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.
Covalent Modifiers
A blog highlighting recent publications in the area of covalent modification of proteins, particularly relating to covalent-modifier drugs. @CovalentMod on Twitter, @covalentmod@mstdn.science on Mastodon, and @covalentmod.bsky.social on BlueSky
Tuesday, October 15, 2024
Redirecting the pioneering function of FOXA1 with covalent small molecules
Potent Inhibition and Rapid Photoactivation of Endogenous Bruton’s Tyrosine Kinase Activity in Native Cells via Opto-Covalent Modulators
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., 2024Reversible 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.
Saturday, October 12, 2024
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.
Thursday, October 10, 2024
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.
Wednesday, October 9, 2024
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.
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. Remsb...
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Linqi Cheng Yixian Wang, Yiming Guo, Sophie S. Zhang Han Xiao C ell Chemical Biology, 2024 Volume 31, 3, 428 - 445 https://doi.org/10.10...
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Nathalie M. Grob, Clint Remarcik, Simon L. Rössler, Jeffrey Y. K. Wong, John C. K. Wang, Jason Tao, Corey L. Smith, Andrei Loas, Stephen L. ...
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Guanghui Tang , Wei Wang , Chengjun Zhu , Huisi Huang , Peng Chen , Xuan Wang , Manyi Xu , Jie Sun , Chong-Jing Zhang , Qicai Xiao ...