Expanding the Chemistry of Dihaloacetamides as Tunable Electrophiles for Reversible Covalent Targeting of Cysteines

Daiki Yamane, Ryo Tetsukawa, Naoki Zenmyo, Kaori Tabata, Yuya Yoshida, Naoya Matsunaga, Naoya Shindo, and Akio Ojida

Journal of Medicinal Chemistry 2023

DOI: 10.1021/acs.jmedchem.3c00737

The choice of an appropriate electrophile is crucial in the design of targeted covalent inhibitors (TCIs). In this report, we systematically investigated the glutathione (GSH) reactivity of various haloacetamides and the aqueous stability of their thiol adducts. Our findings revealed that dihaloacetamides cover a broad range of GSH reactivity depending on the combination of the halogen atoms and the structure of the amine scaffold. Among the dihaloacetamides, dichloroacetamide (DCA) exhibited slightly lower GSH reactivity than chlorofluoroacetamide (CFA). The DCA–thiol adduct is readily hydrolyzed under aqueous conditions, but it can stably exist in the solvent-sequestered binding pocket of the protein. These reactivity profiles of DCA were successfully exploited in the design of TCIs targeting noncatalytic cysteines of KRASG12C and EGFRL858R/T790M. These inhibitors exhibited strong antiproliferative activities against cancer cells. Our findings provide valuable insights for designing dihaloacetamide-based reversible covalent inhibitors.

Electrophile Scanning Reveals Reactivity Hotspots for the Design of Covalent Peptide Binders

Grob, N. M.; Remarcik, C.; Rössler, S. L.; Wong, J. Y. K.; Wang, J. C. K.; Tao, J.; Smith, C. L.; Loas, A.; Buchwald, S. L.; Eaton, D. L.; Preciado López, M.; Pentelute, B. L. 

ChemRxiv 2023.

https://doi.org/10.26434/chemrxiv-2023-hvq1k

Protein–protein interactions (PPIs) are intriguing targets in drug discovery and development. Peptides are well suited to target PPIs, which typically present with large surface areas lacking distinct features and deep binding pockets. To improve binding interactions to these topologies by PPI-focused therapeutics and advance their development, potential ligands can be equipped with electrophilic groups to enable binding through covalent mechanisms of action. We report a strategy termed electrophile scanning to identify reactivity hotspots in a known peptide ligand. Cysteine mutants of the ligand are used to install protein-reactive modifiers via a palladium oxidative addition complex (Pd-OAC). Reactivity hotspots are revealed by cross-linking reactions with the target protein under physiological conditions. In a system with the 9-mer peptide antigen VL9 and MHC class I receptor HLA-E, we identify two reactivity hotspots that afford up to 87% conversion to the protein–peptide conjugate within 4 hours. The reactions are specific to the target protein in vitro and dependent on the peptide sequence. Moreover, the cross-linked peptide successfully inhibits molecular recognition of HLA-E by CD94─NKG2A possibly due to structural changes enacted at the PPI interface. The results illustrate the potential of electrophile scanning as a tool for rapid discovery and development of covalent peptide binders.

Enhancing the Equilibrium of Dynamic Thia-Michael Reactions through Heterocyclic Design

Alex E. Crolais, Neil D. Dolinski, Nicholas R. Boynton, Julia M. Radhakrishnan, Scott A. Snyder, and Stuart J. Rowan

Journal of the American Chemical Society 2023

DOI: 10.1021/jacs.3c03643

Although the catalyst-free dynamic thia-Michael (tM) reaction has been leveraged for a range of significant applications in materials science and pharmaceutical development, exploiting its full potential has been limited by relatively low equilibrium constants. To address this shortcoming, a new series of catalyst-free, room-temperature dynamic thia-Michael acceptors bearing an isoxazolone motif were developed and utilized to access both dynamic covalent networks and linear polymers. By leveraging the generation of aromaticity upon thiol addition and tuning the electronic-withdrawing/donating nature of the acceptor at two different sites, a wide range of equilibrium constants (Keq ∼1000 to ∼100,000 M–1) were obtained, constituting a 2 orders of magnitude increase compared to their noncyclic benzalcyanoacetate analogues. Integration into a ditopic isoxazolone-based Michael acceptor allowed access to both bulk dynamic networks and linear polymers; these materials not only exhibited tailorable thermomechanical properties based on thia-Michael acceptor composition, but the higher Keq tM bonds resulted in more mechanically robust materials relative to past designs. Furthermore, solution-state formation of linear polymers was achieved thanks to the increased Keq of the isoxazolone-based acceptors.

Comprehensive Mapping of Electrophilic Small Molecule-Protein Interactions in Human Cells

Cravatt, B.; Njomen, E.; Hayward, R.; DeMeester, K.; Ogasawara, D.; Dix, M.; Nguyen, T.; Ashby, P.; Simon, G.; Schreiber, S.; Melillo, B. 

ChemRxiv 2023

https://doi.org/10.26434/chemrxiv-2023-s446n

Covalent chemistry is a versatile approach for expanding the ligandability of the human proteome. Activity-based protein profiling (ABPP) can infer the specific residues modified by electrophilic compounds through competition with broadly reactive probes. Nonetheless, the extent to which such residue-directed ABPP platforms fully assess the protein targets of electrophilic compounds in human cells remains unclear. Here, we introduce a complementary approach that directly identifies proteins showing stereoselective reactivity with focused libraries of stereochemically-defined, alkynylated electrophilic compounds. Integration of protein- and cysteine-directed ABPP data from compound-treated human cancer cells revealed generally well-correlated target maps and highlighted specific features, such as protein size and the proteotypicity of cysteine-containing peptides, that help to explain gaps in each ABPP platform. The integrated ABPP strategy furnished stereoselective, high-engagement covalent ligands for > 300 structurally and functionally diverse human proteins, including compounds that modulate enzymes by canonical (active-site cysteine) and non-canonical (isotype-restricted and non-catalytic cysteines) mechanisms.

Structure-Based Design and Characterization of the Highly Potent and Selective Covalent Inhibitors Targeting the Lysine Methyltransferases G9a/GLP

Zongbo Feng, Chunju Yang, Yi Zhang, Huaxuan Li, Wei Fang, Junhua Wang, Yichu Nie, Chang-Yun Wang, Zhiqing Liu, Zhimin Jiang, Junjian Wang, and Yuanxiang Wang

Journal of Medicinal Chemistry 2023

DOI: 10.1021/acs.jmedchem.3c00411

Protein lysine methyltransferases G9a and GLP, which catalyze mono- and di-methylation of histone H3K9 and nonhistone proteins, play important roles in diverse cellular processes. Overexpression or dysregulation of G9a and GLP has been identified in various types of cancer. Here, we report the discovery of a highly potent and selective covalent inhibitor 27 of G9a/GLP via the structure-based drug design approach following structure–activity relationship exploration and cellular potency optimization. Mass spectrometry assays and washout experiments confirmed its covalent inhibition mechanism. Compound 27 displayed improved potency in inhibiting the proliferation and colony formation of PANC-1 and MDA-MB-231 cell lines and exhibited enhanced potency in reducing the levels of H3K9me2 in cells compared to noncovalent inhibitor 26. In vivo, 27 showed significant antitumor efficacy in the PANC-1 xenograft model with good safety. These results clearly indicate that 27 is a highly potent and selective covalent inhibitor of G9a/GLP.

Characterization of a Potent and Orally Bioavailable Lys-Covalent Inhibitor of Apoptosis Protein (IAP) Antagonist

Parima Udompholkul, Ana Garza-Granados, Giulia Alboreggia, Carlo Baggio, Jack McGuire, Scott D. Pegan, and Maurizio Pellecchia

Journal of Medicinal Chemistry 2023

DOI: 10.1021/acs.jmedchem.3c00467

We have recently reported on the use of aryl-fluorosulfates in designing water- and plasma-stable agents that covalently target Lys, Tyr, or His residues in the BIR3 domain of the inhibitor of the apoptosis protein (IAP) family. Here, we report further structural, cellular, and pharmacological characterizations of this agent, including the high-resolution structure of the complex between the Lys-covalent agent and its target, the BIR3 domain of X-linked IAP (XIAP). We also compared the cellular efficacy of the agent in two-dimensional (2D) and three-dimensional (3D) cell cultures, side by side with the clinical candidate reversible IAP inhibitor LCL161. Finally, in vivo pharmacokinetic studies indicated that the agent was long-lived and orally bioavailable. Collectively our data further corroborate that aryl-fluorosulfates, when incorporated correctly in a ligand, can result in Lys-covalent agents with pharmacodynamic and pharmacokinetic properties that warrant their use in the design of pharmacological probes or even therapeutics.

 

Covalent Inhibition by a Natural Product-Inspired Latent Electrophile

David P. Byun, Jennifer Ritchie, Yejin Jung, Ronald Holewinski, Hong-Rae Kim, Ravichandra Tagirasa, Joseph Ivanic, Claire M. Weekley, Michael W. Parker, Thorkell Andresson, and Euna Yoo

Journal of the American Chemical Society 2023 145 (20), 11097-11109

DOI: 10.1021/jacs.3c00598

Strategies to target specific protein cysteines are critical to covalent probe and drug discovery. 3-Bromo-4,5-dihydroisoxazole (BDHI) is a natural product-inspired, synthetically accessible electrophilic moiety that has previously been shown to react with nucleophilic cysteines in the active site of purified enzymes. Here, we define the global cysteine reactivity and selectivity of a set of BDHI-functionalized chemical fragments using competitive chemoproteomic profiling methods. Our study demonstrates that BDHIs capably engage reactive cysteine residues in the human proteome and the selectivity landscape of cysteines liganded by BDHI is distinct from that of haloacetamide electrophiles. Given its tempered reactivity, BDHIs showed restricted, selective engagement with proteins driven by interactions between a tunable binding element and the complementary protein sites. We validate that BDHI forms covalent conjugates with glutathione S-transferase Pi (GSTP1) and peptidyl-prolyl cis–trans isomerase NIMA-interacting 1 (PIN1), emerging anticancer targets. BDHI electrophile was further exploited in Bruton’s tyrosine kinase (BTK) inhibitor design using a single-step late-stage installation of the warhead onto acrylamide-containing compounds. Together, this study expands the spectrum of optimizable chemical tools for covalent ligand discovery and highlights the utility of 3-bromo-4,5-dihydroisoxazole as a cysteine-reactive electrophile.