The pathogenic role of c‑Kit+ mast cells in the spinal motor neuron‑vascular niche in ALS

Degeneration of motor neurons, glial cell reactivity, and vascular alterations in the CNS are important neuropathological features of amyotrophic lateral sclerosis (ALS). Immune cells trafficking from the blood also infiltrate the affected CNS parenchyma and contribute to neuroinflammation. Mast cells (MCs) are hematopoietic-derived immune cells whose precursors differentiate upon migration into tissues. Upon activation, MCs undergo degranulation with the ability to increase vascular permeability, orchestrate neuroinflammation and modulate the neuroimmune response. However, the prevalence, pathological significance, and pharmacology of MCs in the CNS of ALS patients remain largely unknown. In autopsy ALS spinal cords, we identified for the first time that MCs express c-Kit together with chymase, tryptase, and Cox-2 and display granular or degranulating morphology, as compared with scarce MCs in control cords. In ALS, MCs were mainly found in the niche between spinal motor neuron somas and nearby microvascular elements, and they displayed remarkable pathological abnormalities. Similarly, MCs accumulated in the motor neuron-vascular niche of ALS murine models, in the vicinity of astrocytes and motor neurons expressing the c-Kit ligand stem cell factor (SCF), suggesting an SCF/c-Kit-dependent mechanism of MC differentiation from precursors. Mechanistically, we provide evidence that fully differentiated MCs in cell cultures can be generated from the murine ALS spinal cord tissue, further supporting the presence of c-Kit+ MC precursors. Moreover, intravenous administration of bone marrow-derived c-Kit+ MC precursors infiltrated the spinal cord in ALS mice but not in controls, consistent with aberrant trafficking through a defective microvasculature. Pharmacological inhibition of c-Kit with masitinib in ALS mice reduced the MC number and the influx of MC precursors from the periphery. Our results suggest a previously unknown pathogenic mechanism triggered by MCs in the ALS motor neuron-vascular niche that might be targeted pharmacologically

Substrate Recognition Properties from an Intermediate Structural State of the UreA Transporter

Through a combination of comparative modeling, site-directed and classical random mutagenesis approaches, we previously identified critical residues for binding, recognition, and translocation of urea, and its inhibition by 2-thiourea and acetamide in the Aspergillus nidulans urea transporter, UreA. To deepen the structural characterization of UreA, we employed the artificial intelligence (AI) based AlphaFold2 (AF2) program. In this analysis, the resulting AF2 models lacked inward- and outward-facing cavities, suggesting a structural intermediate state of UreA. Moreover, the orientation of the W82, W84, N279, and T282 side chains showed a large variability, which in the case of W82 and W84, may operate as a gating mechanism in the ligand pathway. To test this hypothesis non-conservative and conservative substitutions of these amino acids were introduced, and binding and transport assessed for urea and its toxic analogue 2-thiourea, as well as binding of the structural analogue acetamide. As a result, residues W82, W84, N279, and T282 were implicated in substrate identification, selection, and translocation. Using molecular docking with Autodock Vina with flexible side chains, we corroborated the AF2 theoretical intermediate model, showing a remarkable correlation between docking scores and experimental affinities determined in wild-type and UreA mutants. The combination of AI-based modeling with classical docking, validated by comprehensive mutational analysis at the binding region, would suggest an unforeseen option to determine structural level details on a challenging family of proteins

Closo-Carboranyl- and Metallacarboranyl [1,2,3]triazolyl-Decorated Lapatinib-Scaffold for Cancer Therapy Combining Tyrosine Kinase Inhibition and Boron Neutron Capture Therapy

© 2020 by the authors.One of the driving forces of carcinogenesis in humans is the aberrant activation of receptors; consequently, one of the most promising mechanisms for cancer treatment is receptor inhibition by chemotherapy. Although a variety of cancers are initially susceptible to chemotherapy, they eventually develop multi-drug resistance. Anti-tumor agents overcoming resistance and acting through two or more ways offer greater therapeutic benefits over single-mechanism entities. In this study, we report on a new family of bifunctional compounds that, offering the possibility of dual action (drug + radiotherapy combinations), may result in significant clinical benefits. This new family of compounds combines two fragments: the drug fragment is a lapatinib group, which inhibits the tyrosine kinase receptor activity, and an icosahedral boron cluster used as agents for neutron capture therapy (BNCT). The developed compounds were evaluated in vitro against different tyrosine kinase receptors (TKRs)-expressing tumoral cells, and in vitro–BNCT experiments were performed for two of the most promising hybrids, 19 and 22. We identified hybrid 19 with excellent selectivity to inhibit cell proliferation and ability to induce necrosis/apoptosis of glioblastoma U87 MG cell line. Furthermore, derivative 22, bearing a water-solubility-enhancing moiety, showed moderate inhibition of cell proliferation in both U87 MG and colorectal HT-29 cell lines. Additionally, the HT-29 cells accumulated adequate levels of boron after hybrids 19 and 22 incubations rendering, and after neutron irradiation, higher BNCT-effects than BPA. The attractive profile of developed hybrids makes them interesting agents for combined therapy.This research was funded by FCE-ANII (FCE_3_2018_1_148288), Institut Pasteur de Montevideo— FOCEM. M.C., M.F.G., E.T., and H.C. are Sistema Nacional de Investigadores- Agencia Nacional de Investigación e Innovación (ANII) researchers. M.C. thanks CSIC-Universidad de la República (UdelaR) (Grupo I + D, CSIC-421) for his scholarships. M.C. thanks funding from ANII for his doctoral-scholarship (POS_NAC_2015_1_110068). C.V. and F.T. thanks MINECO (CTQ2016-75150-R) for financial support.Peer reviewe

Sunitinib-Containing Carborane Pharmacophore with the Ability to Inhibit Tyrosine Kinases Receptors FLT3, KIT and PDGFR-β, Exhibits Powerful In Vivo Anti-Glioblastoma Activity

Malignant gliomas are the most common malignant and aggressive primary brain tumors in adults, the prognosis being—especially for glioblastomas—extremely poor. There are no effective treatments yet. However, tyrosine kinase receptor (TKR) inhibitors and boron neutron capture therapy (BNCT), together, have been proposed as future therapeutic strategies. In this sense in our ongoing project of developing new anti-glioblastoma drugs, we identified a sunitinib-carborane hybrid agent, 1, with both in vitro selective cytotoxicity and excellent BNCT-behavior. Consequently, we studied the ability of compound 1 to inhibit TKRs, its promotion of cellular death processes, and its effects on the cell cycle. Moreover, we analyzed some relevant drug-like properties of 1, i.e., mutagenicity and ability to cross the blood–brain barrier. These results encouraged us to perform an in vivo anti-glioblastoma proof of concept assay. It turned out to be a selective FLT3, KIT, and PDGFR-β inhibitor and increased the apoptotic glioma-cell numbers and arrested sub-G1-phase cell cycle. Its in vivo activity in immunosuppressed mice bearing U87 MG human glioblastoma evidenced excellent anti-tumor behavior.This research was funded by Agencia Nacional de Investigación e Innovación (ANII, Uruguay), grant numbers FCE_3_2018_1_148288 and POS_NAC_2015_1_110068, Institut Pasteur de Montevideo—FOCEM, and Comisión Sectorail de Investigación Científica-Universidad de la República (Uruguay). M.C., M.F.G., E.T., L.B., and H.C. are Sistema Nacional de Investigadores-ANII researchers.Peer reviewe

Bimodal Therapeutic Agents Against Glioblastoma, One of the Most Lethal Forms of Cancer

About 95 % of people diagnosed with glioblastoma die within five years. Glioblastoma is the most aggressive central nervous system tumour. It is necessary to make progress in the glioblastoma treatment so that advanced chemotherapy drugs or radiation therapy or, ideally, two‐in‐one hybrid systems should be implemented. Tyrosine kinase receptors–inhibitors and boron neutron capture therapy (BNCT), together, could provide a therapeutic strategy. In this work, sunitinib decorated‐carborane hybrids were prepared and biologically evaluated identifying excellent antitumoral‐ and BNCT‐agents. One of the selected hybrids was studied against glioma‐cells and found to be 4 times more cytotoxic than sunitinib and 1.7 times more effective than 10B‐boronophenylalanine fructose complex when the cells were irradiated with neutrons.This work was supported in part by grant by FCE‐ANII (FCE_3_2018_1_148288). M.C., P.C., and H.C. are Sistema Nacional de Investigadores‐Agencia Nacional de Investigación e Innovación (ANII) researchers. M.C. thanks CSIC‐Universidad de la República (UdelaR) (Grupo I+D, CSIC‐421) for his scholarships. M.C. thanks funding from ANII for his doctoral‐scholarship (POS_NAC_2015_1_110068).Peer reviewe

Carboranylanilinoquinazoline EGFR-inhibitors: toward ‘lead-to-candidate’ stage in the drug-development pipeline

Peer reviewe

Bimodal therapeutic agents against Glioblastoma, one of the most lethal forms of cancer

About 95 % of people diagnosed with glioblastoma die within five years. Glioblastoma is the most aggressive central nervous system tumour. It is necessary to make progress in the glioblastoma treatment so that advanced chemotherapy drugs or radiation therapy or, ideally, two-in-one hybrid systems should be implemented. Tyrosine kinase receptors–inhibitors and boron neutron capture therapy (BNCT), together, could provide a therapeutic strategy. In this work, sunitinib decorated-carborane hybrids were prepared and biologically evaluated identifying excellent antitumoral- and BNCT-agents. One of the selected hybrids was studied against glioma-cells and found to be 4 times more cytotoxic than sunitinib and 1.7 times more effective than 10B-boronophenylalanine fructose complex when the cells were irradiated with neutrons.Fil: Couto, Marcos. Universidad de la República. Facultad de Ciencias; Uruguay. Consejo Superior de Investigaciones Científicas; España. Universitat Autònoma de Barcelona; EspañaFil: Alamón, Catalina. Universidad de la República. Facultad de Ciencias; UruguayFil: Nievas, Susana Isabel. Comisión Nacional de Energía Atómica; ArgentinaFil: Perona, Marina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Comisión Nacional de Energía Atómica. Gerencia de Área de Aplicaciones de la Tecnología Nuclear. Departamento de Radiobiología; ArgentinaFil: Dagrosa, María Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Comisión Nacional de Energía Atómica. Gerencia de Área de Aplicaciones de la Tecnología Nuclear. Departamento de Radiobiología; ArgentinaFil: Teixidor, Francesc. Consejo Superior de Investigaciones Científicas; España. Universitat Autònoma de Barcelona; EspañaFil: Cabral, Pablo. Universidad de la República. Facultad de Ciencias; UruguayFil: Viñas, Clara. Consejo Superior de Investigaciones Científicas; España. Universitat Autònoma de Barcelona; EspañaFil: Cerecetto, Hugo. Universidad de la República. Facultad de Ciencias; Urugua

Informe final del proyecto: Profundización en la identificación de determinantes estructurales y funcionales de UreA

En el hongo filamentoso Aspergillus nidulans el transporte de urea tiene lugar a través de UreA, un simportador urea/H+ con ortólogos en hongos y plantas, perteneciente a la familia de los simportadores de sodio (SSS). Nuestro grupo llevó a cabo un estudio de la relación estructura- función de UreA mediante una estrategia de mutagénesis y modelado tridimensional. El mismo permitió identificar una serie de aminoácidos implicados en la unión, reconocimiento y translocación de la urea por parte del transportador. En este proyecto se propuso ahondar en el conocimiento de los determinantes estructurales y funcionales de UreA, mediante la identificación de nuevos residuos o regiones de UreA implicadas en la unión al sustrato y la selectividad por el mismo. En este sentido pudimos determinar que los aminoácidos W82, W84, N279 y T282 cumplen un rol en la interacción y/o selectividad por el sustrato, proponiendo a su vez, que la región en donde éstos se encuentran comprendería el sitio de unión al sustrato de UreA. Asimismo se realizó un análisis de los “loops” extracelulares 3 e intracelular 7, determinando que éstos poseen un rol estructural y funcional en UreA. Ya que la oligomerización de transportadores es un fenómeno que aparece con frecuencia en la biología de los mismos, pero no se conoce cuán general es ésta ni cuál es su rol para cada clase de proteínas de transporte, nos propusimos aportar a esta temática determinando si UreA es capaz de formar oligómeros. Se obtuvieron resultados preliminares que indicarían que UreA oligomeriza. Actualmente estamos realizando experimentos adicionales para confirmar ésto. Creemos que los resultados obtenidos en este proyecto contribuyen al conocimiento de la relación estructura/función de transportadores de hongos y plantas, así como en la determinación de la especificidad de transportadores en general.Agencia Nacional de Investigación e Innovació

Informe final del proyecto: Profundización en la identificación de determinantes estructurales y funcionales de UreA

En el hongo filamentoso Aspergillus nidulans el transporte de urea tiene lugar a través de UreA, un simportador urea/H+ con ortólogos en hongos y plantas, perteneciente a la familia de los simportadores de sodio (SSS). Nuestro grupo llevó a cabo un estudio de la relación estructura- función de UreA mediante una estrategia de mutagénesis y modelado tridimensional. El mismo permitió identificar una serie de aminoácidos implicados en la unión, reconocimiento y translocación de la urea por parte del transportador. En este proyecto se propuso ahondar en el conocimiento de los determinantes estructurales y funcionales de UreA, mediante la identificación de nuevos residuos o regiones de UreA implicadas en la unión al sustrato y la selectividad por el mismo. En este sentido pudimos determinar que los aminoácidos W82, W84, N279 y T282 cumplen un rol en la interacción y/o selectividad por el sustrato, proponiendo a su vez, que la región en donde éstos se encuentran comprendería el sitio de unión al sustrato de UreA. Asimismo se realizó un análisis de los “loops” extracelulares 3 e intracelular 7, determinando que éstos poseen un rol estructural y funcional en UreA. Ya que la oligomerización de transportadores es un fenómeno que aparece con frecuencia en la biología de los mismos, pero no se conoce cuán general es ésta ni cuál es su rol para cada clase de proteínas de transporte, nos propusimos aportar a esta temática determinando si UreA es capaz de formar oligómeros. Se obtuvieron resultados preliminares que indicarían que UreA oligomeriza. Actualmente estamos realizando experimentos adicionales para confirmar ésto. Creemos que los resultados obtenidos en este proyecto contribuyen al conocimiento de la relación estructura/función de transportadores de hongos y plantas, así como en la determinación de la especificidad de transportadores en general.Agencia Nacional de Investigación e Innovació