Biocompatible covalent reactive groups (CRGs) play pivotal roles in several areas of chemical biology and the life sciences, including targeted covalent inhibitor design and preparation of advanced biologic drugs, such as antibody–drug conjugates. In this study, we present the discovery that the small, chlorinated polyketide natural product cyclohelminthiol II (CHM-II) acts as a new type of cysteine/thiol-targeting CRG incorporating both reversible and irreversible reactivity. We devise the first syntheses of four simple cyclohelminthols, (±)-cyclohelminthol I–IV, with selective chlorinations (at C2 and C5) and a Ni-catalyzed reductive cross coupling between an enone, a vinyl bromide and triethylsilyl chloride as the key steps. Unbiased biological profiling (cell painting) was used to discover a putative covalent mechanism for CHM-II in cells with subsequent validation experiments demonstrating mechanistic similarity to dimethyl fumarate (DMF) – a known (covalent) drug used in the treatment of multiple sclerosis. Focused biochemical experiments revealed divergent thiol-reactivity inherent to the CHM-II scaffold and through further chemical derivatization of CHM-II we applied activity-based protein profiling (ABPP)-workflows to show exclusive cysteine-labelling in cell lysate. Overall, this study provides both efficient synthetic access to the CHM-II chemotype – and neighboring chemical space – and proof-of-concept for several potential applications of this new privileged CRG-class within covalent chemical biology.
A blog highlighting recent publications in the area of covalent modification of proteins, particularly relating to covalent-modifier drugs. @CovalentMod on Twitter, @covalentmod@mstdn.science on Mastodon, and @covalentmod.bsky.social on BlueSky
Thursday, January 30, 2025
Total syntheses of cyclohelminthol I–IV reveal a new cysteine-selective covalent reactive group
Highly Optimized CNS Penetrant Inhibitors of EGFR Exon20 Insertion Mutations
William McCoull, Clare Thomson, Erin Braybrooke, Christina Chan, Nicola Colclough, Miguel A. Cortés González, Sabina Cosulich, Nichola L. Davies, Nicolas Floc’h, Ryan Greenwood, David Hargreaves, Peng Huang, Thomas A. Hunt, Tony Johnson, Peter Johnström, Jason G. Kettle, Mikhail Kondrashov, Demetrios H. Kostomiris, Songlei Li, Andrew Lister, Scott Martin, Darren McKerrecher, Neville McLean, J. Willem M. Nissink, Jonathan P. Orme, Paige Orwig, Martin J. Packer, Stuart Pearson, Lina Qin, Catarina Felisberto-Rodrigues, Adriana Savoca, Magnus Schou, Stephen Stokes, Aisha M. Swaih, Sara Talbot, Michael J. Tucker, Richard A. Ward, Emma Wadforth, Chunli Wang, Joanne Wilson, and Yawen Yang
Tuesday, January 21, 2025
Discovery of STX-721, a Covalent, Potent, and Highly Mutant-Selective EGFR/HER2 Exon20 Insertion Inhibitor for the Treatment of Non-Small Cell Lung Cancer
Benjamin C. Milgram, Deanna R. Borrelli, Natasja Brooijmans, Jack A. Henderson, Brendan J. Hilbert, Michael R. Huff, Takahiro Ito, Erica L. Jackson, Philip Jonsson, Brendon Ladd, Erin L. O’Hearn, Raymond A. Pagliarini, Simon A. Roberts, Sébastien Ronseaux, Darrin D. Stuart, Weixue Wang, and Angel Guzman-Perez
Journal of Medicinal Chemistry 2025
DOI: 10.1021/acs.jmedchem.4c02377Saturday, January 18, 2025
Species Dependent Metabolism of a Covalent nsP2 Protease Inhibitor with in Vivo Anti-alphaviral Activity
Mohammad Anwar Hossain, Abigail K. Mayo, Anirban Ghoshal, Sharon A. Taft-Benz, Elizabeth J. Anderson, Noah L. Morales, Katia D. Pressey, Ava M. Vargason, Kim L. R. Brouwer, Nathaniel J. Moorman, Mark T. Heise, Timothy M. Willson
bioRxiv 2025.01.13.632788;
doi: https://doi.org/10.1101/2025.01.13.632788
Monday, January 13, 2025
Covalent Plant Natural Product that Potentiates Antitumor Immunity
Saturday, January 11, 2025
CovCysPredictor: Predicting Selective Covalently Modifiable Cysteines Using Protein Structure and Interpretable Machine Learning
Bryn Marie Reimer, Ernest Awoonor-Williams, Andrei A. Golosov, and Viktor Hornak
Journal of Chemical Information and Modeling 2025
Targeted covalent inhibition is a powerful therapeutic modality in the drug discoverer’s toolbox. Recent advances in covalent drug discovery, in particular, targeting cysteines, have led to significant breakthroughs for traditionally challenging targets such as mutant KRAS, which is implicated in diverse human cancers. However, identifying cysteines for targeted covalent inhibition is a difficult task, as experimental and in silico tools have shown limited accuracy. Using the recently released CovPDB and CovBinderInPDB databases, we have trained and tested interpretable machine learning (ML) models to identify cysteines that are liable to be covalently modified (i.e., “ligandable” cysteines). We explored myriad physicochemical features (pKa, solvent exposure, residue electrostatics, etc.) and protein–ligand pocket descriptors in our ML models. Our final logistic regression model achieved a median F1 score of 0.73 on held-out test sets. When tested on a small sample of holo proteins, our model also showed reasonable performance, accurately predicting the most ligandable cysteine in most cases. Taken together, these results indicate that we can accurately predict potential ligandable cysteines for targeted covalent drug discovery, privileging cysteines that are more likely to be selective rather than purely reactive. We release this tool to the scientific community as CovCysPredictor.
Identification of Novel Organo-Se BTSA-Based Derivatives as Potent, Reversible, and Selective PPARγ Covalent Modulators for Antidiabetic Drug DiscoveryClick to copy article link
Fangyuan Chen, Qingmei Liu, Lei Ma, Cuishi Yan, Haiman Zhang, Zhi Zhou, and Wei Yi
Journal of Medicinal Chemistry 2025 68 (1), 819-831
DOI: 10.1021/acs.jmedchem.4c02803
Recent studies have identified selective peroxisome proliferator-activated receptor γ (PPARγ) modulators, which synergistically engage in the inhibition mechanism of PPARγ-Ser273 phosphorylation, as a promising approach for developing safer and more effective antidiabetic drugs. Herein, we present the design, synthesis, and evaluation of a new class of organo-Se compounds, namely, benzothiaselenazole-1-oxides (BTSAs), acting as potent, reversible, and selective PPARγ covalent modulators. Notably, 2n, especially (R)-2n, displayed a high binding affinity and superior antidiabetic effects with diminished side effects. This is mainly because it can reversibly form a unique covalent bond with the Cys285 residue in PPARγ-LBD. Further mechanistic investigations revealed that it manifested such desired pharmacological profiles primarily by effectively suppressing PPARγ-Ser273 phosphorylation, enhancing glucose metabolism, and selectively upregulating the expression of insulin-sensitive genes. Collectively, our results suggest that (R)-2n holds promise as a lead compound for treating T2DM and also provides an innovative reversible covalent warhead reference for future covalent drug design.
Friday, January 10, 2025
Selective Protein (Post-)modifications through Dynamic Covalent Chemistry: Self-activated SNAr Reactions
Ferran Esteve, Jean-Louis Schmitt, Sergii Kolodych, Oleksandr Koniev, and Jean-Marie Lehn
Journal of the American Chemical Society 2025
DOI: 10.1021/jacs.4c15421
Thursday, January 9, 2025
Targeted Covalent Modification Strategies for Drugging the Undruggable Targets
DOI: 10.1021/acs.chemrev.4c00745
The term “undruggable” refers to proteins or other biological targets that have been historically challenging to target with conventional drugs or therapeutic strategies because of their structural, functional, or dynamic properties. Drugging such undruggable targets is essential to develop new therapies for diseases where current treatment options are limited or nonexistent. Thus, investigating methods to achieve such drugging is an important challenge in medicinal chemistry. Among the numerous methodologies for drug discovery, covalent modification of therapeutic targets has emerged as a transformative strategy. The covalent attachment of diverse functional molecules to targets provides a powerful platform for creating highly potent drugs and chemical tools as well the ability to provide valuable information on the structures and dynamics of undruggable targets. In this review, we summarize recent examples of chemical methods for the covalent modification of proteins and other biomolecules for the development of new therapeutics and to overcome drug discovery challenges and highlight how such methods contribute toward the drugging of undruggable targets. In particular, we focus on the use of covalent chemistry methods for the development of covalent drugs, target identification, drug screening, artificial modulation of post-translational modifications, cancer specific chemotherapies, and nucleic acid-based therapeutics.
Covalent-fragment screening identifies selective inhibitors of multiple Staphylococcus aureus serine hydrolases important for growth and biofilm formation
Research Square Preprint 2025
Staphylococcus aureus is a leading cause of bacteria-associated mortality worldwide. This is largely because infection sites are often difficult to localize and the bacteria forms biofilms which are not effectively cleared using classical antibiotics. Therefore, there is a need for new tools to both image and treat S. aureus infections. We previously identified a group of S. aureus serine hydrolases known as fluorophosphonate-binding hydrolases (Fphs), which regulate aspects of virulence and lipid metabolism. However, because their structures are similar and their functions overlap, it remains challenging to distinguish the specific roles of individual members of this family. In this study, we applied a high-throughput screening approach using a library of covalent electrophiles to identify inhibitors for FphB, FphE, and FphH. We identified inhibitors that irreversibly bind to the active-site serine residue of each enzyme with high potency and selectivity without requiring extensive medicinal chemistry optimization. Structural and biochemical analysis identified novel binding modes for several of the inhibitors. Selective inhibitors of FphH impaired both bacterial growth and biofilm formation while Inhibitors of FphB and FphE had no impact on cell growth and only limited impact on biofilm formation. These results suggest that all three hydrolases likely play functional, but non-equivalent roles in biofilm formation and FphH is a potential target for development of therapeutics that have both antibiotic and anti-biofilm activity. Overall, we demonstrate that focused covalent fragment screening can be used to rapidly identify highly potent and selective electrophiles targeting bacterial serine hydrolases. This approach could be applied to other classes of lipid hydrolases in diverse pathogens or higher eukaryotes.
Tuesday, January 7, 2025
N-Acyl-N-alkyl/aryl Sulfonamide Chemistry Assisted by Proximity for Modification and Covalent Inhibition of Endogenous Proteins in Living Systems
Tomonori Tamura and Itaru Hamachi
Accounts of Chemical Research 2025 58 (1), 87-100
DOI: 10.1021/acs.accounts.4c00628
Selective chemical modification of endogenous proteins in living systems with synthetic small molecular probes is a central challenge in chemical biology. Such modification has a variety of applications important for biological and pharmaceutical research, including protein visualization, protein functionalization, proteome-wide profiling of enzyme activity, and irreversible inhibition of protein activity. Traditional chemistry for selective protein modification in cells largely relies on the high nucleophilicity of cysteine residues to ensure target-selectivity and site-specificity of modification. More recently, lysine residues, which are more abundant on protein surfaces, have attracted attention for the covalent modification of proteins. However, it has been difficult to efficiently modify the ε-amino groups of lysine side-chains, which are mostly (∼99.9%) protonated and thus exhibit low nucleophilicity at physiological pH. Our group revealed that N-acyl-N-alkyl sulfonamide (NASA) moieties can rapidly and efficiently acylate noncatalytic (i.e., less reactive) lysine residues in proteins by leveraging a reaction acceleration effect via proximity. The excellent reaction kinetics and selectivity for lysine of the NASA chemistry enable covalent modification of natural intracellular and cell-surface proteins, which is intractable using conventional chemistries. Moreover, recently developed N-acyl-N-aryl sulfonamide (ArNASA) scaffolds overcome some problems faced by the first-generation NASA compounds. In this Account, we summarize our recent works in the development of NASA/ArNASA chemistry and several applications reported by ourselves and other groups. First, we characterize the basic properties of NASA/ArNASA chemistry, including the labeling kinetics, amino acid preference, and biocompatibility, and compare this approach with other ligand-directed chemistries. This section also describes the principles of nucleophilic organocatalyst-mediated protein acylation, another important protein labeling strategy using the NASA reactive group, and its application to neurotransmitter receptor labeling in brain slices. Second, we highlight various recent examples of protein functionalization using NASA/ArNASA chemistry, such as visualization of membrane proteins including therapeutically important G-protein coupled receptors, gel-based ligand screening assays, photochemical control of protein activity, and targeted protein degradation. Third, we survey covalent inhibition of proteins by NASA/ArNASA-based lysine-targeting. The unprecedented reactivity of NASA/ArNASA toward lysine allows highly potent, irreversible inhibition of several drug targets for the treatment of cancer, including HSP90, HDM2–p53 protein–protein interaction, and a Bruton’s tyrosine kinase mutant that has developed resistance to cysteine-targeted covalent-binding drugs. Finally, current limitations of, and future perspectives on, this research field are discussed. The new chemical labeling techniques offered by NASA/ArNASA chemistry and its derivatives create a valuable molecular toolbox for studying numerous biomolecules in living cells and even in vivo.
Monday, January 6, 2025
Selective Protein (Post-)modifications through Dynamic Covalent Chemistry: Self-activated SNAr Reactions
Ferran Esteve, Jean-Louis Schmitt, Sergii Kolodych, Oleksandr Koniev, and Jean-Marie Lehn
Journal of the American Chemical Society 2025
DOI: 10.1021/jacs.4c15421
SNAr reactions were remarkably accelerated using a pretargeting and activating unit based on dynamic covalent chemistry (DCvC). A Cys attack at the C–F bond on the aromatic ring of salicylaldehyde derivatives was only observed upon iminium formation with a neighboring Lys residue of model small peptides. Such self-activation was ascribed to the stronger electron-withdrawing capability of the iminium bond with respect to that of the parent aldehyde that stabilized the transition state of the reaction, together with the higher preorganization of the reactive groups in the cationic aldiminium species. This approach was further applied for the functionalization of two antibodies. In both cases, the presence of the aldehyde group in close proximity to the reactive C–F bond resulted in a noteworthy increase in bioconjugation yields, with excellent chemo-selectivity. Whereas the modification of an IgG1 antibody led to stochastic product distributions, microenvironment selectivity was noted when employing IgG4, in line with the lower number of Lys residues in the hinge region of the latter. Additionally, the postfunctionalization of the modified antibodies was attained through the dynamic covalent exchange of the tethered iminium derivative with hydrazides, representing an unprecedented “tag and modify” selective bioconjugation strategy based on DCvC.
Friday, January 3, 2025
Vivek Kumar, Jiyun Zhu, Bala C. Chenna, Zoe A. Hoffpauir, Andrew Rademacher, Ashley M. Rogers, Chien-Te Tseng, Aleksandra Drelich, Sharfa Farzandh, Audrey L. Lamb, and Thomas D. Meek
Journal of the American Chemical Society Article 2024
DOI: 10.1021/jacs.4c11620
SARS-CoV-2 3CL protease (Main protease) and human cathepsin L are proteases that play unique roles in the infection of human cells by SARS-CoV-2, the causative agent of COVID-19. Both proteases recognize leucine and other hydrophobic amino acids at the P2 position of a peptidomimetic inhibitor. At the P1 position, cathepsin L accepts many amino acid side chains, with a partial preference for phenylalanine, while 3CL-PR protease has a stringent specificity for glutamine or glutamine analogues. We have designed, synthesized, and evaluated peptidomimetic aldehyde dual-target (dual-acting) inhibitors using two peptide scaffolds based on those of two Pfizer 3CL-PR inhibitors, Nirmatrelvir, and PF-835321. Our inhibitors contain glutamine isosteres at the P1 position, including 2-pyridon-3-yl-alanine, 3-pyridinyl-alanine, and 1,3-oxazo-4-yl-alanine groups. Inhibition constants for these new inhibitors ranged from Ki = 0.6–18 nM (cathepsin L) and Ki = 2.6–124 nM (3CL-PR), for which inhibitors with the 2-pyridon-3-yl-alanal substituent were the most potent for 3CL-PR. The anti-CoV-2 activity of these inhibitors ranged from EC50 = 0.47–15 μM. X-ray structures of the peptidomimetic aldehyde inhibitors of 3CL-PR with similar scaffolds all demonstrated the formation of thiohemiacetals with Cys145, and hydrogen-bonding interactions with the heteroatoms of the pyridon-3-yl-alanyl group, as well as the nitrogen of the N-terminal indole and its appended carbonyl group at the P3 position. The absence of these hydrogen bonds for the inhibitors containing the 3-pyridinyl-alanyl and 1,3-oxazo-4-yl-alanyl groups was reflected in the less potent inhibition of the inhibitors with 3CL-PR. In summary, our studies demonstrate the value of a second generation of cysteine protease inhibitors that comprise a single agent that acts on both human cathepsin L and SARS-CoV-2 3CL protease. Such dual-target inhibitors will provide anti-COVID-19 drugs that remain active despite the development of resistance due to mutation of the viral protease. Such dual-target inhibitors are more likely to remain useful therapeutics despite the emergence of inactivating mutations in the viral protease because the human cathepsin L will not develop resistance. This particular dual-target approach is innovative since one of the targets is viral (3CL-PR) required for viral protein maturation and the other is human (hCatL) which enables viral infection.
Wednesday, January 1, 2025
Hydralazine inhibits cysteamine dioxygenase to treat preeclampsia and senesce glioblastoma [@MegaMatthewsLab]
Kyosuke Shishikura, Jiasong Li, Yiming Chen, Nate R McKnight, Katelyn A Bustin, Eric W Barr, Snehil R Chilkamari, Mahaa Ayub, Sun Woo Kim, Zongtao Lin, Ren-Ming Hu, Kelly Hicks, Xie Wang, Donald M O'Rourke, J. Martin Bollinger Jr., Zev A Binder, William H Parsons, Kirill A Martemyanov, Aimin Liu, Megan L Matthews
bioRxiv 2024.12.19.629450;
doi: https://doi.org/10.1101/2024.12.19.629450
The vasodilator hydralazine (HYZ) has been used clinically for ~ 70 years and remains on the World Health Organization's List of Essential Medicines as a therapy for preeclampsia. Despite its longstanding use and the concomitant progress toward a general understanding of vasodilation, the target and mechanism of HYZ have remained unknown. We show that HYZ selectively targets 2-aminoethanethiol dioxygenase (ADO) by chelating its metal cofactor and alkylating one of its ligands. This covalent inactivation slows entry of proteins into the Cys/N-degron pathway that ADO initiates. HYZ's capacity to stabilize regulators of G-protein signaling (RGS4/5) normally marked for degradation by ADO explains its effect on blood vessel tension and comports with prior associations of insufficient RGS levels with human preeclampsia and analogous symptoms in mice. The established importance of ADO in glioblastoma led us to test HYZ in these cell types. Indeed, a single treatment induced senescence, suggesting a potential new HYZ-based therapy for this deadly brain cancer.
Total syntheses of cyclohelminthol I–IV reveal a new cysteine-selective covalent reactive group
DOI Thomas T. Paulsen, Anders E. Kiib, Gustav J. Wørmer, Stephan M. Hacker and Thomas B. Poulsen Chemical Science, 2025 https://doi.org/10...
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Linqi Cheng Yixian Wang, Yiming Guo, Sophie S. Zhang Han Xiao C ell Chemical Biology, 2024 Volume 31, 3, 428 - 445 https://doi.org/10.10...
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Mariko Takahashi, Harrison B. Chong,Siwen Zhang, Tzu-Yi Yang,Matthew J. Lazarov,Stefan Harry,Michelle Maynard, Brendan Hilbert,Ryan D. White...
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Özge Ünsal, Z. Selin Bacaksiz, Vladislav Khamraev, Vittorio Montanari, Martin Beinborn, and Krishna Kumar ACS Chemical Biology 2024 DOI: ...