Fast Cysteine Bioconjugation Chemistry

Chem. Eur. J. 2022, e202201843

https://doi.org/10.1002/chem.202201843

Cysteine bioconjugation serves as a powerful tool in biological research and has been widely used for the chemical modification of proteins, constructing antibody-drug conjugates, and enabling cell imaging studies. Cysteine conjugation reactions with fast kinetics and exquisite selectivity have been under heavy pursuit as they would allow clean protein modification with just stoichiometric amount of reagents, which minimizes side reaction, simplifies purification and broadens the functional group tolerance. In this concept, we summarize the recent advances of fast cysteine bioconjugation, and discuss the mechanism and chemical principles that underlie the high efficiencies of the newly developed cysteine reactive reagents.

Chemoselective Covalent Modification of K-Ras(G12R) with a Small Molecule Electrophile

Ziyang Zhang, Johannes Morstein, Andrew K. Ecker, Keelan Z. Guiley, and Kevan M. Shokat

Journal of the American Chemical Society 2022

DOI: 10.1021/jacs.2c05377

KRAS mutations are one of the most common oncogenic drivers in human cancer. While small molecule inhibitors for the G12C mutant have been successfully developed, allele-specific inhibition for other KRAS hotspot mutants remains challenging. Here we report the discovery of covalent chemical ligands for the common oncogenic mutant K-Ras(G12R). These ligands bind in the Switch II pocket and irreversibly react with the mutant arginine residue. An X-ray crystal structure reveals an imidazolium condensation product formed between the α,β-diketoamide ligand and the ε- and η-nitrogens of arginine 12. Our results show that arginine residues can be selectively targeted with small molecule electrophiles despite their weak nucleophilicity and provide the basis for the development of mutant-specific therapies for K-Ras(G12R)-driven cancer.

Advances in covalent drug discovery

Boike, L., Henning, N.J. & Nomura, D.K.  Nat Rev Drug Discov (2022). 

https://doi.org/10.1038/s41573-022-00542-z

Covalent drugs have been used to treat diseases for more than a century, but tools that facilitate the rational design of covalent drugs have emerged more recently. The purposeful addition of reactive functional groups to existing ligands can enable potent and selective inhibition of target proteins, as demonstrated by the covalent epidermal growth factor receptor (EGFR) and Bruton’s tyrosine kinase (BTK) inhibitors used to treat various cancers. Moreover, the identification of covalent ligands through ‘electrophile-first’ approaches has also led to the discovery of covalent drugs, such as covalent inhibitors for KRAS(G12C) and SARS-CoV-2 main protease. In particular, the discovery of KRAS(G12C) inhibitors validates the use of covalent screening technologies, which have become more powerful and widespread over the past decade. Chemoproteomics platforms have emerged to complement covalent ligand screening and assist in ligand discovery, selectivity profiling and target identification. This Review showcases covalent drug discovery milestones with emphasis on the lessons learned from these programmes and how an evolving toolbox of covalent drug discovery techniques facilitates success in this field.

Systematic exploration of privileged warheads for covalent kinase drug discovery

Zhao, Z.; Bourne, P. E. ChemRxiv 2022.

https://doi.org/10.26434/chemrxiv-2022-nlb0m

Kinase-targeted drug discovery for cancer therapy has advanced significantly in the last three decades. Currently, diverse kinase inhibitors or degraders have been reported, such as allosteric inhibitors, covalent inhibitors, macrocyclic inhibitors, and PROTAC degraders. Out of these, covalent kinase inhibitors (CKIs) have been attracting attention due to their enhanced selectivity and exceptionally strong affinity. Eight covalent kinase drugs have been FDA approved thus far. Here, we review current developments in CKIs. We explore the characteristics of the CKIs: the features of nucleophilic amino acids and the preferences of electrophilic warheads. We provide systematic insights into privileged warheads for repurposing to other kinase targets. Finally, we discuss trends in CKI development across the whole proteome. 

Lysine-Targeted Reversible Covalent Ligand Discovery for Proteins via Phage Display

Mengmeng Zheng, Fa-Jie Chen, Kaicheng Li, Rahi M. Reja, Fredrik Haeffner, and Jianmin Gao

Journal of the American Chemical Society 2022

DOI: 10.1021/jacs.2c07375

Binding via reversible covalent bond formation presents a novel and powerful mechanism to enhance the potency of synthetic inhibitors for therapeutically important proteins. Work on this front has yielded the anticancer drug bortezomib as well as the antisickling drug voxelotor. However, the rational design of reversible covalent inhibitors remains difficult even when noncovalent inhibitors are available as a scaffold. Herein, we report chemically modified phage libraries, both linear and cyclic, that incorporate 2-acetylphenylboronic acid (APBA) as a warhead to bind lysines via reversible iminoboronate formation. To demonstrate their utility, these APBA-presenting phage libraries were screened against sortase A of Staphylococcus aureus, as well as the spike protein of SARS-CoV-2. For both protein targets, peptide ligands were readily identified with single-digit micromolar potency and excellent specificity, enabling live-cell sortase inhibition and highly sensitive spike protein detection, respectively. Furthermore, our structure–activity studies unambiguously demonstrate the benefit of the APBA warhead for protein binding. Overall, this contribution shows for the first time that reversible covalent inhibitors can be developed via phage display for a protein of interest. The phage display platform should be widely applicable to proteins including those involved in protein–protein interactions.

Covalent Proteomimetic Inhibitor of the Bacterial FtsQB Divisome Complex

Felix M. Paulussen, Gina K. Schouten, Carolin Moertl, Jolanda Verheul, Irma Hoekstra, Gregory M. Koningstein, George H. Hutchins, Aslihan Alkir, Rosa A. Luirink, Daan P. Geerke, Peter van Ulsen, Tanneke den Blaauwen, Joen Luirink, and Tom N. Grossmann

Journal of the American Chemical Society 2022

DOI: 10.1021/jacs.2c06304

The use of antibiotics is threatened by the emergence and spread of multidrug-resistant strains of bacteria. Thus, there is a need to develop antibiotics that address new targets. In this respect, the bacterial divisome, a multi-protein complex central to cell division, represents a potentially attractive target. Of particular interest is the FtsQB subcomplex that plays a decisive role in divisome assembly and peptidoglycan biogenesis in E. coli. Here, we report the structure-based design of a macrocyclic covalent inhibitor derived from a periplasmic region of FtsB that mediates its binding to FtsQ. The bioactive conformation of this motif was stabilized by a customized cross-link resulting in a tertiary structure mimetic with increased affinity for FtsQ. To increase activity, a covalent handle was incorporated, providing an inhibitor that impedes the interaction between FtsQ and FtsB irreversibly. The covalent inhibitor reduced the growth of an outer membrane-permeable E. coli strain, concurrent with the expected loss of FtsB localization, and also affected the infection of zebrafish larvae by a clinical E. coli strain. This first-in-class inhibitor of a divisome protein–protein interaction highlights the potential of proteomimetic molecules as inhibitors of challenging targets. In particular, the covalent mode-of-action can serve as an inspiration for future antibiotics that target protein–protein interactions.

Thiol Reactivity of N-Aryl α-Methylene-γ-lactams: Influence of the Guaianolide Structure [@KayBrummond]

 Daniel P. Dempe, Chong-Lei Ji, Peng Liu, and Kay M. Brummond

The Journal of Organic Chemistry, 2020

DOI: 10.1021/acs.joc.2c01530

The α-methylene-γ-lactam offers promise as a complementary warhead for the development of targeted covalent inhibitors. However, an understanding of the factors governing its electrophilic reactivity is needed to promote the development of lead compounds utilizing this motif. Herein we synthesize a series of N-aryl-substituted α-methylene-γ-lactams installed within the framework of a bioactive guaianolide analog. To determine the effects of the guaianolide structure on the electrophilic reactivity, these compounds were reacted with glutathione under biomimetic conditions, and the rate constants were measured. A linear free-energy relationship was observed with the Hammett parameter of the N-aryl group within the cis- or trans-annulated isomeric series of compounds. However, the trans-annulated compounds exhibited a ca. 10-fold increase in reactivity relative to both the cis-annulated compounds and the corresponding N-arylated 3-methylene-2-pyrrolidinones. Density functional theory calculations revealed that the reactivity of the trans-annulated stereoisomers is promoted by the partial release of the ring strain of the fused seven-membered ring in the thio-Michael addition transition state.