RESUMO
Human drug metabolites (HDMs) are important chemicals widely used in drug-related studies. However, acquiring these enzyme-derived and regio-/stereo-selectively modified compounds through chemical approaches is complicated. PikC is a biosynthetic P450 enzyme involved in pikromycin biosynthesis from the bacterium Streptomyces venezuelae. Here, we identify the mutant PikCD50N as a potential biocatalyst, with a broad substrate scope, diversified product profile, and high catalytic efficiency, for preparation of HDMs. Remarkably, PikCD50N can mediate the drug-metabolizing reactions using the low-cost H2O2 as a direct electron and oxygen donor.
Assuntos
Peróxido de Hidrogênio , Preparações Farmacêuticas , Sistema Enzimático do Citocromo P-450/genética , Humanos , MacrolídeosRESUMO
Natural killer T cells (NKT cells) respond to presentation of specific glycolipids with release of a variety of proinflammatory and immunomodulatory cytokines. The repertoire of glycolipid antigens for these cells includes alpha-glycosylceramides, alpha-glycosyldiacylglycerols, and the triglycosylceramide iGb3. Two features of iGb3 set it apart from these other antigens: (i) three sugars are required for stimulation and (ii) the glycosidic bond between ceramide and the proximal sugar is beta in iGb3, whereas it is alpha in other antigens. We have synthesized the alpha versions of iGb3 and Gb3 and demonstrate that they are effective antigens for NKT cells and that they do not require lysosomal processing to the monoglycosylceramides for stimulation. These triglycosylceramides constitute a new class of antigen that stimulates NKT cells comparably to monoglycosylceramides.
Assuntos
Globosídeos/farmacologia , Interleucina-2/biossíntese , Células T Matadoras Naturais/efeitos dos fármacos , Triexosilceramidas/farmacologia , Animais , Antígenos CD1d/metabolismo , Células Dendríticas/imunologia , Globosídeos/síntese química , Globosídeos/química , Hibridomas , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Camundongos , Células T Matadoras Naturais/imunologia , Saposinas/imunologia , Triexosilceramidas/síntese química , Triexosilceramidas/químicaRESUMO
Glycosphingolipids (GSLs) from the Sphingomonadaceae family of bacteria have been reported to be potent stimulators of natural killer T cells. These glycolipids include mono-, tri- and tetraglycosylceramides. Here we have prepared the GSL-1 to GSL-4 series of glycolipids and tested their abilities to stimulate natural killer T cells. Among these glycolipids, only GSL-1 (1) is a potent stimulator. Using a series of synthetic diglycosylceramides, we show that oligoglycosylceramides from Sphingomonadaceae are not effectively truncated to GSL-1 in lysosomes in antigen-presenting cells, possibly because the higher-order GSLs are poor substrates for lysosomal acyltransfer enzymes.