A chemoproteomic atlas of the human purine interactome for regioselective ligand discovery

Zhihong Li, Hsiao-Kuei Tsai, Adam H. Libby, Michael W. Founds, Olivia L. Murtagh, Madeleine L. Ware, David M. Leace, Wesley J. Wolfe, Phillip W. Gingrich, Bissan Al-Lazikani, Chin-Yuan Chang & Ku-Lung Hsu

Nat Commun (2026). 

https://doi.org/10.1038/s41467-026-73407-3

Purines are essential bioactive molecules that interact with a large fraction of the human proteome. Despite their importance, the scope of actionable purine-binding pockets for ligand discovery remains limited. Here, we develop a quantitative chemoproteomics platform using sulfonyl-purine (SuPUR) chemistry to produce a massive and functional map of the human purine interactome. The SuPUR platform captures 31,000+ targetable tyrosine and lysine sites, representing the most comprehensive beyond cysteine chemoproteomics database for enabling protein ligand discovery. SuPUR ligands that bind through a regioselective fashion serve as enabling starting points for developing potent (nanomolar) and proteome-wide-selective modulators of enzymatic and protein-protein interaction function. Phenotypic screening identifies a site-specific (Y237) and regioselective SuPUR ligand of ACAT2 to reveal an unexpected metabolic dependency in cancer cells. A crystal structure of SuPUR ligand-bound ACAT2 reveals the purine group binds deep in the CoA pocket forming key interactions with catalytic residues via a water bridge to guide future structure-based ligand design.

Linking of fragments in neighboring binding sites is one of the optimization strategies in fragment-based drug discovery, where additive or even more substantial bioactivity improvements can be realized. However, such efforts present a considerable challenge when one fragment binds covalently to the target protein, as small modifications can influence the correct positioning of the covalent warhead toward the targeted nucleophilic residue. Here, we present a case study of fragment linking that yielded single-digit micromolar, covalent inhibitors of the SARS-CoV-2 main protease, starting from fragments that were inactive in the biochemical assay. Using structural information from a recent, high-throughput crystallographic fragment screen, we show that the success of fragment linking in the design of targeted covalent inhibitors is heavily impacted by several factors, including the warhead type, the labeling chemistry, and even subtle changes in the designed linker. Notably, we observe that induced fit effects might override the original fragment orientations in the linked molecule, highlighting the need for reliable structure verification, especially in consecutive rounds of fragment elaboration.

Levente Kollár, Levente M. Mihalovits, Dávid Bajusz, DamijanKnez, József Simon, Blake H. Balcomb, Daren Fearon, Stanislav Gobec, György M. Keserű,

ChemMedChem 2026, 21, e202501108. 

https://doi.org/10.1002/cmdc.202501108

Linking of fragments in neighboring binding sites is one of the optimization strategies in fragment-based drug discovery, where additive or even more substantial bioactivity improvements can be realized. However, such efforts present a considerable challenge when one fragment binds covalently to the target protein, as small modifications can influence the correct positioning of the covalent warhead toward the targeted nucleophilic residue. Here, we present a case study of fragment linking that yielded single-digit micromolar, covalent inhibitors of the SARS-CoV-2 main protease, starting from fragments that were inactive in the biochemical assay. Using structural information from a recent, high-throughput crystallographic fragment screen, we show that the success of fragment linking in the design of targeted covalent inhibitors is heavily impacted by several factors, including the warhead type, the labeling chemistry, and even subtle changes in the designed linker. Notably, we observe that induced fit effects might override the original fragment orientations in the linked molecule, highlighting the need for reliable structure verification, especially in consecutive rounds of fragment elaboration.

Characterization of the Second-Generation Covalent Fragment Library (CovLib Gen2): Thiol Reactivity Profiling and p53-Y220C Rescue

Schwer, M., Aldea, S. R., Engelhardt, M. U., Stahlecker, J., Rheinganz, J., Langkamp, A., & Boeckler, F. M. 

Drug Design, Development and Therapy, (2026). 20. 

https://doi.org/10.2147/DDDT.S598622

Purpose

Covalent Fragment-Based Drug Discovery (FBDD) has emerged as a powerful strategy for unlocking challenging pharmacological targets and engaging shallow or “cryptic” binding pockets. In this study, we present the design and characterization of the Second Generation Covalent Fragment Library (CovLib Gen2), an expanded collection of 81 structurally diverse electrophiles tailored for Covalent Fragment-Based Drug Discovery (FBDD) using an electrophile-first approach. The library spans five distinct warhead classes, including epoxides, vinyl sulfones, acrylamides, α-cyanoacrylamides, and a core set of SɴAr-reactive heteroarenes.

Methods

We comprehensively profiled the library for physicochemical properties and intrinsic thiol reactivity using high-throughput 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB) and high-performance liquid chromatography (HPLC)-based glutathione (GSH) reactivity assays. To demonstrate the library’s utility, we performed differential scanning fluorimetry (DSF) screening against the oncogenic, thermally unstable p53-Y220C mutant and subsequent specificity testing with two control mutants.

Results

The library exhibited a broad dynamic range of reactivities with a clear correlation between the assay methods. Additionally, we identified 12 fragments with desirable mild reactivity profiles (t1/2GSH = 1–10 h). The DSF screen yielded 15 hits, primarily SɴAr-reactive heteroarenes and vinyl sulfones. Notably, the fragment SN054 emerged as the most potent stabilizer, inducing a maximal thermal shift of 4.5 °C. Specificity was confirmed using a cysteine-light variant (T-p53C-Y220C-CL), where SN054 retained significant stabilizing activity.

Conclusion

Our findings validate CovLib Gen2 as a versatile tool for ligand discovery, including electrophilic fragments covering a broad range of reactivity, and provide tractable starting points for the pharmacological rescue of p53-Y220C.

Accelerating SuFEx Reactions via Aryl Fluorosulfate Structural Engineering for Enhanced Covalent Targeted Cancer Therapy

Gao, W.; Zhang, C.; Li, D.; Liu, Y.; Zhao, M.; Xia, X.-X.; Huang, W.; Xia, X.; Yan, D. 

Angew. Chem. Int. Ed. 2026

https://doi.org/10.1002/anie.4497440

 https://onlinelibrary.wiley.com/doi/full/10.1002/anie.4497440 Aryl fluorosulfate warheads, possessing sulfur(VI) fluoride exchange (SuFEx) reaction, hold significant promise for the development of covalent protein drugs. However, their SuFEx reactivity remains limited within the complex microenvironment of protein interactions. To address this challenge, we sought to enhance their reactivity by adjusting the electronic and steric properties of warheads. Herein, we synthesized various maleimide-functionalized aryl fluorosulfate (MFS) bearing different substituents (e.g., o-F, o-CF3, o-NO2, o-CH3, o-OCH3, o-Cl, o-Br, o-I, 2,6-diF, and m-F), which were then chemically conjugated to Adnectin (an EGFR-targeting protein). The SuFEx reactivity of the resulting xMFS-modified Adnectin was systematically investigated by comparing their covalent cross-linking efficiency to EGFR. Notably, the meta-Fluoro-substituted MFS warhead, featuring moderate electrophilicity and minimal steric hindrance, exhibited the highest reactivity, achieving a 3.5-fold increase in cross-linking efficiency compared to unsubstituted control. The m-F MFS-modified Adnectin was further attached to the surface of albumin-bound DXd. Leveraging its enhanced SuFEx reactivity, the resulting covalent albumin-bound drug exhibited 6.4-fold higher intracellular accumulation, 3.0-fold greater tumor retention, and 4.0-fold higher antitumor efficiency compared to unsubstituted control. Overall, fine-adjusting the electronic and steric properties of warheads significantly enhances their SuFEx reactivity, enabling the rational design of SuFEx-based warheads and facilitating the application in covalent protein drugs.

Covalent Inhibitors of Monoacylglycerol Lipase Induce Conformational Changes and Proteasomal Degradation

Jordan A. Pham, Thanawat Thaingtamtanha, William McLeish, David Lefebvre, Spencer M. Uguccioni, Roxana Filip, Francesco Gentile, and John Paul Pezacki

J. Am. Chem. Soc. 2026

https://doi.org/10.1021/jacs.5c22859

Monoacylglycerol lipase (MGLL) is a key serine hydrolase that regulates 2-arachidonoylglycerol (2-AG) and eicosanoid signaling. Inhibition of MGLL blocks the conversion of 2-AG into arachidonic acid (AA) with broad therapeutic implications in inflammation, cancer, and viral infection. Carbamate/urea inhibitors such as MJN110, JZL184, and SAR629 are widely used to irreversibly inhibit MGLL through covalent modification of the catalytic serine residue. Here, we demonstrate that this inhibitor class also induces proteasome-dependent degradation of MGLL, functioning as monovalent degraders. We show that loss of MGLL following covalent inhibitor treatment is dependent on the 26S proteasome, while detailed simulations of MGLL dynamics following inhibitor binding suggest that these inhibitors do not destabilize their protein target but instead induce conformational changes that likely facilitate polyubiquitination by exposing two lysine residues. Taken together, these findings establish carbamate/urea inhibitors as dual functional molecules with the propensity to both covalently inhibit their target and act as structural degraders, potentially functioning as monovalent molecular glues, highlighting the need to evaluate the degradation potential alongside inhibitory potency in future screening and drug discovery.

Development and Structural Characterization of UTE-156, a Covalent Inhibitor of the VCP/p97 AAA+ ATPase

Tamayo-Jaramillo D, Hegde S, Jia X, Coffman K, Vankayalapati H, Bearss D, Jones KB, Stark AW, Shen PS. 

Adv Sci. 2026; 13(25):e20545. 

https://doi.org/10.1002/advs.202520545

The AAA+ ATPase valosin-containing protein (VCP/p97) is a central regulator of protein homeostasis that is well characterized for its role in extracting and remodeling ubiquitinated substrates. Dysregulation of VCP activity contributes to the pathogenesis of neurodegenerative diseases and cancer, making it an important therapeutic target. Here, we report the development and characterization of UTE-156, a novel covalent small-molecule inhibitor that modifies Cys522 within the D2 ATPase domain of VCP. UTE-156 potently inhibits VCP ATPase activity, while losing activity against a C522A mutant, supporting a covalent mechanism of action. High-resolution cryo-electron microscopy (cryo-EM) structures reveal that UTE-156 occupies the D2 nucleotide-binding site, sterically blocking ATP binding and inducing conformational remodeling of the pocket. Biochemical and cell-based assays demonstrate strong inhibitory potency but limited solubility and rapid metabolic turnover. These pharmacochemical limitations preclude immediate therapeutic use but underscore its value as a chemical probe. Together, these findings establish UTE-156 as a powerful tool for dissecting VCP function and provide a framework for future optimization of covalent modulators of protein homeostasis.

Nitro-Diphenyl Ethers as Emerging Cysteine-Targeting Covalent Warheads Enable Identification of Novel Target LDLRAP1 for Anticoronaviral Activity

Zeyue Huang, Xiuqi Hu, Zheng Liu, Hongxuan Cao, Yunjie Xiang, Jian Wan, Ivailo Slavchev, Li Rao, Ivanka Nikolova, Petar Grozdanov, Nadya Nikolova, Georgi M. Dobrikov, and Yanliang Ren

Journal of Medicinal Chemistry 2026

DOI: 10.1021/acs.jmedchem.5c03394

Targeted covalent inhibitors (TCIs) are powerful tools in drug discovery, but the high intrinsic reactivity of conventional warheads often compromises selectivity and increases the off-target liability. Here, we reported nitrodiphenyl-ether compounds as a novel irreversible and released-type covalent warhead with exceptionally low reactivity that potently inhibits coronavirus HCoV-OC43 infection. To identify their molecular targets, we designed a panel of active and inactive alkyne-tagged probes and performed chemical proteomic profiling in human host cells. An integrated approach combining activity- and inactivity-based proteome profiling (AIBPP), competitive ABPP, LC–MS/MS, and fluorescence polarization (FP) assays identified low-density lipoprotein receptor adapter protein 1 (LDLRAP1) as the primary target, modified selectively at C119, thereby disrupting the LDLR–LDLRAP1 interaction. Inhibition of this interaction strongly correlated with antiviral efficacy, confirming LDLRAP1 as the functional target. Collectively, this study establishes LDLRAP1 as an unexploited host antiviral target and expands the repertoire of cysteine-targeted covalent warheads for host-directed therapy.