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Discovery of a 29-Amino-Acid Reactive Abiotic Peptide for Selective Cysteine Arylation.

ABSTRACT: The regio- and chemoselective modification of proteins or peptides with chemical reagents is often challenging. One approach to overcome this problem involves identifying abiotic polypeptide sequences that react with specific small molecules. Toward this goal, we profiled ?5 × 1013 randomized 30-mer peptides using mRNA display and high-throughput sequencing in search of polypeptides that can undergo cysteine arylation with a water-soluble perfluoroarene. Within this vast chemical space, we discovered a cysteine-containing sequence with a second-order rate constant of 0.29 M-1 s-1 for arylation. An N- and C-terminal truncation reduced the reaction rate, as did the addition of denaturants. When the reactive peptide was covalently fused to the enzyme Sortase A, we observed regiospecific arylation at a single cysteine site, leaving the enzyme's active site cysteine unchanged. Taken together, these results demonstrate that long polypeptides of defined sequence, when matched with the appropriate reactive group, can be used for selective arylation of cysteine in water.

SUBMITTER: Evans ED

PROVIDER: S-EPMC5856623 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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Discovery of a 29-Amino-Acid Reactive Abiotic Peptide for Selective Cysteine Arylation.

Evans Ethan D ED Pentelute Bradley L BL

ACS chemical biology 20180109 3

The regio- and chemoselective modification of proteins or peptides with chemical reagents is often challenging. One approach to overcome this problem involves identifying abiotic polypeptide sequences that react with specific small molecules. Toward this goal, we profiled ∼5 × 10<sup>13</sup> randomized 30-mer peptides using mRNA display and high-throughput sequencing in search of polypeptides that can undergo cysteine arylation with a water-soluble perfluoroarene. Within this vast chemical spac ...[more]

PMID: 29283243

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Project description:Synthetic glucocorticoids (GC) are the mainstay therapy for treatment of acute and chronic inflammatory disorders. Due to the high adverse effects associated with long-term use, GC pharmacology has focused since the nineties on more selective GC ligand-binding strategies, classified as selective glucocorticoid receptor (GR) agonists (SEGRAs) or selective glucocorticoid receptor modulators (SEGRMs). In the current study, GSK866 analogs with electrophilic covalent-binding warheads were developed with potential SEGRA properties to improve their clinical safety profile for long-lasting topical skin disease applications. Since the off-rate of a covalently binding drug is negligible compared to that of a non-covalent drug, its therapeutic effects can be prolonged and typically, smaller doses of the drug are necessary to reach the same level of therapeutic efficacy, thereby potentially reducing systemic side effects. Different analogs of SEGRA GSK866 coupled to cysteine reactive warheads were characterized for GR potency and selectivity in various biochemical and cellular assays. GR- and NF?B-dependent reporter gene studies show favorable anti-inflammatory properties with reduced GR transactivation of two non-steroidal GSK866 analogs UAMC-1217 and UAMC-1218, whereas UAMC-1158 and UAMC-1159 compounds failed to modulate cellular GR activity. These results were further supported by GR immuno-localization and S211 phospho-GR western analysis, illustrating significant GR phosphoactivation and nuclear translocation upon treatment of GSK866, UAMC-1217, or UAMC-1218, but not in case of UAMC-1158 or UAMC-1159. Furthermore, mass spectrometry analysis of tryptic peptides of recombinant GR ligand-binding domain (LBD) bound to UAMC-1217 or UAMC-1218 confirmed covalent cysteine-dependent GR binding. Finally, molecular dynamics simulations, as well as glucocorticoid receptor ligand-binding domain (GR-LBD) coregulator interaction profiling of the GR-LBD bound to GSK866 or its covalently binding analogs UAMC-1217 or UAMC-1218 revealed subtle conformational differences that might underlie their SEGRA properties. Altogether, GSK866 analogs UAMC-1217 and UAMC-1218 hold promise as a novel class of covalent-binding SEGRA ligands for the treatment of topical inflammatory skin disorders.

Dataset Information

Discovery of a 29-Amino-Acid Reactive Abiotic Peptide for Selective Cysteine Arylation.

Publications

Discovery of a 29-Amino-Acid Reactive Abiotic Peptide for Selective Cysteine Arylation.

Similar Datasets

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