Browsing by Author "Sousa, Pedro M. F."
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- Modulation of Human Phenylalanine Hydroxylase by 3-Hydroxyquinolin-2(1H)-One DerivativesPublication . Lopes, Raquel R.; Tomé, Catarina S.; Russo, Roberto; Paterna, Roberta; Leandro, João; Candeias, Nuno R.; Gonçalves, Lídia; Teixeira, Miguel; Sousa, Pedro M. F.; Guedes, R. C.; Vicente, João B.; Gois, Pedro M. P.; Leandro, PaulaPhenylketonuria (PKU) is a genetic disease caused by deficient activity of human phenylalanine hydroxylase (hPAH) that, when untreated, can lead to severe psychomotor impairment. Protein misfolding is recognized as the main underlying pathogenic mechanism of PKU. Therefore, the use of stabilizers of protein structure and/or activity is an attractive therapeutic strategy for this condition. Here, we report that 3-hydroxyquinolin-2(1H)-one derivatives can act as protectors of hPAH enzyme activity. Electron paramagnetic resonance spectroscopy demonstrated that the 3-hydroxyquinolin-2(1H)-one compounds affect the coordination of the non-heme ferric center at the enzyme active-site. Moreover, surface plasmon resonance studies showed that these stabilizing compounds can be outcompeted by the natural substrate l-phenylalanine. Two of the designed compounds functionally stabilized hPAH by maintaining protein activity. This effect was observed on the recombinant purified protein and in a cellular model. Besides interacting with the catalytic iron, one of the compounds also binds to the N-terminal regulatory domain, although to a different location from the allosteric l-Phe binding site, as supported by the solution structures obtained by small-angle X-ray scattering.
- Structural and biophysical insights into the mode of covalent binding of rationally designed potent BMX inhibitorsPublication . Seixas, João D.; Sousa, Bárbara B.; Marques, Marta C.; Guerreiro, Ana; Traquete, Rui; Rodrigues, Tiago; Albuquerque, Inês S.; Sousa, Marcos F. Q.; Lemos, Ana R.; Sousa, Pedro M. F.; Bandeiras, Tiago M.; Wu, Di; Doyle, Shelby K.; Robinson, Carol V.; Koehler, Angela N.; Corzana, Francisco; Matias, Pedro M.; Bernardes, Gonçalo J. L.The bone marrow tyrosine kinase in chromosome X (BMX) is pursued as a drug target because of its role in various pathophysiological processes. We designed BMX covalent inhibitors with single-digit nanomolar potency with unexploited topological pharmacophore patterns. Importantly, we reveal the first X-ray crystal structure of covalently inhibited BMX at Cys496, which displays key interactions with Lys445, responsible for hampering ATP catalysis and the DFG-out-like motif, typical of an inactive conformation. Molecular dynamic simulations also showed this interaction for two ligand/BMX complexes. Kinome selectivity profiling showed that the most potent compound is the strongest binder, displays intracellular target engagement in BMX-transfected cells with two-digit nanomolar inhibitory potency, and leads to BMX degradation PC3 in cells. The new inhibitors displayed anti-proliferative effects in androgen-receptor positive prostate cancer cells that where further increased when combined with known inhibitors of related signaling pathways, such as PI3K, AKT and Androgen Receptor. We expect these findings to guide development of new selective BMX therapeutic approaches.