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Chemoselective modification of specific residues within a given protein poses a significant challenge, as the microenvironment of amino acid residues in proteins is variable. Developing a universal molecular platform with tunable chemical warheads can provide powerful tools for precisely labeling specific amino acids in proteins. Cysteine and lysine are hot targets for chemoselective modification, but current cysteine/lysine‐selective warheads face challenges due to cross‐reactivity and unstable reaction products. In this study, a versatile fluorescent platform is developed for highly selective modification of cysteine/lysine under biocompatible conditions. Chloro‐ or phenoxy‐substituted NBSe derivatives effectively labeled cysteine residues in the cellular proteome with high specificity. This finding also led to the development of phenoxy‐NBSe phototheragnostic for the diagnosis and activatable photodynamic therapy of GSH‐overexpressed cancer cells. Conversely, alkoxy‐NBSe derivatives are engineered to selectively react with lysine residues in the cellular environment, exhibiting excellent anti‐interfering ability against thiols. Leveraging a proximity‐driven approach, alkoxy‐NBSe probes are successfully designed to demonstrate their utility in bioimaging of lysine deacetylase activity. This study also achieves integrating a small photosensitizer into lysine residues of proteins in a regioselective manner, achieving photoablation of cancer cells activated by overexpressed proteins. [ABSTRACT FROM AUTHOR]

Remarkable progress has been made in the development of cysteine‐targeted covalent inhibitors. In kinase drug discovery, covalent inhibitors capable of targeting other nucleophilic residues (i.e. lysine, or K) have emerged in recent years. Besides a highly conserved catalytic lysine, almost all human protein kinases possess an equally conserved glutamate/aspartate (e.g. E/D) that forms a K–E/D salt bridge within the enzyme's active site. Electrophilic ynamides were previously used as effective peptide coupling reagents and to develop E/D‐targeting covalent protein inhibitors/probes. In the present study, we report the first ynamide‐based small‐molecule inhibitors capable of inducing intramolecular cross‐linking of various protein kinases, leading to subsequent irreversible inhibition of kinase activity. Our strategy took advantage of the close distance between the highly conserved catalytic K and E/D residues in a targeted kinase, thus providing a conceptually general approach to achieve irreversible kinase inhibition with high specificity and desirable cellular potency. Finally, this ynamide‐facilitated, ligand‐induced mechanism leading to intramolecular kinase cross‐linking and inhibition was unequivocally established by using recombinant ABL kinase as a representative. [ABSTRACT FROM AUTHOR]

Herein, we report a salicylaldehyde-based, reversible covalent inhibitor (A2) that possesses moderate cellular activity against AURKA with a prolonged residence time and shows significant non-covalent inhibition towards LRRK2. Our results indicated that this multitarget kinase inhibitor may be used as the starting point for future development of more potent, selective and dual-targeting covalent kinase inhibitors against AURKA and LRRK2 for mitophagy. [ABSTRACT FROM AUTHOR]

The human endocannabinoid system regulates a myriad of physiological processes through a complex lipid signaling network involving cannabinoids and their respective receptors, cannabinoid receptor 1 (hCB1R) and cannabinoid receptor 2 (hCB2R). Anandamide (AEA) and cannabidiol (CBD) are classical examples of cannabinoids that elicit a variety of effects, both beneficial and detrimental, through these receptors. Mounting evidence suggested the presence of other potential cannabinoid targets that may be responsible for other observable effects. However, prior pharmacological studies on these cannabinoid compounds provided scant evidence of direct engagement to these proposed targets. Moreover, to the best of our knowledge, no chemoproteomic studies have been demonstrated on CBD. Here we showed that, by taking advantage of a recently developed 'label‐free' 2D‐TPP (2 Dimensional‐Thermal Protein Profiling) approach, we have identified several new putative targets of both AEA and CBD. Comparison of these interaction landscapes with those obtained from well‐established affinity‐based protein profiling (AfBPP) platforms has led to the discovery of both shared and unique protein targets. Subsequent target validation of selected proteins led us to conclude that this 2D‐TPP strategy complements well with AfBPP. [ABSTRACT FROM AUTHOR]

Drugs and bioactive natural products exert their pharmacological effects by engaging numerous cellular targets in our body. Identification of these protein targets is essential for understanding the mechanism-of-action of these compounds, thus contributing to improved drug design in drug discovery programs. Termed "in situ drug profiling", a common strategy for studying these bioactive compounds centralized on the covalent capture of protein targets along with a reporter tag to facilitate downstream proteomic analyses. Though highly successful, such reliance on innate electrophilic traps to facilitate covalent capture restricted its applications to covalent acting compounds. Late-stage C--H functionalization (LSF) may resolve this by substituting biologically inert C--H bonds with desired electrophilic groups. Herein, we demonstrated this concept by arming a diverse range of electron-rich aromatic drugs and natural products with α,β-unsaturated esters, via late-stage C--H olefination with an arylthio-based carboxylic acid ligand developed by Ibanez and co-workers. We also showed that covalent probes generated from this LSF approach could be applied for "in situ drug profiling" of Δ8-THC, as exemplified by the successful target engagement of α-4db, a Δ8-THC-based probe, to its native target hCB2R. In combination with AfBP 7, a photoaffinity-based derivative of Δ8-THC, we identified several novel putative targets that could account for some of the effects in THC consumption. We anticipate our C--H LSF strategy to be widely adopted for future studies of noncovalent drugs. [ABSTRACT FROM AUTHOR]