Many biological experiments are not compatible with the use of immunofluorescence or genetically-encoded fluorescent tags or FRET-based reporters. Conjugation of existing kinase inhibitors to cell-permeable fluorophores can provide a generalized approach to develop fluorescent probes of intracellular kinases. Here, we report the development of a small molecule probe of Src through conjugation of BODIPY to two well-established, dual Src-Abl kinase inhibitors, dasatinib and saracatinib. We show that this approach is not successful for saracatinib, but that largely dasatinib-BODIPY retains the biological activity of its parent compound and can be used to monitor the presence of Src kinase in individual cells by flow cytometry and to track the localization of Src by fixed and live-cell fluorescence microscopy. This strategy may enable generation of additional kinase-specific probes useful in systems not amenable to genetic manipulation or used together with fluorescent proteins to enable a multiplexed assay read-out.
We previously reported that the halogenase RebH catalyzes selective halogenation of several heterocycles and carbocycles, but product yields were limited by enzyme instability. Here, we use directed evolution to engineer a RebH variant, 3-LR, with a Toptover 5 °C higher than that of wild type, and 3-LSR, with a Tm18 °C higher than that of wild type. These enzymes provided significantly (up to 4-fold) improved conversions for halogenation of tryptophan and several non-natural substrates. This initial demonstration of RebH evolution not only provides improved enzymes for immediate synthetic applications, but also establishes a robust protocol for further halogenase evolution.
By combining a riboswitch with a cell-permeable photocaged small molecule ligand, an optochemical gene control element was constructed, enabling spatial and temporal control of gene expression in bacterial cells. Because of the simplicity of this strategy, coupled with the ability to create synthetic riboswitches with tailored ligand specificities and output in a variety of microorganisms, plants, and fungi, this approach may afford a general strategy to photo-control gene expression in vivo. The ability to activate riboswitches using light enables the interrogation and manipulation of a wide range of biological processes with high precision, and will have broad utility in regulation of artificial genetic circuits.
The reversible post-translational modification (PTM) of eukaryotic proteins by ubiquitin (Ub) regulates key cellular processes including protein degradation and gene transcription. Studies of the mechanistic roles for protein ubiquitylation require quantities of homogenously modified substrates that are typically inaccessible from natural sources or by enzymatic ubiquitylation in vitro. Therefore, we developed a facile and scalable methodology for site-specific chemical ubiquitylation. Our semisynthetic strategy utilized a temporary ligation auxiliary, 2-(aminooxy)ethanethiol, to direct ubiquitylation at specific lysines in peptide substrates. Mild reductive removal of the auxiliary after ligation yielded ubiquitylated peptides with the native isopeptide linkage. Alternatively, retention of the ligation auxiliary yielded protease-resistant analogues of ubiquitylated peptides. Importantly, our strategy was fully compatible with protein sulfhydryl groups, as demonstrated by the synthesis of peptides modified by the human small ubiquitin-related modifier 3 (SUMO-3) protein.
A new strategy that combines the concepts of fragment-based drug design and dynamic combinatorial chemistry (DCC) for targeting adenosine recognition sites on enzymes is reported. We demonstrate the use of 5′-deoxy-5′-thioadenosine as a noncovalent anchor fragment in dynamic combinatorial libraries templated byMycobacterium tuberculosispantothenate synthetase. A benzyl disulfide derivative was identified upon library analysis by HPLC. Structural and binding studies of protein–ligand complexes by X-ray crystallography and isothermal titration calorimetry informed the subsequent optimisation of the DCC hit into a disulfide containing the novelmeta-nitrobenzyl fragment that targets the pantoate binding site of pantothenate synthetase. Given the prevalence of adenosine-recognition motifs in enzymes, our results provide a proof-of-concept for using this strategy to probe adjacent pockets for a range of adenosine binding enzymes, including other related adenylate-forming ligases, kinases, and ATPases, as well as NAD(P)(H), CoA and FAD(H2) binding proteins.
Combinatorial alanine substitution of active site residues in a thermostable cytochrome P450BM3(BM3) variant was used to generate BM3 variants with activity on large substrates. Selective hydroxylation of methoxymethylated monosaccharides, alkaloids, and steroids was thus made possible. This approach could be generally useful for improving the activity of enzymes that show only limited activity on larger substrates.