There has been tremendous progress in covalent inhibitors as evidenced by the ascent of innovative electrophilic warheads with suppressed non-specific reactivity but enhanced capacity for proximity-driven covalent reactions with nucleophilic residues in the targeted site. Kinases, a central player in cancers, autoimmune disorders and chronic diseases, represent a highly targeted class of enzymes by covalent inhibitors. However, innovative strategies to afford high selectivity in target recognition remain a pressing need. This minireview focuses on four promising strategies to achieve superior target selectivity through rational design of the covalent engagement. Special emphasis is placed on examples where the selectivity had arisen by complementing the reactivity of protein cysteines with electrophilic warheads specified for distinct covalent chemistry, or inspired from native electrophile signalling in cells.[a] I. Guan
Cells employ post-translational modifications (PTMs) as key mechanisms to expand proteome diversity beyond the inherent limitations of a concise genome. The ability to incorporate post-translationally modified amino acids into protein targets via chemical ligation of peptide fragments has enabled the access to homogeneous proteins bearing discrete PTM patterns and empowered functional elucidation of individual modification sites. Native chemical ligation (NCL) represents a powerful and robust means for convergent assembly of two homogeneous, unprotected peptides bearing an N-terminal cysteine residue and a C-terminal thioester, respectively. The subsequent discovery that protein cysteine residues can be chemoselectively desulfurized to alanine has ignited tremendous interest in preparing unnatural thiol-derived variants of proteogenic amino acids for chemical protein synthesis following the ligation-desulfurization logic. Recently, the 21st amino acid selenocysteine, together with other selenyl derivatives of amino acids, have been shown to facilitate ultrafast ligation with peptidyl selenoesters, while the advancement in deselenization chemistry has provided reliable bio-orthogonality to PTMs and other amino acids. The combination of these ligation techniques and desulfurization/deselenization chemistries has led to streamlined synthesis of multiple structurally-complex, post-translationally modified proteins. In this review, we aim to summarize the latest chemical synthesis of thiolated and selenylated amino-acid building blocks and exemplify their important roles in conquering challenging protein targets with distinct PTM patterns.
A large variety of dietary phytochemicals have been shown to improve thrombosis and stroke outcomes in preclinical studies. Many of these compounds feature electrophilic functionalities that potentially undergo covalent addition to the sulfhydryl side chain of cysteine residues within proteins. However, the impact of such covalent modifications on platelet activity and function remains unclear. In this study, we evaluated phenotypes associated with irreversible protein engagement of twenty-three electrophilic phytochemicals. This revealed a novel antiplatelet selectivity profile of the isothiocyanate-containing natural product sulforaphane (SFN), whereby the response of platelets to adenosine diphosphate (ADP) and a thromboxane A2 receptor agonist was impaired without affecting thrombin and collagen-related peptide (CRP) activation. SFN also substantially reduces the formation of platelet thrombi on surfaces coated with collagen under arterial flow conditions. Activity-based protein profiling identified protein disulfide isomerase A6 (PDIA6) as a rapid kinetic responder of SFN. Mechanistic profiling studies revealed how SFN fine-tunes the enzymatic activity and substrate specificity of PDIA6. In an electrolytic injury model of thrombosis, SFN enhanced the thrombolytic activity of recombinant tissue plasminogen activator (rtPA) without increasing blood loss. Our results serve as a catalyst for further investigations into the preventive and therapeutic mechanisms of dietary antiplatelets, with a view to develop more effective and safer adjunctive treatments to improve the clot-busting power of rtPA – currently the sole approved therapeutic for stroke recanalization that has significant limitations.
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