KRAS, a frequently mutated oncogene, has been challenging to target therapeutically. Although covalent inhibitors like sotorasib against KRASG12C have been developed, their efficacy is often limited by acquired resistance. Targeted protein degradation offers a potential solution but has largely relied on large PROTAC molecules. Here, we report DJX-A-KM, a small-molecule degrader of KRASG12C, designed by incorporating an acrylamide warhead into the MRTX849 scaffold. It induces potent and sustained degradation of KRASG12C in cells and in vivo. Mechanistic investigation reveal that degradation is mediated by the ubiquitin-proteasome system, facilitated by covalent engagement with a E3 ligase, FBXO28, at cysteine 98. Antiproliferation assays demonstrate its potent inhibitory effects across multiple KRASG12C-mutant cancer models. This strategy also enables the development of pan-KRAS degraders against a broader spectrum of KRAS mutations. Our work presents a small-molecule degrader recruiting FBXO28 and provides a blueprint for exploring E3 ligases in protein degradation.

Targeted protein degradation (TPD) is a promising therapeutic strategy, yet its application remains constrained by the limited repertoire of available E3 ubiquitin ligases, primarily CRBN and VHL. Here, we identify RNF213 as a recruitable E3 ligase that mediates protein degradation induced by molecular glue degraders. We developed CYB-5067 by equipping the pan-FGFR inhibitor Infigratinib with a minimal dibromoacetamide covalent warhead. This covalent molecular glue recruits RNF213 to potently degrade FGFR1-4, with the strongest effect on FGFR2 (DC50 = 27 nM, Dmax = 96%). CYB-5067 outperforms parent inhibitors in vitro (IC50 = 3.8 nM) and shows comparable antitumor efficacy in vivo (TGI = 94.6%), with sustained target suppression and no apparent hook effects under the tested conditions. Notably, the dibromoacetamide warhead is transplantable, enabling selective degradation of other challenging targets such as WEE1 and CDK12, which regulate cell-cycle progression and transcription. This offers a rational strategy for creating molecular glues. Our work identifies RNF213 as an exploitable ligase for TPD and establishes covalent molecular glues as a modular platform. This strategy expands the scope of degrader design beyond conventional E3 ligases, offering an avenue for developing potent and selective therapeutics.

Targeted protein degradation represents a promising therapeutic strategy, yet its broader application is often limited by the scarcity of usable E3 ligases. Glutathione peroxidase 4 (GPX4) is a key target for inducing ferroptosis, but achieving sustained and potent inhibition remains challenging with conventional enzymatic inhibitors. Herein, we report the first small-molecule GPX4 degraders that incorporate either electrophilic warheads or a minimal azide group as an E3 recruitment ligand. The azide-based degrader DK-5070 effectively drives potent GPX4 degradation, achieving a DC50 of 17.4 nM and a Dmax of 84%, thereby outperforming larger PROTAC-based degraders. Notably, DK‑5070 exhibits potent antitumor activity both in vitro (IC50 = 47.21 nM) and in vivo (TGI = 41.8%), demonstrating significant efficacy as a GPX4 degrader. Mechanistic studies reveal that degradation is mediated through recruitment of the oncogenic E3 ligase UHRF1, which is frequently overexpressed in tumors, underscoring the potential for tumor-specific protein degradation. This demonstrated small-molecule degraders that recruit UHRF1 to facilitate targeted degradation of GPX4. In this system, the azide group functions as a minimal recruitment ligand, thereby expanding the E3 ligase toolbox and offering a promising strategy for targeted cancer therapy.

Protein N-terminal cysteines (NCys) are valuable for selective modification and posttranslational regulation, but their proteome-wide landscape remains unexplored. We constructed a library of electrophilic probes, including cyanobenzothiazole (CBT), to systematically map ligandable native NCys residues. Modifiable NCys sites were identified in proteins such as GFPT1 and CSTB, revealing accessible NCys in the human proteome. Integrating CBT into a BRD4-targeting ligand generated a reversible covalent degrader (DC50 = 16.7 nM, Dmax = 98%). Mechanistic studies showed that CBT modifies DCAF16 at Cys58, promoting ternary complex formation and enabling efficient ubiquitination and degradation. The platform achieved potent and selective degradation of challenging targets like EGFRL858R/T790M/C797S and HER2 without hook effects, outperforming clinical inhibitors. This work provides the first proteome-wide map of ligandable NCys residues and establishes CBT as a versatile platform for covalent targeted protein degradation (TPD), opening new avenues for precision therapeutics.

Targeted protein degradation (TPD) has been recognized as a powerful therapeutic strategy for the treatment of a wide range of diseases. However, the application of existing degraders is constrained by their dependence on a limited number of E3 ubiquitin ligases, such as CRBN and VHL. To address this limitation, we developed a suite of novel small‐molecule degraders by integrating an ynamide electrophile into protein‐targeting ligands. These compounds demonstrated remarkable target degradation capability. Subsequent proteome profiling and functional validation revealed that Cys97 residue of retinoblastoma binding protein 7 (RBBP7) E3 ligase was covalently engaged and responsible for the degradation mechanism. Furthermore, the ynamide motif has proved to be a versatile and transplantable chemical handle, facilitating the development of degraders targeting a wide range of proteins, including CDK4, PDE5, PI3K, AKT, BCR‐ABL, BRD4, EGFRL858R, and EGFRL858R/T790M/C797S. Notably, incorporation of ynamide into the “pan‐kinase” inhibitor XO44 yielded degraders capable of simultaneously degrading various kinases, such as PI3K, Syk, AKT, and GSK‐3β, further highlighting the general feasibility of this approach. Importantly, the ynamide‐containing degraders demonstrated significantly enhanced anticancer potency compared to their parent inhibitors.

Nitrogen-nitrogen (N-N) bond formation is integrated into the biosynthetic pathways of various classes of natural products, some of which exhibit intriguing biological activities. While recent studies have identified several distinct groups of enzymes responsible for N-N bond formation, the underlying catalytic mechanisms are largely unknown. Here, we report the dimeric structure of the N-N bond forming enzyme LnzB (Streptomyces spp.), which relies on a heme-iron to catalyze the formation of intramolecular N-N bonds using N-hydroxyornithine as a substrate. The structure reveals the molecular architecture of its active sites and heme-interacting pocket. In combination with MD simulation, site-directed mutagenesis, and kinetic activity assays, we have identified key residues responsible for ligand binding and N-N bond formation activity. Phylogenetic analysis and structural comparison reveal that LnzB and its homologues may have evolved from the transcriptional regulator PaiB by altering the substrate binding pocket. Our study extends the limited knowledge of N-N bond formation catalyzed by a heme iron-dependent enzyme in natural products. Nitrogen–nitrogen bond formation is crucial in the biosynthesis of various classes of natural products with significant biological activities, yet the catalytic mechanisms remain largely unknown. Here, the authors elucidate the dimeric structure of the heme-iron-dependent enzyme LnzB, revealing key residues for N-N bond formation and suggesting evolutionary links to transcriptional regulators, advancing our understanding of enzymatic catalysis.

Bioconjugation chemistry has been a powerful avenue in expanding the repertoire of druggable proteome, as well as in identifying new E3 ligases to support targeted protein degradation (TPD). However, a large fraction of proteome remains inaccessible with existing covalent probes. Herein, we incorporated various electron-withdrawing groups into styrene derivatives and identified β-nitrostyrene as a cysteine-targeting reversible covalent warhead for target discovery. Through phenotypic screening and chemoproteomics platforms, we identified new ligandable sites such as C96 of SND1, C110 of PTGES2, modulating cell proliferation in an acute myeloid leukemia cell line. Moreover, incorporation of this warhead into the BRD4 inhibitor (+)-JQ1 demonstrated that the covalent handle engages the novel E3 ligase tripartite motif-containing 28 (TRIM28) at Cys232 residue, thereby promoting the targeted degradation. Notably, when transplanted into other protein-targeting ligands, the β-nitrostyrene warhead effectively induced protein degradation of EGFRL858R/T790M/C797S, PDE5, BTK, LRRK2, and BCR-ABL/c-ABL without eliciting a hook effect. Importantly, the degraders demonstrate significantly enhanced antcancer effects compared to corresponding inhibitors. To our knowledge, this is the first report of small-molecular degraders engaging TRIM28 to support targeted protein degradation, and provides a rational pathway for design and development of potent monovalent degraders.

Targeted protein degradation (TPD) has emerged as a promising therapeutic strategy for treating various diseases. However, current small molecule degraders predominantly rely on a limited set of E3 ubiquitin ligases, such as CRBN and VHL, which restricts their applications. Here, we report that incorporation of the 2H-azirine chemical handle into the EGFRL858R/T790M/C797S inhibitor induced remarkable degradation of the targeted protein. Proteomic profiling and functional validation confirmed that the NEDD4 E3 ligase was covalently recruited by 2H-azirine through engagement of C1286 residue, facilitating target degradation. Furthermore, the 2H-azirine moiety demonstrated versatility by acting as a small molecular degrader when conjugated to various ligands, effectively mediating the degradation of CDK4, PDE5, BTK and Brd4. More importantly, using the identical protein ligand scaffold, we demonstrated that the 2H-azirine based probe can degrade proteins resistant to degradation by CRBN or VHL recruitment. This approach provides a rational strategy for developing novel small molecular degraders that target alternative E3 ubiquitin ligases. Notably, these degraders significantly outperformed their parent kinase inhibitor in suppressing cancer cell growth.

Due to the widespread distribution and critical roles of lysine residues, the development of new methods to characterize their reactivity and ligandability could significantly expand the landscape of druggable targets. Herein, we report a new class of five-membered heterocyclic electrophiles, dioxazolones, which function as masked isocyanate electrophiles for in-depth profiling of functional lysine residues. These probes demonstrated remarkable chemoselectivity and exceptional reaction kinetics across the proteome, enabling the identification of over 3000 covalently modifiable lysine residues. And up to 30% of them were undetected by previously reported probes. Leveraging fragment-based ligand discovery, we identified dioxazolone-based fragments that covalently engage previously uncharacterized or functional lysines, such as K33 of SLC25A5, K69 of CSNK2A2, and K97 of MAP2K1, modulating protein function and substantially expanding the content of ligandable lysines. Furthermore, we demonstrated the utility of the dioxazolone moiety as a latent electrophilic warhead in the design of Bruton's tyrosine kinase (BTK) inhibitors, enabling covalent modification of the conserved catalytic lysine residue (K430) and offering a potential strategy to overcome drug resistance. These findings establish dioxazolones as versatile tools for lysine-targeted covalent chemistry.

Covalent probes integrated with chemical proteomics have been an efficient method for disclosing new druggable targets and E3 ubiquitin ligases supporting targeted protein degradation. However, a large fraction of the proteome including E3 ligases remains inaccessible with existing electrophiles. In this work, we developed a new reactive warhead, terminal azoacetylene, which can be generated by in situ desilylation for proteome profiling under cellular conditions. A series of uncharacterized targets and E3 ubiquitin ligases were covalently engaged. Fragment-based ligand discovery (FBLD) showed that the azoacetylene-containing fragments can covalently bind a series of essential protein hits at the active sites such as C130 of TUFM probably modulating the protein functions. Incorporation of this warhead into BRD4 targeting inhibitor JQ1 led to generation of novel small molecular degraders that degrade BRD4 without inducing the hook effect. This provides a new method for ligand and target discovery, as well as the development of new types of small molecular degraders.

Glutathione peroxidase 4 (GPX4) emerges as a promising target for the treatment of therapy-resistant cancer through ferroptosis. Thus, there is a broad interest in the development of GPX4 inhibitors. However, a majority of reported GPX4 inhibitors utilize chloroacetamide as a reactive electrophilic warhead, and the selectivity and pharmacokinetic properties still need to be improved. Herein, we developed a compound library based on a novel electrophilic warhead, the sulfonyl ynamide, and executed phenotypic screening against pancreatic cancer cell lines. Notably, one compound A16 exhibiting potent cell toxicity was identified. Further chemical proteomics investigations have demonstrated that A16 specifically targets GPX4 under both in situ and in vivo conditions, inducing ferroptosis. Importantly, A16 exhibited superior selectivity and potency compared to reported GPX4 inhibitors, ML210 and ML162. This provides the structural diversity of tool probes for unraveling the fundamental biology of GPX4 and exploring the therapeutic potential of pancreatic cancer via ferroptosis induction.

The evolving use of covalent ligands as chemical probes and therapeutic agents could greatly benefit from an expanded array of cysteine-reactive electrophiles for efficient and versatile proteome profiling. Herein, to expand the current repertoire of cysteine-reactive electrophiles, we developed a new class of strain-enabled electrophiles based on cyclopropanes. Proteome profiling has unveiled that C163 of lactate dehydrogenase A (LDHA) and C88 of adhesion regulating molecule 1 (ADRM1) are ligandable residues to modulate the protein functions. Moreover, fragment-based ligand discovery (FBLD) has revealed that one fragment (Y-35) shows strong reactivity toward C66 of thioredoxin domain-containing protein 12 (TXD12), and its covalent binding has been demonstrated to impact its downstream signal pathways. TXD12 plays a pivotal role in enabling Y-35 to exhibit its antisurvival and antiproliferative effects. Finally, dicarbonitrile-cyclopropane has been demonstrated to be an electrophilic warhead in the development of GSTO1-involved dual covalent inhibitors, which is promising to alleviate drug resistance.

Due to the high abundance and diverse functions of lysine residues, both in the interior and on the surface of proteins, the development of new methods to characterize their reactivity and ligandability could significantly expand the pool of druggable targets. To date, only a limited number of aminophilic electrophiles have been assessed for interactions with the lysine proteome, resulting in a substantial fraction remaining inaccessible to current probes. Here, to the best of our knowledge, we report the first oxidant-triggered bioconjugation platform for in-depth profiling of lysines. We quantified over 7000 covalently modifiable lysine residues, which significantly expands the coverage of ligandable lysines in the whole proteome. Chemical proteomics enabled the mapping of more than 100 endogenous kinases, thus providing a comprehensive landscape of ligandable catalytic lysines within the kinome. Moreover, we identified a suite of new ligandable lysines such as K60 of ENO1 and K31 of PPIA, offering insights for exploring new functional and targetable residues. These findings could provide valuable clues for the development of targeted covalent inhibitors (TCIs).

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 Mg 2+ , 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).

Owing to their remarkable pharmaceutical properties compared to those of noncovalent inhibitors, the development of targeted covalent inhibitors (TCIs) has emerged as a powerful method for cancer treatment. The K-Ras mutant, which is prevalent in multiple cancers, has been confirmed to be a crucial drug target in the treatment of various malignancies. However, although the K-Ras(G12D) mutation is present in up to 33% of K-Ras mutations, no covalent inhibitors targeting K-Ras(G12D) have been developed to date. The relatively weak nucleophilicity of the acquired aspartic acid (12D) residue in K-Ras may be the reason for this. Herein, we present the first compound capable of covalently engaging both K-Ras(G12D) and K-Ras(G12C) mutants. Proteome profiling revealed that this compound effectively conjugates with G12C and G12D residues, modulating the protein functions in situ. These findings offer a unique pathway for the development of novel dual covalent inhibitors.

Chemical synthesis of hydrophobic proteins presents a formidable task as they are often difficultly achieved via peptide synthesis, purification, and peptide ligation. Thus, peptide solubilizing strategies are needed to integrate with peptide ligation to achieve protein total synthesis. Herein, we report a tunable backbone modification strategy, taking advantage of the tunable stability of the Cys/Pen ligation intermediate, which allows for readily introducing a solubilizing tag for both peptide purification and ligation processes. The effectiveness of this strategy was demonstrated by the chemical synthesis of interleukin-2.

HMGB1 (high-mobility group box 1) is a non-histone chromatin-associated protein that has been widely reported as a representative damage-associated molecular pattern (DAMP) and to play a pivotal role in the proinflammatory process once it is in an extracellular location. Accumulating evidence has shown that HMGB1 undergoes extensive post-translational modifications (PTMs) that actively regulate its conformation, localization, and intermolecular interactions. However, fully characterizing the functional implications of these PTMs has been challenging due to the difficulty in accessing homogeneous HMGB1 with site-specific PTMs of interest. In this study, we developed a streamlined protein semi-synthesis strategy via salicylaldehyde ester-mediated chemical ligations (Ser/Thr ligation and Cys/Pen ligation, STL/CPL). This methodology enabled us to generate a series of N-terminal region acetylated HMGB1 proteins. Further studies revealed that acetylation regulates HMGB1-heparin interaction and modulates HMGB1's stability against thrombin, representing a regulatory switch to control HMGB1's extracellular activity.

Covalent inhibitors with an electrophilic warhead have received considerable attention due to their remarkable pharmacological properties. However, the electrophilic warhead in covalent drugs is often an α, β-unsaturated amide, and the targets are mainly cysteine or lysine residues. Thus, the development of novel electrophiles that can target other amino acids is highly desirable. Ynamide, a useful and versatile building block, is commonly employed in the construction of various compounds in organic synthesis. The performance of this functional group in a proteome-wide environment has been studied here for the first time, and it has been shown that it can efficiently modify carboxyl residues in situ and in vitro. Upon incorporation of this ynamide warhead into the pharmacophores of kinase inhibitors, the resulting compound showed moderate inhibition against the EGFR L858R mutant but not against EGFR WT. This novel electrophilic group can be used in the development of new types of covalent inhibitors.

Chemical synthesis of proteins with poor solubility presents a challenging task. The existing solubilizing tag strategies are not suitable for the expressed protein segment. To address this issue, we report herein that solubilizing tags could be introduced at the side chain of the peptide and C-terminal peptide salicylaldehyde esters via a disulfide linker. Such reducible solubilizing tags (RSTs) are compatible with peptide salicylaldehyde ester-mediated Ser/Thr ligation and Cys/Pen ligation for purifying and ligating peptides with poor solubility. This strategy features operational simplicity and readily accessible materials. Both the protein 2B4 cytoplasmic tail and FCER1G protein have been successfully synthesized via this strategy. Of particular note, the RST strategy could be used for solubilizing the expressed protein segment for protein semi-synthesis of the HMGB1 protein.