4-aminopyridyl-based lead compounds targeting CYP51 prevent spontaneous parasite relapse in a chronic model and improve cardiac pathology in an acute model of Trypanosoma cruzi infection.

BackgroundChagas disease, caused by the protozoan Trypanosoma cruzi, is the leading cause of heart failure in Latin America. The clinical treatment of Chagas disease is limited to two 60 year-old drugs, nifurtimox and benznidazole, that have variable efficacy against different strains of the parasite and may lead to severe side effects. CYP51 is an enzyme in the sterol biosynthesis pathway that has been exploited for the development of therapeutics for fungal and parasitic infections. In a target-based drug discovery program guided by x-ray crystallography, we identified the 4-aminopyridyl-based series of CYP51 inhibitors as being efficacious versus T.cruzi in vitro; two of the most potent leads, 9 and 12, have now been evaluated for toxicity and efficacy in mice.Methodology/principal findingsBoth acute and chronic animal models infected with wild type or transgenic T. cruzi strains were evaluated. There was no evidence of toxicity in the 28-day dosing study of uninfected animals, as judged by the monitoring of multiple serum and histological parameters. In two acute models of Chagas disease, 9 and 12 drastically reduced parasitemia, increased survival of mice, and prevented liver and heart injury. None of the compounds produced long term sterile cure. In the less severe acute model using the transgenic CL-Brenner strain of T.cruzi, parasitemia relapsed upon drug withdrawal. In the chronic model, parasitemia fell to a background level and, as evidenced by the bioluminescence detection of T. cruzi expressing the red-shifted luciferase marker, mice remained negative for 4 weeks after drug withdrawal. Two immunosuppression cycles with cyclophosphamide were required to re-activate the parasites. Although no sterile cure was achieved, the suppression of parasitemia in acutely infected mice resulted in drastically reduced inflammation in the heart.Conclusions/significanceThe positive outcomes achieved in the absence of sterile cure suggest that the target product profile in anti-Chagasic drug discovery should be revised in favor of safe re-administration of the medication during the lifespan of a Chagas disease patient. A medication that reduces parasite burden may halt or slow progression of cardiomyopathy and therefore improve both life expectancy and quality of life

Characterization of the in vivo telomeric single-strand binding proteins from Leishmania amazonensis

Orientador: Maria Isabel Nogueira CanoTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: A leishmaniose é uma parasitose humana emergente e ainda não controlada, causada por protozoários pertencentes ao gênero Leishmania. Atualmente, a doença atinge mais de 12 milhões de pessoas, não existindo ainda métodos eficientes para seu controle e erradicação. Por estas razões a Organização Mundial da Saúde classifica a leishmaniose como doença de categoria I e incentiva o desenvolvimento de novos métodos para controlar a doença para buscar novos alvos para drogas contra parasita. No presente trabalho, estudou-se as proteínas LaRBP38 e LaRPA-1 previamente identificadas por se associarem in vitro com a simples-fita telomérica rica em ¿G¿ de L. amazonensis. Os telomeros são extremidades físicas dos cromossomos de eucariotos, formados por complexos nucleoproteicos. São responsáveis por conferir estabilidade aos cromossomos, envitando a degradação pela maquina de reparo celular e a fusão entre extremidades cromossomais. Instabilidades no telômero causam normalmente danos irreparáveis à célula podendo levar à senescência e morte celular. As proteínas que se mantém complexadas ao telômero são responsáveis por mantê-lo funcioal. Cada proteína desempenha um papel importante, seja na proteção, processo replicativo ou manutenção da estabilidade estrutural do telômero, sendo portanto, alvos potenciais para o desenvolvimento de terapias antiparasitárias. A proteína RPA é conservada em toda escala evolutiva e cumpre importantes papéis nas maquinarias de replicação, recombinação e reparo do DNA genômico ...Observação: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digitalAbstract: Leishmaniasis is an emerging and non-controlled human disease, caused by protozoan belonging to the Leishmania genera. More than 12 million people are infected and there are no efficient methods for the controlling or eradication of the disease. For those reasons, the World Health Organization classifies leishmaniasis as category I disease and encourages the development of new methods to control the disease and to find new targets for drugs against the parasite. In the present work, we studied the proteins LaRBP38 and LaRPA-1, prior identifield by in vitro assays as proteins that associates with the Leishmania amazonensis G-rich single-stranded telomeric DNA. Telomeres are the physical ends of eukaryote chromosomes formed by proteins and DNA complexes. They are responsible for the chromosome stability, avoiding degradation by the rapair machinery end-to-end fusion. Telomere instability may cause irreversible damage in the cell, leading to senescence and cell death. The proteins that interact with the telomeres are responsible for the functional dynamics of theses structures. Each protein has an important role in the protection, replication process or in the stability maintenance. Therefore, telomeric protein could be considered good targets for the development of new therapies. RPA is an evolutionary conserved protein and plays important roles in replication, recombination and repair machineries. At the telomeres, RPA recruits telomerase, the protein responsible for telomeric elengation ...Note: The complete abstract is available with the full electronic digital thesis or dissertationsDoutoradoGenetica de MicroorganismosDoutor em Genetica e Biologia Molecula

Metabolite Profiling Of Experimental Cutaneous Leishmaniasis Lesions Demonstrates Significant Perturbations In Tissue Glycerophosphocholines

Each year 700,000 to 1.2 million new cases of cutaneous leishmaniasis (CL) are reported and yet CL remains one of thirteen diseases classified as neglected tropical diseases (NTDs). Leishmania major is one of several different species of that same genus that can cause CL. Current CL treatments are limited by adverse effects and rising resistance. Studying disease metabolism at the site of infection can lead to new drug targets. In this study, samples were collected from mice infected in the ear and footpad with L. major and analyzed by untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS). Significant differences in overall metabolite profiles were noted in the ear at the site of the lesion. Interestingly, lesion-adjacent, macroscopically healthy sites also showed alterations in specific metabolites, including select phosphocholines (PCs). Host-derived PCs in the lower m/z range (m/z 200-799) showed an increase with infection in the ear at the lesion site, while those in the higher m/z range (m/z 800-899) were decreased with infection at the lesion site. Overall, our results expanded our understanding of the mechanisms of CL pathogenesis through the host metabolism and may lead to new curative measures against infection with Leishmania

Mass Spectrometry-Based Chemical Cartography of a Cardiac Parasitic Infection

Trypanosoma cruzi parasites are the causative agents of Chagas disease, a leading infectious form of heart failure whose pathogenesis is still not fully characterized. In this work, we applied untargeted liquid chromatography-tandem mass spectrometry to heart sections from T. cruzi-infected and uninfected mice. We combined molecular networking and three-dimensional modeling to generate chemical cartographical heart models. This approach revealed for the first time preferential parasite localization to the base of the heart and regiospecific distributions of nucleoside derivatives and eicosanoids, which we correlated to tissue-damaging immune responses. We further detected novel cardiac chemical signatures related to the severity and ultimate outcome of the infection. These signatures included differential representation of higher- vs lower-molecular-weight carnitine and phosphatidylcholine family members in specific cardiac regions of mice infected with lethal or nonlethal T. cruzi strains and doses. Overall, this work provides new insights into Chagas disease pathogenesis and presents an analytical chemistry approach that can be broadly applied to the study of host-microbe interactions

Discovery of Triple Inhibitors of Both SARS-CoV-2 Proteases and Human Cathepsin L

One inhibitor of the main SARS-CoV-2 protease has been approved recently by the FDA, yet it targets only SARS-CoV-2 main protease (Mpro). Here, we discovered inhibitors containing thiuram disulfide or dithiobis-(thioformate) tested against three key proteases involved in SARS-CoV-2 replication, including Mpro, SARS-CoV-2 papain-like protease (PLpro), and human cathepsin L. The use of thiuram disulfide and dithiobis-(thioformate) covalent inhibitor warheads was inspired by an idea to find a better alternative than disulfiram, an approved treatment for chronic alcoholism that is currently in phase 2 clinical trials against SARS-CoV-2. Our goal was to find more potent inhibitors that target both viral proteases and one essential human protease to reduce the dosage, improve the efficacy, and minimize the adverse effects associated with these agents. We found that compounds coded as RI175, RI173, and RI172 were the most potent inhibitors in an enzymatic assay against SARS-CoV-2 Mpro, SARS-CoV-2 PLpro, and human cathepsin L, with IC50s of 300, 200, and 200 nM, which is about 5-, 19-, and 11-fold more potent than disulfiram, respectively. In addition, RI173 was tested against SARS-CoV-2 in a cell-based and toxicity assay and was shown to have a greater antiviral effect than disulfiram. The identified compounds demonstrated the promising potential of thiuram disulfide or dithiobis-(thioformate) as a reactive functional group in small molecules that could be further developed for treatment of the COVID-19 virus or related variants

Discovery of Triple Inhibitors of Both SARS-CoV-2 Proteases and Human Cathepsin L.

One inhibitor of the main SARS-CoV-2 protease has been approved recently by the FDA, yet it targets only SARS-CoV-2 main protease (Mpro). Here, we discovered inhibitors containing thiuram disulfide or dithiobis-(thioformate) tested against three key proteases involved in SARS-CoV-2 replication, including Mpro, SARS-CoV-2 papain-like protease (PLpro), and human cathepsin L. The use of thiuram disulfide and dithiobis-(thioformate) covalent inhibitor warheads was inspired by an idea to find a better alternative than disulfiram, an approved treatment for chronic alcoholism that is currently in phase 2 clinical trials against SARS-CoV-2. Our goal was to find more potent inhibitors that target both viral proteases and one essential human protease to reduce the dosage, improve the efficacy, and minimize the adverse effects associated with these agents. We found that compounds coded as RI175, RI173, and RI172 were the most potent inhibitors in an enzymatic assay against SARS-CoV-2 Mpro, SARS-CoV-2 PLpro, and human cathepsin L, with IC50s of 300, 200, and 200 nM, which is about 5-, 19-, and 11-fold more potent than disulfiram, respectively. In addition, RI173 was tested against SARS-CoV-2 in a cell-based and toxicity assay and was shown to have a greater antiviral effect than disulfiram. The identified compounds demonstrated the promising potential of thiuram disulfide or dithiobis-(thioformate) as a reactive functional group in small molecules that could be further developed for treatment of the COVID-19 virus or related variants

Machine Learning Models and Pathway Genome Data Base for <i>Trypanosoma cruzi</i> Drug Discovery

<div><p>Background</p><p>Chagas disease is a neglected tropical disease (NTD) caused by the eukaryotic parasite <i>Trypanosoma cruzi</i>. The current clinical and preclinical pipeline for <i>T</i>. <i>cruzi</i> is extremely sparse and lacks drug target diversity.</p><p>Methodology/Principal Findings</p><p>In the present study we developed a computational approach that utilized data from several public whole-cell, phenotypic high throughput screens that have been completed for <i>T</i>. <i>cruzi</i> by the Broad Institute, including a single screen of over 300,000 molecules in the search for chemical probes as part of the NIH Molecular Libraries program. We have also compiled and curated relevant biological and chemical compound screening data including (i) compounds and biological activity data from the literature, (ii) high throughput screening datasets, and (iii) predicted metabolites of <i>T</i>. <i>cruzi</i> metabolic pathways. This information was used to help us identify compounds and their potential targets. We have constructed a Pathway Genome Data Base for <i>T</i>. <i>cruzi</i>. In addition, we have developed Bayesian machine learning models that were used to virtually screen libraries of compounds. Ninety-seven compounds were selected for <i>in vitro</i> testing, and 11 of these were found to have EC₅₀ < 10μM. We progressed five compounds to an <i>in vivo</i> mouse efficacy model of Chagas disease and validated that the machine learning model could identify <i>in vitro</i> active compounds not in the training set, as well as known positive controls. The antimalarial pyronaridine possessed 85.2% efficacy in the acute Chagas mouse model. We have also proposed potential targets (for future verification) for this compound based on structural similarity to known compounds with targets in <i>T</i>. <i>cruzi</i>.</p><p>Conclusions/ Significance</p><p>We have demonstrated how combining chemoinformatics and bioinformatics for <i>T</i>. <i>cruzi</i> drug discovery can bring interesting <i>in vivo</i> active molecules to light that may have been overlooked. The approach we have taken is broadly applicable to other NTDs.</p></div

Leave-out cross validation data for <i>T</i>.<i>cruzi</i> Bayesian models.

<p>Leave-out cross validation data for <i>T</i>.<i>cruzi</i> Bayesian models.</p