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.