Prolonged Activation of the GLP-1 Receptor via Covalent Capture

Özge Ünsal, Z. Selin Bacaksiz, Vladislav Khamraev, Vittorio Montanari, Martin Beinborn, and Krishna Kumar

ACS Chemical Biology 2024

DOI: https://doi.org/10.1021/acschembio.4c00039

The incretin gut hormone glucagon-like peptide-1 (GLP-1) has become a household name because of its ability to induce glucose-dependent insulin release with accompanying weight loss in patients. Indeed, derivatives of the peptide exert numerous pleiotropic actions that favorably affect other metabolic functions, and consequently, such compounds are being considered as treatments for a variety of ailments. The ability of native GLP-1 to function as a clinical drug is severely limited because of its short half-life in vivo. All of the beneficial effects of GLP-1 come from its agonism at the cognate receptor, GLP-1R. In our quest for long-lived activation of the receptor, we hypothesized that an agonist that had the ability to covalently cross-link with GLP-1R would prove useful. We here report the structure-guided design of peptide analogues containing an electrophilic warhead that could be covalently captured by a resident native nucleophile on the receptor. The compounds were evaluated using washout experiments, and resistance to such washing serves as an index of prolonged activation and covalent capture, which we use to tabulate longevity and robust long-lived GLP-1R agonism. The addition of SulF (cross-linkable warhead), an N-terminal trifluoroethyl group (for protease protection), and a C18 diacid lipid (protractor) all contributed to the increased wash resistance of GLP-1. The most effective compound based on the wash resistance metric, C2K26DAC18_K34SulF, has all three elements outlined and may serve as a blueprint and a proof-of-concept scaffold for the design of clinically useful molecules.

N-Acyl-N-Alkyl Sulfonamide Probes for Ligand-Directed Covalent Labeling of GPCRs: The Adenosine A2B Receptor as Case Study

Bert L. H. Beerkens, Vasiliki Andrianopoulou, Xuesong Wang, Rongfang Liu, Gerard J. P. van Westen, Willem Jespers, Adriaan P. IJzerman, Laura H. Heitman, and Daan van der Es

ACS Chemical Biology 2024

DOI: 10.1021/acschembio.4c00210

Small molecular tool compounds play an essential role in the study of G protein-coupled receptors (GPCRs). However, tool compounds most often occupy the orthosteric binding site, hampering the study of GPCRs upon ligand binding. To overcome this problem, ligand-directed labeling techniques have been developed that leave a reporter group covalently bound to the GPCR, while allowing subsequent orthosteric ligands to bind. In this work, we applied such a labeling strategy to the adenosine A2B receptor (A2BAR). We have synthetically implemented the recently reported N-acyl-N-alkyl sulfonamide (NASA) warhead into a previously developed ligand and show that the binding of the A2BAR is not restricted by NASA incorporation. Furthermore, we have investigated ligand-directed labeling of the A2BAR using SDS-PAGE, flow cytometric, and mass spectrometry techniques. We have found one of the synthesized probes to specifically label the A2BAR, although detection was hindered by nonspecific protein labeling most likely due to the intrinsic reactivity of the NASA warhead. Altogether, this work aids the future development of ligand-directed probes for the detection of GPCRs.

Development of a Dual Factor Activatable Covalent Targeted Photoacoustic Imaging Probe for Tumor Imaging

Jiho Song, Tianqu Zhai, Heung Sik Hahm, Yuancheng Li, Hui Mao, Xueding Wang, Janggun Jo, Jae Won Chang, 

Angew. Chem. Int. Ed. 2024, e202410645.

 https://doi.org/10.1002/anie.202410645

Photoacoustic imaging (PAI) is an emerging modality in biomedical imaging with superior imaging depth and specificity. However, PAI still has significant limitations, such as the background noise from endogenous chromophores. To overcome these limitations, we developed a covalent activity-based PAI probe, NOx-JS013, targeting NCEH1. NCEH1, a highly expressed and activated serine hydrolase in aggressive cancers, has the potential to be employed for the diagnosis of cancers. We show that NOx-JS013 labels active NCEH1 in live cells with high selectivity relative to other serine hydrolases. NOx-JS013 also presents its efficacy as a hypoxia-responsive imaging probe in live cells. Finally, NOx-JS013 successfully visualizes aggressive prostate cancer tumors in mouse models of PC3, while negligibly detected in tumors of non-aggressive LNCaP mouse models. These findings show that NOx-JS013 has the potential to be used to develop precision PAI reagents for detecting metastatic progression in various cancers.

Design of a covalent protein-protein interaction inhibitor of SRPKs to suppress angiogenesis and invasion of cancer cells

 Cai, G., Bao, Y., Li, Q. et al. 

Commun Chem 7, 144 (2024). 

https://doi.org/10.1038/s42004-024-01230-2

Serine–arginine (SR) proteins are splicing factors that play essential roles in both constitutive and alternative pre-mRNA splicing. Phosphorylation of their C-terminal RS domains by SR protein kinases (SRPKs) regulates their localization and diverse cellular activities. Dysregulation of phosphorylation has been implicated in many human diseases, including cancers. Here, we report the development of a covalent protein–protein interaction inhibitor, C-DBS, that targets a lysine residue within the SRPK-specific docking groove to block the interaction and phosphorylation of the prototypic SR protein SRSF1. C-DBS exhibits high specificity and conjugation efficiency both in vitro and in cellulo. This self-cell-penetrating inhibitor attenuates the phosphorylation of endogenous SR proteins and subsequently inhibits the angiogenesis, migration, and invasion of cancer cells. These findings provide a new foundation for the development of covalent SRPK inhibitors for combatting diseases such as cancer and viral infections and overcoming the resistance encountered by ATP-competitive inhibitors.

Discovery of a Covalent Inhibitor That Overcame Resistance to Venetoclax in AML Cells Overexpressing BFL-1

Jianfeng Lou, Qianqian Zhou, Xilin Lyu, Xinyi Cen, Chen Liu, Ziqin Yan, Yan Li, Haotian Tang, Qiupei Liu, Jian Ding, Ye Lu, He Huang, Hua Xie, and Yujun Zhao

Journal of Medicinal Chemistry 2024

DOI: 10.1021/acs.jmedchem.4c00291

Clinical and biological studies have shown that overexpression of BFL-1 is one contributing factor to venetoclax resistance. The resistance might be overcome by a potent BFL-1 inhibitor, but such an inhibitor is rare. In this study, we show that 56, featuring an acrylamide moiety, inhibited the BFL-1/BID interaction with a Ki value of 105 nM. More interestingly, 56 formed an irreversible conjugation adduct at the C55 residue of BFL-1. 56 was a selective BFL-1 inhibitor, and its MCL-1 binding affinity was 10-fold weaker, while it did not bind BCL-2 and BCL-xL. Mechanistic studies showed that 56 overcame venetoclax resistance in isogenic AML cell lines MOLM-13-OE and MV4-11-OE, which both overexpressed BFL-1. More importantly, 56 and venetoclax combination promoted stronger apoptosis induction than either single agent. Collectively, our data show that 56 overcame resistance to venetoclax in AML cells overexpressing BFL-1. These attributes make 56 a promising candidate for future optimization.

Electrophilic proximity-inducing synthetic adapters enhance universal T cell function by covalently enforcing immune receptor signalling

Nickolas J. Serniuck, Eden Kapcan, Duane Moogk, Allyson E. Moore, Benjamin P.M. Lake, Galina Denisova, Joanne A. Hammill, Jonathan L. Bramson, Anthony F. Rullo,

Molecular Therapy: Oncology, 2024, 200842

https://doi.org/10.1016/j.omton.2024.200842

Proximity-induction of cell-cell interactions via small molecules represents an emerging field in basic and translational sciences. Covalent anchoring of these small molecules represents a useful chemical strategy to enforce proximity, however it remains largely unexplored for driving cell-cell interactions. In immunotherapeutic applications, bifunctional small molecules are attractive tools for inducing proximity between immune effector cells like T cells and tumor cells to induce tumoricidal function. We describe a two-component system comprised of electrophilic bifunctional small molecules and paired synthetic antigen receptors (SARs) that elicit T cell activation. The molecules, termed covalent immune recruiters (CIRs), were designed to affinity label and covalently engage SARs. We evaluated the utility of CIRs to direct anti-tumor function of human T cells engineered with three biologically distinct classes of SAR. Irrespective of the electrophilic chemistry, tumor targeting moiety, or SAR design, CIRs outperformed equivalent non-covalent bifunctional adapters, establishing a key role for covalency in maximizing functionality. We determined that covalent linkage enforced early T cell activation events in a manner that was dependent upon each SARs biology and signalling threshold. These results provide a platform to optimize universal SAR T cell functionality and more broadly reveal new insights into how covalent adapters modulate cell-cell proximity-induction.

Covalent Degrader of the Oncogenic Transcription Factor β-Catenin

 Flor A. Gowans, Nafsika Forte, Justin Hatcher, Oscar W. Huang, Yangzhi Wang, Belen E. Altamirano Poblano, Ingrid E. Wertz, and Daniel K. Nomura

Journal of the American Chemical Society 2024

DOI: 10.1021/jacs.4c05174

β-catenin (CTNNB1) is an oncogenic transcription factor that is important in cell–cell adhesion and transcription of cell proliferation and survival genes that drive the pathogenesis of many different types of cancers. However, direct pharmacological targeting of CTNNB1 has remained challenging. Here, we have performed a screen with a library of cysteine-reactive covalent ligands to identify the monovalent degrader EN83 that depletes CTNNB1 in a ubiquitin-proteasome-dependent manner. We show that EN83 directly and covalently targets CTNNB1 three cysteines C466, C520, and C619, leading to destabilization and degradation of CTNNB1. Through structural optimization, we generate a highly potent and relatively selective destabilizing degrader that acts through the targeting of only C619 on CTNNB1. Our results show that chemoproteomic approaches can be used to covalently target and degrade challenging transcription factors like CTNNB1 through destabilization-mediated degradation.