Molecular Characterization of Two Known SRD5A2 Gene Variants in Mexican Patients With Disorder of Sexual Development

Background: The 5 alpha-reductase type 2 deficiency (5 alpha-RD2) is a specific form of disorder of sexual development (DSD). Pathogenic variants in the SRD5A2 gene, which encodes this enzyme, are responsible for 46,XY DSD.Objective: The objective of the study was to investigate the genetic etiology of 46,XY DSD in two Mexican families with affected children.Materials and methods: The SRD5A2 gene of the parents and affected children was screened in both families via polymerase chain reaction amplification and DNA direct sequencing. The role of genetic variants in enzymatic activity was tested by site-directed mutagenesis.Results: Subject 1 presented two variants: p.Glu197Asp and p.Pro212Arg. Subject 2 was homozygous for the variant p.Glu197Asp. The two variants were reported in early studies. The directed mutagenesis study showed that the p.Glu197Asp and p.Pro212Arg variants lead to a total loss of enzymatic activity and, consequently, abnormal genitalia development in the patients.Conclusion: These results suggest that p.Glu197Asp and p.Pro212Arg are pathogenic variants that lead to the phenotypic expression of DSD. 5 alpha-RD2 is of extreme importance not only because of its frequency (it is rare) but also because of its significance in understanding the mechanism of androgen action, the process of sexual differentiation, and the factors that influence normal sexual behavior.Peer reviewe

Pranlukast Antagonizes CD49f and Reduces Sternness in Triple-Negative Breast Cancer Cells

Introduction: Cancer stem cells (CSCs) drive the initiation, maintenance, and therapy response of breast tumors. CD49f is expressed in breast CSCs and functions in the maintenance of stemness. Thus, blockade of CD49f is a potential therapeutic approach for targeting breast CSCs. In the present study, we aimed to repurpose drugs as CD49f antagonists. Materials and Methods: We performed consensus molecular docking using a subdomain of CD49f that is critical for heterodimerization and a collection of pharmochemicals clini-cally tested. Molecular dynamics simulations were employed to further characterize drug-target binding. Using MDA-MB-231 cells, we evaluated the effects of potential CD49f antagonists on 1) cell adhesion to laminin; 2) mammosphere formation; and 3) cell viability. We analyzed the effects of the drug with better CSC-selectivity on the activation of CD49f-downstream signaling by Western blot (WB) and co-immunoprecipitation. Expressions of the stem cell markers CD44 and SOX2 were analyzed by flow cytometry and WB, respectively. Transactivation of SOX2 promoter was evaluated by luciferase reporter assays. Changes in the number of CSCs were assessed by limiting-dilution xenotransplantation. Results: Pranlukast, a drug used to treat asthma, bound to CD49f in silico and inhibited the adhesion of CD49f+ MDA-MB-231 cells to laminin, indicating that it antagonizes CD49f-containing integrins. Molecular dynamics analysis showed that pranlukast binding induces con-formational changes in CD49f that affect its interaction with β1-integrin subunit and constrained the conformational dynamics of the heterodimer. Pranlukast decreased the clonogenicity of breast cancer cells on mammosphere formation assay but had no impact on the viability of bulk tumor cells. Brief exposure of MDA-MB-231 cells to pranlukast altered CD49f-dependent signaling, reducing focal adhesion kinase (FAK) and phosphatidylinositol 3-kinase (PI3K) activation. Further, pranlukast-treated cells showed decreased CD44 and SOX2 expression, SOX2 promoter transacti-vation, and in vivo tumorigenicity, supporting that this drug reduces the frequency of CSC. Conclusion: Our results support the function of pranlukast as a CD49f antagonist that reduces the CSC population in triple-negative breast cancer cells. The pharmacokinetics and toxicology of this drug have already been established, rendering a potential adjuvant therapy for breast cancer patients

Polyamines Influence Mouse Sperm Channels Activity

Polyamines are ubiquitous polycationic compounds that are highly charged at physiological pH. While passing through the epididymis, sperm lose their capacity to synthesize the polyamines and, upon ejaculation, again come into contact with the polyamines contained in the seminal fluid, unleashing physiological events that improve sperm motility and capacitation. In the present work, we hypothesize about the influence of polyamines, namely, spermine, spermidine, and putrescine, on the activity of sperm channels, evaluating the intracellular concentrations of chloride [Cl−]i, calcium [Ca2+]i, sodium [Na+]i, potassium [K+]i, the membrane Vm, and pHi. The aim of this is to identify the possible regulatory mechanisms mediated by the polyamines on sperm-specific channels under capacitation and non-capacitation conditions. The results showed that the presence of polyamines did not directly influence the activity of calcium and chloride channels. However, the results suggested an interaction of polyamines with sodium and potassium channels, which may contribute to the membrane Vm during capacitation. In addition, alkalization of the pHi revealed the possible activation of sperm-specific Na+/H+ exchangers (NHEs) by the increased levels of cyclic AMP (cAMP), which were produced by soluble adenylate cyclase (sAC) and interact with the polyamines, evidence that is supported by in silico analysis

Structure-Based Virtual Screening and In Vitro Evaluation of New <i>Trypanosoma cruzi</i> Cruzain Inhibitors

Chagas disease (CD), or American trypanosomiasis, causes more than 10,000 deaths per year in the Americas. Current medical therapy for CD has low efficacy in the chronic phase of the disease and serious adverse effects; therefore, it is necessary to search for new pharmacological treatments. In this work, the ZINC15 database was filtered using the N-acylhydrazone moiety and a subsequent structure-based virtual screening was performed using the cruzain enzyme of Trypanosoma cruzi to predict new potential cruzain inhibitors. After a rational selection process, four compounds, Z2 (ZINC9873043), Z3 (ZINC9870651), Z5 (ZINC9715287), and Z6 (ZINC9861447), were chosen to evaluate their in vitro trypanocidal activity and enzyme inhibition. Compound Z5 showed the best trypanocidal activity against epimatigote (IC50 = 36.26 &#177; 9.9 &#956;M) and trypomastigote (IC50 = 166.21 &#177; 14.5 &#956;M and 185.1 &#177; 8.5 &#956;M on NINOA and INC-5 strains, respectively) forms of Trypanosoma cruzi. In addition, Z5 showed a better inhibitory effect on Trypanosoma cruzi proteases than S1 (STK552090, 8-chloro-N-(3-morpholinopropyl)-5H-pyrimido[5,4-b]-indol-4-amine), a known cruzain inhibitor. This study encourages the use of computational tools for the rational search for trypanocidal drugs

Favipiravir and/or nitazoxanide: a randomized, double-blind, 2x2 design, placebo-controlled trial of early therapy in COVID-19 in health workers, their household members, and patients treated at IMSS (FANTAZE)

BackgroundThe 2020 pandemic of SARS-CoV-2 causing COVID-19 disease is an unprecedented global emergency. COVID-19 appears to be a disease with an early phase where the virus replicates, coinciding with the first presentation of symptoms, followed by a later 'inflammatory' phase which results in severe disease in some individuals. It is known from other rapidly progressive infections such as sepsis and influenza that early treatment with antimicrobials is associated with a better outcome. The hypothesis is that this holds for COVID-19 and that early antiviral treatment may prevent progression to the later phase of the disease.MethodsTrial design: Phase IIA randomised, double-blind, 2 × 2 design, placebo-controlled, interventional trial.RandomisationParticipants will be randomised 1:1 by stratification, with the following factors: gender, obesity, symptomatic or asymptomatic, current smoking status presence or absence of comorbidity, and if the participant has or has not been vaccinated.BlindingParticipants and investigators will both be blinded to treatment allocation (double-blind).DiscussionWe propose to conduct a proof-of-principle placebo-controlled clinical trial of favipiravir plus or minus nitazoxanide in health workers, their household members and patients treated at the Mexican Social Security Institute (IMSS) facilities. Participants with or without symptomatic COVID-19 or who tested positive will be assigned to receive favipiravir plus nitazoxanide or favipiravir plus nitazoxanide placebo. The primary outcome will be the difference in the amount of virus ('viral load') in the upper respiratory tract after 5 days of therapy. Secondary outcomes will include hospitalization, major morbidity and mortality, pharmacokinetics, and impact of antiviral therapy on viral genetic mutation rate. If favipiravir with nitazoxanide demonstrates important antiviral effects without significant toxicity, there will be a strong case for a larger trial in people at high risk of hospitalization or intensive care admission, for example older patients and/or those with comorbidities and with early disease.Trial registrationClinicalTrials.gov NCT04918927 . Registered on June 9, 2021