4 research outputs found

    Site-Specific Incorporation of Selenocysteine by Genetic Encoding as a Photocaged Unnatural Amino Acid

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    Selenocysteine (Sec) is a naturally occurring amino acid that is also referred to as the 21st amino acid. Site-specific incorporation of Sec into proteins is attractive, because the reactivity of a selenol group exceeds that of a thiol group and thus allows site-specific protein modifications. It is incorporated into proteins by an unusual enzymatic mechanism which, in E. coli and other organisms, involves the recognition of a selenocysteine insertion sequence (SECIS) in the mRNA of the target protein. Reengineering of the natural machinery for Sec incorporation at arbitrary sites independent of SECIS elements, however, is challenging. Here we demonstrate an alternative route, whereby a photocaged selenocysteine (PSc) is incorporated as an unnatural amino acid in response to an amber stop codon, using a mutant Methanosarcina mazei pyrrolysyl-tRNA synthetase, <i>Mm</i> PCC2RS, and its cognate tRNA<sub>CUA</sub>. Following decaging by UV irradiation, proteins synthesized with PSc are readily tagged, e.g., with NMR probes to study ligand binding by NMR spectroscopy. The approach provides a facile route for genetically encoded Sec incorporation. It allows the production of pure selenoproteins and the Sec residue enables site-specific covalent protein modification with reagents that would usually react first with naturally occurring cysteine residues. The much greater reactivity of Sec residues allows their selective alkylation in the presence of highly solvent-exposed cysteine residues

    Rapid Elaboration of Fragments into Leads Applied to Bromodomain‑3 Extra-Terminal Domain

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    The development of low-affinity fragment hits into higher-affinity leads is a major hurdle in fragment-based drug design. Here, we demonstrate the Rapid Elaboration of Fragments into Leads (REFiL) by applying an integrated workflow that provides a systematic approach to generate higher-affinity binders without the need for structural information. The workflow involves the selection of commercial analogues of fragment hits to generate preliminary structure–activity relationships. This is followed by parallel microscale chemistry using chemoinformatically designed reagent libraries to rapidly explore chemical diversity. After a fragment screen against bromodomain-3 extra-terminal (BRD3-ET) domain, we applied the REFiL workflow, which allowed us to develop a series of ligands that bind to BRD3-ET. With REFiL, we were able to rapidly improve binding affinity > 30-fold. REFiL can be applied readily to a broad range of proteins without the need for a structure, allowing the efficient evolution of low-affinity fragments into higher-affinity leads and chemical probes

    Rapid Elaboration of Fragments into Leads Applied to Bromodomain‑3 Extra-Terminal Domain

    No full text
    The development of low-affinity fragment hits into higher-affinity leads is a major hurdle in fragment-based drug design. Here, we demonstrate the Rapid Elaboration of Fragments into Leads (REFiL) by applying an integrated workflow that provides a systematic approach to generate higher-affinity binders without the need for structural information. The workflow involves the selection of commercial analogues of fragment hits to generate preliminary structure–activity relationships. This is followed by parallel microscale chemistry using chemoinformatically designed reagent libraries to rapidly explore chemical diversity. After a fragment screen against bromodomain-3 extra-terminal (BRD3-ET) domain, we applied the REFiL workflow, which allowed us to develop a series of ligands that bind to BRD3-ET. With REFiL, we were able to rapidly improve binding affinity > 30-fold. REFiL can be applied readily to a broad range of proteins without the need for a structure, allowing the efficient evolution of low-affinity fragments into higher-affinity leads and chemical probes
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