Structure-based rational design of covalent probes

Holcomb, M., Llanos, M., Hansel-Harris, A. et al. 

Commun Chem 8, 242 (2025). 

https://doi.org/10.1038/s42004-025-01606-y

Covalent probes are becoming increasingly important to both fundamental biology and drug design, as what was once an idiosyncrasy at best and a source of toxicity at worst has transitioned to a paradigm for drug discovery. However, these covalent probes offer different challenges for the incorporation of structural data into the design process than do non-covalent probes, whose activity may be captured by very few bound states. In this review, we discuss the role of structure-based design in the discovery and optimization of covalent probes, with a special emphasis on computational methods to leverage structural data.

Potent Inducers of Paraptosis through Electronic Tuning of Hemicyanine Electrophiles

uan F. Tamez-Fernández, Craig F. Steven, Jade Nguyen, and Pablo Rivera-Fuentes

Journal of the American Chemical Society 2025

DOI: 10.1021/jacs.5c07109

Paraptosis is a distinct form of programmed cell death characterized by cytoplasmic vacuolization, mitochondrial swelling, and endoplasmic reticulum (ER) dilation, offering an alternative to apoptosis for therapeutic applications. In this study, we identified a hemicyanine derivative that is a potent paraptosis inducer in two cancer cell lines. This compound triggers hallmark paraptotic features, including ER swelling, mitochondrial morphological changes, increased superoxide production, and caspase-independent cell death. This activity is dependent on the ability of the probe to modify thiols covalently. Proteomic analysis using a biotinylated, activity-based probe revealed Sec23 homologue A and GDP-dissociation inhibitor alpha as potential targets implicated in paraptosis activation. This lead compound already displayed some degree of selectivity, exemplified by its minimal interaction with well-known nucleophilic protein targets such as protein disulfide isomerases. These findings establish the hemicyanine chemical family as a promising scaffold for paraptosis research and suggest potential as a therapeutic lead for diseases where traditional apoptosis pathways are dysregulated.

Profiling the proteome-wide selectivity of diverse electrophiles

Zanon, P. R. A.; Yu, F.; Musacchio, P.; Lewald, L.; Zollo, M.; Krauskopf, K.; Mrdović, D.; Raunft, P.; Maher, T. E.; Cigler, M.; Chang, C.; Lang, K.; Toste, F. D.; Nesvizhskii, A. I.; Hacker, S. M. 

Nature Chemistry volume 17, pages1712–1721 (2025)

https://www.nature.com/articles/s41557-025-01902-z

https://doi.org/10.26434/chemrxiv-2021-w7rss-v2

Targeted covalent inhibitors are powerful entities in drug discovery, but their application has so far mainly been limited to addressing cysteine residues. The development of cysteine-directed covalent inhibitors has largely profited from determining their proteome-wide selectivity using competitive residue-specific proteomics. Several probes have recently been described to monitor other amino acids using this technology and many more electrophiles exist to modify proteins. Nevertheless, a direct, proteome-wide comparison of the selectivity of diverse probes is still entirely missing. Here, we developed a completely unbiased workflow to analyse electrophile selectivity proteome-wide and applied it to directly compare 54 alkyne probes containing diverse reactive groups. In this way, we verified and newly identified probes to monitor a total of nine different amino acids as well as the N-terminus proteome-wide. This selection includes the first probes to globally monitor tryptophans, histidines and arginines as well as novel tailored probes for methionines, aspartates and glutamates.

Discovery of an Orally Bioavailable Reversible Covalent SARS-CoV-2 Mpro Inhibitor with Pan-Coronavirus Activity

Qian Wen Tan, Subramanyam Vankadara, Jia Yi Fong, Yi Yang See, Nithya Baburajendran, Pearly Shuyi Ng, Weijun Xu, Yee Khoon Yeo, Weiling Wang, Choon Heng Low, Li Hong Tan, Eileen Gui Ju Tay, Yun Xuan Wong, Chuhui Huang, Sandra Sim, Shi Hua Ang, Hannah Hui Min Toh, Juliana Mohammad, Gang Wang, Boping Liu, Shu Ting Tan, Perlyn Zekui Kwek, Monique Danielle Dawson, Qin Yao Oh, Xiaoying Koh, Joma Joy, May Ann Lee, Walter Stunkel, Vishal Pendharkar, Hannes Hentze, Siew Pheng Lim, Kantharaj Ethirajulu, C. S. Brian Chia, and Joseph Cherian

J. Med. Chem. 2025

https://doi.org/10.1021/acs.jmedchem.5c00581

Resulting in several million deaths globally, the COVID-19 pandemic has highlighted the criticality of antiviral drugs during a viral pandemic. Herein, we describe our efforts toward targeting SARS-CoV-2 Mpro, a key viral protease, which led to the discovery of compound 18, a reversible covalent inhibitor with potent antiviral activity against several clinical variants of SARS-CoV-2. Compound 18 demonstrated dose-dependent efficacy in a mouse-adapted SARS-CoV-2 infection model, with favorable pharmacokinetic profiles in mice, rats, dogs, and monkeys.

 

A twist in the tale: shifting from covalent targeting of a tyrosine in JAK3 to a lysine in MK2

Laura Hillebrand, Guiqun Wang, Alexander Rasch, Benedikt Masberg, Apirat Chaikuad, Thales Kronenberger, Ellen Günther, Michael Forster, Antti Poso, Michael Lämmerhofer, Stefan A. Laufer, Stefan Knapp, Matthias Gehringer

RSC Med. Chem., 2025

https://doi.org/10.1039/D5MD00440C

While cysteine targeting in kinases is well established and widely used, covalent interactions with other amino acids remain much less explored. We aimed to develop covalent inhibitors targeting tyrosine residues in the protein kinases JAK3 and MK2 using structure-based design principles to generate small sets of ligands containing tyrosine-reactive sulfonyl fluoride and the less-explored fluorosulfate warheads. While the JAK3 inhibitors failed to achieve covalent binding, the fluorosulfate-bearing MK2 inhibitor 42, which had been designed as an allosteric binder, unexpectedly formed a bond with the “catalytic” lysine, additionally uncovering a unique interaction at the hinge region. This highlights the untapped potential of fluorosulfates and provides a rare example of the use of this electrophile for lysine targeting in kinases. Our results highlight the limitations of traditional design methods and support the integration of fragment/lead-like covalent library screening to discover unanticipated interactions.

AGPAT4 targeted covalent inhibitor potentiates targeted therapy to overcome cancer cell plasticity in hepatocellular carcinoma mouse models

Ng, K.-Y.; Koo, T.-Y.; Huang, I. B.; Lee, T. K.-W.; Fong, T.-L.; Gao, Y.; Wong, T.-L.; Gao, Y.; Yun, J.-P.; Guan, X.-Y.; Liu, M.; Chung, C. Y.-S.; Ma, S.

Sci. Transl. Med.17,eadn9472(2025).

DOI:10.1126/scitranslmed.adn9472

The development of cancerous cells leads to considerable changes in metabolic processes to meet the demands of tumor growth. Tumor lineage plasticity has been identified as a key factor in therapy resistance and tumor recurrence. Herein, we showed one aspect of this plasticity to be abnormal glycerophospholipid metabolism, specifically the presence of a metabolic protein called 1-acylglycerol-3-phosphate o-acyltransferase 4 (AGPAT4). We identified AGPAT4 as an oncofetal protein that is abundant in embryonic stem cells and hepatocellular carcinoma (HCC) tumor cells but is low or absent in most normal tissues. We demonstrated that AGPAT4 is a functional regulator of tumor lineage plasticity, which correlates with enhanced metastasis and resistance to sorafenib. Heightened plasticity was induced as a result of increased AGPAT4-mediated conversion of LPA (lysophosphatidic acid) to phosphatidic acid (PA), which then acts on its downstream mTOR/S6K/S6 signaling pathway. Inhibition of Agpat4 by the AAV8-mediated liver-directed strategy in an immunocompetent HCC mouse model reduced tumorigenicity and stemness and sensitized tumors to sorafenib. Through a chemical biology approach, a cysteine-reacting compound that specifically targets AGPAT4 at the Cys228 residue and therefore hinders its acyltransferase activity was identified and found to work synergistically with sorafenib in suppressing HCC in tumor xenograft models derived from patients with preclinical HCC and sorafenib-resistant HCC. Toxicological analysis revealed minimal side effects associated with the covalent inhibitor. In conclusion, the plasticity of tumor lineages induced by AGPAT4 represents a potential target for HCC treatment and could expand the effectiveness of sorafenib treatment, offering new possibilities for HCC therapy.

A Covalent Self-Reporting Peptide Degrader Enables Real-Time Monitoring of Targeted Protein Degradation In Vivo

Wei Zhang, Lizhen Yuan, Rui Liu, Yanbo Jing, Shijun Lin, Hao Fang, Yuxuan Li, Xiaohui Zhang, Jun Dai, Tao Liu, Fan Xia, and Xiaoding Lou

Journal of the American Chemical Society 2025

DOI: 10.1021/jacs.5c07041

Peptides have demonstrated great potential in drug development. However, their broader application in modalities such as proteolysis-targeting chimeras (PROTACs) remains limited by the lack of real-time efficacy feedback and poor pharmacokinetic stability. Herein, we develop a covalent self-reporting peptide degrader (Co-SPeD) by integrating a fluorine-substituted aryl fluorosulfate warhead and a rotor fluorophore derived from stilbene derivatives, which allows for covalent binding to target proteins via sulfur(VI) fluoride exchange chemistry and emitting activatable fluorescence. Co-SPeD is found to covalently bind to the K51 residue of the MDM2 protein, enabling real-time monitoring of targeted MDM2 degradation. By swapping the targeting peptide and screening rotor fluorophores, the Co-SPeD platform is successfully extended to other oncogenic proteins, including BCL-xL, GRP78, and KRAS (G12D). Additionally, Co-SPeD demonstrates significant antitumor efficacy in preclinical tumor models. More importantly, real-time in vivo monitoring of MDM2 degradation using Co-SPeD plays a crucial role in guiding cisplatin combination administration, leading to a 50% increase in tumor growth inhibition compared to nonguided treatment groups. This approach provides a targeted endogenous protein degradation strategy with real-time monitoring, offering a powerful and generalizable platform for next-generation PROTAC design, the advancement of peptide-based therapeutics, and the rational optimization of cancer therapy.