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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]

Recent advances in proteomics have provided impetus towards the development of robust technologies for high-throughput studies of enzymes. The term “catalomics” defines an emerging ‘-omics’ field in which high-throughput studies of enzymes are carried out by using advanced chemical proteomics approaches. Of the various available methods, microarrays have emerged as a powerful and versatile platform to accelerate not only the functional annotation but also the substrate and inhibitor specificity (e.g. substrate and inhibitor fingerprinting, respectively) of enzymes. Herein, we review recent developments in the fabrication of various types of microarray technologies (protein-, peptide- and small-molecule-based microarrays) and their applications in high-throughput characterizations of enzymes. [ABSTRACT FROM AUTHOR]

MLN8237 is a highly potent and presumably selective inhibitor of Aurora kinase A (AKA) and has shown promising antitumor activities. Like other kinase inhibitors which target the ATP-binding site of kinases, MLN8237 might be expected to have potential cellular off-targets. Herein, we report the first photoaffinity-based, small molecule AKA probe capable of both live-cell imaging of AKA activities and in situ proteome profiling of potential off-targets of MLN8237 (including AKA-associating proteins). By using two mutually compatible, bioorthogonal reactions (copper-catalyzed azide-alkyne cycloaddition chemistry and TCO-tetrazine ligation), we demostrate small molecule-based multiplex bioimaging for simultaneous in situ monitoring of two important cell-cycle regulating kinases (AKA and CDK1). A broad range of proteins, as potential off-targets of MLN8237 and AKA's-interacting partners, is subsequently identified by affinity-based proteome profiling coupled with large-scale LC-MS/MS analysis. From these studies, we discover novel AKA interactions which were further validated by cell-based immunoprecipitation (IP) experiments. [ABSTRACT FROM AUTHOR]

The cis-trans peptidyiprolyl isomerase Pin1 plays a critical role in regulating a subset of phosphoproteins by catalyzing conformational changes on the phosphorylated Ser/Thr-Pro motifs. The phosphorylation-directed ubiquitination is one of the major mechanisms to regulate the abundance of p27Kip1. In this study, we demonstrate that Pin1 catalyzes the cis-trans conformational changes of p27KiP1 and further mediates its stability through the polyubiquitination mechanism. Our results show that the phosphorylated Thr-187-Pro motif in p27Kip1 is a key Pin1-binding site. In addition, NMR analyses show that this phosphorylated Thr-187-Pro site undergoes conformational change catalyzed by Pin1. Moreover, in Pini knock-out mouse embryonic fibroblasts, p27Kip1 has a shorter lifetime and displays a higher degree of polyubiquitination than in Pin1 wild-type mouse embryonic fibroblasts, suggesting that Pin1 plays a critical role in regulating p27Kip1 degradation. Additionally, Pin1 dramatically reduces the interaction between p27Kip1 and Cks1, possibly via isomerizing the cis-trans conformation of p27Kip1. Our study thus reveals a novel regulatory mechanism for p27Kip1 stability and sheds new light on the biological function of Pin1 as a general regulator of protein stability. [ABSTRACT FROM AUTHOR]

Background: Chronic Myeloid Leukemia (CML) is a blood cancer that remains challenging to cure due to drug resistance and side effects from current BCR-ABL inhibitors. There is an urgent need for novel and more effective BCR-ABL targeting inhibitors and therapeutic strategies to combat this deadly disease., Method: We disclose an "OH-implant" strategy to improve a noncovalent BCR-ABL inhibitor, PPY-A, by adding a hydroxyl group to its scaffold. By taking advantage of this OH "hot spot", we designed a panel of irreversible covalent kinase inhibitors and hypoxia-responsive pro-/dual-drugs, and their biological activities were studied in vitro, in cellulo and in vivo., Result: The resulting compound B1 showed enhanced solubility and biological activity. B4 achieved sustained BCR-ABL inhibition by forming a stable covalent bond with ABL kinase. Hypoxia-responsive prodrug P1 and dual-drugs D1/D2/D3 demonstrated significant anti-tumor effects under hypoxic conditions. The in vivo studies using K562-xenografted mice showed that B1 displayed superior antitumor activity than PPY-A, while P1 and D3 offered better safety profiles alongside significant tumor control., Conclusion: We have successfully developed a chemical biology approach to convert a known noncovalent BCR-ABL inhibitor into more potent and safer inhibitors through covalent and pro-/dual-drug targeting strategies. Our "OH-implant" approach and the resulting drug design strategies have general applicability and hold promise for improvement the performance of various other reported drugs/drug candidates, thereby providing advanced medicines for disease treatment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Production and hosting by Elsevier B.V.)

Activity-based protein profiling (ABPP) is a technique that uses highly selective active-site targeted chemical probes to label and monitor the state of proteins. ABPP integrates the strengths of both chemical and biological disciplines. By utilizing chemically synthesized or modified bioactive molecules, ABPP is able to reveal complex physiological and pathological enzyme-substrate interactions at molecular and cellular levels. It is also able to provide critical information of the catalytic activity changes of enzymes, annotate new functions of enzymes, discover new substrates of enzymes, and allow real-time monitoring of the cellular location of enzymes. Based on the mechanism of probe-enzyme interaction, two types of probes that have been used in ABPP are activity-based probes (ABPs) and affinity-based probes (A f BPs). This review highlights the recent advances in the use of ABPs and A f BPs, and summarizes their design strategies (based on inhibitors and substrates) and detection approaches., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)