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.

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.

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.

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.