochondria-mediated apoptosis and DNA damage in p53 mutant leukemic T cells.

DNA methyltransferase (DNMT)-inhibiting nucleoside analogs reactivate the expression of tumor suppressor genes and apoptosis-related genes silenced by methylation, thus favoring the induction of apoptosis in tumor cells. Moreover, induction of DNA damage seems to contribute to their antitumor effect. However, the apoptotic signaling pathway induced by these demethylating drugs is not well understood. Here, we have investigated the induction of apoptosis by two nucleoside DNMT inhibitors, decitabine and zebularine, in leukemic T cells. Both inhibitors induced caspase-dependent apoptosis in Jurkat, CEM-6 and MOLT-4 leukemia T cell lines, all with mutant p53, whereas resting and activated normal T lymphocytes were highly resistant to these demethylating agents. Although decitabine and zebularine showed different ability to induce apoptosis and cell cycle arrest among the three cell lines, they similarly activated the intrinsic apoptotic pathway by inducing mitochondrial alterations such as Bak activation, loss of transmembrane potential and generation of reactive oxygen species (ROS). Accordingly, Bcl-2- and Bcl-x(L) -overexpressing Jurkat cells, as well as caspase-9-deficient Jurkat cells, were resistant to apoptosis induced by decitabine and zebularine. Interestingly, ROS production seemed to be necessary for the induction of apoptosis. Apoptotic events, such as Bak and caspase activation, started as soon as 20 hr after treatment with either decitabine or zebularine. In addition, progression of apoptosis triggered by both DNMT inhibitors was paralleled by the induction of DNA damage. Our results suggest that the mitochondrial apoptotic pathway activated by decitabine and zebularine in p53 mutant leukemic T cells depends mainly on the induction of DNA damage.

CLK1/CLK2-driven signalling at the Leishmania kinetochore is captured by spatially referenced proximity phosphoproteomics.

Kinetochores in the parasite Leishmania and related kinetoplastids appear to be unique amongst eukaryotes and contain protein kinases as core components. Using the kinetochore kinases KKT2, KKT3 and CLK2 as baits, we developed a BirA* proximity biotinylation methodology optimised for sensitivity, XL-BioID, to investigate the composition and function of the Leishmania kinetochore. We could detect many of the predicted components and also discovered two novel kinetochore proteins, KKT24 and KKT26. Using KKT3 tagged with a fast-acting promiscuous biotin ligase variant, we took proximity biotinylation snapshots of the kinetochore in synchronised parasites. To quantify proximal phosphosites at the kinetochore as the parasite progressed through the cell cycle, we further developed a spatially referenced proximity phosphoproteomics approach. This revealed a group of phosphosites at the kinetochore that were highly dynamic during kinetochore assembly. We show that the kinase inhibitor AB1 targets CLK1/CLK2 (KKT10/KKT19) in Leishmania leading to defective cytokinesis. Using AB1 to uncover CLK1/CLK2 driven signalling pathways important for kinetochore function at G2/M, we found a set of 16 inhibitor responsive kinetochore-proximal phosphosites. Our results exploit new proximity labelling approaches to provide a direct analysis of the Leishmania kinetochore, which is emerging as a promising drug target.

A CLK1-KKT2 Signaling Pathway Regulating Kinetochore Assembly in Trypanosoma brucei.

During mitosis, eukaryotic cells must duplicate and separate their chromosomes in a precise and timely manner. The apparatus responsible for this is the kinetochore, which is a large protein structure that links chromosomal DNA and spindle microtubules to facilitate chromosome alignment and segregation. The proteins that comprise the kinetochore in the protozoan parasite Trypanosoma brucei are divergent from yeast and mammals and comprise an inner kinetochore complex composed of 24 distinct proteins (KKT1 to KKT23, KKT25) that include four protein kinases, CLK1 (KKT10), CLK2 (KKT19), KKT2, and KKT3. We recently reported the identification of a specific trypanocidal inhibitor of T. brucei CLK1, an amidobenzimidazole, AB1. We now show that chemical inhibition of CLK1 with AB1 impairs inner kinetochore recruitment and compromises cell cycle progression, leading to cell death. Here, we show that KKT2 is a substrate for CLK1 and identify phosphorylation of S508 by CLK1 to be essential for KKT2 function and for kinetochore assembly. Additionally, KKT2 protein kinase activity is required for parasite proliferation but not for assembly of the inner kinetochore complex. We also show that chemical inhibition of the aurora kinase AUK1 does not affect CLK1 phosphorylation of KKT2, indicating that AUK1 and CLK1 are in separate regulatory pathways. We propose that CLK1 is part of a divergent signaling cascade that controls kinetochore function via phosphorylation of the inner kinetochore protein kinase KKT2. IMPORTANCE In eukaryotic cells, kinetochores are large protein complexes that link chromosomes to dynamic microtubule tips, ensuring proper segregation and genomic stability during cell division. Several proteins tightly coordinate kinetochore functions, including the protein kinase aurora kinase B. The kinetochore has diverse evolutionary roots. For example, trypanosomatids, single-cell parasitic protozoa that cause several neglected tropical diseases, possess a unique repertoire of kinetochore components whose regulation during the cell cycle remains unclear. Here, we shed light on trypanosomatid kinetochore biology by showing that the protein kinase CLK1 coordinates the assembly of the inner kinetochore by phosphorylating one of its components, KKT2, allowing the timely spatial recruitment of the rest of the kinetochore proteins and posterior attachment to microtubules in a process that is aurora kinase B independent.

Targeting the trypanosome kinetochore with CLK1 protein kinase inhibitors.

The kinetochore is a macromolecular structure that assembles on the centromeres of chromosomes and provides the major attachment point for spindle microtubules during mitosis. In Trypanosoma brucei, the proteins that make up the kinetochore are highly divergent; the inner kinetochore comprises at least 20 distinct and essential proteins (KKT1-20) that include four protein kinases-CLK1 (also known as KKT10), CLK2 (also known as KKT19), KKT2 and KKT3. Here, we report the identification and characterization of the amidobenzimidazoles (AB) protein kinase inhibitors that show nanomolar potency against T. brucei bloodstream forms, Leishmania and Trypanosoma cruzi. We performed target deconvolution analysis using a selection of 29 T. brucei mutants that overexpress known essential protein kinases, and identified CLK1 as a primary target. Biochemical studies and the co-crystal structure of CLK1 in complex with AB1 show that the irreversible competitive inhibition of CLK1 is dependent on a Michael acceptor forming an irreversible bond with Cys 215 in the ATP-binding pocket, a residue that is not present in human CLK1, thereby providing selectivity. Chemical inhibition of CLK1 impairs inner kinetochore recruitment and compromises cell-cycle progression, leading to cell death. This research highlights a unique drug target for trypanosomatid parasitic protozoa and a new chemical tool for investigating the function of their divergent kinetochores.

The AMPKα1 Pathway Positively Regulates the Developmental Transition from Proliferation to Quiescence in Trypanosoma brucei.

During infection in mammals, the protozoan parasite Trypanosoma brucei transforms from a proliferative bloodstream form to a quiescent form that is pre-adapted to host transition. AMP analogs are known to induce quiescence and also inhibit TbTOR4. To examine the role of AMP-activated kinase (AMPK) in the regulation of this developmental transition, we characterized trypanosome TbAMPK complexes. Expression of a constitutively active AMPKα1 induces quiescence of the infective form, and TbAMPKα1 phosphorylation occurs during differentiation of wild-type pleomorphic trypanosomes to the quiescent stumpy form in vivo. Compound C, a well-known AMPK inhibitor, inhibits parasite differentiation in mice. We also provide evidence linking oxidative stress to TbAMPKα1 activation and quiescent differentiation, suggesting that TbAMPKα1 activation balances quiescence, proliferation, and differentiation.

Establishment of a structure-activity relationship of 1H-imidazo[4,5-c]quinoline-based kinase inhibitor NVP-BEZ235 as a lead for African sleeping sickness.

Compound NVP-BEZ235 (1) is a potent inhibitor of human phospoinositide-3-kinases and mammalian target of rapamycin (mTOR) that also showed high inhibitory potency against Trypanosoma brucei cultures. With an eye toward using 1 as a starting point for anti-trypanosomal drug discovery, we report efforts to reduce host cell toxicity, to improve the physicochemical properties, and to improve the selectivity profile over human kinases. In this work, we have developed structure-activity relationships for analogues of 1 and have prepared analogues of 1 with improved solubility properties and good predicted central nervous system exposure. In this way, we have identified 4e, 9, 16e, and 16g as the most promising leads to date. We also report cell phenotype and phospholipidomic studies that suggest that these compounds exert their anti-trypanosomal effects, at least in part, by inhibition of lipid kinases.

The susceptibility of trypanosomatid pathogens to PI3/mTOR kinase inhibitors affords a new opportunity for drug repurposing.

BACKGROUND: Target repurposing utilizes knowledge of "druggable" targets obtained in one organism and exploits this information to pursue new potential drug targets in other organisms. Here we describe such studies to evaluate whether inhibitors targeting the kinase domain of the mammalian Target of Rapamycin (mTOR) and human phosphoinositide-3-kinases (PI3Ks) show promise against the kinetoplastid parasites Trypanosoma brucei, T. cruzi, Leishmania major, and L. donovani. The genomes of trypanosomatids encode at least 12 proteins belonging to the PI3K protein superfamily, some of which are unique to parasites. Moreover, the shared PI3Ks differ greatly in sequence from those of the human host, thereby providing opportunities for selective inhibition. METHODOLOGY/PRINCIPAL FINDINGS: We focused on 8 inhibitors targeting mTOR and/or PI3Ks selected from various stages of pre-clinical and clinical development, and tested them against in vitro parasite cultures and in vivo models of infection. Several inhibitors showed micromolar or better efficacy against these organisms in culture. One compound, NVP-BEZ235, displayed sub-nanomolar potency, efficacy against cultured parasites, and an ability to clear parasitemia in an animal model of T. brucei rhodesiense infection. CONCLUSIONS/SIGNIFICANCE: These studies strongly suggest that mammalian PI3/TOR kinase inhibitors are a productive starting point for anti-trypanosomal drug discovery. Our data suggest that NVP-BEZ235, an advanced clinical candidate against solid tumors, merits further investigation as an agent for treating African sleeping sickness.

Concentración espermática mínima requerida para inseminación intrauterina mediante capacitación por migración ascendente / Low sperm concentration required to IUI through training by upward migration

Objetivo: Determinar cuál es la cantidad mínima necesaria de espermatozoides móviles que se requiere para realizar la inseminación intrauterina y evaluar la morfología estricta de Kruger y la movilidad espermática antes y después de la capacitación por migración ascendente. Métodos: Estudio prospectivo de 35 muestras de semen de hombres infértiles, se lavaron alícuotas de 1 mL de semen fresco, se centrifugaron y sobre el centrifugado se colocó una capa de medio de capacitación para lograr una migración ascendente. Resultados: Los valores de movilidad y formas normales espermáticas se observaron significativamente aumentados en las muestras después de la capacitación. Fue posible recuperar ≥ 2 x 106 espermatozoides móviles aun en muestras aparentemente inapropiadas caracterizadas por hipospermia u oligozoospermia severa, pero contenían en el total del eyaculado al menos 5 millones de espermatozoides móviles que permitieron un elevado porcentaje de recuperación espermática. Conclusiones: La posibilidad de obtener altos porcentajes de recuperación de espermatozoides móviles en el total del eyaculado permite la inseminación intrauterina como técnica de reproducción asistida en pacientes oligozoospérmicos antes de elegir fertilización in vitro o inyección citoplasmática del espermatozoide cuando el factor masculino es la causa de infertilidad.

Objective: To determine what is the minimum necessary amount of motile sperm required for intrauterine insemination and to evaluate the Kruger strict morphology test and sperm motility before and after training sperm by swim up. Methods: Prospective study of 35 semen samples from infertile men, aliquots of 1 mL of fresh semen was washed, centrifuged and over the pellet was placed a layer of capacitation medium to achieve an upward migration. Results: The values of motility and normal sperm forms were observed in the samples significantly increased after the training. It was possible to recover ≥2 x 106 motile sperm even in seemingly inappropriate samples with hypospermia or severe oligozoospermia, but these contained in the total ejaculate at least 5 million of motile spermatozoa that allowed a high percentage of retrieval. Conclusions: The possibility of obtaining high recoveries of motile sperm in the total ejaculate allows IUI as assisted reproduction technique in oligozoospermic patients before choosing IVF or cytoplasmic sperm injection when the male factor is cause of infertility.