RESUMO
Molecules containing an (cyanovinyl)arene moiety are known as tyrphostins because of their ability to inhibit proteins from the tyrosine kinase family, an interesting target for the development of anticancer and trypanocidal drugs. In the present work, (E)-(cyanovinyl)benzeneboronic acids were synthesized by Knoevenagel condensations without the use of any catalysts in water through a simple protocol that completely avoided the use of organic solvents in the synthesis and workup process. The inâ vitro anticancer and trypanocidal activities of the synthesized boronic acids were also evaluated, and it was discovered that the introduction of the boronic acid functionality improved the activity of the boronic tyrphostins. Inâ silico target fishing with the use of a chemogenomic approach suggested that tyrosine-phosphorylation-regulated kinaseâ 1a (DYRK1A) was a potential target for some of the designed compounds.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Compostos de Boro/química , Compostos de Boro/farmacologia , Tripanossomicidas/química , Tripanossomicidas/farmacologia , Tirfostinas/química , Tirfostinas/farmacologia , Animais , Antineoplásicos/síntese química , Compostos de Boro/síntese química , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , Doença de Chagas/tratamento farmacológico , Desenho de Fármacos , Humanos , Camundongos , Modelos Moleculares , Neoplasias/tratamento farmacológico , Proteínas Tirosina Quinases/antagonistas & inibidores , Tripanossomicidas/síntese química , Trypanosoma cruzi/efeitos dos fármacos , Tirfostinas/síntese químicaRESUMO
The facilitative hexose transporter GLUT1 activity is blocked by tyrosine kinase inhibitors that include natural products such as flavones and isoflavones and synthetic compounds such as tyrphostins, molecules that are structurally unrelated to the transported substrates [Vera, et al. (2001) Biochemistry, 40, 777-790]. Here we analyzed the interaction of GLUT1 with quercetin (a flavone), genistein (an isoflavone), and tyrphostin A47 and B46 to evaluate if they share one common or have several binding sites on the protein. Kinetic assays showed that genistein, quercetin, and tyrphostin B46 behave as competitive inhibitors of equilibrium exchange and zero-trans uptake transport and noncompetitive inhibitors of net sugar exit out of human red cells, suggesting that they interact with the external surface of the GLUT1 molecule. In contrast, tyrphostin A47 was a competitive inhibitor of equilibrium exchange and zero-trans exit transport and a noncompetitive inhibitor of net sugar entry into red cells, suggesting that it interacts with the cytoplasmic surface of the transporter. Genistein protected GLUT1 against iodide-elicited fluorescence quenching and also decreased the affinity of d-glucose for its external binding site, while quercetin and tyrphostins B46 and A47 promoted fluorescence quenching and did not affect the external d-glucose binding site. These findings are explained by a carrier that presents at least three binding sites for tyrosine kinase inhibitors, in which (i) genistein interacts with the transporter in a conformation that binds glucose on the external surface (outward-facing conformation), in a site which overlaps with the external binding site for d-glucose, (ii) quercetin and tyrphostin B46 interact with the GLUT1 conformation which binds glucose by the internal side of the membrane (inward-facing conformation), but to a site accessible from the external surface of the protein, and (iii) the binding site for tyrphostin A47 is accessible from the inner surface of GLUT1 by binding to the inward-facing conformation of the transporter. These data provide groundwork for a molecular understanding of how the tyrosine kinase inhibitors directly affect glucose transport in animal cells.