Sulfinyl Aziridines as Stereoselective Covalent Destabilizing Degraders of the Oncogenic Transcription Factor MYC

H. T. Rosen, K. Li, C. E. Stieger, E. L. Li, B. Currier, S. M. Brittain, F. J. Garcia, D. C. Beard, M. D. Jones, S. Haenni-Holzinger, D. Dovala, J. M. McKenna, M. Schirle, T. J. Maimone, D. K. Nomura, Angew. Chem. Int. Ed.. 2025, e202508518.

https://doi.org/10.1002/anie.202508518

Although MYC is a significant oncogenic transcription factor driver of cancer, directly targeting MYC has remained challenging due to its intrinsic disorder and poorly defined structure, deeming it “undruggable.” Whether transient pockets formed within unstructured regions of proteins can be selectively targeted with small molecules remains an outstanding challenge. Here, we developed a stereochemically paired spirocyclic oxindole aziridine covalent library and screened this library for degradation of MYC. We identified a hit covalent ligand, KL2-236, bearing a unique sulfinyl aziridine warhead, that engaged MYC as a pure MYC/MAX protein complex, and in cancer cells to destabilize MYC, inhibit MYC transcriptional activity and degrade MYC in a proteasome-dependent manner through targeting intrinsically disordered C203 and D205 residues. Notably, this reactivity was most pronounced for specific stereoisomers of KL2-236 with a diastereomer, KL4-019, that was largely inactive. Mutagenesis of both C203 and D205 completely attenuated KL2-236-mediated MYC degradation. We also optimized our KL2-236 hit compound to generate a more potent, selective, and durable MYC degrader, KL4-219A. Our results reveal a novel ligandable site within MYC and indicate that certain intrinsically disordered regions within transcription factors, such as MYC, can be interrogated by isomerically unique chiral small molecules, leading to destabilization and degradation.

Covalent Ligand Electrophiles Are Differentially Activated by Proximity Effects Which Govern Latent Protein Reactivit

 Tomas V. Frankovich, Harrison M. McCann, Kyle S. Hoffman, and Anthony F. Rullo

ACS Central Science Article ASAP

DOI: 10.1021/acscentsci.5c00699

Covalent ligands contain an electrophilic moiety that reacts with a nucleophilic residue on a target protein, following an initial reversible binding event. Covalent ligand development typically involves efforts to increase on-target selectivity by maximizing the ligand binding affinity and minimizing intrinsic electrophile reactivity. Problematically, this limits labeling kinetics and requires high affinity ligands. The concept of “latency” describes the potential for “turn-on” activation of electrophiles upon target engagement. Here, we investigate the potential intrinsic latency of covalent electrophiles and test the hypothesis that diverse electrophiles can be differentially activated by proximity effects. We develop a kinetic effective molarity (EMk) approach to quantitatively characterize kinetics associated with diverse electrophilic reaction mechanisms, both with and without binding proximity effects. We observe that different electrophiles are associated with significantly different EMk parameters, with SuFEx and acrylamide electrophiles associated with the highest intrinsic latency. Eyring transition state analysis revealed that all covalent ligands, independent of electrophile, benefit from significant transition state entropic stabilization. Strikingly, electrophiles associated with the highest latency are associated with greater relative transition state stabilization with different enthalpic and entropic contributions. These findings quantitatively describe electrophile latency and will aid the mechanism-guided development of next-generation covalent ligands associated with “turn-on” reactivity.

A covalent inhibitor targeting Cys16 on RhoA in colorectal cancer

Tin-Yan Koo, Jason Ying Ki Li, Nga-Sze Lee, Jintian Chen, Hillary Yui-Yan Yip, Ianto Bosheng Huang, Kai-Yu Ng, Helen H.N. Yan, Suet Yi Leung, Stephanie Ma, Jingying Zhou, Clive Yik-Sham Chung

https://www.cell.com/cell-chemical-biology/fulltext/S2451-9456%2825%2900255-7

RhoA is a key cancer driver and potential colorectal cancer (CRC) therapy target but remains undrugged clinically. Using activity-based protein profiling (ABPP) and mass spectrometry (MS), we identified CL16, a covalent inhibitor targeting the unique Cys16 on RhoA subfamily, which confers high specificity over other Rho family proteins. Cys16 is adjacent to the nucleotide-binding pocket and switch regions, which are critical for RhoA function. The binding by CL16 effectively disrupts GTP binding and inhibits RhoA activity in CRC cells, leading to cytotoxic killing of CRC cells through cell-cycle arrest and apoptosis. In mouse CRC models, CL16 exhibits strong antitumor and antimetastatic effects, promotes T cell infiltration into the tumor microenvironment, and shows no observable toxicity. Our findings suggest that covalent targeting of the druggable Cys16 on RhoA offers a promising strategy for CRC treatment, providing a foundation for developing specific RhoA inhibitors for clinical application.

Covalent Irreversible Inhibitors of Tetracycline Destructases

Ruihao Li, Yao-Peng Xue, Steven T. Le, Wai Kwan Tang, Niraj H. Tolia, Gautam Dantas, and Timothy A. Wencewicz

ACS Infectious Diseases 2025

DOI: 10.1021/acsinfecdis.5c00322

 Next-generation tetracycline antibiotics are threatened by an emerging resistance mechanism ─ enzymatic inactivation. The relevant enzymes ─ tetracycline destructases (TDases) ─ are structural homologues of class A flavin monooxygenase (FMO) that oxidize tetracycline antibiotics, leading to various inactive degradation products. Small molecule inhibitors of antibiotic-inactivating enzymes are critical clinical therapeutics used to manage bacterial resistance with combination therapy. While reversible TDase inhibitors have been reported, we sought to develop covalent inhibitors that are better aligned with clinically effective covalent β-lactamase inhibitors. Here, we report the design, chemical synthesis, and biochemical characterization of the first covalent irreversible inhibitors of TDases based on C9-derivatives of anhydrotetracycline (aTC). The reactive warheads were installed via a one-step Mannich reaction linking either an N-(1-methyl)cyclopropylamine or N-propargylamine group to the C9-position of the aTC D-ring via an amino methylene linkage. We also synthesized two nonspecific FMO inhibitors, N-(1-methyl)cyclopropylbenzylamine (1) and N-methyl-N-benzyl-propargylamine (2) as mechanistic probes to distinguish reactivity with the essential FAD cofactor in TDases via one- or two-electron transfer pathways, respectively. We evaluated the compounds as potential inhibitors of representative TDases from the two major classes─Type 1 (TetX6 and TetX7) and Type 2 (Tet50). The aTC-based compounds 3-5 inhibited both Type 1 and Type 2 TDases with notable differences in potency and inhibition mechanism. The inhibition of Type 1 TDases was more potent but reversible with no time dependence. The inhibition of Type 2 TDases was time-dependent and irreversible even after exhaustive dialysis, consistent with a covalent mechanism of inhibition. Molecular modeling of the inhibitors supports unique inhibitor binding modes for Type 1 and Type 2 TDases that are consistent with the observed differences in the inhibition modes. Blue light irradiation of the Type 2 TDase enhanced this reactivity. Treatment of Tet50 with probe molecules 1 and 2 under blue light exposure enabled the identification of covalent FAD adducts via mass spectrometry that are consistent with the expected one- and two-electron transfer reaction modes of the cyclopropylamine and propargylamine warheads with the FAD cofactor. At concentrations as low as 2 μg/mL, the aTC-based covalent inhibitors 3-5 recovered tetracycline activity against E. coli overexpressing TDases. Our findings suggest that the inhibition of TDases through covalent trapping of the FAD cofactor is a viable strategy for overcoming TDase-mediated antibiotic resistance.

Covalent chemical probes

Backus, K.M., Wilson, A.J. 

Commun Chem 8, 266 (2025). 

https://doi.org/10.1038/s42004-025-01652-6

Cell-Active, Irreversible Covalent Inhibitors Targeting a Surface-Exposed Non-Catalytic Lysine on Aurora a Kinase by Using Squarate Chemistry

Z. Wang, X. Wang, Y. Li, P. Li, S. Huang, P. Chen, G. Tang, X. Ding, Z. Zhang, Z.-M. Zhang, Y. Zhou, S. Q. Yao, X. Lu, 

Angew. Chem. Int. Ed. 2025, e202510763

https://doi.org/10.1002/anie.202510763

Targeting surface-exposed lysines in kinases through covalent modification presents a major challenge due to their high pKa and inherently low reactivity. While current research primarily targets more reactive catalytic lysines buried in the ATP-binding pocket, no systematic rational strategy has yet been developed for selectively engaging surface-exposed lysines. Herein, we present a versatile strategy for developing cell-active covalent kinase inhibitors (CKIs) by selectively targeting unique surface-exposed lysines using squarate chemistry. By using Aurora A (AURKA) as a proof-of-concept, we systematically evaluated this approach against other well-known lysine-reactive warheads (e.g., EBA, SO2F, and OSO2F) and demonstrated, for the first time, squarates’ superior efficacy in engaging these challenging low-reactivity lysines. Amongst various AURKA CKIs, AL8 emerged as the first-in-class squarate-based, cell-active inhibitor, exhibiting excellent selectivity in both biochemical and cellular assays with long-residence time in covalently engaging endogenous AURKA. Detailed investigation of effects of leaving groups on squaric esters provided valuable insights for future development of lysine-reactive CKIs. Our finding has established squarate-containing ligands as a unique and readily tunable platform for covalent modification of surface-exposed, non-catalytic lysines in targeted kinase drug discovery.

Small-Molecule Covalent Stabilization and Inhibition of the TEAD·YAP1 Transcription Factor in Cancer Cells

I-Ju Yeh, Khuchtumur Bum-Erdene, Mona K. Ghozayel, Giovanni Gonzalez-Gutierrez, and Samy O. Meroueh

ACS Chemical Biology 2025

https://pubs.acs.org/doi/10.1021/acschembio.5c00283

Transcriptional enhanced associate domain transcription factors (TEAD1 to TEAD4) bind to transcriptional coactivator Yes-Associated Protein (YAP1) or its paralog transcriptional coactivator with PDZ-binding motif (TAZ) to regulate Hippo pathway target genes. The Hippo pathway is a conserved signaling pathway that regulates organ size and cell fate by controlling cell proliferation and apoptosis. Here we report small acrylamide molecules that form a covalent bond with a conserved cysteine at the TEAD palmitate pocket. Binding studies showed profound stabilization of TEADs by the small molecules, and cocrystal structures reveal that the compounds mimic the binding mode of palmitate. The small molecules achieved submicromolar binding constants and subhour reaction half-lives for all four TEADs. In mammalian cells, the compounds stabilize the TEAD•YAP1 interaction yet inhibit the TEAD transcription factor activity. Unexpectedly, several compounds degraded TEAD and YAP1 proteins and inhibited cancer cell viability. This work suggests that degradation of TEAD and YAP1 may amplify the antitumor effects of small molecules targeting the TEAD palmitate pocket, with implications for other cancer targets featuring allosteric lipid-binding sites.