RESUMO
A solubilizing Trt-K10 tag was developed for the effective chemical preparation of peptides/proteins with low solubility. The Trt-K10 tag comprises a hydrophilic oligo-Lys sequence and a trityl anchor, and can be selectively introduced to a side chain thiol of Cys of deprotected peptides/proteins with a trityl alcohol-type introducing reagent Trt(OH)-K10 under acidic conditions. Significantly, the ligation product in the reaction mixture of a thiol-additive-free native chemical ligation can be modified directly in a one-pot manner to facilitate the isolation of the product by high-performance liquid chromatography. Finally, the Trt-K10 tag can be readily removed with a standard trifluoroacetic acid cocktail. Using this easy-to-attach/detach tag-aided method, a hepatitisâ B virus capsid protein that is usually difficult to handle was synthesized successfully.
Assuntos
Proteínas do Capsídeo/síntese química , Sequência de Aminoácidos , Proteínas do Capsídeo/química , Cisteína/química , Vírus da Hepatite B/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Polilisina/química , Solubilidade , Compostos de Sulfidrila/químicaRESUMO
Grb2 is an adaptor protein that mediates cellular signal transduction. Grb2 contains an SH2 domain that interacts with phosphotyrosine-containing sequences in EGFR and other signaling molecules, and it is a promising molecular target for anticancer agents. To identify novel inhibitors of the Grb2 SH2 domain from natural products and their mirror-image isomers, screening systems using both enantiomers of a synthetic Grb2 SH2 domain protein were established. A pair of synthetic procedures for the proteins were investigated: one employed a single native chemical ligation (NCL) of two segment peptides, and the other used the N-to-C-directed NCL of three segment peptides for easier preparation. Labeling at the N-terminus or the Ala115 residue of the Grb2 SH2 domain provided functional probes to detect binding to a phosphotyrosine-containing peptide. The resulting synthetic-protein-based probes were applied to bioassays, including chemical array analysis and enzyme-linked immunosorbent assays.
Assuntos
Descoberta de Drogas/métodos , Proteína Adaptadora GRB2/síntese química , Domínios de Homologia de src/efeitos dos fármacos , Sequência de Aminoácidos , Ensaio de Imunoadsorção Enzimática , Proteína Adaptadora GRB2/antagonistas & inibidores , Proteína Adaptadora GRB2/química , Proteína Adaptadora GRB2/metabolismo , Humanos , Modelos Moleculares , Peptídeos/química , Peptídeos/farmacologiaRESUMO
Mirror-image screening using d-proteins is a powerful approach to provide mirror-image structures of chiral natural products for drug screening. During the course of our screening study for novel MDM2-p53 interaction inhibitors, we identified that NPD6878 (R-(-)-apomorphine) inhibited both the native l-MDM2-l-p53 interaction and the mirror-image d-MDM2-d-p53 interaction at equipotent doses. In addition, both enantiomers of apomorphine showed potent inhibitory activity against the native MDM2-p53 interaction. In this study, we investigated the inhibitory mechanism of both enantiomers of apomorphine against the MDM2-p53 interaction. Achiral oxoapomorphine, which was converted from chiral apomorphines under aerobic conditions, served as the reactive species to form a covalent bond at Cys77 of MDM2, leading to the inhibitory effect against the binding to p53.