Nucleophilic covalent ligand discovery for the cysteine redoxome

Fu, L., Jung, Y., Tian, C. et al. 

Nat Chem Biol (2023). 

https://doi.org/10.1038/s41589-023-01330-5

With an eye toward expanding chemistries used for covalent ligand discovery, we elaborated an umpolung strategy that exploits the ‘polarity reversal’ of sulfur when cysteine is oxidized to sulfenic acid, a widespread post-translational modification, for selective bioconjugation with C-nucleophiles. Here we present a global map of a human sulfenome that is susceptible to covalent modification by members of a nucleophilic fragment library. More than 500 liganded sulfenic acids were identified on proteins across diverse functional classes, and, of these, more than 80% were not targeted by electrophilic fragment analogs. We further show that members of our nucleophilic fragment library can impair functional protein–protein interactions involved in nuclear oncoprotein transport and DNA damage repair. Our findings reveal a vast expanse of ligandable sulfenic acids in the human proteome and highlight the utility of nucleophilic small molecules in the fragment-based covalent ligand discovery pipeline, presaging further opportunities using non-traditional chemistries for targeting proteins.

Covalent drug discovery using sulfur(VI) fluoride exchange warheads

Huang Huang, Lyn H. Jones

Expert Opinion on Drug Discovery, 2023

https://doi.org/10.1080/17460441.2023.2218642

Covalent drug discovery has traditionally focused on targeting cysteine, but the amino acid is often absent in protein binding sites. This review makes the case to move beyond cysteine labeling using sulfur (VI) fluoride exchange (SuFEx) chemistry to expand the druggable proteome.

Selective control of parasitic nematodes using bioactivated nematicide

Burns, A.R., Baker, R.J., Kitner, M. et al. Selective control of parasitic nematodes using bioactivated nematicides. 

Nature (2023)

https://doi.org/10.1038/s41586-023-06105-5

Parasitic nematodes are a major threat to global food security, particularly as the world amasses 10 billion people amid limited arable land. Most traditional nematicides have been banned owing to poor nematode selectivity, leaving farmers with inadequate means of pest control. Here we use the model nematode Caenorhabditis elegans to identify a family of selective imidazothiazole nematicides, called selectivins, that undergo cytochrome-p450-mediated bioactivation in nematodes. At low parts-per-million concentrations, selectivins perform comparably well with commercial nematicides to control root infection by Meloidogyne incognita, a highly destructive plant-parasitic nematode. Tests against numerous phylogenetically diverse non-target systems demonstrate that selectivins are more nematode-selective than most marketed nematicides. Selectivins are first-in-class bioactivated nematode controls that provide efficacy and nematode selectivity.

Data-Driven Global Assessment of Protein Kinase Inhibitors with Emphasis on Covalent Compounds

Elena Xerxa, Filip Miljković, and Jürgen Bajorath

Journal of Medicinal Chemistry  2023

DOI: 10.1021/acs.jmedchem.3c00621

Large-scale analysis of public human and mouse protein kinase inhibitor (PKI) data identified more than 155,000 human PKIs (and ∼3000 murine PKIs), for which reliable activity measurements were available. Human PKIs were active against 440 kinases (85% coverage of the kinome). Over the past years, there has been substantial growth of human PKIs, dominated by inhibitors with single-kinase annotations and high core structure diversity. Human PKIs included an unexpectedly large number of nearly 14,000 covalent PKIs (CPKIs), ∼87% of which contained acrylamide or heterocyclic urea warheads. These CPKIs were active against a large number of 369 human kinases. The promiscuity of PKIs and CPKIs was overall comparable. However, there was a notable enrichment of acrylamide- but not heterocyclic urea-containing CPKIs among most promiscuous inhibitors. Furthermore, CPKIs with both warheads had significantly higher potency than structurally analogous PKIs. Taken together, these findings have several implications for medicinal chemistry that are discussed.

Discovery of a New-Generation S-Adenosylmethionine-Noncompetitive Covalent Inhibitor Targeting the Lysine Methyltransferase Enhancer of Zeste Homologue 2

Yi Zhang, Hong Yang, Bingbing Li, Jiayi Li, Huaxuan Li, Qiongyu Shi, Bang Li, Zekun Wang, Jiahong Zheng, Ying Zhang, Hui Dong, Xun Huang, and Yuanxiang Wang

Journal of Medicinal Chemistry 2023

DOI: 10.1021/acs.jmedchem.3c00504

The first-generation enhancer of zeste homologue 2 (EZH2) inhibitors suffer from several limitations, such as high dosage, cofactor S-adenosylmethionine (SAM) competition, and acquired drug resistance. Development of covalent EZH2 inhibitors that are noncompetitive with cofactor SAM offers an opportunity to overcome these disadvantages. The structure-based design of compound 16 (BBDDL2059) as a highly potent and selective covalent inhibitor of EZH2 is presented in this context. 16 inhibits EZH2 enzymatic activity at sub-nanomolar concentrations and achieves low nanomolar potencies in cell growth inhibition. The kinetic assay revealed that 16 is noncompetitive with the cofactor SAM, providing the basis for its superior activity over noncovalent and positive controls by reducing competition with cofactor SAM and offering a preliminary proof for its covalent inhibition nature. Mass spectrometric analysis and washout experiments firmly establish its covalent inhibition mechanism. This study demonstrates that covalent inhibition of EZH2 can offer a new opportunity for the development of promising new-generation drug candidates.

Targeted Proximity-Labelling of Protein Tyrosines via Flavin-Dependent Photoredox Catalysis with Mechanistic Evidence for a Radical-Radical Recombination Pathway

Taylor O. Hope, Tamara Reyes-Robles, Keun Ah Ryu, Steven Mauries, Nicole Removski, Jacinthe Maisonneuve, Rob C. Oslund,* Olugbeminiyi O. Fadeyi,* and Mathieu Frenette*

Chemical Science, 2023

DOI: 10.1039/D3SC00638G

Flavin-based photocatalysts such as riboflavin tetraacetate (RFT) serve as a robust platform for light-mediated protein labelling via phenoxy radical-mediated tyrosine-biotin phenol coupling on live cells. To gain insight into this coupling reaction, we conducted detailed mechanistic analysis for RFT-photomediated activation of phenols for tyrosine labelling. Contrary to previously proposed mechanisms, we find that the initial covalent binding step between the tag and tyrosine is not radical addition, but rather radical-radical recombination. The proposed mechanism may also explain the mechanism of other reported tyrosine-tagging approaches. Competitive kinetics experiments show that phenoxyl radicals are generated with several reactive intermediates in the proposed mechanism—primarily with the excited riboflavin-photocatalyst or singlet oxygen—and these multiple pathways for phenoxyl radical generation from phenols increase the likelihood of radical-radical recombination.

Discovery of a first-in-class Aurora A covalent inhibitor for the treatment of triple negative breast cancer

Bin Zhang, Chengchen Zhu, Albert S.C. Chan, Gui Lu

European Journal of Medicinal Chemistry, 2023

https://doi.org/10.1016/j.ejmech.2023.115457

Aurora kinases, which belong to the serine/threonine protein family, play critical roles in the regulation of the cell cycle and mitotic spindle assembly. They are frequently highly expressed in various types of tumors, and the use of selective Aurora kinase inhibitors has become a potential treatment option for cancer therapy. Despite the development of some reversible Aurora kinase inhibitors, none has been approved for clinical use yet. In this study, we report the discovery of the first-in-class irreversible Aurora A covalent inhibitors that target a cysteine residue at the substrate binding site. These inhibitors were characterized in enzymatic and cellular assays, and 11c exhibited selective inhibition to normal and cancer cells, as well as to Aurora A and B kinases. The covalent binding of 11c to Aurora A was confirmed by SPR, MS, and enzyme kinetic analysis, and Cys290-mediated covalent inhibition was supported through a bottom-up analysis of inhibitor-modified targets. Moreover, Western blotting assays were conducted on cells and tissues, and cellular thermal shift assays (CETSA) were further performed on cells to demonstrate the selectivity to Aurora A kinase. 11c displayed comparable therapeutic efficacy in an MDA-MB-231 xenograft mouse model relative to the positive control ENMD-2076, while requiring only half the dose of ENMD-2076. These results confirmed that 11c may be a promising drug candidate for the treatment of triple negative breast cancer (TNBC). Our work may provide a new perspective on the design of covalent inhibitors of Aurora kinase.

Thiomethyltetrazines are Reversible Covalent Cysteine Warheads whose Dynamic Behavior can be "Switched off” via Bioorthogonal Chemistry Inside Live Cells

Amanda Tallon, Yingrong Xu, Graham West, Christopher am Ende, Joseph Fox

ChemRXiv, 2023

https://doi.org/10.26434/chemrxiv-2023-w0p5m

Electrophilic small molecules that can reversibly modify proteins are of growing interest in drug discovery. However, the ability to study reversible covalent probes in live cells can be limited by their reversible reactivity after cell lysis and in proteomic workflows, leading to scrambling and signal loss. We describe how thiomethyltetrazines function as reversible covalent warheads for cysteine modification and this dynamic labeling behavior can be "switched off” via bioorthogonal chemistry inside live cells. Simultaneously, the tetrazine serves as bioorthogonal reporter enabling the introduction of tags for fluorescent imaging or affinity purification. Thiomethyltetrazines can label isolated proteins, proteins in cellular lysates, and proteins in live cells with second-order rate constants spanning two orders of magnitude (k2 1–100 M-1s-1). Reversible modification by thiomethyltetrazines can be switched off upon the addition of trans-cyclooctene in live cells, converting the dynamic thiomethyltetrazine tag into a Diels-Alder adduct which is stable to lysis and proteomic workflows. Time-course quenching experiments were used to demonstrate temporal control over electrophilic modification. Moreover, it is shown that “locking in” the tag through Diels-Alder chemistry enables the identification of protein targets that are otherwise lost during sample processing. Three probes were further evaluated to identify unique pathways in a live-cell proteomic study. We anticipate that discovery efforts will be enabled by the trifold function of thiomethyltetrazines as electrophilic warheads, bioorthogonal reporters, and switches for “locking in” stability.

Global Discovery of Covalent Modulators of Ribonucleoprotein Granules

Anthony M. Ciancone, Kyung W. Seo, Miaomiao Chen, Adam L. Borne, Adam H. Libby, Dina L. Bai, Ralph E. Kleiner*, and Ku-Lung Hsu*

J. Am. Chem. Soc., 2023

https://doi.org/10.1021/jacs.3c00165

Stress granules (SGs) and processing-bodies (PBs, P-bodies) are ubiquitous and widely studied ribonucleoprotein (RNP) granules involved in cellular stress response, viral infection, and the tumor microenvironment. While proteomic and transcriptomic investigations of SGs and PBs have provided insights into molecular composition, chemical tools to probe and modulate RNP granules remain lacking. Herein, we combine an immunofluorescence (IF)-based phenotypic screen with chemoproteomics to identify sulfonyl-triazoles (SuTEx) capable of preventing or inducing SG and PB formation through liganding of tyrosine (Tyr) and lysine (Lys) sites in stressed cells. Liganded sites were enriched for RNA-binding and protein–protein interaction (PPI) domains, including several sites found in RNP granule-forming proteins. Among these, we functionally validate G3BP1 Y40, located in the NTF2 dimerization domain, as a ligandable site that can disrupt arsenite-induced SG formation in cells. In summary, we present a chemical strategy for the systematic discovery of condensate-modulating covalent small molecules.

α-Lactam Electrophiles for Covalent Chemical Biology

Mahía, Alejandro, Kiib, Anders, Nisavíc, Marija, Svenningsen, Esben, Palmfeldt, Johan, Poulsen, Thomas Bjørnskov, Angew. Chem. Int. Ed. 2023, e202304142

https://doi.org/10.1002/anie.202304142

Electrophilic groups are one of the key pillars of contemporary chemical biology and medicinal chemistry. For instance, the group of 3-membered N-heterocyclic compounds – such as aziridines, azirines, and oxaziridines – possess unique electronic and structural properties which underlie their potential and applicability as covalent tools. The α-lactams are also members of this group of compounds, however, their utility within the field remains unexplored. Here, we demonstrate an α-lactam reagent (AM2) that is tolerant to aqueous buffers while being reactive towards biologically relevant nucleophiles. Interestingly, carboxylesterases 1 and 2 (CES1/2), both serine hydrolases with key roles in endo- and xenobiotic metabolism, were found as primary covalent targets for AM2 in HepG2 liver cancer cells. All in all, our study constitutes the starting point for the further development and exploration of α-lactam-based electrophilic probes in covalent chemical biology.

Benchmarking In Silico Tools for Cysteine pKa Prediction

Ernest Awoonor-Williams*, Andrei A. Golosov, and Viktor Hornak

Journal of Chemical Information and Modeling 2023 63 (7), 2170-2180

DOI: 10.1021/acs.jcim.3c00004

Accurate estimation of the pKa’s of cysteine residues in proteins could inform targeted approaches in hit discovery. The pKa of a targetable cysteine residue in a disease-related protein is an important physiochemical parameter in covalent drug discovery, as it influences the fraction of nucleophilic thiolate amenable to chemical protein modification. Traditional structure-based in silico tools are limited in their predictive accuracy of cysteine pKa’s relative to other titratable residues. Additionally, there are limited comprehensive benchmark assessments for cysteine pKa predictive tools. This raises the need for extensive assessment and evaluation of methods for cysteine pKa prediction. Here, we report the performance of several computational pKa methods, including single-structure and ensemble-based approaches, on a diverse test set of experimental cysteine pKa’s retrieved from the PKAD database. The dataset consisted of 16 wildtype and 10 mutant proteins with experimentally measured cysteine pKa values. Our results highlight that these methods are varied in their overall predictive accuracies. Among the test set of wildtype proteins evaluated, the best method (MOE) yielded a mean absolute error of 2.3 pK units, highlighting the need for improvement of existing pKa methods for accurate cysteine pKa estimation. Given the limited accuracy of these methods, further development is needed before these approaches can be routinely employed to drive design decisions in early drug discovery efforts.