Daniel Gedder Silva.

With the aim of creating a consortium of researchers interested in designing and fastly synthesizing compounds with the required potency, safety and DMPK properties, the central idea of the project consists of a miniaturized and accelerated library synthesis and the screening of the unpurified compounds in the same format yielding rapid, high-quality hit findings. To test whether unpurified reaction mixtures can give useful screening results against parasite panels, identified hits will be resynthesized, purified and further characterized. Quality control will be followed by HPLC, mass spectrometry and quantitative NMR. As soon as the methods are fully validated, we will progress with the applicability of the Microscale Parallel Synthesis approach for late-stage functionalization to diversify the new anti-Infectives agents. The implementation and validation of the synthetic methods will demonstrate that large compound libraries can be produced without purification to an initial biological screening in a very fast fashion on a nanomole scale. Additionally, the method will contribute to reducing the costs of drug design and development and to implementing a greener process. This strong and original research proposal will establish in Brazil a new area of Medicinal Chemistry since institutions in São Paulo state do not yet have a tradition in microscale synthesis linked to biological assays as well as DMPK and mode of action for infectious diseases.

Trypanosoma cruzi is the etiological agent of Chagas disease, a pathogenesis that affects 6 to 7 million people worldwide, mainly in Latin America. There is no effective treatment for chronic Chagas disease and resistant strains to the current frontline treatment have already emerged. There is a growing need for new pharmacological alternatives to treat this disease. This proposal exploits a target-based approach to search for lead compounds against Chagas disease. Trypanosoma cruzi dihydroorotate dehydrogenase (TcDHODH) is a flavin mononucleotide containing enzyme, which catalyzes the oxidation of L-dihydroorotate to orotate, the fourth step and only redox reaction in the de novo biosynthesis of pyrimidine nucleotides. TcDHODH was also described as a soluble fumarate reductase playing a role in connecting succinate/fumarate metabolism to de novo pyrimidine biosynthesis. DHODH has already been extensively exploited as a drug target for proliferative and parasitic diseases. Genetic studies have shown that DHODH is essential for T. cruzi survival providing evidence that this enzyme is an attractive target for the development of antichagasic drugs. The goal of this project is to chemically validate TcDHODH as a new drug target for Chagas disease and to provide leads for drug development. Our approach combines multiple techniques including computational chemistry, enzymology, structural biology, parasitology, and medicinal chemistry to develop selective and covalent inhibitors of TcDHODH. Initially, we will seek to gain a greater understanding of the DHODH hot spots by using solvent mapping to characterize the binding pockets with the flexible active-site loop open and closed. This information will help strengthen the computational modeling, and subsequent compound design. Covalent inhibition of enzymes based on the identification of a set of cysteine targeting covalent warheads including acrylamides, vinyl sulfones and nitriles, has been shown to be a successful approach in drug discovery pipelines. In our proposal, an initial set of putative inhibitors that incorporate an acrylamide warhead have already been rationally designed to target the TcDHODH active-site cysteine. Predicted inhibitors will be evaluated for inhibitory potency and mechanism of inhibition against TcDHODH, cytotoxicity and its anti-parasitic effects in cell culture. X-ray crystallography, fragment screening and medicinal chemistry will be combined to provide the chemical basis for the synthesis of a new generation of potent, selective, and drug-like inhibitors. The top 3 inhibitors that meet lead criteria (including PK and tolerability) will have their efficacy evaluated by DNDi via an in-kind contribution to the project. This proposal represents an unprecedent initiative to significantly contribute to building capacity in the field of drug discovery in Brazil, a field of research still very incipient in developing countries.

Uma série de novos compostos heterociclos foi sintetizada e testada como potenciais agentes tripanossomicidas, bactericidas e citotoxicidade. O conhecimento do alvo molecular destes compostos fenotipicamente ativos foi estudado, o que facilitará a próxima fase do processo de descoberta de fármacos: projetos baseados em alvos mais bem validados, acelerando a subsequente otimização de compostos líderes. Uma abordagem baseada no alvo pode ser usada para contornar problemas farmacocinéticos ou toxicológicos, usando técnicas como scaffold hopping. Para atingir esses objetivos, foi planejado e sintetizado uma nova série de compostos heterocíclicos com propriedades farmacocinéticas calculadas melhoradas que exploram o espaço químico inacessível às abordagens atuais. Todos os compostos foram avaliados em ensaios de inibição do crescimento parasitários e bacterianos. A estabilidade dos compostos selecionados foram investigadas com microssomas de camundongo, ensaios de solubilidade e ligação a proteínas. Esses resultados nos permitiram selecionar os compostos mais promissores para o futuro trabalho de translação em direção a novas terapias muito necessárias para doenças infecciosas. Dois artigos foram publicados na ACS Infectious Diseases e todos os dados do projeto foram disponibilizados publicamente na plataforma GitHub, permitindo que outros pesquisadores contribuam com a pesquisa. Esta abordagem de "ciência aberta" maximiza o impacto da pesquisa financiada.

A serie of isoindolone-containing compounds have been synthesized and identified as having potent activity against T. b. rhodesiense, no cytotoxicity, excellent metabolic stability, but low solubility.The low solubility of the most potent compounds need to be improved as well as to explore a greater chemical diversity in this series of compounds. In addition, knowledge of the molecular target of phenotypically active compounds can facilitate the drug discovery process. It opens up the possibility of target (structure)-based projects against better validated targets, derisking medicinal chemistry optimization of compounds. It can be used to circumvent pharmacokinetic or toxicological issues using techniques such as scaffold hopping. In order to achieve these goals: i) we aim to design, synthesize and identify new heterocyclic compounds with antitrypanosomal activity; ii) to employ and modify synthetic routes to obtain the proposed heterocyclic compounds; iii) to carry out the T. cruzi and T. brucei growth inhibition assays, to evaluate the stability of selected compounds to mouse microsomes, solubility, protein binding, and in vivo efficacy studies against T.cruzi and T. brucei infections; iv) study the new candidates with improved bioavailability profile for further Drug Metabolism and PharmacoKinetics (DMPK) and their putative mode of action (MoA). These studies will be through a partnership with the researchers at the University of Dundee. Based on all these results select the best compound that can be transitioned into clinical trials for these parasitic infections.

Development, SAR studies and primary in#8197;vivo evaluation of novel isoindolone-based agents against Trypanosomiasis-induced HAT infections are presented. The most potent derivative displays selective potency for Trypanosoma brucei rhodesiense (IC50=40#8197;nm), no detectable side activity against T. cruzi and Leishmania infantum, no cytotoxicity against MRC-5 and PMM cell lines and high in#8197;vitro metabolic stability, making it a promising lead for further drug development. In addition, highly selective CCR2 antagonists have been designed and synthesized as 18F-labeled PET tracers and evaluated extensively for their off-target profile targets. A promising profile for further preclinical development was demonstrated by the most potent and selective candidate in an in#8197;vivo biodistribution study. This compound is the first potential nonpeptidic PET tracer for imaging CCR2 receptors.

Significant effort has put into designing and developing new medicines for infectious diseases. With collaborations all around the world, the focus of the research is the discovery and development of new potent and selective heterocyclic compounds as anti-infective agents. During the period of the postdoc, were synthesized new imidazopyridine/pyrimidine, furopyridine, and isoindole compounds arising from medicinal chemistry optimization. Based on promising results of in vitro activity (EC50 < 100 nM), cytotoxicity, metabolic stability, protein binding and pharmacokinetics (PK) properties, imidazopyrimidine compound was selected as a candidate for in vivo efficacy studies. In addition, the novel compound can cross the blood-brain barrier and are effective inhibitors of neurological pathogens such as trypanosomes. It represents a potential lead for the development of drugs to treat trypanosomiases. A novel isoindolone-based agents against Trypanosomiasis-induced HAT infections were discovered. The most potent derivative displays selective potency for Trypanosoma brucei rhodesiense (IC50 = 40 nM), no detectable side activity against T. cruzi and Leishmania infantum, no cytotoxicity against MRC-5 and PMM cell lines and high in vitro metabolic stability, making it a promising lead for further drug development. However, due the low solubility of the most potent compounds and the absence of the target identification, we could not select the most promising compounds for future translational work towards much-needed new therapies for infectious diseases. Understanding the PK/PD is critical and can help to understand what is required for a drug to progress. Also, seeking for the target is important to help with rational modifications of the lead compounds to improve PK/PD properties.

The present work describes the synthesis of 22 new imidazopyridine analogues arising from medicinal chemistry optimization at different sites on the molecule. Seven and twelve compounds exhibited an in vitro EC50 &#8804; 1µM against Trypanosoma brucei (T. brucei) and Trypanosoma cruzi (T. cruzi) parasites, respectively. All compounds were tested for toxicity against human lymphocytes CRL-8155 and human hepatocytes HepG2. Only compounds 2, 3, 6, 10, 12 and 21 exhibited detectable toxicity to CRL-8155 cell line (EC50 range from 14.50 to 47.42 &#956;M). Select compounds were assayed for stability to pooled mouse liver microsomes, for solubility in three different pHs (7.4, 6.5 and 2.0) and for plasma protein-binding. The half-lives of compounds 6, 9, 12 and 20 were greater than 60 min with a range of 84&#8722;100% of the test compounds remaining at the 60 min time point. Compounds 6, 9 and 12 showed significantly higher solubility (about 50 µM) than analogues 20 and 21 in all pH conditions, with the exception of compound 9 at pH 7.4 and pH 6.5. Select compounds 6, 9 and 12 showed the percent fraction unbound of 3.0%, 2.6% and 4.0% respectively. Analogue 21 exhibited the lowest percent of fraction unbound drug (FU = 0.7%).The greatest unbound fraction was observed in the most active compound 20 (FU = 9.9 %; EC50 value of 93.32 nM for T. cruzi; EC50 value of 18.12 nM for T. brucei). Brain penetration studies were performed upon compounds 12 and 20. Compound 12 exhibited a mean brain concentration of 0.55 &#956;M and a mean plasma concentration of 4.84 &#956;M. Compound 20 had a mean brain concentration of 1.12 &#956;M and a mean plasma concentration of 1.31 &#956;M. This gave a brain:plasma ratio of 0.114 for compound 12 and 0.856 for compound 20, a 7.5 fold change. Pharmacokinetic studies were performed in mice to evaluate if compound 20 was suitable for in vivo studies. Compound 20 showed an (average ± SEM) CMAX of 5.74 ± 0.86 µM, an average AUC of 3584.95 ± 669.84 min*µM. This CMAX is almost 60 fold higher than the T. cruzi EC50, and maintained a concentration higher than the EC50 well past the 12 h timepoint. This compound was screened in an acute model against T.cruzi TcTC2/Tulahuen. Mice were dosed orally during 6 weeks (42 days). Concentrations were analyzed and the pre-dose concentration of compound 20 was found to be (average ± SEM) 1.60 ± 1.60 (n=3) µM, where 1 of the 3 mice showed 4.81 µM of compound 20 still in the blood before the dose. For 1 h and 6 h post dose, the concentrations were found to be 9.10 ± 1.43 (n=3) µM and 5.83 ± 1.93 (n=3) µM respectively. This demonstrates that through repeated dosing, compound 20 was still able to achieve concentrations in the blood as expected from the single dose PK data. Overall, compound 20 represents a potential lead for the development of drugs to treat Trypanosomiasis.

O projeto foi desenvolvido em 3 etapas. Na etapa 1, a modelagem HQSAR, a docagem e os estudos de ROCS foram construídos utilizando uma série de 57 inibidores de cruzaína. O melhor modelo HQSAR (q2 = 0,70, r2 = 0,95, r2test = 0,62, q2rand. = 0,09 and r2rand. = 0,26) foi utilizado para predizer a potência de 121 compostos extraídos da literatura (conjunto de dados V1), resultando em um valor de r2 satisfatório de 0,65 para essa validação externa. Uma validação externa adicional foi empregada utilizando uma série de 1223 compostos extraído dos bancos de dados ChEMBL e CDD (conjunto de dados V3); nessa validação externa o valor de AUC (área sob a curva) para a curva ROC foi de 0,70. Os mapas de contribuição, obtidos para o melhor modelo HQSAR 3.4, estão de acordo com as predições do modo de interação e com as bioatividades dos compostos estudados. Nos estudos de ROCS, a forma molecular utilizada como filtro, foi útil na rápida identificação de modificações moleculares promissoras para inibidores de cruzaína. O valor de AUC obtido com a curva ROC foi de 0,72, isso indica que o método foi muito eficiente na distinção entre inibidores ativos e inativos da enzima cruzaína. Em seguida, o melhor modelo HQSAR foi utilizado para predizer os valores de pIC50 para novos compostos. Alguns dos compostos identificados, utilizando esse método, demonstraram valores de potência calculada maior do que a série de treinamento em estudo. No etapa 2, os efeitos sobre a potência na inibição da enzima cruzaína pela substituição de um grupo nitrila como warhead por outros grupos foi avaliada. Com a síntese de 20 compostos do tipo dipeptidil, avaliou-se a relação estrutura-atividade (SAR), baseado na troca do grupo warhead na porção P1'. O grupo oxima foi mais potente que o grupo correspondente nitrila em 0,7 unidades logarítmicas. Os compostos do tipo dipeptidil aldeídos e azanitrila obtiveram potências mais elevadas do que o correspondente dipeptidil nitrila em duas de magnitude. Os compostos dipeptidil alfa-beta insaturados foram menos potentes do que o correspondente dipeptidil nitrila. Na etapa 3, estratégias de química medicinal foram empregadas nas sínteses de 23 novos análogos, contendo o esqueleto básico de imidazopiridina. Sete e doze compostos sintetizados exibiram EC50 <= 1M in vitro contra os parasitos Tripanosoma cruzi (T. cruzi) e brucei (T. brucei), respectivamente. Com os resultados promissores de atividade biológica in vitro, citotoxicidade, estabilidade metabólica, ligação proteica e propriedades farmacocinéticas, o composto 41 foi selecionado como candidato para os estudos de eficácia in vivo. Esse composto foi submetido em um modelo agudo da infecção com T. cruzi em ratos (cepa Tulahuen). Depois de estabelecida a infecção, os ratos foram dosados duas vezes ao dia, durante 5 dias; e monitorados por 6 semanas usando um sistema de imagem in vivo IVIS (do inglês, &quot;In Vivo Imaging System&quot;). O composto 41 demonstrou inibição parasitária comparável com o grupo de treinamento dosado com benzonidazol. O composto 41 representa um potencial líder para o desenvolvimento de novos fármacos para o tratamento de tripanossomíases.

Técnicas computacionais (QSAR e ancoramento molecular) associadas à estratégia de se combinarem subestruturas químicas de moléculas altamente bioativas como inibidores da enzima conversora de angiotensina (ECA) foram usadas para obter novos dipeptídeos modificados como inibidores da ECA. Além da elevada potência, alguns dos inibidores da ECA propostos também exibiram parâmetros farmacocinéticos calculados bastante satisfatórios. Link da Dissertação: http://repositorio.ufla.br/jspui/bitstream/1/673/1/DISSERTA%C3%87%C3%83O_Modelagem%20racional%20de%20novos%20dipept%C3%ADdeos%20modificados%20como%20inibidores%20da%20enzima%20conversora%20de%20angiotensina.pdf

Estudos têm mostrado que é comum após os vinte e cinco anos o aparecimento de manchas na pele, um elemento importante para auxiliar na resolução desse problema é a hidroquinona, pois é reconhecida como o agente despigmentante mais eficiente para tal ocorrência. Essa substância, contudo, possui dois inconvenientes: capacidade irritante da pele e instabilidade química, pois ela é facilmente oxidada quando exposta à luz, na presença de oxigênio e pH elevado originando produtos de cor marrom escuro. Está forma, além de não exercer ação despigmentante, torna o aspecto da formulação desagradável. Por isso o presente trabalho teve como objetivo a avaliação de parâmetros físico-químicos de cremes contendo hidroquinona 2 % manipulados no município de Lavras - MG. Ao avaliar os rótulos e embalagens verifica ? se que não há uma padronização na rotulagem das amostras, o ideal seria que não houvesse ausência de informações na embalagem, pois todas são importantes, e que os cremes fossem armazenados somente em bisnagas de alumínio. Nos ensaios organolépticos e no teste de centrifugação nenhuma alteração importante foi observada, e ao medir o pH das amostras notou ? se que duas amostras estavam fora do pH ideal, comprometendo a eficácia do produto, e ao realizar o doseamento do teor de hidroquinona nas amostras de creme apenas uma amostra estava dentro do patamar estabelecido pela USP (2002), podendo concluir que os ensaios organolépticos não são suficientes para afirmar que a hidroquinona não sofreu processo de oxidação-redução.