RESUMO
Serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT) is a member of the GCN5 N-acetyltransferase (GNAT) superfamily and catalyzes the penultimate step in the biosynthesis of melatonin; a large daily rhythm in AANAT activity drives the daily rhythm in circulating melatonin. We have used a structure-based computational approach to identify the first druglike and selective inhibitors of AANAT. Approximately 1.2 million compounds were virtually screened by 3D high-throughput docking into the active site of X-ray structures for AANAT, and in total 241 compounds were tested as inhibitors. One compound class, containing a rhodanine scaffold, exhibited low micromolar competitive inhibition against acetyl-CoA (AcCoA) and proved to be effective in blocking melatonin production in pineal cells. Compounds from this class are predicted to bind as bisubstrate inhibitors through interactions with the AcCoA and serotonin binding sites. Overall, this study demonstrates the feasibility of using virtual screening to identify small molecules that are selective inhibitors of AANAT.
Assuntos
Arilalquilamina N-Acetiltransferase/antagonistas & inibidores , Arilalquilamina N-Acetiltransferase/química , Inibidores Enzimáticos/química , Modelos Moleculares , Relação Quantitativa Estrutura-Atividade , Acetilcoenzima A/antagonistas & inibidores , Acetilcoenzima A/química , Animais , Arilalquilamina N-Acetiltransferase/biossíntese , Sítios de Ligação , Células Cultivadas , Cristalografia por Raios X , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Melatonina/antagonistas & inibidores , Melatonina/biossíntese , Glândula Pineal/citologia , Ligação Proteica , Conformação Proteica , Ratos , Rodanina/análogos & derivados , Rodanina/química , Rodanina/farmacologia , Triptaminas/química , Triptaminas/farmacologiaRESUMO
Recently, Na(+)/K(+)-ATPase has been detected in the luminal membrane of the anterior midgut of larval yellow fever mosquitoes (Aedes aegypti) with immunohistochemical techniques. In this study, the possible involvement of this ATPase in strong alkalinization was investigated on the level of whole larvae, isolated and perfused midgut preparations and on the molecular level of the Na(+)/K(+)-ATPase protein. Ouabain (5 mM) did not inhibit the capability of intact larval mosquitoes to alkalinize their anterior midgut. Also in isolated and perfused midgut preparations the perfusion of the lumen with ouabain (5 mM) did not result in a significant change of the transepithelial voltage or the capacity of luminal alkalinization. Na(+)/K(+)-ATPase activity was completely abolished when KCl was substituted with choline chloride, suggesting that the enzyme cannot act as an ATP-driven Na(+)/H(+)-exchanger. Altogether the results of the present investigation indicate that apical Na(+)/K(+)-ATPase is not of direct importance for strong luminal alkalinization in the anterior midgut of larval yellow fever mosquitoes.