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Nuclear receptors (NRs) are a superfamily of ligand-dependent transcription factors that modulate several biological processes. Traditionally, modulation of NRs has been focused on the development of ligands that recognize and bind to the ligand binding domain (LBD), resulting in activation or repression of transcription through the recruitment of coregulators. However, for more severe diseases, such as breast and prostate cancer, the conventional treatment addressing LBD modulation is not always successful, due to tumor resistance. To overcome these challenges and aiming to modulate NR activity by inhibiting the NR-DNA interaction, new studies focus on the development of molecules targeting alternative sites and domains on NRs. Here, we discuss two different approaches for this alternative NR modulation: one targeting the NR DNA binding domain (DBD); and the other targeting the DNA sites recognized by NRs. Our aim is to present the challenges and perspectives for developing specific inhibitors for each purpose, alongside with already reported examples.

Orientador: Ana Carolina Migliorini Figueira Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia Resumo: Os Receptores Ativadores da Proliferacao de Peroxissomos (PPAR) sao fatores de transcricao modulados por ligantes e pertencentes a superfamilia dos receptores nucleares. O isotipo PPAR¿À/¿Â esta envolvido em processos metabolicos como metabolismo energetico, adipogenese e metabolismo de lipidios, sendo um alvo interessante para o desenvolvimento de farmacos para doencas metabolicas. Alem disso, esse receptor e o isotipo mais expresso em pele e esta envolvido em diversos processos nesse orgao, como manutencao da barreira epidermal, sintese de lipidios, diferenciacao epidermal e cicatrizacao, o que o torna um alvo para o desenvolvimento de farmacos ou dermocosmeticos que possam atuar em processos de regeneracao de pele. O objetivo geral deste trabalho foi prospectar compostos que possam atuar como ligantes ativadores do PPAR¿À/¿Â e investigar os efeitos da modulacao deste receptor por ligantes em celulas epiteliais. Foi desenvolvido um protocolo de triagem de agonistas para este receptor, composto por um ensaio de transativacao celular seguido por dois ensaios de caracterizacao biofisica da interacao ligante:proteina - ensaio de termoestabilidade e ensaio de supressao da fluorescencia da sonda 8-anilino-1-naftalenosulfonico. O protocolo desenvolvido apresenta melhorias em relacao aos ensaios de transativacao celular comumente utilizados para triagem de ligantes de PPARs: (i) semi-automatizacao; (ii) analise da citotoxicidade aparente; (iii) uso de metodologias biofisicas para confirmacao da interacao direta entre ligante:proteina; (iv) e afericao da afinidade entre os hits e o receptor por meio do calculo da constante de dissociacao. Apos padronizacao, este racional foi utilizado para triar 121 compostos isolados enviados por colaboradores, 560 extratos de plantas brasileiras da biblioteca Phytobios, 719 compostos da biblioteca drug-like NIH-NCC e 80 derivados semi-sinteticos de produtos naturais da biblioteca TimTec NDL-3,000 pre-selecionados por virtual screening. Ao final do pipeline de busca de agonistas, o Extrato 2 da biblioteca Phytobios e sua fracao Extrato 2-18 foram selecionados como hits de PPAR¿À/¿Â, pois apresentaram componentes que ativam e estabilizam a estrutura terciaria do receptor, com constantes de dissociacao de 0,011 } 0,007 mg/mL e 0,016 } 0,007 mg/mL, respectivamente. Alem do protocolo de triagem, foram padronizados metodos de migracao, proliferacao e diferenciacao de queratinocitos humanos (HaCaT) para caracterizacao da modulacao do PPAR¿À/¿Â por ligantes em processos de regeneracao de pele. Nestes ensaios, os agonistas comerciais GW0742, GW501516 e L-165,041 aumentaram a taxa de migracao 2D de queratinocitos humanos (HaCaT), e os agonistas GW501516 e L-165,041 diminuiram a proliferacao deste tipo celular. Em relacao a diferenciacao de HaCaT, os agonistas GW0742 e L-165,041 nao alteraram a expressao genica dos marcadores de diferenciacao celular queratina 14, queratina 1 e involucrina. Avaliando os niveis de expressao de mRNA de componentes da matriz extracelular da pele (colageno, fibronectina e colagenases), nao foram observadas alteracoes no padrao de expressao desses alvos apos a ativacao do PPAR¿À/¿Â em celulas HaCaT, ou apos a supressao da expressao do receptor em camundongos knockouts com estimulo de reparo de feridas cutaneas. Em resumo, neste trabalho foi desenvolvido um pipeline de selecao de agonistas para o PPAR¿À/¿Â que ativem e a estabilizem a estrutura do receptor, alem de possibilitar o calculo da constante de dissociacao entre as amostras testadas e a proteina. Por fim, foram acoplados ensaios a este pipeline para posterior caracterizacao dos agonistas de PPA Abstract: Peroxisome Proliferator Activated Receptors (PPAR) are transcriptional factors regulated by ligands and members of the nuclear receptor superfamily. The PPARâ/ä is involved in metabolic processes such as energetic metabolism, adipogenesis and lipid metabolisms, which made it an interesting target for the development of drugs for metabolic disorders. In addition, this isotype is the most expressed in skin and, in this organ, it is involved in processes such as epidermal barrier maintenance, lipid synthesis, epidermal differentiation and wound healing, which made it a target for the development of dermocosmetics or drugs for skin repair. Aiming to select new activators (agonists) for PPARâ/ä, for future drug development, in this work, we developed a pipeline for screening of agonists for this receptor. The pipeline is composed of a cellular transactivation assay followed by two biophysical assays (Thermal Shift Assay and 8-Anilino-1-naphthalenesulfonic acid (ANS) fluorescence quenching assay) for characterization of the direct interaction of ligand:protein. The pipeline brings improvements if compared to traditional methods for PPAR ligand-screening: semi-automatization; parameter of apparent cytotoxicity; biophysical methodologies for confirmation of direct interaction ligand:protein; and calculation of dissociation constant between hits and the receptor. With the pipeline, we screened 121 compounds/extracts given by collaborators, 560 Brazilian plant extracts from Phytobios library, 719 drug-like compounds from NIH-NCC library, and 80 semi-synthetic natural products derivatives from TimTec NDL-3,000 library pre-selected via virtual screening. At the end of the pipeline, Extract 2 and one of its fraction (Extract 2-18) from Phytobios library showed to activate PPARâ/ä, stabilize its tertiary structure and had a dissociantion constant of, respectively, 0.011 ± 0.007 mg/mL e 0.016 ± 0.007 mg/mL, being selected as PPARâ/ä agonists. Besides the screening pipeline, methods of migration, proliferation and differentiation of keratinocytes (HaCaT cells) were also developed to characterize PPARâ/ä modulation by ligands in skin regeneration processes. These methods will be incorporated to the pipeline for agonist screening to futher characterize new PPARâ/ä ligands. The commercial agonists GW0742, GW501516 and L-165,041 increased the migration rate of HaCaT, and GW501516 and L-165,041 decreased proliferation in this cell type. Regarding HaCaT differentiation, gene expression of the analysed markers (keratin 14, keratin 1 and involucrin) were not changed after ligand treatment in the selected protocols. The RNA expression levels of extracellular matrix proteins (collagens, fibronectin and collagenases) were not altered after keratinocyte activation by PPARâ/ä ligand GW501516, nor in PPARâ/ä- knockout compared to wild type after wound healing stimuli. In summary, in this work we developed a pipeline to screening for PPARâ/ä agonists and further characterize them in skin regeneration processes Doutorado Fármacos, Medicamentos e Insumos para Saúde Doutora em Ciências FAPESP 2013/22648-2, 2016/16476-2 CNPQ 403433/2016-9

Peroxisome proliferator-activated receptor beta/delta (PPARß/δ) is considered a therapeutic target for metabolic disorders, cancer, and cardiovascular diseases. Here, we developed one pipeline for the screening of PPARß/δ agonists, which reduces the cost, time, and false-positive hits. The first step is an optimized 3-day long cellular transactivation assay based on reporter-gene technology, which is supported by automated liquid-handlers. This primary screening is followed by a confirmatory transactivation assay and by two biophysical validation methods (thermal shift assay (TSA) and (ANS) fluorescence quenching), which allow the calculation of the affinity constant, giving more information about the selected hits. All of the assays were validated using well-known commercial agonists providing trustworthy data. Furthermore, to validate and test this pipeline, we screened a natural extract library (560 extracts), and we found one plant extract that might be interesting for PPARß/δ modulation. In conclusion, our results suggested that we developed a cheaper and more robust pipeline that goes beyond the single activation screening, as it also evaluates PPARß/δ tertiary structure stabilization and the ligand affinity constant, selecting only molecules that directly bind to the receptor. Moreover, this approach might improve the effectiveness of the screening for agonists that target PPARß/δ for drug development.

Nuclear receptors (NRs) comprise a superfamily of proteins modulated by ligands that regulate the expression of target genes. These proteins share a multidomain structure harboring an N-terminal domain, a highly conserved DNA binding domain, and a ligand binding domain, which has ligand dependent activation function. They play key roles in development, metabolism, and physiology being closely related to diseases. Most of the knowledge about this superfamily emerges from investigations on new ligands and are mostly centered in the ligand binding domain. However, more investigation focusing on interactions between DNA and DNA binding domain is necessary to shed light on important roles of NRs' participation in transcriptional mechanisms and in specific genes network. Here, our goal is to discuss some nuances of NRs-DNA interaction, describing details of the most used techniques in this sort of study, such as gel shift (EMSA), DNA footprinting, reporter gene assay, ChIP-Seq, 3C, and fluorescence anisotropy. Additionally, we aim to provide tools, presenting advantages and disadvantages of these common methods, when choosing the most suitable one to study NRs-DNA interactions to answer specific questions.