RESUMO
The 3C-like protease (3CL(pro)) of severe acute respiratory syndrome associated coronavirus (SARS-CoV) is vital for SARS-CoV replication and is a promising drug target. Recombinant 3CL(pro) was expressed in Pichia pastoris GS115 as a 42 kDa protein that displayed a K ( m ) of 15 ± 2 µM with Dabcyl-KTSAVLQSGFRKME-Edans as substrate. Purified 3CL(pro) was used for inhibition and kinetic assays with seven flavonoid compounds. The IC(50) of six flavonoid compounds were 47-381 µM. Quercetin, epigallocatechin gallate and gallocatechin gallate (GCG) displayed good inhibition toward 3CL(pro) with IC(50) values of 73, 73 and 47 µM, respectively. GCG showed a competitive inhibition pattern with K ( i ) value of 25 ± 1.7 µM. In molecular docking experiments, GCG displayed a binding energy of -14 kcal mol(-1) to the active site of 3CL(pro) and the galloyl moiety at 3-OH position was required for 3CL(pro) inhibition activity.
Assuntos
Flavonoides/metabolismo , Inibidores de Proteases/metabolismo , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/enzimologia , Proteínas Virais/antagonistas & inibidores , Catequina/análogos & derivados , Catequina/metabolismo , Proteases 3C de Coronavírus , Cisteína Endopeptidases/biossíntese , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Expressão Gênica , Hidrólise , Concentração Inibidora 50 , Cinética , Peso Molecular , Pichia/genética , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/genética , Proteínas Virais/biossíntese , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
Astragalin (kaempferol-3-O-ß-D-glucopyranoside, Ast) glucosides were synthesized by the acceptor reaction of a dextransucrase produced by Leuconostoc mesenteroides B-512FMCM with astragalin and sucrose. Each glucoside was purified and their structures were assigned as kaempferol-3-O-ß-D-glucopyranosyl-(1â3)-O-α-D-glucopyranoside (or kaempferol-3-O-ß-D-nigeroside, Ast-G1') and kaempferol-3-O-ß-D-glucopyranosyl-(1â6)-O-α-D-glucopyranoside (or kaempferol-3-O-ß-D-isomaltoside, Ast-G1) for one glucose transferred, and kaempferol-3-O-ß-D-isomaltooligosacharide (Ast-IMO or Ast-Gn; n=2-8). The astragalin glucosides exhibited 8.3-60.6% higher inhibitory effects on matrix metalloproteinase-1 expression, 18.8-20.3% increased antioxidant effects, and 3.8-18.8% increased inhibition activity of melanin synthesis compared to control (without the addition of compound), depending on the number of glucosyl residues linked to astragalin. These novel compounds could be used to further expand the industrial applications of astragalin glucosides, in particular in the cosmetics industry.