RESUMO
Proteomics has played a central role in the identification of reliable disease biomarkers, which are the basis of precision medicine, a promising approach for tackling recalcitrant diseases such as cancer, that elude conventional treatments. Among proteomic methodologies, targeted proteomics employing stable isotope-labeled (SIL) internal standards is particularly suited for the clinical translation of biomarker information owing to its high throughput and accuracy in the quantitative analysis of patient-derived proteomes. Using SIL internal standards ensures the utmost level of confidence in detection and precision in targeted MS experiments. For successfully establishing assays based on targeted proteomics, it is crucial to secure broad coverage when selecting the SIL standard peptide panel. However, cysteinyl peptides have often been excluded because of cysteine's high chemical reactivity. To address this limitation, a new cysteine building block was developed by incorporating a sulfhydryl group configured with an S-carbamidomethyl group, which is commonly used in proteome sampling. This compound was found to be chemically stable and applicable to a variety of solid-phase peptide synthesis (SPPS) campaigns. Furthermore, a direct comparison of the synthesized SIL peptides and tryptic endogenous peptides demonstrated the potential utility of an SPPS flow based on the new cysteine building block for improving the success of targeted proteomic applications.
Assuntos
Cisteína , Proteômica , Humanos , Compostos de Sulfidrila , Bioensaio , Peptídeos , ProteomaRESUMO
Concerns about antibiotic-resistant Gram-negative pathogens are escalating, and accordingly siderophore-based intracellular antibiotic delivery is attracting more attention as an effective means to overcome these infections. Despite the successful clinical translation of this strategy, the delivery potential of siderophores has been limited to periplasm targeting, and this has appreciably restricted the repertoire of applicable antibiotics. To overcome this shortcoming of the current technology, this study focused on investigating the capability of simple bidentate catechol analogs to function as vehicles for cytoplasmic antibiotic delivery. Specifically, by employing trimethoprim, an inhibitor of dihydrofolate reductase located in the cytoplasm, as a model antibiotic, a chemical library of chelator-antibiotic conjugates featuring four different catechol analogs was prepared. Then, their various pharmacological properties and antimicrobial activities were evaluated. Analysis of these characterization data led to the identification of the active conjugates exhibiting notable iron- and trimethoprim-dependent potency against Escherichia coli. Further characterization of these hit molecules using E. coli mutant strains revealed that 2,3-dihydroxybenzoate could effectively deliver several corresponding conjugates to the cytoplasm by exploiting the siderophore uptake machineries present across the outer and inner membranes, originally designated for the native siderophore of E. coli, enterobactin. Considering the synthetic simplicity, such a catechol analog could have appreciable usage in potentiating cytoplasm-active antibiotics against recalcitrant Gram-negative pathogens.
Assuntos
Antibacterianos , Sideróforos , Sideróforos/química , Antibacterianos/farmacologia , Antibacterianos/química , Trimetoprima/farmacologia , Escherichia coli , Catecóis/farmacologia , CitoplasmaRESUMO
This study presents a Pd(II)-catalyzed method for the ß-C(sp3)-H arylation of N-Cbz- or N-Fmoc-protected N-methyl alanines, providing ready access to building blocks for N-methylated peptide synthesis. For this transformation, the native carboxylate was exploited as the directing group, attributing its success to the use of a monoprotected amino-pyridine ligand. Its synthetic utility was demonstrated by facile generation of nine analogues of the naturally occurring N-methylated cyclic peptide cycloaspeptide A.