Structure-based discovery and in vitro validation of inhibitors of chloride intracellular channel 4 protein

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.csbj.2022.12.040.Acknowledgment We would like to thank the Center for High-Performance Computing, Cape Town, South Africa for providing computational resources and Dr Nabil Hajji for his kind contributions in enhancing the staining protocol.The use of computer-aided methods have continued to propel accelerated drug discovery across various disease models, interestingly allowing the specific inhibition of pathogenic targets. Chloride Intracellular Channel Protein 4 (CLIC4) is a novel class of intracellular ion channel highly implicated in tumor and vascular biology. It regulates cell proliferation, apoptosis and angiogenesis; and is involved in multiple pathologic signaling pathways. Absence of specific inhibitors however impedes its advancement to translational research. Here, we integrate structural bioinformatics and experimental research approaches for the discovery and validation of small-molecule inhibitors of CLIC4. High-affinity allosteric binders were identified from a library of 1615 Food and Drug Administration (FDA)-approved drugs via a high-performance computing-powered blind-docking approach, resulting in the selection of amphotericin B and rapamycin. NMR assays confirmed the binding and conformational disruptive effects of both drugs while they also reversed stress-induced membrane translocation of CLIC4 and inhibited endothelial cell migration. Structural and dynamics simulation studies further revealed that the inhibitory mechanisms of these compounds were hinged on the allosteric modulation of the catalytic glutathione (GSH)-like site loop and the extended catalytic β loop which may elicit interference with the catalytic activities of CLIC4. Structurebased insights from this study provide the basis for the selective targeting of CLIC4 to treat the associated pathologies

An Updated View of the Trypanosoma cruzi Life Cycle: Intervention Points for an Effective Treatment

Chagas disease (CD) is a parasitic, systemic, chronic, and often fatal illness caused by infection with the protozoan Trypanosoma cruzi. The World Health Organization classifies CD as the most prevalent of povertypromoting neglected tropical diseases, the most important parasitic one, and the third most infectious disease in Latin America. Currently, CD is a global public health issue that affects 6−8 million people. However, the current approved treatments are limited to two nitroheterocyclic drugs developed more than 50 years ago. Many efforts have been made in recent decades to find new therapies, but our limited understanding of the infection process, pathology development, and long-term nature of this disease has made it impossible to develop new drugs, effective treatment, or vaccines. This Review aims to provide a comprehensive update on our understanding of the current life cycle, new morphological forms, and genetic diversity of T. cruzi, as well as identify intervention points in the life cycle where new drugs and treatments could achieve a parasitic cure.Junta de Andalucia E-BIO-464-UGR20Junta de Andalucia (Proyectos I+D+I FEDER Andalucia 2014-2020)Alfonso Martin Escudero Foundatio

Características clínicas y urodinámicas de las mujeres con detrusor hipoactivo

Objective: To describe the clinical and urodynamic features of the hypoactive detrusor (DU) in women. Methods: Cross sectional comparative study between two groups of women with lower tract urinary symptoms. Group 1 were patients with Pdet Qmax 30 or with the bladder emptying efficiency (BVE) criteria, and Group 2 were patients without DU. Central tendency measures and proportions were used to report results and parametric tests were performed to compare groups. Results: 155 patients were included, 44 with DU (Group 1) and 111 without DU (Group 2); mean age was 60,8 ± 16,3 and 55,7 ± 13,4 respectively (p=0,0468). Nocturia (p=0,0061) and micturition effort (p=0,000) were the only variables identified in the bivariate analysis who achieved statistical significance. Mean Watts factor (WF) was 3,2 ± 1,0 y 6,0 ± 2,2 in Group 1 and 2, respectively (p=0,000), while mean projected isovolumetric pressure 1 (PIP1) was 28,0 ± 7,5 and 45,9 ± 11,2, respectively (p=0,000). The multivariate analysis identified age above 60 years, nocturia and micturition effort associated with DU. Conclusion: Urodynamic parameters such as PIP1, BCI, BE and WF showed significant difference in the bivariate analysis, WF<5 and BCI <80 are best predictors of DU.Objetivo: Determinar las características clínicas y urodinámicas en mujeres con detrusor hipoactivo (DU). Material y métodos: Estudio de serie de casos de corte transversal, retrospectivo, comparativo, entre 2 grupos de pacientes femeninos con síntomas del tracto urinario inferior (STUI). Grupo 1: pacientes con DU según criterios urodinámicos, presión del del detrusor en el flujo máximo de 30 cm H20 (PdetQmax30) o eficiencia del vaciado de la vejiga (BVE) y grupo 2: pacientes sin DU. Se utilizaron medidas de tendencia central y proporciones para la descripción de los datos y pruebas paramétricas para la comparación entre grupos. Resultados: Ciento cincuenta y cinco pacientes fueron incluidos, 44 con DU (grupo 1) y 111 sin DU (grupo 2), con una media de edad de 60,8 ± 16,3 y 55,7 ± 13,4 respectivamente (p=0,0468). La nicturia (p=0,0061) y el esfuerzo miccional (p=0,000) fueron las únicas variables clínicas que presentaron una diferencia significativa en el análisis bivariado. La media de watts factor (WF) fue 3,2 ± 1,0 y 6,0 ± 2,2 en el grupo 1 y 2 respectivamente (p=0,000), mientras que el promedio de la presión isovolumétrica proyectada 1 (PIP1) fue de 28,0 ± 7,5 y 45,9 ± 11,2 respectivamente (p=0,000).En el análisis multivariado, la edad > 60 años, la nicturia, el esfuerzo miccional, tuvieron asociación significativa con DU. Conclusión: Las variables urodinámicas como PIP1, índice de contracción vesical (BCI), BVE y WF mostraron una diferencia significativa en el análisis bivariado, siendo el WF <5 y BCI <80 las que mejor identifican la presencia de DU en mujeres

Site-to-site interdomain communication may mediate different loss-of-function mechanisms in a cancer-associated NQO1 polymorphism

Disease associated genetic variations often cause intracellular enzyme inactivation, dysregulationand instability. However, allosteric communication of mutational effects to distant functional sites leading to loss-of-function remains poorly understood. We characterize here interdomain site-to-site communication by which a common cancer-associated single nucleotide polymorphism (c.C609T/p. P187S) reduces the activity and stability in vivo of NAD(P)H:quinone oxidoreductase 1 (NQO1). NQO1 is a FAD-dependent, two-domain multifunctional stress protein acting as a Phase II enzyme, activating cancer pro-drugs and stabilizing p53 and p73α oncosuppressors. We show that p.P187S causes structural and dynamic changes communicated to functional sites far from the mutated site, affecting the FAD binding site located at the N-terminal domain (NTD) and accelerating proteasomal degradation through dynamic effects on the C-terminal domain (CTD). Structural protein:protein interactionstudies reveal that the cancer-associated polymorphism does not abolish the interaction with p73α, indicating that oncosuppressor destabilization largely mirrors the low intracellular stability of p.P187S. In conclusion, we show how a single disease associated amino acid change may allosterically perturb several functional sites in an oligomeric and multidomain protein. These results have important implications for the understanding of loss-of-function genetic diseases and the identification of novelstructural hot spots as targets for pharmacological intervention

Conformational dynamics is key to understanding loss-of-function of NQO1 cancer-associated polymorphisms and its correction by pharmacological ligands

Protein dynamics is essential to understand protein function and stability, even though is rarely investigated as the origin of loss-of-function due to genetic variations. Here, we use biochemical, biophysical, cell and computational biology tools to study two loss-of-function and cancer-associated polymorphisms (p.R139W and p.P187S) in human NAD(P)H quinone oxidoreductase 1 (NQO1), a FAD-dependent enzyme which activates cancer pro-drugs and stabilizes several oncosuppressors. We show that p.P187S strongly destabilizes the NQO1 dimer in vitro and increases the flexibility of the C-terminal domain, while a combination of FAD and the inhibitor dicoumarol overcome these alterations. Additionally, changes in global stability due to polymorphisms and ligand binding are linked to the dynamics of the dimer interface, whereas the low activity and affinity for FAD in p.P187S is caused by increased fluctuations at the FAD binding site. Importantly, NQO1 steady-state protein levels in cell cultures correlate primarily with the dynamics of the C-terminal domain, supporting a directional preference in NQO1 proteasomal degradation and the use of ligands binding to this domain to stabilize p.P187S in vivo. In conclusion, protein dynamics are fundamental to understanding loss-of-function in p.P187S, and to develop new pharmacological therapies to rescue this function

Enhanced vulnerability of human proteins towards disease-associated inactivation through divergent evolution

Human proteins are vulnerable towards disease-associated single amino acid replacements affecting protein stability and function. Interestingly, a few studies have shown that consensus amino acids from mammals or vertebrates can enhance protein stability when incorporated into human proteins. Here, we investigate yet unexplored relationships between the high vulnerability of human proteins towards disease-associated inactivation and recent evolutionary site-specific divergence of stabilizing amino acids. Using phylogenetic, structural and experimental analyses, we show that divergence from the consensus amino acids at several sites during mammalian evolution has caused local protein destabilization in two human proteins linked to disease: cancer-associated NQO1 and alanine:glyoxylate aminotransferase, mutated in primary hyperoxaluria type I. We demonstrate that a single consensus mutation (H80R) acts as a disease suppressor on the most common cancer-associated polymorphism in NQO1 (P187S). The H80R mutation reactivates P187S by enhancing FAD binding affinity through local and dynamic stabilization of its binding site. Furthermore, we show how a second suppressor mutation (E247Q) cooperates with H80R in protecting the P187S polymorphism towards inactivation through long-range allosteric communication within the structural ensemble of the protein. Our results support that recent divergence of consensus amino acids may have occurred with neutral effects on many functional and regulatory traits of wild-type human proteins. However, divergence at certain sites may have increased the propensity of some human proteins towards inactivation due to disease-associated mutations and polymorphisms. Consensus mutations also emerge as a potential strategy to identify structural hot-spots in proteins as targets for pharmacological rescue in loss-of-function genetic diseases

The role of protein dynamics in loss-of-function disease mechanisms: a NQO1 cancer-associated polymorphism as model

Tesis Univ. Granada.Programa Oficial de Doctorado en QuímicaWe thank Prof. José Manuel Sánchez-Ruíz for kindly providing the home-built software to determine solvent accessible surface areas. This work was supported by grants from MINECO (BIO2012-34937, CSD2009-00088 and SAF2011-23933), Junta de Andalucia (P11-CTS-07187), European Union (FP7-REGPOT-CT2012-31637- IMBRAIN) and FEDER Funds. A.L.P. is supported by a Ramón y Cajal research contract from MINECO/ University of Granada (RYC-2009-04147). N.M.-T. is supported by FPI predoctoral fellowships from MINECO. E.M.-C. is supported by a predoctoral contract from Junta de Andalucia.ALP thanks Prof. Jose Manuel Sanchez-Ruiz for support. This work was supported by grants from MINECO (BIO2015 66426-R to JMSR, CTQ2015-64445-R to JLN and SAF2015-69796 to ES), Junta de Andalucia (P11- CTS-07187 to ALP) and FEDER funds. EMC acknowledges a pre-doctoral fellowship from Junta de Andalucia.ALP thanks Prof. Jose Manuel Sanchez-Ruiz for support. JLN thanks C. Arrowsmith for the kind gift of the SAMp73 vector. We acknowledge the ESRF for the provision of synchrotron radiation time at beam lines ID29 and ID30A-1, and the staff for their helpful support. EMC acknowledges a pre-doctoral fellow- ship from Junta de Andalucía.ALP thanks Prof Jose Manuel Sánchez-Ruiz and BM thanks Prof Francisco Conejero-Lara for support. This work was supported by the Spanish Ministry of Economy and Competitiveness (BIO2015-66426-R to JMSR and BIO2013-40697-R to FCL), Junta de Andalucía (P11-CTS-07187toALP) and FEDER funds. EMC acknowledges a predoctoral fellowship from Junta de Andalucía. We acknowledge the Diamond Light Source for provision of synchrotron radiation time at beam line B21, and to Robert Rambo and Nathan Cowieson for assistance during data collection. The sponsors had no role in the design of this study

Enhanced vulnerability of human proteins towards disease-associated inactivation through divergent evolution

Human proteins are vulnerable towards disease-associated single amino acid replacements affecting protein stability and function. Interestingly, a few studies have shown that consensus amino acids from mammals or vertebrates can enhance protein stability when incorporated into human proteins. Here, we investigate yet unexplored relationships between the high vulnerability of human proteins towards disease-associated inactivation and recent evolutionary site-specific divergence of stabilizing amino acids. Using phylogenetic, structural and experimental analyses, we show that divergence from the consensus amino acids at several sites during mammalian evolution has caused local protein destabilization in two human proteins linked to disease: cancer-associated NQO1 and alanine: glyoxylate aminotransferase, mutated in primary hyperoxaluria type I. We demonstrate that a single consensus mutation (H80R) acts as a disease suppressor on the most common cancer-associated polymorphism in NQO1 (P187S). The H80R mutation reactivates P187S by enhancing FAD binding affinity through local and dynamic stabilization of its binding site. Furthermore, we show how a second suppressor mutation (E247Q) cooperates with H80R in protecting the P187S polymorphism towards inactivation through long-range allosteric communication within the structural ensemble of the protein. Our results support that recent divergence of consensus amino acids may have occurred with neutral effects on many functional and regulatory traits of wild-type human proteins. However, divergence at certain sites may have increased the propensity of some human proteins towards inactivation due to disease-associated mutations and polymorphisms. Consensus mutations also emerge as a potential strategy to identify structural hot-spots in proteins as targets for pharmacological rescue in loss-of-function genetic diseases.Spanish Ministry of Economy and Competitiveness, MINECO (BIO 2015 66426-R to JMSR, CTQ 2015-64445-R to JLN, ‘Factoría Española de Cristalización’, Consolider-Ingenio 2010 to JAG and SAF2015-69796 to ES), Junta de Andalucia (P11-CTS-07187 to ALP) and FEDER fun

Validación del Cuestionario de Preferencias de Soporte Vital (LSPQ) para su uso en la población española

ObjetivoValidar el Cuestionario de Preferencias de Soporte Vital (LSPQ) para su uso en población española.DiseñoEstudio realizado en tres fases: a) traducción y adaptación idiomática; b) evaluación de su validez, y c) validación de sus propiedades psicométricas.EmplazamientoEl estudio se ha llevado a cabo en 9 consultas de enfermería de atención primaria y en 2 aulas universitarias de Granada.ParticipantesSe incluyeron pacientes mayores de edad y estudiantes universitarios de primer y segundo cursos, sin deterioro cognitivo.Mediciones principalesEquivalencia de la traducción con el original verificada por bilingües. Corrección lingüística y gramatical evaluada por expertos en lengua española. Validez del cuestionario evaluada por 15 jueces-expertos y 20 pacientes.Complejidad lingüística (índice de Szigriszt). Estabilidad temporal (test de McNemar). Consistencia interna (alfa de Cronbach).Resultadosa) 2 personas bilingües realizaron una traducción que fue revisada por 6 expertos en lengua española. Tras las modificaciones que sugirieron, las personas bilingües aseguraron su equivalencia con el original; b) 15 expertos afirmaron que cada ítem era buen indicador de las preferencias de cuidado al final de la vida; c) 20 usuarios confirmaron la aceptabilidad y comprensibilidad del cuestionario; d) en relación con la fiabilidad, completaron el cuestionario 299 participantes (de los 369 inicialmente seleccionados), y e) la estabilidad del cuestionario es de 0,92 y su consistencia interna de 0,85.ConclusiónEl cuestionario LSPQen español (LSPQ-e) es una herramienta fiable diseñada para ayudar a los pacientes a clarificar sus preferencias de cuidado y tratamiento al final de la vida.ObjectiveTo validate the Life-Support Preferences Questionnaire (LSPQ) for its use in Spain.DesignThree-stage study: a) translation and linguistic adaptation; b) evaluation of its validity; and c) validation of its psychometric properties.SettingNine primary care nursing clinics and 2 university classes in Granada, Spain.ParticipantsPatients who were of age and were first- or second-year university students, without any cognitive deterioration.Main measurementsThe equivalence of the translation and the original was checked by bilingual people; its linguistic and grammatical standard was appraised by experts in Spanish. The validity of the questionnaire was evaluated by 15 judges-experts and 20 patients; linguistic complexity (Szigriszt index); stability in time (McNemar test); internal consistency (Cronbach's alpha).Resultsa) 2 bilingual people did a translation that was reviewed by 6 experts in Spanish. After alterations suggested by the experts, the bilingual people ratified its equivalence with the original; b) 15 experts affirmed that each item was a good indicator of care preferences atthe end of life; c) 20 users confirmed the acceptability and comprehensibility of the questionnaire; d) reliability: 299 participants out of the 369 initially selected filled in the questionnaire; and e) its stability was 0.92; and its internal consistency, 0.85.ConclusionThe LSPQ questionnaire in Spanish (LSPQ-e) is a reliable tool, designed to help patients to clarify their care and treatment preferences at the end of their lives