Covalent Plant Natural Product that Potentiates Antitumor Immunity

Misao Takemoto, Sara Delghandi, Masahiro Abo, Keiko Yurimoto, Minami Odagi, Vaibhav Pal Singh, Jun Wang, Reiko Nakagawa, Shin-ichi Sato, Yasushi Takemoto, Asmaa M. A. S. Farrag, Yoshimasa Kawaguchi, Kazuo Nagasawa, Tasuku Honjo, Kenji Chamoto, and Motonari Uesugi

Journal of the American Chemical Society 2025

DOI: 10.1021/jacs.4c17837

Despite the unprecedented therapeutic potential of immune checkpoint antibody therapies, their efficacy is limited partly by the dysfunction of T cells within the cancer microenvironment. Combination therapies with small molecules have also been explored, but their clinical implementation has been met with significant challenges. To search for antitumor immunity activators, the present study developed a cell-based system that emulates cancer-attenuated T cells. The cell-based screening of 232 natural products containing electrophilic reactive functional groups led to the identification of arvenin I, also known as cucurbitacin B 2-O-β-d-glucoside (CuBg), as a plant natural product that activates T cells within the cancer-competitive environment. Chemoproteomic and mechanistic analyses indicated that arvenin I covalently reacts with and hyperactivates MKK3, thereby reviving the mitochondrial fitness of exhausted T cells through the activation of the p38MAPK pathway. In mice, administration of arvenin I enhanced the efficacy of cancer immunotherapy when used alone or in combination with an immune checkpoint inhibitor. These findings highlight the potential of arvenin I as a covalent kinase activator that potentiates antitumor immunity.

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

DOI: 10.1021/acs.jcim.4c01281

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.

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.

Targeted Covalent Modification Strategies for Drugging the Undruggable Targets

Tomonori Tamura, Masaharu Kawano, and Itaru Hamachi

Chemical Reviews 2024

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

Matthew Bogyo, Tulsi Upadhyay, Emily Woods, Stephen Ahator. Kjersti Julin, Franco Faucher, Marijn Hollander, Nichole Pedowitz, Daniel Abegg, Isabella Hammond, Ifeanyichukwu Eke, Sijie Wang, Shiyu Chen, John Bennett, Jeyun Jo, Christian Lentz, Alex Adibekian, Matthias Fellner

Research Square Preprint 2025

https://doi.org/10.21203/rs.3.rs-5494070/v1

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.

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.