Catalyst-free late-stage functionalization to assemble α-acyloxyenamide electrophiles for selectively profiling conserved lysine residues.

Covalent probes coupled with chemical proteomics represent a powerful method for investigating small molecule and protein interactions. However, the creation of a reactive warhead within various ligands to form covalent probes has been a major obstacle. Herein, we report a convenient and robust process to assemble a unique electrophile, an α-acyloxyenamide, through a one-step late-stage coupling reaction. This procedure demonstrates remarkable tolerance towards other functional groups and facilitates ligand-directed labeling in proteins of interest. The reactive group has been successfully incorporated into a clinical drug targeting the EGFR L858R mutant, erlotinib, and a pan-kinase inhibitor. The resulting probes have been shown to be able to covalently engage a lysine residue proximal to the ATP-binding pocket of the EGFR L858R mutant. A series of active sites, and Mg2+, ATP-binding sites of kinases, such as K33 of CDK1, CDK2, CDK5 were detected. This is the first report of engaging these conserved catalytic lysine residues in kinases with covalent inhibition. Further application of this methodology to natural products has demonstrated its success in profiling ligandable conserved lysine residues in whole proteome. These findings offer insights for the development of new targeted covalent inhibitors (TCIs).

Cyclopropenone, Cyclopropeniminium Ion, and Cyclopropenethione as Novel Electrophilic Warheads for Potential Target Discovery of Triple-Negative Breast Cancer.

Because very few targets are currently available for drug development, triple-negative breast cancer (TNBC) has been defined as one of the most difficult diseases for chemotherapy. Herein, we describe a suite of novel electrophilic warheads, which we have used in chemical proteomics studies in a search for potential targets for TNBC. Binding site analysis revealed that these warheads can modify not only highly nucleophilic residues, including cysteine and lysine, but also weakly nucleophilic residues. Cys12 of Kirsten rat sarcoma (KRASG12C) was successfully labeled by cyclopropenone and the cyclopropeniminium ions. Moderate inhibitory activity against TNBC cells was achieved with these novel electrophile-based probes. Activity-based protein profiling reveals that these electrophiles can covalently label a series of essential protein targets, including ALDH2, LRPPRC, and FABP5 from MDA-MB-231 cells. Further functional validation experiments demonstrated that FABP5 might be a potential target for TNBC.

Blockade of NMT1 enzymatic activity inhibits N-myristoylation of VILIP3 protein and suppresses liver cancer progression.

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors. Identification of the underlying mechanism of HCC progression and exploration of new therapeutic drugs are urgently needed. Here, a compound library consisting of 419 FDA-approved drugs was taken to screen potential anticancer drugs. A series of functional assays showed that desloratadine, an antiallergic drug, can repress proliferation in HCC cell lines, cell-derived xenograft (CDX), patient-derived organoid (PDO) and patient-derived xenograft (PDX) models. N-myristoyl transferase 1 (NMT1) was identified as a target protein of desloratadine by drug affinity responsive target stability (DARTS) and surface plasmon resonance (SPR) assays. Upregulation of NMT1 expression enhanced but NMT1 knockdown suppressed tumor growth in vitro and in vivo. Metabolic labeling and mass spectrometry analyses revealed that Visinin-like protein 3 (VILIP3) was a new substrate of NMT1 in protein N-myristoylation modification, and high NMT1 or VILIP3 expression was associated with advanced stages and poor survival in HCC. Mechanistically, desloratadine binds to Asn-246 in NMT1 and inhibits its enzymatic activity, disrupting the NMT1-mediated myristoylation of the VILIP3 protein and subsequent NFκB/Bcl-2 signaling. Conclusively, this study demonstrates that desloratadine may be a novel anticancer drug and that NMT1-mediated myristoylation contributes to HCC progression and is a potential biomarker and therapeutic target in HCC.

Novel Electrophilic Warhead Targeting a Triple-Negative Breast Cancer Driver in Live Cells Revealed by "Inverse Drug Discovery".

The "inverse drug discovery" strategy is a potent means of exploring the cellular targets of latent electrophiles not typically used in medicinal chemistry. Cyclopropenone, a powerful electrophile, is generally used in bio-orthogonal reactions mediated by triarylphosphine or in photo-triggered cycloaddition reactions. Here, we have studied, for the first time, the proteome reactivity of cyclopropenones in live cells and discovered that the cyclopropenone warhead can specifically and efficiently modify a triple-negative breast cancer driver, glutathione S-transferase pi-1 (GSTP1), by covalently binding at the catalytic active site. Further structure optimization and signaling pathway validation have led to the discovery of potent inhibitors of GSTP1.

AFM detects the effects of acidic condition on the size and biomechanical properties of native/oxidized low-density lipoprotein.

Solution acidification exists under some physiological conditions (e.g. lysosomes in cells) and diseases (e.g. atherosclerosis, tumors, etc.). It is poorly understood whether and how acidification influences the size and biomechanical (stiffness and stickiness) properties of native Low-density lipoprotein (LDL) and its oxidized form (oxLDL) which plays a vital role in atherogenesis and tumorigenesis. Atomic force microscopy (AFM) evaluated that gradient acidification from pH 7.4 to pH 4.4 caused an expanding-first-and-then-shrinking decrease in size and a dramatic decrease in stiffness (but no statistically significant changes in stickiness) of LDL/oxLDL particles by influencing secondary/tertiary structures and lipid release detected by infrared spectral analysis and cholesterol detection, respectively. The smaller and softer characteristics of LDL/oxLDL at acidic conditions versus at the neutral pH partially explains the atherogenic role of acidification. The data may provide important information for a better understanding of LDL/oxLDL and some diseases (e.g. atherosclerosis and tumors).

2H-Azirine-Based Reagents for Chemoselective Bioconjugation at Carboxyl Residues Inside Live Cells.

Protein modification by chemical reagents has played an essential role in the treatment of human diseases. However, the reagents currently used are limited to the covalent modification of cysteine and lysine residues. It is thus desirable to develop novel methods that can covalently modify other residues. Despite the fact that the carboxyl residues are crucial for maintaining the protein function, few selective labeling reactions are currently available. Here, we describe a novel reactive probe, 3-phenyl-2H-azirine, that enables chemoselective modification of carboxyl groups in proteins under both in vitro and in situ conditions with excellent efficiency. Furthermore, proteome-wide profiling of reactive carboxyl residues was performed with a quantitative chemoproteomic platform.