Search

The lack of robustly validated drug targets in Leishmania spp. is a major impediment to the discovery of new drugs and effective treatments for leishmaniasis. Here, 6 phenotypically-active anti-leishmanial compounds were selected from GSKs Kinetobox compound collections for subsequent target deconvolution studies. The principal aim of these studies was to identify molecular targets that can be exploited by subsequent target-focused anti-leishmanial drug discovery. Using multiple orthogonal genome-wide genetic, chemical proteomic and biochemistry-based approaches, two compounds were found to target novel anti-leishmanial targets, namely oxidosqualene cyclase, a key enzyme of sterol biosynthesis, and Sec23, a component of the COPII coatomer, a vesicular coat protein involved in vesicular trafficking and secretion. Future studies to further investigate and exploit these novel targets are discussed. Remarkably, three of the four remaining compounds studied were confirmed as chelators of divalent metals. Compounds acting via this generic and potentially toxic mechanism of action are not considered progressible for drug discovery. Therefore, the significant number of chelators identified in this random selection of the Kinetobox is concerning and may be indicative of high levels of chelators more generally in screening libraries. Strategies to identify chelators at an early stage of the drug discovery process are proposed.

SGC-AAK1-1 is presented as a chemical probe for the understudied kinases (adaptor protein 2)-associated kinase (AAK1) and (bone morphogenetic protein 2)-inducible kinase (BIKE/BMP2K). SGC-AAK1-1 is potent with submicromolar cellular target engagement with AAK1. The compound shows > 30-fold selectivity in binding relative to all other kinases profiled with the exception of RIOK1, RIOK3, and PIP5K1C. A closely related molecule that is significantly less potent with AAK1 and BIKE/BMP2K, SGC-AAK1-1N, is included as a negative control., The SGC is a registered charity (number 1097737) that receives funds from AbbVie, Bayer Pharma AG, Boehringer Ingelheim, Canada Foundation for Innovation, Eshelman Institute for Innovation, Genome Canada, Innovative Medicines Initiative (EU/EFPIA), Janssen, Merck & Co., Novartis Pharma AG, Ontario Ministry of Economic Development and Innovation, Pfizer, São Paulo Research Foundation-FAPESP, Takeda, and Wellcome Trust.

Orientador: Sandra Martha Gomes Dias Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia Resumo: A descoberta de fármacos é um processo longo e complexo. Em muitos casos, ele é composto pela identificação e validação inicial de alvo, seguida pelo desenvolvimento de ensaios bioquímicos ou celulares, rastreio de alto rendimento (High Throughput Screening, HTS) de uma biblioteca de compostos, identificação de compostos hits, otimização de moléculas leads e finalmente seleção de um candidato para seu desenvolvimento clínico. Glutaminase é uma enzima que desempenha um papel fundamental nas células tumorais e sua inibição ou atenuação da expressão demonstraram ser citotóxicas para vários tipos de tumor, em especial ao câncer de mama triplo-negativo (TN) (sem expressão dos receptores de estrógeno -ER, progesterona -PR e da proteína de membrana Her2). Mesmo sendo um importante alvo terapêutico, até a presente data, não há inibidores de glutaminase aprovados para o uso na clínica. Nosso grupo desenvolveu e realizou um ensaio bioquímico de HTS utilizando uma biblioteca de 30.000 compostos filtrados para seguirem as cinco regras de Lipinski. Neste trabalho, descrevemos no primeiro capítulo a avaliação de 11 dos compostos hits obtida em ensaios secundários celulares com o objetivo de verificar se os mesmos têm ação seletiva para tumores de mama TN em contraste a tumores não-T, células de tumor benigno (fibrose cística) e epitélio de mama imortalizado não tumorigênico. Dentre os compostos avaliados verificamos que os compostos C1 e C10 exibiram potência e seletividade tumoral medidos pela inibição do crescimento celular. Além disto, estes compostos inibiram em 50% o consumo de glutamina nas células tumorais TN MDA-MB-231 e diminuíram, nestas células, a porcentagem de núcleos marcados com Edu, indicando também ação citoestática. Entretanto, ambos compostos não apresentaram ação antitumoral dependente de GLS. Dando continuidade a busca por inibidores de glutaminase, no segundo capítulo desta dissertação, abordamos o estabelecimento de uma nova metodologia de triagem alvo-direcionada baseada em células, ensaio o qual foi empregado para o screening de duas coleções de bibliotecas de produtos naturais. Após validação e realização das triagens foi possível obter um total de 6 extratos/frações com potencial de serem inibidores seletivos de GLS Abstract: The drug discovery process is a long and complex process composed of an initial target identification and validation, followed by the development of a biochemical or cellular assays, high throughput screening (HTS) of a compound library, hit identification, lead optimization and ?nally the selection of a candidate molecule for clinical development. Glutaminase is an enzyme that plays a key role on Myc-driven tumor cells and its expression inhibition or attenuation has been shown to be cytotoxic for various tumor types, especially triple-negative (TN) breast cancer (without expression of estrogen receptors-ER, Progesterone-PR and HER-2 membrane protein). Although it is an important therapeutic target, until nowadays, there are no glutamine inhibitors approved for use in the clinic. Our group developed and performed a biochemical HTS assay using a 30,000 compounds library filtered to follow the five Lipinsky rule. In this work, we describe in the first chapter the evaluation of 11 hits compounds obtained in secondary cell assays in order to verify if they have selective action against TN breast tumors in contrast to non-TN tumors, benign tumor cells (cystic fibrosis) and non-tumorigenic immortalized breast epithelium. Among the evaluated compounds, we verified that the compounds C1 and C10 exhibited potency and tumor selectivity measured by cell growth inhibition. In addition, these compounds inhibited the consumption of glutamine in TN tumor cells MDA-MB-231 by 50% and decreased the percentage of nuclei labeled with Edu, indicating also its cytostatic action. However, both compounds did not present GLS-dependent antitumor action. Continuing the search for glutaminase inhibitors in the second chapter of this work, we approached the establishment of a new target cell-based screening methodology, which was used to screen two collections of natural compounds libraries. After screening validation and execution, it was possible obtain a total of 6 extracts/fractions with potential to be selective GLS inhibitors Mestrado Genética Animal e Evolução Mestra em Genética e Biologia Molecular CAPES