Mutant-selective AKT inhibition through lysine targeting and neo-zinc chelation

Gregory B. Craven, Hang Chu, Jessica D. Sun, Jordan D. Carelli, Brittany Coyne, Hao Chen, Ying Chen, Xiaolei Ma, Subhamoy Das, Wayne Kong, Adam D. Zajdlik, Kin S. Yang, Solomon H. Reisberg, Peter A. Thompson, J. Russell Lipford & Jack Taunton 

Nature, 2024

https://doi.org/10.1038/s41586-024-08176-4

Somatic alterations in the oncogenic kinase AKT1 have been identified in a broad spectrum of solid tumours. The most common AKT1 alteration replaces Glu17 with Lys (E17K) in the regulatory pleckstrin homology domain1, resulting in constitutive membrane localization and activation of oncogenic signalling. In clinical studies, pan-AKT inhibitors have been found to cause dose-limiting hyperglycaemia, which has motivated the search for mutant-selective inhibitors. We exploited the E17K mutation to design allosteric, lysine-targeted salicylaldehyde inhibitors with selectivity for AKT1 (E17K) over wild-type AKT paralogues, a major challenge given the presence of three conserved lysines near the allosteric site. Crystallographic analysis of the covalent inhibitor complex unexpectedly revealed an adventitious tetrahedral zinc ion that coordinates two proximal cysteines in the kinase activation loop while simultaneously engaging the E17K–imine conjugate. The salicylaldimine complex with AKT1 (E17K), but not that with wild-type AKT1, recruits endogenous Zn2+ in cells, resulting in sustained inhibition. A salicylaldehyde-based inhibitor was efficacious in AKT1 (E17K) tumour xenograft models at doses that did not induce hyperglycaemia. Our study demonstrates the potential to achieve exquisite residence-time-based selectivity for AKT1 (E17K) by targeting the mutant lysine together with Zn2+ chelation by the resulting salicylaldimine adduct.

Target Ligand Separation and Identification of Isoforsythiaside as a Histone Lysine-Specific Demethylase 1 Covalent Inhibitor Against Breast Cancer Metastasis

Mengzhen Gu, Xiaoqing Xu, Xiaoping Wang, Yun Wang, Yu Zhao, Xiaoxian Hu, Lu Zhu, Zhenzhong Deng, and Chao Han

Journal of Medicinal Chemistry 2024

DOI: 10.1021/acs.jmedchem.4c02277

Histone lysine-specific demethylase 1 (LSD1) is hyperactive in breast cancer, which is associated with the metastasis of the tumor. Current irreversible LSD1 inhibitors are all synthesized by covalently binding to the flavin adenine dinucleotide cofactor, which often have side effects due to the high affinity for a variety of targets. Here, we identified isoforsythiaside (IFA), a natural phenylpropanoid glycoside isolated from Forsythia suspensa, as a novel covalent inhibitor of LSD1. The target ligand fishing technique and LC–MS/MS analysis identified that IFA could covalently bind to the Ser817 residue of LSD1 by α,β-unsaturated ketone moiety to block the amine oxidase-like domain of LSD1. Moreover, RBMS3/Twist1/MMP2, the downstream signaling pathway of LSD1, was activated after IFA treatment to inhibit the metastasis of MDA-MB-231 cells in vitro and in vivo. This study provided novel molecular templates for development of LSD1 covalence-binding inhibitor and laid a foundation for developing agents against breast carcinoma metastasis for targeting LSD1.

Covalency in PROTACs: Mechanisms and applications [@RPNowak]

Thomas M. Geiger, Radosław P. Nowak

Annual Reports in Medicinal Chemistry, 2024

https://doi.org/10.1016/bs.armc.2024.10.001

Proteolysis targeting chimeras (PROTACs) are hetero-bifunctional molecules that remove disease-causing proteins through the means of targeted protein degradation (TPD). Since their proof-of-concept over 20 years ago, PROTACs emerged as new modality in drug discovery and chemical biology. Historically, the vast majority of PROTACs use reversible-binding recruiters for both target and E3 ligase. However, in recent years more covalent PROTACs have been developed to harness the advantages of covalency such as unlocking the “undruggable” proteome to expand the repertoire of addressable targets and recruitable E3 ligases. Here, we review recent advances in covalent PROTACs, discuss their distinct mechanism of action and outline the key differences of this approach.

A Practical Guide for the Assay-Dependent Characterisation of Irreversible Inhibitors

Lavleen Mader,   Jessica Borean  and  Jeffrey W Keillor

RSC Med. Chem. 2024 

DOI 10.1039/D4MD00707G

Irreversible targeted covalent inhibitors, in the past regarded as inappropriately reactive and toxic, have seen a recent resurgence in clinical interest. This paradigm shift is attributed to the exploitation of the two-step mechanism, in which a high affinity and selectivity (i.e., low KI) scaffold binds the target and only then does a pendant low intrinsic reactivity warhead react with the target (moderate kinact). This highlights the importance of evaluating inhibitors by deriving both their KI and kinact values. The development of methods to evaluate these inhibitors by accounting for their time-dependent nature has been crucial to the discovery of promising clinical candidates. Herein, we report all the practical kinetic methods available to date to derive kinact and KI values. These methods include direct observation of covalent modification, continuous assay (Kitz & Wilson) evaluation, and discontinuous incubation and pre-incubation time-dependent IC50 assays. We also provide practical guidelines and examples for performing these assays, comparison of their utility, and perspectives for their extended applications. This review aims to provide clarity about the use of these methods for reporting complete inhibitor kinetic profiles, guiding irreversible drug development towards increased target affinity and selectivity, while modulating in-vivo stability and on-target reactivity.

Pan-Transcriptional Enhanced Associated Domain Palmitoylation Pocket Covalent Inhibitor

Jinhyuk Kim, Hadong Kim, Jongwan Kim, Seon Yeon Cho, Sungho Moon, Youngki Yoo, Hanseong Kim, Jin Kwan Kim, Hyejin Jeon, Wan Namkung, Gyoonhee Han, and Kyoung Tai No

Journal of Medicinal Chemistry 2024

DOI: 10.1021/acs.jmedchem.4c01393

In the Hippo signaling pathway, the palmitoylated transcriptional enhanced associated domain (TEAD) protein interacts with the coactivator Yes-associated protein/PDZ-binding motif, leading to transcriptional upregulation of oncogenes such as Ctgf and Cyr61. Consequently, targeting the palmitoylation sites of TEAD has emerged as a promising strategy for treating TEAD-dependent cancers. Compound 1 was identified using a structure-based drug design approach, leveraging the molecular insights gained from the known TEAD palmitoylation site inhibitor, K-975. Optimization of the initial hit compound resulted in the development of compound 3, a covalent pan-TEAD inhibitor characterized by high potency and oral bioavailability.

Total Synthesis of Tagitinins, Goyazensolide and RelatedFuranoheliangolides and their Covalent Interaction withImportin-5 (IPO5)

W Liu, R Patouret, E Peev, S Barluenga, N Winssinger

Helvetica Chimica Acta, 2024 
https://doi.org/10.1002/hlca.202400122

Herein, we detail an extension of our research on the synthesis of a small library of furanoheliangolides and the characterization of the covalent interaction between goyazensolide and IPO5. Using a build‐couple‐pair strategy, we assembled a small library of germacrene‐type lactones and diversified them into eight groups of structurally different analogues. The germacrene lactones were synthesized using Sonogashira coupling and Barbier‐type macrocyclization, while the furanoheliangolides were further elaborated through gold‐catalyzed transannulation followed by esterification. This synthetic approach enabled the generation of a goyazensolide alkyne‐tagged cellular probe, which was used to identify the selective binding between goyazensolide and the oncoprotein importin‐5 (IPO5). Mass spectrometry analysis of the proteolytic digest from the reaction between the goyazensolide probe and a recombinant IPO5 indicated a covalent engagement at Cys560 of IPO5, which was confirmed by site‐directed mutagenesis.

Slow-Binding and Covalent HDAC Inhibition: A New Paradigm?

Yasir S. Raouf and Carlos Moreno-Yruela

JACS Au, 2024

DOI: 10.1021/jacsau.4c00828

The dysregulated post-translational modification of proteins is an established hallmark of human disease. Through Zn2+-dependent hydrolysis of acyl-lysine modifications, histone deacetylases (HDACs) are key regulators of disease-implicated signaling pathways and tractable drug targets in the clinic. Early targeting of this family of 11 enzymes (HDAC1–11) afforded a first generation of broadly acting inhibitors with medicinal applications in oncology, specifically in cutaneous and peripheral T-cell lymphomas and in multiple myeloma. However, first-generation HDAC inhibitors are often associated with weak-to-modest patient benefits, dose-limited efficacies, pharmacokinetic liabilities, and recurring clinical toxicities. Alternative inhibitor design to target single enzymes and avoid toxic Zn2+-binding moieties have not overcome these limitations. Instead, recent literature has seen a shift toward noncanonical mechanistic approaches focused on slow-binding and covalent inhibition. Such compounds hold the potential of improving the pharmacokinetic and pharmacodynamic profiles of HDAC inhibitors through the extension of the drug–target residence time. This perspective aims to capture this emerging paradigm and discuss its potential to improve the preclinical/clinical outlook of HDAC inhibitors in the coming years.

Discovery and development of Krazati (adagrasib/MRTX849), a potent, selective, orally bioavailable, covalent KRASG12C(OFF) inhibitor

Adrian L. Gill, Mathew A. Marx

RAS Drug Discovery Past, Present and Future 2025, 205-227

https://doi.org/10.1016/B978-0-443-21861-3.00017-6

Krazati (adagrasib/MRTX849) is a potent, selective, and covalent KRASG12C inhibitor, representing a significant breakthrough in directly targeting KRAS. Adagrasib demonstrates favorable drug-like properties, selectively modifying mutant cysteine 12 in GDP-bound KRASG12C, leading to the inhibition of KRAS-dependent signaling both in vitro and in vivo, and tumor regression in many KRASG12C-positive cell lines and patient-derived xenograft models across various tumor types. Objective responses have been observed in clinical trials, particularly in lung and colon adenocarcinoma patients with KRASG12C mutations. Comprehensive pharmacodynamic and pharmacogenomic profiling in both sensitive and partially resistant nonclinical models has shed light on the mechanisms limiting adagrasib antitumor activity. These resistance mechanisms include contributing factors related to KRAS nucleotide cycling, feedback reactivation pathways through activation of receptor tyrosine kinases, instances where tumors bypass KRAS dependence, and genetic dysregulation of the cell cycle. The ongoing characterization of adagrasib's activity, along with insights into response and resistance mechanisms, provides valuable understanding of KRAS dependence and opened a long-awaited opportunity to selectively target KRASG12C in patients. Furthermore, the identification of effective preclinical combinations, such as adagrasib with agents targeting RTKs, SHP2, mTOR, or the cell cycle, demonstrates enhanced responses and marked tumor regression. These findings contribute to the rational development of this class of agents, holding promise for improving therapeutic outcomes in KRASG12C-mutant human cancers.

Tuning isatoic anhydrides’ lysine ligation chemistry for bioconjugation and drug delivery

Tiwari, Sona, Senthil, Sathyapriya, Khanna, Shweta, Duraisamy, Santhosh, Vechalapu, Sai Kumari, Mallojjala, Sharath Chandra, Allimuthu, Dharmaraja,

Cell Reports Physical Science, 2024

DOI: https://doi.org/10.1016/j.xcrp.2024.102260

The discovery of new chemical entities for the selective modification of protein lysines is a recent interest in the development of unique covalent chemical probes. Isatoic anhydride (benzoxauracil), possessing aminophilic reactivity, was employed for the profiling of ligandable lysines in the cellular proteome. Our reactivity evaluation of benzoxauracil with proteins using mass spectral peptide mapping revealed a biased reactivity profile with nearly all the nucleophilic amino acids. The chemoselective reactivity of electrophilic tags is a key determinant of their idiosyncratic reactions. We applied the hard-soft-acid-base (HSAB) principle for tuning isatoic anhydride’s reactivity through systematic chemical modifications for lysine-dominant reactivity. We demonstrated the employability of ring-opening chemistry in isatoic anhydride as a drug delivery modality for the release of a small molecule and doxorubicin in cancer cells. Broadly, the tunable reactivity of isatoic anhydride could be leveraged for developing lysine-selective probes and drug delivery cargos.

CovalentInDB 2.0: an updated comprehensive database for structure-based and ligand-based covalent inhibitor design and screening

Hongyan Du, Xujun Zhang, Zhenxing Wu, Odin Zhang, Shukai Gu, Mingyang Wang, Feng Zhu, Dan Li, Tingjun Hou, Peichen Pan 

Nucleic Acids Research, 2024, gkae946

 https://doi.org/10.1093/nar/gkae946

The rational design of targeted covalent inhibitors (TCIs) has emerged as a powerful strategy in drug discovery, known for its ability to achieve strong binding affinity and prolonged target engagement. However, the development of covalent drugs is often challenged by the need to optimize both covalent warhead and non-covalent interactions, alongside the limitations of existing compound libraries. To address these challenges, we present CovalentInDB 2.0, an updated online database designed to support covalent drug discovery. This updated version includes 8303 inhibitors and 368 targets, supplemented by 3445 newly added cocrystal structures, providing detailed analyses of non-covalent interactions. Furthermore, we have employed an AI-based model to profile the ligandability of 144 864 cysteines across the human proteome. CovalentInDB 2.0 also features the largest covalent virtual screening library with 2 030 192 commercially available compounds and a natural product library with 105 901 molecules, crucial for covalent drug screening and discovery. To enhance the utility of these compounds, we performed structural similarity analysis and drug-likeness predictions. Additionally, a new user data upload feature enables efficient data contribution and continuous updates. CovalentInDB 2.0 is freely accessible at http://cadd.zju.edu.cn/cidb/.

Delineating cysteine-reactive compound modulation of cellular proteostasis processes

Ashley R. Julio, Flowreen Shikwana, Cindy Truong, Nikolas R. Burton, Emil R. Dominguez III, Alexandra C. Turmon, Jian Cao & Keriann M. Backus 

Nat Chem Biol 2024

https://doi.org/10.1038/s41589-024-01760-9

Covalent modulators and covalent degrader molecules have emerged as drug modalities with tremendous therapeutic potential. Toward realizing this potential, mass spectrometry-based chemoproteomic screens have generated proteome-wide maps of potential druggable cysteine residues. However, beyond these direct cysteine-target maps, the full scope of direct and indirect activities of these molecules on cellular processes and how such activities contribute to reported modes of action, such as degrader activity, remains to be fully understood. Using chemoproteomics, we identified a cysteine-reactive small molecule degrader of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nonstructural protein 14 (nsp14), which effects degradation through direct modification of cysteines in both nsp14 and in host protein disulfide isomerases. This degrader activity was further potentiated by generalized electrophile-induced global protein ubiquitylation, proteasome activation and widespread aggregation and depletion of host proteins, including the formation of stress granules. Collectively, we delineate the wide-ranging impacts of cysteine-reactive electrophilic compounds on cellular proteostasis processes.

Expanding the Library of Covalent Cysteine Cathepsin Probes Featuring Sulfoxonium Ylide Electrophiles

Bangyan Xu, Simon J. Mountford, Philip E. Thompson, and Laura E. Edgington-Mitchell

ACS Omega 2024

DOI: 10.1021/acsomega.4c07604

Covalent activity-based probes are invaluable tools to monitor protease activity in vitro and in vivo. We recently discovered that dimethyl sulfoxonium ylides (SYs) bind selectively to cysteine cathepsin proteases in a mechanism-dependent manner. Herein, we present the synthetic routes and characterization of an expanded library of SY probes with a greater diversity in recognition sequences. The probes exhibit a range of potency and selectivity for the cathepsin family members. We also investigated the impact of fluorophore positioning on probes bearing P1 lysine. When sulfonated cyanine 5 was attached via the lysine side chain, the resulting probe was selective for cathepsin S. When attached to the α-amine, with the side chain amine either free or Boc-protected, the probes reacted with both cathepsin S and X. Bulk in the P1 position is thus well tolerated by cathepsin S but not cathepsin X. We examined the impact of Cy5 sulfonation on probe properties, demonstrating that unsulfonated probes exhibit greater cellular uptake, which affects their relative selectivity. Finally, we demonstrated that SY probes exhibit minimal labeling of cathepsin S in freshly prepared lysates, but this increases during the prolonged incubation of lysates. This work extends our understanding of SY probes and informs future probe development.

Redirecting the pioneering function of FOXA1 with covalent small molecules

Sang Joon Won, Yuxiang Zhang, Christopher J. Reinhardt,Lauren M. Hargis, Nicole S. MacRae,Kristen E. DeMeester,Evert Njomen,Jarrett R. Remsberg,Bruno Melillo, Benjamin F. Cravatt, Michael A. Erb

Molecular Cell, 2024

https://www.cell.com/molecular-cell/abstract/S1097-2765(24)00780-9

Pioneer transcription factors (TFs) bind to and open closed chromatin, facilitating engagement by other regulatory factors involved in gene activation or repression. Chemical probes are lacking for pioneer TFs, which has hindered their mechanistic investigation in cells. Here, we report the chemical proteomic discovery of electrophilic compounds that stereoselectively and site-specifically bind the pioneer TF forkhead box protein A1 (FOXA1) at a cysteine (C258) within the forkhead DNA-binding domain. We show that these covalent ligands react with FOXA1 in a DNA-dependent manner and rapidly remodel its pioneer activity in prostate cancer cells reflected in redistribution of FOXA1 binding across the genome and directionally correlated changes in chromatin accessibility. Motif analysis supports a mechanism where the ligands relax the canonical DNA-binding preference of FOXA1 by strengthening interactions with suboptimal sequences in predicted proximity to C258. Our findings reveal a striking plasticity underpinning the pioneering function of FOXA1 that can be controlled by small molecules.

Potent Inhibition and Rapid Photoactivation of Endogenous Bruton’s Tyrosine Kinase Activity in Native Cells via Opto-Covalent Modulators

Weizhi Weng, Ping Zhang, and Zhengying Pan

Journal of the American Chemical Society 2024

DOI: 10.1021/jacs.4c06459

Naturally, kinases exert their activities in a highly regulated fashion. A number of ingenious approaches have been developed to artificially control kinase activity by external stimuli, such as the incorporation of unnatural amino acids or the fusion of additional protein domains; however, methods that directly modulate endogenous kinases in native cells are lacking. Herein, we present a facile and potent method that takes advantage of recent developments in targeted covalent inhibitors and rapid light-mediated uncaging chemistry. Using an important drug target, Bruton’s tyrosine kinase (BTK), as an example, these opto-covalent modulators successfully blocked the activity of endogenous BTK in native cells after simple incubation and washout steps. However, upon a few minutes of light irradiation, BTK activity was cleanly restored, and could be blocked again by conventional inhibitors. Promisingly, this photoactivation strategy easily worked in human peripheral blood mononuclear cells (hPBMCs).

 

New electrophiles targeting thiols in a reversible covalent manner

Xingyu Ma,   Manyi Xu,   Fengge Wang,  Tingting Hu,  Xinyuan Chena  and  Chong-Jing Zhang

Chem. Commun., 2024

DOI

https://doi.org/10.1039/D4CC04612A

Reversible covalent electrophiles with the advantages of both reversible and covalent interactions receive much attention in the fields of chemical biology and medicinal chemistry. Here, we report two electron-deficient olefins activated by amide and ester, amide-substituted acrylamide and methyl ester-substituted acrylamide, targeting thiols in a reversible covalent manner.

Identification of a cell-active chikungunya virus nsP2 protease inhibitor using a covalent fragment-based screening approach

Eric M. Merten  and John D. Sears  and Tina M. Leisner  and P. Brian Hardy  and Anirban Ghoshal  and Mohammad Anwar Hossain  and Kesatebrhan Haile Asressu  and Peter J. Brown  and Edwin G. Tse  and Michael A. Stashko  and Kelin Li  and Jacqueline L. Norris-Drouin  and Laura E. Herring  and Angie L. Mordant  and Thomas S. Webb  and Christine A. Mills  and Natalie K. Barker  and Zachary J. Streblow  and Sumera Perveen  and Cheryl H. Arrowsmith  and Rafael Miguez Couñago  and Jamie J. Arnold  and Craig E. Cameron  and Daniel N. Streblow  and Nathaniel J. Moorman  and Mark T. Heise  and Timothy M. Willson  and Konstantin I. Popov  and Kenneth H. Pearce 

Proc. Natl. Acad. Sci. U.S.A. 2024 121 (42) e2409166121

https://doi.org/10.1073/pnas.2409166121

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that has been responsible for numerous large-scale outbreaks in the last twenty years. Currently, there are no FDA-approved therapeutics for any alphavirus infection. CHIKV nonstructural protein 2 (nsP2), which contains a cysteine protease domain, is essential for viral replication, making it an attractive target for a drug discovery campaign. Here, we optimized a CHIKV nsP2 protease (nsP2pro) biochemical assay for the screening of a 6,120-compound cysteine-directed covalent fragment library. Using a 50% inhibition threshold, we identified 153 hits (2.5% hit rate). In dose–response follow-up, RA-0002034, a covalent fragment that contains a vinyl sulfone warhead, inhibited CHIKV nsP2pro with an IC50 of 58 ± 17 nM, and further analysis with time-dependent inhibition studies yielded a kinact /KI of 6.4 × 103 M−1s−1. LC-MS/MS analysis determined that RA-0002034 covalently modified the catalytic cysteine in a site-specific manner. Additionally, RA-0002034 showed no significant off-target reactivity in proteomic experiments or against a panel of cysteine proteases. In addition to the potent biochemical inhibition of CHIKV nsP2pro activity and exceptional selectivity, RA-0002034 was tested in cellular models of alphavirus infection and effectively inhibited viral replication of both CHIKV and related alphaviruses. This study highlights the identification and characterization of the chemical probe RA-0002034 as a promising hit compound from covalent fragment-based screening for development toward a CHIKV or pan-alphavirus therapeutic.

Reversible covalent c-Jun N-terminal kinase inhibitors targeting a specific cysteine by precision-guided Michael-acceptor warheads

Bálint, D., Póti, Á.L., Alexa, A. et al. 

Nat Commun 15, 8606 (2024). 

https://doi.org/10.1038/s41467-024-52573-2

There has been a surge of interest in covalent inhibitors for protein kinases in recent years. Despite success in oncology, the off-target reactivity of these molecules is still hampering the use of covalent warhead-based strategies. Herein, we disclose the development of precision-guided warheads to mitigate the off-target challenge. These reversible warheads have a complex and cyclic structure with optional chirality center and tailored steric and electronic properties. To validate our proof-of-concept, we modified acrylamide-based covalent inhibitors of c-Jun N-terminal kinases (JNKs). We show that the cyclic warheads have high resilience against off-target thiols. Additionally, the binding affinity, residence time, and even JNK isoform specificity can be fine-tuned by adjusting the substitution pattern or using divergent and orthogonal synthetic elaboration of the warhead. Taken together, the cyclic warheads presented in this study will be a useful tool for medicinal chemists for the deliberate design of safer and functionally fine-tuned covalent inhibitors.

Targeting a key protein-protein interaction surface on mitogen-activated protein kinases by a precision-guided warhead scaffold

Póti, Á.L., Bálint, D., Alexa, A. et al. 

Nat Commun 15, 8607 (2024). 

https://doi.org/10.1038/s41467-024-52574-1

For mitogen-activated protein kinases (MAPKs) a shallow surface—distinct from the substrate binding pocket—called the D(ocking)-groove governs partner protein binding. Screening of broad range of Michael acceptor compounds identified a double-activated, sterically crowded cyclohexenone moiety as a promising scaffold. We show that compounds bearing this structurally complex chiral warhead are able to target the conserved MAPK D-groove cysteine via reversible covalent modification and interfere with the protein-protein interactions of MAPKs. The electronic and steric properties of the Michael acceptor can be tailored via different substitution patterns. The inversion of the chiral center of the warhead can reroute chemical bond formation with the targeted cysteine towards the neighboring, but less nucleophilic histidine. Compounds bind to the shallow MAPK D-groove with low micromolar affinity in vitro and perturb MAPK signaling networks in the cell. This class of chiral, cyclic and enhanced 3D shaped Michael acceptor scaffolds offers an alternative to conventional ATP-competitive drugs modulating MAPK signaling pathways.

Development of an orally bioavailable covalent STING inhibitor

NIU, G.-H., Hsiao, W.-C., Lee, P.-H., Zheng, L.-G., Yang, Y.-S., Huang, W.-C., Hsieh, C.-C., Chiu, T.-Y., Wang, J.-Y., Chen, C.-P., Huang, C.-L., You, M.-S., Kuo, Y.-P., Wang, C.-M., Wen, Z.-H., Yu, G.-Y., Chen, C.-T., Chi, Y.-H., Tung, C.-W., Hsu, S.-C., Yeh, T.-K., Sung, P.-J., Zhang, M. M., and Tsou, L. K. 

ChemRxiv, 2024

https://doi.org/10.26434/chemrxiv-2024-62g35

Pharmacological inhibition of cGAS-STING-controlled innate immune pathway is an emerging therapeutic strategy for a myriad of inflammatory diseases, including autoimmune disease, ulcerative colitis, non-alcoholic fatty liver disease and aging-related neurodegeneration. Here we report GHN105 as an orally bioavailable covalent STING inhibitor. Late-stage diversification of the briarane-type diterpenoid excavatolide B allowed the installation of solubility-enhancing functional groups while enhancing its activity as a covalent STING inhibitor against multiple human STING variants, including the S154 variant responsible for a genetic autoimmune disease. Selectively engaging the membrane-proximal Cys91 residue of STING, GHN105 dose-dependently inhibited cGAS-STING signaling and type I interferon responses in cells and in vivo. Orally administered GHN105 exerted marked therapeutic efficacy and reversed key pathological features in a delayed-treatment acute colitis mouse model. Notably, we also showed that GHN105 covalently engaged STING in the colon tissues. Our study provided proof of concept that synthetic briarane analog GHN105 serves as a safe and orally active covalent STING inhibitor. With a growing number of chronic inflammatory diseases linked to aberrant STING activation, orally bioavailable STING inhibitors would benefit patients by lowering the infection risk from frequent injections while allowing long-term systemic administration.

Discovery of electrophilic degraders that exploit SNAr chemistry

Zhe Zhuang, Woong Sub Byun, Zuzanna Kozicka, Brendan G. Dwyer, Katherine A. Donovan, Zixuan Jiang, Hannah M. Jones, Dinah M. Abeja, Meredith N. Nix, Jianing Zhong, Mikołaj Słabicki, Eric S. Fischer, Benjamin L. Ebert, Nathanael S. Gray

bioRxiv 2024.09.25.615094; 

doi: https://doi.org/10.1101/2024.09.25.615094

Targeted covalent inhibition (TCI) and targeted protein degradation (TPD) have proven effective in pharmacologically addressing formerly ‘undruggable’ targets. Integration of both methodologies has resulted in the development of electrophilic degraders where recruitment of a suitable E3 ubiquitin ligase is achieved through formation of a covalent bond with a cysteine nucleophile. Expanding the scope of electrophilic degraders requires the development of electrophiles with tempered reactivity that enable selective ligase recruitment and reduce cross-reactivity with other cellular nucleophiles. In this study, we report the use of chemical moieties that enable nucleophilic aromatic substitution (SNAr) reactions in the rational design of electrophilic protein degraders. Appending an SNAr covalent warhead to several preexisting small molecule inhibitors transformed them into degraders, obviating the need for a defined E3 ligase recruiter. The SNAr covalent warhead is versatile; it can recruit various E3 ligases, including DDB1 and CUL4 associated factor 11 (DCAF11), DDB1 and CUL4 associated factor 16 (DCAF16), and possibly others. The incorporation of an SNAr covalent warhead into the BRD4 inhibitor led to the discovery of degraders with low picomolar degradation potency. Furthermore, we demonstrate the broad applicability of this approach through rational functional switching from kinase inhibitors into potent degraders.

Multi-tiered chemical proteomic maps of tryptoline acrylamide–protein interactions in cancer cells

Njomen, E., Hayward, R.E., DeMeester, K.E. et al. Multi-tiered chemical proteomic maps of tryptoline acrylamide–protein interactions in cancer cells. Nat. Chem. (2024). 

https://doi.org/10.1038/s41557-024-01601-1

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. However, the extent to which such residue-directed platforms fully assess the protein targets of electrophilic compounds in cells remains unclear. Here we evaluate a complementary protein-directed ABPP method that identifies proteins showing stereoselective reactivity with alkynylated, chiral electrophilic compounds—termed stereoprobes. Integration of protein- and cysteine-directed data from cancer cells treated with tryptoline acrylamide stereoprobes revealed generally well-correlated ligandability maps and highlighted features, such as protein size and the proteotypicity of cysteine-containing peptides, that explain gaps in each ABPP platform. In total, we identified stereoprobe binding events for >300 structurally and functionally diverse proteins, including compounds that stereoselectively and site-specifically disrupt MAD2L1BP interactions with the spindle assembly checkpoint complex leading to delayed mitotic exit in cancer cells.

Activity-Based Acylome Profiling with N-(Cyanomethyl)-N-(phenylsulfonyl)amides for Targeted Lysine Acylation and Post-Translational Control of Protein Function in Cells

Elizabeth M. Ryan, Michael A. Norinskiy, Amy K. Bracken, Emma E. Lueders, Xueer Chen, Qin Fu, Elizabeth T. Anderson, Sheng Zhang, and Mikail E. Abbasov

Journal of the American Chemical Society 2024

DOI: 10.1021/jacs.4c09073

Lysine acylations are ubiquitous and structurally diverse post-translational modifications that vastly expand the functional heterogeneity of the human proteome. Hence, the targeted acylation of lysine residues has emerged as a strategic approach to exert biomimetic control over the protein function. However, existing strategies for targeted lysine acylation in cells often rely on genetic intervention, recruitment of endogenous acylation machinery, or nonspecific acylating agents and lack methods to quantify the magnitude of specific acylations on a global level. In this study, we develop activity-based acylome profiling (ABAP), a chemoproteomic strategy that exploits elaborate N-(cyanomethyl)-N-(phenylsulfonyl)amides and lysine-centric probes for site-specific introduction and proteome-wide mapping of posttranslational lysine acylations in human cells. Harnessing this framework, we quantify various artificial acylations and rediscover numerous endogenous lysine acylations. We validate site-specific acetylation of target lysines and establish a structure–activity relationship for N-(cyanomethyl)-N-(phenylsulfonyl)amides in proteins from diverse structural and functional classes. We identify paralog-selective chemical probes that acetylate conserved lysines within interferon-stimulated antiviral RNA-binding proteins, generating de novo proteoforms with obstructed RNA interactions. We further demonstrate that targeted acetylation of a key enzyme in retinoid metabolism engenders a proteoform with a conformational change in the protein structure, leading to a gain-of-function phenotype and reduced drug potency. These findings underscore the versatility of our strategy in biomimetic control over protein function through targeted delivery and global profiling of endogenous and artificial lysine acylations, potentially advancing therapeutic modalities and our understanding of biological processes orchestrated by these post-translational modifications.

Isocyanides inhibit bacterial pathogens by covalent targeting of essential metabolic enzymes

Alexandra Geißler, Howard Junca , Andreas M. Kany , Lena J. Daumann, Anna K. H. Hirsch  Dietmar H. Pieper b and Stephan A. Sieber 

Chem. Sci., 2024, 15, 11946-11955

 https://doi.org/10.1039/D4SC01940G

Isonitrile natural products, also known as isocyanides, demonstrate potent antimicrobial activities, yet our understanding of their molecular targets remains limited. Here, we focus on the so far neglected group of monoisonitriles to gain further insights into their antimicrobial mode of action (MoA). Screening a focused monoisonitrile library revealed a potent S. aureus growth inhibitor with a different MoA compared to previously described isonitrile antibiotics. Chemical proteomics via competitive cysteine reactivity profiling, uncovered covalent modifications of two essential metabolic enzymes involved in the fatty acid biosynthetic process (FabF) and the hexosamine pathway (GlmS) at their active site cysteines. In-depth studies with the recombinant enzymes demonstrated concentration-dependent labeling, covalent binding to the catalytic site and corresponding functional inhibition by the isocyanide. Thermal proteome profiling and full proteome studies of compound-treated S. aureus further highlighted the destabilization and dysregulation of proteins related to the targeted pathways. Cytotoxicity and the inhibition of cytochrome P450 enzymes require optimization of the hit molecule prior to therapeutic application. The here described novel, covalent isocyanide MoA highlights the versatility of the functional group, making it a useful tool and out-of-the-box starting point for the development of innovative antibiotics.

Peptide and Protein Cysteine Modification Enabled by Hydrosulfuration of Ynamide

Changliu Wang, Zhenguang Zhao, Reem Ghadir, Dechun Yang, Zhenjia Zhang, Zhe Ding, Yuan Cao, Yuqing Li, Rosi Fassler, Dana Reichmann, Yujie Zhang, Yongli Zhao, Can Liu, Xiaobao Bi, Norman Metanis, and Junfeng Zhao

ACS Central Science 2024 10 (9), 1742-1754

DOI: 10.1021/acscentsci.4c01148

Efficient functionalization of peptides and proteins has widespread applications in chemical biology and drug discovery. However, the chemoselective and site-selective modification of proteins remains a daunting task. Herein, a highly efficient chemo-, regio-, and stereoselective hydrosulfuration of ynamide was identified as an efficient method for the precise modification of peptides and proteins by uniquely targeting the thiol group of cysteine (Cys) residues. This novel method could be facilely operated in aqueous buffer and was fully compatible with a wide range of proteins, including small model proteins and large full-length antibodies, without compromising their integrity and functions. Importantly, this reaction provides the Z-isomer of the corresponding conjugates exclusively with superior stability, offering a precise approach to peptide and protein therapeutics. The potential application of this method in peptide and protein chemical biology was further exemplified by Cys-bioconjugation with a variety of ynamide-bearing functional molecules such as small molecule drugs, fluorescent/affinity tags, and PEG polymers. It also proved efficient in redox proteomic analysis through Cys-alkenylation. Overall, this study provides a novel bioorthogonal tool for Cys-specific functionalization, which will find broad applications in the synthesis of peptide/protein conjugates.

An allosteric cyclin E-CDK2 site mapped by paralog hopping with covalent probes

Yuanjin Zhang, Zhonglin Liu, Marscha Hirschi, Oleg Brodsky, Eric Johnson, Sang Joon Won, Asako Nagata, Divya Bezwada, Matthew D. Petroski, Jaimeen D. Majmudar, Sherry Niessen, Todd VanArsdale, Adam M. Gilbert, Matthew M. Hayward, Al E. Stewart, Andrew R. Nager, Bruno Melillo & Benjamin F. Cravatt 

Nat Chem Biol, 2024 

https://doi.org/10.1038/s41589-024-01738-7

More than half of the ~20,000 protein-encoding human genes have paralogs. Chemical proteomics has uncovered many electrophile-sensitive cysteines that are exclusive to subsets of paralogous proteins. Here we explore whether such covalent compound–cysteine interactions can be used to discover ligandable pockets in paralogs lacking the cysteine. Leveraging the covalent ligandability of C109 in the cyclin CCNE2, we substituted the corresponding residue in paralog CCNE1 to cysteine (N112C) and found through activity-based protein profiling that this mutant reacts stereoselectively and site-specifically with tryptoline acrylamides. We then converted the tryptoline acrylamide–CCNE1-N112C interaction into in vitro NanoBRET (bioluminescence resonance energy transfer) and in cellulo activity-based protein profiling assays capable of identifying compounds that reversibly inhibit both the N112C mutant and wild-type CCNE1:CDK2 (cyclin-dependent kinase 2) complexes. X-ray crystallography revealed a cryptic allosteric pocket at the CCNE1:CDK2 interface adjacent to N112 that binds the reversible inhibitors. Our findings, thus, show how electrophile–cysteine interactions mapped by chemical proteomics can extend the understanding of protein ligandability beyond covalent chemistry.

Nuclear Receptors: A new mode of inhibition

1. Andrew D Huber, 

1. Taosheng Chen
 

eLife 2024, 13:e101446.

https://doi.org/10.7554/eLife.101446

The article presents the discussion on interaction of covalent inhibitors GW9662 and T0070907 with the peroxisome proliferator-activated receptor gamma (PPARγ), revealing new insights into how these compounds influence receptor activity. Topics include the role of PPARγ in regulating fatty tissue and glucose levels; and the effects of covalent inhibitors on PPARγ's ligand-binding site and its interaction with co-activators and co-repressors.

Selective Covalent Inhibiting JNK3 by Small Molecules for Parkinson's Diseases

Liang Ouyang, Wen Shuai, Panpan Yang, Huan Xiao, Yumeng Zhu, Faqian Bu, Aoxue Wang, Qiu Sun, Guan Wang

Angewandte Chemie 2024 e202411037

https://doi.org/10.1002/ange.202411037

c-Jun N-terminal kinases (JNKs) including JNK1/2/3 are key members of mitogen-activated protein kinase family. Wherein JNK3 is specifically expressed in brain and emerges as therapeutic target, especially for neurodegenerative diseases. However, developing JNK3 selective inhibitors as chemical probes to investigate its therapeutic potential in diseases remains challenging. Here, we adopted the covalent strategy for identifying JNK3-selective covalent inhibitorJC16I, with high inhibitory activity against JNK3. Despite targeting a conserved cysteine the vicinity of ATP pocket in JNK family, JC16I exerted a greater than 160-fold selectivity for JNK3 over JNK1/2. Importantly, even at low concentration, JC16I showed enhanced and long-lasting inhibition against cellular JNK3. In addition, its alkyne-containing probe JC-P1 could label JNK3 in SH-SY5Y cell lysate and living cells, with goodproteome-wide selectivity. Furthermore, JC16I selectively suppressed the abnormal activation of JNK3 signaling and sufficiently exhibited neuroprotective effect in Parkinson's diseases (PD) models. Overall, our findings highlight the potential of developing isoform-selective and cell-active JNK3 inhibitors by covalent drug design strategy targeting a conserved cysteine. This work not only provides a valuable chemical probe for JNK3-targeted investigations in vitro and in vivo but also opens new avenues for the treatment of PD.

Development of ketalized unsaturated saccharides as multifunctional cysteine-targeting covalent warheads

Dong, S., Huang, H., Li, J. et al.

Commun Chem 7, 201 (2024). 

https://doi.org/10.1038/s42004-024-01279-z

Multi-functional cysteine-targeting covalent warheads possess significant therapeutic potential in medicinal chemistry and chemical biology. Herein, we present novel unsaturated and asymmetric ketone (oxazolinosene) scaffolds that selectively conjugate cysteine residues of peptides and bovine serum albumin under normal physiological conditions. This unsaturated saccharide depletes GSH in NCI-H1299 cells, leading to anti-tumor effects in vitro. The acetyl group of the ketal moiety on the saccharide ring can be converted to other carboxylic acids in a one-pot synthesis. In this way, the loaded acid can be click-released during cysteine conjugation, making the oxazolinosene a potential multifunctional therapeutic agent. The reaction kinetic model for oxazolinosene conjugation to GSH is well established and was used to evaluate oxazolinosene reactivity. The aforementioned oxazolinosenes were stereoselectively synthesized via a one-step reaction of nitriles with saccharides and conveniently converted into a series of α, β-unsaturated ketone N-glycosides as prevalent synthetic building blocks. The reaction mechanisms of oxazolinosene synthesis were investigated through calculations and validated with control experiments. Overall, these oxazolinosenes can be easily synthesized and developed as cysteine-targeted covalent warheads carrying useful click-releasing groups.

From Mechanism-Based Retaining Glycosidase Inhibitors to Activity-Based Glycosidase Profiling

 Marta Artola, Johannes M. F. G. Aerts, Gijsbert A. van der Marel, Carme Rovira, Jeroen D. C. Codée, Gideon J. Davies, and Herman S. Overkleeft

Journal of the American Chemical Society 2024

DOI: 10.1021/jacs.4c08840

Activity-based protein profiling (ABPP) is an effective technology for the identification and functional annotation of enzymes in complex biological samples. ABP designs are normally directed to an enzyme active site nucleophile, and within the field of Carbohydrate-Active Enzymes (CAZymes), ABPP has been most successful for those enzymes that feature such a residue: retaining glycosidases (GHs). Several mechanism-based covalent and irreversible retaining GH inhibitors have emerged over the past sixty years. ABP designs based on these inhibitor chemistries appeared since the turn of the millennium, and we contributed to the field by designing a suite of retaining GH ABPs modeled on the structure and mode of action of the natural product, cyclophellitol. These ABPs enable the study of both exo- and endo-acting retaining GHs in human health and disease, for instance in genetic metabolic disorders in which retaining GHs are deficient. They are also finding increasing use in the study of GHs in gut microbiota and environmental microorganisms, both in the context of drug (de)toxification in the gut and that of biomass polysaccharide processing for future sustainable energy and chemistries. This account comprises the authors’ view on the history of mechanism-based retaining GH inhibitor design and discovery, on how these inhibitors served as blueprints for retaining GH ABP design, and on some current and future developments on how cyclophellitol-based ABPs may drive the discovery of retaining GHs and their inhibitors.

SuFEx Chemistry Enables Covalent Assembly of a 280-kDa 18-Subunit Pore-Forming Complex

Lee Schnaider, Sophia Tan, Pratik R. Singh, Floriana Capuano, Alistair J. Scott, Richard Hambley, Lei Lu, Hyunjun Yang, E. Jayne Wallace, Hyunil Jo, and William F. DeGrado

Journal of the American Chemical Society 2024

DOI: 10.1021/jacs.4c07920

Proximity-enhanced chemical cross-linking is an invaluable tool for probing protein–protein interactions and enhancing the potency of potential peptide and protein drugs. Here, we extend this approach to covalently stabilize large macromolecular assemblies. We used SuFEx chemistry to covalently stabilize an 18-subunit pore-forming complex, CsgG:CsgF, consisting of nine CsgG membrane protein subunits that noncovalently associate with nine CsgF peptides. Derivatives of the CsgG:CsgF pore have been used for DNA sequencing, which places high demands on the structural stability and homogeneity of the complex. To increase the robustness of the pore, we designed and synthesized derivatives of CsgF-bearing sulfonyl fluorides, which react with CsgG in very high yield to form a covalently stabilized CsgG:CsgF complex. The resulting pores formed highly homogeneous channels when added to artificial membranes. The high yield and rapid reaction rate of the SuFEx reaction prompted molecular dynamics simulations, which revealed that the SO2F groups in the initially formed complex are poised for nucleophilic reaction with a targeted Tyr. These results demonstrate the utility of SuFEx chemistry to structurally stabilize very large (here, 280 kDa) assemblies.

Targeted Protein Localization by Covalent 14–3–3 Recruitment

 Qian Shao, Tuong Nghi Duong, Inji Park, Lauren M. Orr, and Daniel K. Nomura

Journal of the American Chemical Society 2024

DOI: 10.1021/jacs.3c12389

14–3–3 proteins have a unique ability to bind and sequester a multitude of diverse phosphorylated signaling proteins and transcription factors. Many previous studies have shown that interactions of 14–3–3 with specific phosphorylated substrate proteins can be enhanced through small-molecule natural products or fully synthetic molecular glue interactions. However, enhancing 14–3–3 interactions with both therapeutically intractable transcription factor substrates and potential neo-substrates to sequester and inhibit their function remains elusive. One of the 14–3–3 proteins, 14–3–3σ or SFN, has cysteine C38 at the substrate-binding interface, near the sites where previous 14–3–3 molecular glues have been found to bind. In this study, we screen a fully synthetic cysteine-reactive covalent ligand library to identify molecular glues that enhance the interaction of 14–3–3σ with not only druggable transcription factors such as estrogen receptor (ERα) but also challenging oncogenic transcription factors such as YAP and TAZ, which are part of the Hippo transducer pathway. We identify a hit EN171 that covalently targets both C38 and C96 on 14–3–3 to enhance 14–3–3 interactions with ERα, YAP, and TAZ, leading to impaired estrogen receptor and Hippo pathway transcriptional activity. We further demonstrate that EN171 could not only be used as a molecular glue to enhance native protein interactions but could also be used as a covalent 14–3–3 recruiter in heterobifunctional molecules to sequester nuclear neo-substrates such as BRD4 and BLC6 into the cytosol. Overall, our study reveals a covalent ligand that acts as a novel 14–3–3 molecular glue for challenging transcription factors such as YAP and TAZ and demonstrates that these glues can be potentially utilized in heterobifunctional molecules to sequester nuclear neo-substrates out of the nucleus and into the cytosol to enable targeted protein localization.

Aminomethyl Salicylaldehydes Lock onto a Surface Lysine by Forming an Extended Intramolecular Hydrogen Bond Network

Jacqueline Weaver, Gregory B. Craven, Linh Tram, Hao Chen, and Jack Taunton

Journal of the American Chemical Society 2024

DOI: 10.1021/jacs.4c04314

The development of electrophilic ligands that rapidly modify specific lysine residues remains a major challenge. Salicylaldehyde-based inhibitors have been reported to form stable imine adducts with the catalytic lysine of protein kinases. However, the targeted lysine in these examples is buried in a hydrophobic environment. A key unanswered question is whether this strategy can be applied to a lysine on the surface of a protein, where rapid hydrolysis of the resulting salicylaldimine is more likely. Here, we describe a series of aminomethyl-substituted salicylaldehydes that target a fully solvated lysine on the surface of the ATPase domain of Hsp90. By systematically varying the orientation of the salicylaldehyde, we discovered ligands with long residence times, the best of which engages Hsp90 in a quasi-irreversible manner. Crystallographic analysis revealed a daisy-chain network of intramolecular hydrogen bonds in which the salicylaldimine is locked into position by the adjacent piperidine linker. This study highlights the potential of aminomethyl salicylaldehydes to generate conformationally stabilized, hydrolysis-resistant imines, even when the targeted lysine is far from the ligand binding site and is exposed to bulk solvent.

Discovery of a Covalent Inhibitor of Pro-Caspase-1 Zymogen Blocking NLRP3 Inflammasome Activation and Pyroptosis

Dongyi Cao, Ruiying Xi, Hongye Li, Zhonghui Zhang, Xiaoke Shi, Shanshan Li, Yujie Jin, Wanli Liu, Guolin Zhang, Xiaohua Liu, Shunxi Dong, Xiaoming Feng, and Fei Wang

Journal of Medicinal Chemistry 2024

DOI https://doi.org/10.1021/acs.jmedchem.4c01558

Caspase-1 plays a central role in innate immunity, as its activation by inflammasomes induces the production of proinflammatory cytokines and pyroptosis. However, specific inhibition of the enzymatic activity of this protease is not effective in suppressing inflammation, owing to its enzyme-independent function. Herein, we identified a cyclohexenyl isothiocyanate compound (CIB-1476) that potently inhibited caspase-1 activity and suppressed the assembly and activation of the NLRP3 inflammasome and gasdermin-D-mediated pyroptosis. Mechanistically, CIB-1476 directly targeted pro-caspase-1 as an irreversible covalent inhibitor by binding to Cys285 and Cys397, resulting in more durable anti-inflammatory effects in the suppression of enzyme-dependent IL-1β production and enzyme-independent nuclear factor κB activation. Chemoproteomic profiling demonstrated the engagement of CIB-1476 with caspase-1. CIB-1476 showed potent therapeutic effects by suppressing inflammasome activation in mice, which was abolished in Casp1–/– mice. These results warrant further development of CIB-1476 along with its analogues as a novel strategy for caspase-1 inhibitors.

Discovering Covalent Cyclic Peptide Inhibitors of 2 Peptidyl Arginine Deiminase 4 (PADI4) Using mRNA3 Display with a Genetically Encoded Electrophilic 4 Warhead

 Isabel Mathiesen, Ewen Calder, Simone Kunzelmann, Louise Walport 

ChemRxiv, 2024

https://doi.org/10.26434/chemrxiv-2024-w8nbl

Covalent drugs can achieve high potency with long dosing intervals. However, concerns remain about side-effects associated with off-target reactivity. Combining macrocyclic peptides with covalent warheads provides a solution to minimise off-target reactivity: the peptide enables highly specific target binding, positioning a weakly reactive warhead proximal to a suitable residue in the target. Here we demonstrate direct discovery of covalent cyclic peptides using encoded libraries containing a weakly electrophilic cysteine-reactive fluoroamidine warhead. We combine direct incorporation of the warhead into peptide libraries using the flexible in vitro translation system with a peptide selection approach that identifies only covalent target binders. Using this approach, we identify potent covalent inhibitors of the peptidyl arginine deiminase, PADI4 or PAD4, that react exclusively at the active site cysteine. We envisage this approach will enable covalent peptide inhibitor discovery for a range of related enzymes and expansion to alternative warheads in the future.

Electrophilic proximity-inducing synthetic adapters enhance universal T cell function by covalently enforcing immune receptor signaling [@RulloLab]

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 2024

DOI: 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 composed 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 signaling 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.