The Cysteinome of Protein Kinases as a Target in Drug Development

Chaikuad, A.; Koch, Pierre; Laufer, Stefan; Knapp, Stefan

doi:10.1002/anie.201707875

Cited by 197 publications

(257 citation statements)

References 110 publications

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“…[3] This interest has led to the recent clinical approval of several covalent kinase inhibitors. [4] Especially in the field of antibiotics,covalent inhibitors are prevalent as exemplified by b-lactams, [3] fosfomycin, [5] showdomycin, [6] and optimized arylomycins. [7] Recently,m ethods have emerged to globally identify the exact interaction site of covalent inhibitors in ac ompetitive fashion.…”

Section: Introductionmentioning

confidence: 99%

Isotopically Labeled Desthiobiotin Azide (isoDTB) Tags Enable Global Profiling of the Bacterial Cysteinome

2020

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Rapid development of bacterial resistance has led to an urgent need to find new druggable targets for antibiotics.In this context, residue-specific chemoproteomic approaches enable proteome-wide identification of binding sites for covalent inhibitors.D escribed here are easily synthesized isotopically labeled desthiobiotin azide (isoDTB) tags that shortened the chemoproteomic workflowa nd allowed an increased coverage of cysteines in bacterial systems.They were used to quantify 59 %o fa ll cysteines in essential proteins in Staphylococcus aureus and enabled the discovery of 88 cysteines that showed high reactivity,w hichc orrelates with functional importance.F urthermore,2 68 cysteines that are engaged by covalent ligands were identified. Inhibition of HMG-CoA synthase was verified and will allow addressing the bacterial mevalonate pathway through an ew target. Overall, ab road map of the bacterial cysteinome was obtained, which will facilitate the development of antibiotics with novel modesof-action.

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Section: Introductionmentioning

confidence: 99%

Isotopically Labeled Desthiobiotin Azide (isoDTB) Tags Enable Global Profiling of the Bacterial Cysteinome

2020

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“…[6] In addition to protein conformational changes,t he rearrangement of water molecules has been discussed as apotential mechanism influencing inhibitor residence time. [10] However, the design of reversibly formed covalent interactions requires the presence of cysteine residues in the drug binding site.M any drug receptors,i ncluding al arge number of kinases,c ontain cysteines in close proximity to their active sites, [11] but the development of covalent inhibitors may not be feasible for all drug targets. [8] In some cases,t he presence of water-shielded hydrogen bonds can also lead to slow dissociation.…”

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confidence: 99%

“…[9] In addition, reversible covalent inhibitors provide an interesting strategy for prolonging target engagement by transient covalent-bond formation. [10] However, the design of reversibly formed covalent interactions requires the presence of cysteine residues in the drug binding site.M any drug receptors,i ncluding al arge number of kinases,c ontain cysteines in close proximity to their active sites, [11] but the development of covalent inhibitors may not be feasible for all drug targets.…”

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confidence: 99%

Halogen–Aromatic π Interactions Modulate Inhibitor Residence Times

et al. 2018

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Prolonged drug residence times may result in longer-lasting drug efficacy, improved pharmacodynamic properties, and "kinetic selectivity" over off-targets with high drug dissociation rates. However, few strategies have been elaborated to rationally modulate drug residence time and thereby to integrate this key property into the drug development process. Herein, we show that the interaction between a halogen moiety on an inhibitor and an aromatic residue in the target protein can significantly increase inhibitor residence time. By using the interaction of the serine/threonine kinase haspin with 5-iodotubercidin (5-iTU) derivatives as a model for an archetypal active-state (type I) kinase-inhibitor binding mode, we demonstrate that inhibitor residence times markedly increase with the size and polarizability of the halogen atom. The halogen-aromatic π interactions in the haspin-inhibitor complexes were characterized by means of kinetic, thermodynamic, and structural measurements along with binding-energy calculations.

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“…In order to conduct a head-to-head comparison of the performance of the azobenzene (AZO) and dialkoxydiphenylsilane (DADPS) chemically-cleavable linkers in characterizing the cellular cysteinome ( Figure 1B), we utilized the cysteine residue-reactive probe iodoacetamide alkyne (IAAyne) as it has been successfully deployed in chemical proteomic applications 11,19 , and serves as a tool to profile cysteine-reactive covalent drugs used for disease indications 29 . We initiated our studies by subjecting 1 mg of lysate derived from K562 chronic myelogenous leukemia cells to alkylation in the presence of IAAyne (100 µM).…”

Section: Resultsmentioning

confidence: 99%

Evaluation and Optimization of Chemically-Cleavable Linkers for Quantitative Mapping of Small Molecule-Protein Interactomes

Rabalski

Bogdan

Baranczak

2019

Preprint

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Numerous reagents have been developed to enable chemical proteomic analysis of small molecule-protein interactomes. However, the performance of these reagents has not been systematically evaluated and compared. Herein, we report our efforts to conduct a parallel assessment of two widely-used chemically-cleavable linkers equipped with dialkoxydiphenylsilane (DADPS linker) and azobenzene (AZO linker) moieties. Profiling a cellular cysteinome using iodoacetamide alkyne probe demonstrated a significant discrepancy between the experimental results obtained through the application of each of the reagents. To better understand the source of observed discrepancy, a mass tolerant database search strategy using MSFragger software was performed. This resulted in identifying a previously unreported artifactual modification on the residual mass of the azobenzene linker. Furthermore, we conducted a comparative analysis of enrichment modes using both cleavable linkers. This effort determined that enrichment of proteolytic digests yielded a far greater number of identified cysteine residues than the enrichment conducted prior to protein digest. Inspired by recent studies where multiplexed quantitative labeling strategies were applied to cleavable biotin linkers, we combined this further optimized protocol using the DADPS cleavable linker with tandem mass tag (TMT) labeling to profile the FDA-approved covalent EGFR kinase inhibitor dacomitinib against the cysteinome of an epidermoid cancer cell line. Our analysis resulted in the detection and quantification of over 10,000 unique cysteine residues, a nearly 3-fold increase over previous studies that used cleavable biotin linkers for enrichment. Critically, cysteine residues corresponding to proteins directly as well as indirectly modulated by dacomitinib treatment were identified. Overall, our study suggests that the dialkoxydiphenylsilane linker could be broadly applied wherever chemically cleavable linkers are required for chemical proteomic characterization of cellular proteomes.

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The Cysteinome of Protein Kinases as a Target in Drug Development

Cited by 197 publications

References 110 publications

Isotopically Labeled Desthiobiotin Azide (isoDTB) Tags Enable Global Profiling of the Bacterial Cysteinome

Isotopically Labeled Desthiobiotin Azide (isoDTB) Tags Enable Global Profiling of the Bacterial Cysteinome

Halogen–Aromatic π Interactions Modulate Inhibitor Residence Times

Evaluation and Optimization of Chemically-Cleavable Linkers for Quantitative Mapping of Small Molecule-Protein Interactomes

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