Chemoselective Stabilized Triphenylphosphonium Probes for Capturing Reactive Carbonyl Species and Regenerating Covalent Inhibitors with Acrylamide Warheads in Cellulo

 

Ai-Lin Chen, Zih-Jheng Lin, Hsiao-Yu Chang, and Tsung-Shing Andrew Wang

Journal of the American Chemical Society, 2025

https://pubs.acs.org/doi/10.1021/jacs.4c09727

Reactive carbonyl species (RCS) are important biomarkers of oxidative stress-related diseases because of their highly reactive electrophilic nature. Despite their potential as triggers for prodrug activation, selective labeling approaches for RCS remain limited. Here, we utilized triphenylphosphonium groups to chemoselectively capture RCS via an aqueous Wittig reaction, forming α,β-unsaturated carbonyls that enable further functionalization. We first designed native (light) and deuterated (heavy) probes to facilitate RCS metabolomic identification through distinct MS isotope patterns. This approach allowed us to capture and relatively quantify several endogenous RCS related to advanced lipoxidation/glycation end products (ALEs/AGEs). Second, we demonstrated that various endogenous RCS can trigger the in situ generation of acrylamide warheads of targeted covalent inhibitors (TCIs) with different substituents. These structural variations influence their protein binding profiles and consequently alter their cytotoxicity, which is beneficial for the development of inhibitor cocktails.

Probing the Protein Kinases' Cysteinome by Covalent Fragments

Guiqun Wang, Nico J. Seidler, Sandra Röhm, Yufeng Pan, Xiaojun Julia Liang, Lisa Haarer, Benedict-Tilman Berger, Saran Aswathaman Sivashanmugam, Valentin R. Wydra, Michael Forster, Stefan A. Laufer, Apirat Chaikuad, Matthias Gehringer, Stefan Knapp

Angew. Chem. Int. Ed. 2024, e202419736

https://doi.org/10.1002/anie.202419736

Protein kinases are important drug targets, yet specific inhibitors have been developed for only a fraction of the more than 500 human kinases. A major challenge in designing inhibitors for highly related kinases is selectivity. Unlike their non-covalent counterparts, covalent inhibitors offer the advantage of selectively targeting structurally similar kinases by modifying specific protein side chains, particularly non-conserved cysteines. Previously, covalent fragment screens yielded potent and selective compounds for individual kinases such as ERK1/2 but have not been applied to the broader kinome. Furthermore, many of the accessible cysteine positions have not been addressed so far. Here, we outline a generalizable approach to sample ATP-site cysteines with fragment-like covalent inhibitors. We present the development of a kinase-focused fragment library and its systematic screening against a curated selection of 47 kinases, with 60 active site-proximal cysteines using LC/MS and differential scanning fluorimetry (DSF) assays, followed by hit validation through various complementary techniques. Our findings expand the repertoire of targetable cysteines within protein kinases, provide insight into unique binding modes identified from crystal structures and deliver isoform-specific hits with promising profiles as starting points for the development of highly potent and selective covalent inhibitors.

Discovery of a Potent Triazole-Based Reversible Targeted Covalent Inhibitor of Cruzipain

Juan Pablo Cerutti, Lucas Abreu Diniz, Viviane Corrêa Santos, Salomé Catalina Vilchez Larrea, Guillermo Daniel Alonso, Rafaela Salgado Ferreira, Mario Alfredo Quevedo, and Wim Dehaen

ACS Medicinal Chemistry Letters 2024

DOI: 10.1021/acsmedchemlett.4c00460

Cruzipain (CZP) is an essential cysteine protease of Trypanosoma cruzi, the etiological agent of Chagas disease, and a promising druggable target. To date, no CZP inhibitors have reached clinical use, with research efforts mostly hampered by insufficient potency, limited target selectivity or lack of bioactivity translation from the isolated enzyme to the parasite in cellular environments. In this study, we report the design of SH-1, a 1,2,3-triazole-based targeted covalent inhibitor with nanomolar potency (IC50 = 28 nM) and null inhibition of human cathepsin L. SH-1 demonstrates bioactivity translation comparable to that of K777 (1–10 μM), a CZP inhibitor previously advanced to clinical trials. Experimental findings indicate that SH-1 forms a reversible covalent bond with Cys25 in CZP, while in silico and structure–activity relationship studies suggest that this interaction is guided by acid–base equilibrium dynamics. The potential of SH-1 for preclinical development is discussed alongside detailed structure–activity relationships for the further optimization of CZP inhibitors.

Identification of Novel Organo-Se BTSA-Based Derivatives as Potent, Reversible, and Selective PPARγ Covalent Modulators for Antidiabetic Drug Discovery

Fangyuan Chen, Qingmei Liu, Lei Ma, Cuishi Yan, Haiman Zhang, Zhi Zhou, and Wei Yi

Journal of Medicinal Chemistry 2024

DOI: 10.1021/acs.jmedchem.4c02803

Recent studies have identified selective peroxisome proliferator-activated receptor γ (PPARγ) modulators, which synergistically engage in the inhibition mechanism of PPARγ-Ser273 phosphorylation, as a promising approach for developing safer and more effective antidiabetic drugs. Herein, we present the design, synthesis, and evaluation of a new class of organo-Se compounds, namely, benzothiaselenazole-1-oxides (BTSAs), acting as potent, reversible, and selective PPARγ covalent modulators. Notably, 2n, especially (R)-2n, displayed a high binding affinity and superior antidiabetic effects with diminished side effects. This is mainly because it can reversibly form a unique covalent bond with the Cys285 residue in PPARγ-LBD. Further mechanistic investigations revealed that it manifested such desired pharmacological profiles primarily by effectively suppressing PPARγ-Ser273 phosphorylation, enhancing glucose metabolism, and selectively upregulating the expression of insulin-sensitive genes. Collectively, our results suggest that (R)-2n holds promise as a lead compound for treating T2DM and also provides an innovative reversible covalent warhead reference for future covalent drug design.

Covalent Proximity Inducers

Nir London

Chemical Reviews Article ASAP

DOI: 10.1021/acs.chemrev.4c00570

Molecules that are able to induce proximity between two proteins are finding ever increasing applications in chemical biology and drug discovery. The ability to introduce an electrophile and make such proximity inducers covalent can offer improved properties such as selectivity, potency, duration of action, and reduced molecular size. This concept has been heavily explored in the context of targeted degradation in particular for bivalent molecules, but recently, additional applications are reported in other contexts, as well as for monovalent molecular glues. This is a comprehensive review of reported covalent proximity inducers, aiming to identify common trends and current gaps in their discovery and application.

Discovery and biological evaluation of potent 2-trifluoromethyl acrylamide warhead-containing inhibitors of protein disulfide isomerase

Jung-Chun Chu, Keng-Chang Tsai, Ting-Yu Wang, Tzu-Yin Chen, Ju-Ying Tsai, Tien Lee, Mei-Hsiang Lin, Yves S.Y. Hsieh, Chin-Chung Wu, Wei-Jan Huang,

European Journal of Medicinal Chemistry, 283, 2025, 117169

https://doi.org/10.1016/j.ejmech.2024.117169

Protein disulfide isomerase (PDI) regulates multiple protein functions by catalyzing the oxidation, reduction, and isomerization of disulfide bonds. The enzyme is considered a potential target for treating thrombosis. We previously developed a potent PDI inhibitor, CPD, which contains the propiolamide as a warhead targeting cysteine residue in PDI. To address its issues with undesirable off-target effects and weak metabolic stability, we replaced the propiolamide group with various electrophiles. Among these, compound 2d, which contains 2-trifluoromethyl acrylamide exhibited potent PDI inhibition compared to the reference PACMA31. Further structural optimization of compound 2d led to a novel series of 2-trifluoromethyl acrylamide derivatives. Several of these compounds displayed substantially improved enzyme inhibition. Notably, compound 14d demonstrated the strongest inhibition against PDI, with an IC50 value of 0.48 ± 0.004 μM. Additionally, compound 14d was found to exhibit a reversible binding mode with PDI enzyme. Further biological evaluations show that 14d suppressed platelet aggregation and thrombus formation by attenuating GPIIb/IIIa activation without significantly causing cytotoxicity. Altogether, these findings suggest PDI inhibitors could be a potential strategy for anti-thrombosis.

Covalent inhibitors of the RAS binding domain of PI3Ka impair tumor growth driven by RAS and HER2

Joseph E Klebba, Nilotpal Roy, Steffen M Bernard, Stephanie Grabow, Melissa A. Hoffman, Hui Miao, Junko Tamiya, Jinwei Wang, Cynthia Berry, Antonio Esparza-Oros, Richard Lin, Yongsheng Liu, Marie Pariollaud, Holly Parker, Igor Mochalkin, Sareena Rana, Aaron N. Snead, Eric J. Walton, Taylor E. Wyrick, Erick Aitichson, Karl Bedke, Jacyln C. Brannon, Joel M. Chick, Kenneth Hee, Benjamin D. Horning, Mohamed Ismail, Kelsey N. Lamb, Wei Lin, Justine Metzger, Martha K. Pastuszka, Jonathan Pollock, John J. Sigler, Mona Tomaschko, Eileen Tran, Todd M. Kinsella, Miriam Molina-Arcas, Gabriel M. Simon, David S. Weinstein, Julian Downward, Matthew P. Patricelli

bioRxiv 2024.12.17.629001; 

doi: https://doi.org/10.1101/2024.12.17.629001

Genetic disruption of the RAS binding domain (RBD) of PI 3-kinase (PI3K) prevents the growth of mutant RAS driven tumors in mice and does not impact PI3Ks role in insulin mediated control of glucose homeostasis. Selectively blocking the RAS-PI3K interaction may represent an attractive strategy for treating RAS-dependent cancers as it would avoid the toxicity associated with inhibitors of PI3K lipid kinase activity such as alpelisib. Here we report compounds that bind covalently to cysteine 242 in the RBD of PI3K p110a and block the ability of RAS to activate PI3K activity. These inhibitors have a profound impact on the growth of RAS mutant and also HER2 over-expressing tumors, particularly when combined with other inhibitors of the RAS/MAPK pathway, without causing hyperglycemia.

Exploring Extended Warheads toward Developing Cysteine-Targeted Covalent Kinase Inhibitors

Zheng Zhao and Philip E. Bourne

Journal of Chemical Information and Modeling 2024

DOI: 10.1021/acs.jcim.4c00890

In designing covalent kinase inhibitors (CKIs), the inclusion of electrophiles as attacking warheads demands careful choreography, ensuring not only their presence on the scaffold moiety but also their precise interaction with nucleophiles in the binding sites. Given the limited number of known electrophiles, exploring adjacent chemical space to broaden the palette of available electrophiles capable of covalent inhibition is desirable. Here, we systematically analyze the characteristics of warheads and the corresponding adjacent fragments for use in CKI design. We first collect all the released cysteine-targeted CKIs from multiple databases and create one CKI data set containing 16,961 kinase-inhibitor data points from 12,381 unique CKIs covering 146 kinases with accessible cysteines in their binding pockets. Then, we analyze this data set, focusing on the extended warheads (i.e., warheads + adjacent fragments)─including 30 common warheads and 1344 unique adjacent fragments. In so doing, we provide structural insights and delineate chemical properties and patterns in these extended warheads. Notably, we highlight the popular patterns observed within reversible CKIs for the popular warheads cyanoacrylamide and aldehyde. This study provides medicinal chemists with novel insights into extended warheads and a comprehensive source of adjacent fragments, thus guiding the design, synthesis, and optimization of CKIs.

Structure-Based Discovery of a Series of Covalent, Orally Bioavailable, and Selective BFL1 Inhibitors

Adeline Palisse, Tony Cheung, Aileen Blokhuis, Thomas Cogswell, Bruna S. Martins, Rick Riemens, Rick Schellekens, Giovanni Battocchio, Chimed Jansen, Matthew A. Cottee, Kimberly Ornell, Claudia Sacchetto, Leonardo Leon, Maaike van Hoek- Emmelot, Mark Bostock, Brooke Leann Brauer, Kevin Beaumont, Simon C. C. Lucas, Samiyah Ahmed, J. Henry Blackwell, Ulf Börjesson, Andrea Gohlke, Iva Monique T. Gramatikov, David Hargreaves, Vera van Hoeven, Vasudev Kantae, Lea Kupcova, Alexander G. Milbradt, Uthpala Seneviratne, Nancy Su, John Vales, Haiyun Wang, Michael J. White, and Olaf Kinzel

Journal of Medicinal Chemistry 2024

DOI: 10.1021/acs.jmedchem.4c01995

BFL1, a member of the antiapoptotic BCL2 family, has been relatively understudied compared to its counterparts despite evidence of its overexpression in various hematological malignancies. Across two articles, we describe the development of BFL1 in vivo tools. The first article describes the hit identification from a covalent fragment library and the subsequent evolution from the hit to compound 6.22 This work reports the structure-based optimization of compound 6 into a series of BFL1 inhibitors selective over the other BCL2 family members, with low nanomolar cellular activity when combined with AZD5991, exemplified by compound 20. Compound 20 demonstrated a cell death phenotype in SUDHL1 and OCILY10 cell lines and in the in vivo study, BFL1 stabilization and cleaved caspase 3 activation were observed in a dose-dependent manner. In addition, the enzymatic turnover studies with the BFL1 protein showed that compound 20 stabilized the protein, extending the half-life to 10.8 h.

Open-source Electrophilic Fragment Screening Platform to Identify Chemical Starting Points for UCHL1 Covalent Inhibitors

Scott B. Ficarro, Zachary H. Marto, Nicholas M. Girardi, Dingyu Deng, Isabella Jaen Maisonet, Guillaume Adelmant, Laura E. Fleming, Mona Sharafi, Isidoro Tavares, Andrew Zhao, HyoJeon Kim, Hyuk-Soo Seo, Sirano Dhe-Paganon, Sara J. Buhrlage, Jarrod A. Marto

SLAS Discovery, 2024

https://doi.org/10.1016/j.slasd.2024.100198

Target-based screening of covalent fragment libraries with mass spectrometry has emerged as a powerful strategy to identify chemical starting points for small molecule inhibitors or find new binding pockets on proteins of interest. These libraries span diverse chemical space with a modest number of compounds. Screening covalent fragments against purified protein targets reduces the demands on the mass spectrometer with respect to absolute throughput, detection limit, and dynamic range. Given these relaxed analytical requirements, we sought to develop an open-source, medium-throughput mass spectrometry system for target-based covalent fragment screening. Our platform comprises automated, dual LC desalting columns integrated with electrospray ionization for rapid sample introduction and mass spectrometry detection. The system is operated through a simple python graphical user interface running on commodity microcontroller boards which allow integration with diverse liquid chromatography and mass spectrometry instruments. We provide scripts for fragment pooling, construction of sample batches, along with routines for data processing and visualization. The system enables primary screening of ∼10,000 covalent fragments per day in pooled format. In a proof-of-concept study we executed primary and secondary screens to identify 27 hit fragments against UCHL1, a deubiquitinating enzyme that is emerging as a drug target of interest across multiple clinical indications. We validated and triaged these covalent compounds through a series of orthogonal biochemical and chemoproteomic assays. The most promising chloroacetamide covalent fragment inhibited UCHL1 activity in vitro (IC50 <5 µM) and exhibited dose-dependent binding along with good selectivity against 57 cellular DUBs as quantified by activity-based protein profiling.