Structure-based design of a phosphotyrosine-masked covalent ligand targeting the E3 ligase SOCS2

Ramachandran, S., Makukhin, N., Haubrich, K. et al.

 Nat Commun 14, 6345 (2023).

https://doi.org/10.1038/s41467-023-41894-3

The Src homology 2 (SH2) domain recognizes phosphotyrosine (pY) post translational modifications in partner proteins to trigger downstream signaling. Drug discovery efforts targeting the SH2 domains have long been stymied by the poor drug-like properties of phosphate and its mimetics. Here, we use structure-based design to target the SH2 domain of the E3 ligase suppressor of cytokine signaling 2 (SOCS2). Starting from the highly ligand-efficient pY amino acid, a fragment growing approach reveals covalent modification of Cys111 in a co-crystal structure, which we leverage to rationally design a cysteine-directed electrophilic covalent inhibitor MN551. We report the prodrug MN714 containing a pivaloyloxymethyl (POM) protecting group and evidence its cell permeability and capping group unmasking using cellular target engagement and in-cell 19F NMR spectroscopy. Covalent engagement at Cys111 competitively blocks recruitment of cellular SOCS2 protein to its native substrate. The qualified inhibitors of SOCS2 could find attractive applications as chemical probes to understand the biology of SOCS2 and its CRL5 complex, and as E3 ligase handles in proteolysis targeting chimera (PROTACs) to induce targeted protein degradation.

Exploration of the Tunability of BRD4 Degradation by DCAF16 Trans-labelling Covalent Glues

Muhammad Murtaza Hassan, Yen-Der Li, Michelle W. Ma, Mingxing Teng, Woong Sub Byun, Kedar Puvar, Ryan Lumpkin, Brittany Sandoval, Justine C. Rutter, Cyrus Y. Jin, Michelle Y. Wang, Shawn Xu, Anna M. Schmoker, Hakyung Cheong, Brian J. Groendyke, Jun Qi, Eric S Fischer, Benjamin L. Ebert, Nathanael Gray

bioRxiv 2023

https://www.biorxiv.org/content/10.1101/2023.10.07.561308v1

Small molecules that can induce protein degradation by inducing proximity between a desired target and an E3 ligase have the potential to greatly expand the number of proteins that can be manipulated pharmacologically. Current strategies for targeted protein degradation are mostly limited in their target scope to proteins with preexisting ligands. Alternate modalities such as molecular glues, as exemplified by the glutarimide class of ligands for the CUL4CRBN ligase, have been mostly discovered serendipitously. We recently reported a trans-labelling covalent glue mechanism which we named Template-assisted covalent modification, where an electrophile decorated small molecule binder of BRD4 was effectively delivered to a cysteine residue on an E3 ligase DCAF16 as a consequence of a BRD4-DCAF16 protein-protein interaction. Herein, we report our medicinal chemistry efforts to evaluate how various electrophilic modifications to the BRD4 binder, JQ1, affect DCAF16 trans-labeling and subsequent BRD4 degradation efficiency. We discovered a decent correlation between the ability of the electrophilic small molecule to induce ternary complex formation between BRD4 and DCAF16 with its ability to induce BRD4 degradation. Moreover, we show that a more solvent-exposed warhead presentation is optimal for DCAF16 recruitment and subsequent BRD4 degradation. Unlike the sensitivity of CUL4CRBN glue degraders to chemical modifications, the diversity of covalent attachments in this class of BRD4 glue degraders suggests a high tolerance and tunability for the BRD4-DCAF16 interaction. This offers a potential new avenue for a rational design of covalent glue degraders by introducing covalent warheads to known binders.

Biomimetic Synthesis and Chemical Proteomics Reveal the Mechanism of Action and Functional Targets of Phloroglucinol Meroterpenoids [@Abbasov_Cornell]

Bracken, A.; Gekko, C.; Suss, N.; Lueders, E.; Cui, Q.; Fu, Q.; Anderson, E.; Zhang, S.; Abbasov, M. 

ChemRxiv 2023

https://doi.org/10.26434/chemrxiv-2023-snx9h

Natural products perennially serve as prolific sources of drug leads and chemical probes, fueling the development of numerous therapeutics. Despite their scarcity, natural products that modulate protein function through covalent interactions with lysine residues hold immense potential to unlock new therapeutic interventions and advance our understanding of the biological processes governed by these modifications. Phloroglucinol meroterpenoids constitute one of the most expansive classes of natural products, displaying a plethora of biological activities. However, their mechanism of action and cellular targets have, until now, remained elusive. In this study, we detail the concise biomimetic synthesis, computational mechanistic insights, physicochemical attributes, kinetic parameters, molecular mechanism of action, and functional cellular targets of several phloroglucinol meroterpenoids. We harness synthetic clickable analogues of natural products to probe their disparate proteome-wide reactivity and subcellular localization through in-gel fluorescence scanning and cell imaging. By implementing sample multiplexing and a redesigned lysine-targeting probe, we streamline a quantitative activity-based protein profiling, enabling the direct mapping of global reactivity and ligandability of proteinaceous lysines in human cells. Leveraging this framework, we identify numerous lysine-meroterpenoid interactions in breast cancer cells at tractable protein sites across diverse structural and functional classes, including those historically deemed undruggable. We validate that phloroglucinol meroterpenoids perturb biochemical functions through stereoselective and site-specific modification of lysines in proteins vital for breast cancer metabolism, including lipid signaling, mitochondrial respiration, and glycolysis. These findings underscore the broad potential of phloroglucinol meroterpenoids for targeting functional lysines in the human proteome 

Advancing protein therapeutics through proximity-induced chemistry

Linqi Cheng, Yixian Wang, Yiming Guo, Sophie S. Zhang, Han Xiao,

Cell Chemical Biology, 2023

https://doi.org/10.1016/j.chembiol.2023.09.004

Recent years have seen a remarkable growth in the field of protein-based medical treatments. Nevertheless, concerns have arisen regarding the cytotoxicity limitations, low affinity, potential immunogenicity, low stability, and challenges to modify these proteins. To overcome these obstacles, proximity-induced chemistry has emerged as a next-generation strategy for advancing protein therapeutics. This method allows site-specific modification of proteins with therapeutic agents, improving their effectiveness without extensive engineering. In addition, this innovative approach enables spatial control of the reaction based on proximity, facilitating the formation of irreversible covalent bonds between therapeutic proteins and their targets. This capability becomes particularly valuable in addressing challenges such as the low affinity frequently encountered between therapeutic proteins and their targets, as well as the limited availability of small molecules for specific protein targets. As a result, proximity-induced chemistry is reshaping the field of protein drug preparation and propelling the revolution in novel protein therapeutics.

Covalent fragment approaches targeting non-cysteine residues

Noémi Csorba,Péter Ábrányi-Balogh,György M. Keserű

Trends in Pharmacological Sciences, 2023

https://doi.org/10.1016/j.tips.2023.08.014

Covalent fragment approaches combine advantages of covalent binders and fragment-based drug discovery (FBDD) for target identification and validation. Although early applications focused mostly on cysteine labeling, the chemistries of available warheads that target other orthosteric and allosteric protein nucleophiles has recently been extended. The range of different warheads and labeling chemistries provide unique opportunities for screening and optimizing warheads necessary for targeting non-cysteine residues. In this review, we discuss these recently developed amino-acid-specific and promiscuous warheads, as well as emerging labeling chemistries, which includes novel transition metal catalyzed, photoactive, electroactive, and noncatalytic methodologies. We also highlight recent applications of covalent fragments for the development of molecular glues and proteolysis-targeting chimeras (PROTACs), and their utility in chemical proteomics-based target identification and validation.

Chemical Specification of E3 Ubiquitin Ligase Engagement by Cysteine-Reactive Chemistry

Roman C. Sarott, Inchul You, Yen-Der Li, Sean T. Toenjes, Katherine A. Donovan, Pooreum Seo, Martha Ordonez, Woong Sub Byun, Muhammad Murtaza Hassan, Franziska Wachter, Edward T. Chouchani, Mikołaj Słabicki, Eric S. Fischer, Benjamin L. Ebert, Stephen M. Hinshaw, and Nathanael S. Gray

Journal of the American Chemical Society Article ASAP

DOI: 10.1021/jacs.3c06622

Targeted protein degradation relies on small molecules that induce new protein–protein interactions between targets and the cellular protein degradation machinery. Most of these small molecules feature specific ligands for ubiquitin ligases. Recently, the attachment of cysteine-reactive chemical groups to pre-existing small molecule inhibitors has been shown to drive specific target degradation. We demonstrate here that different cysteine-reactive groups can specify target degradation via distinct ubiquitin ligases. By focusing on the bromodomain ligand JQ1, we identify cysteine-reactive functional groups that drive BRD4 degradation by either DCAF16 or DCAF11. Unlike proteolysis-targeting chimeric molecules (PROTACs), the new compounds use a single small molecule ligand with a well-positioned cysteine-reactive group to induce protein degradation. The finding that nearly identical compounds can engage multiple ubiquitination pathways suggests that targeting cellular pathways that search for and eliminate chemically reactive proteins is a feasible avenue for converting existing small molecule drugs into protein degrader molecules.

Assigning functionality to cysteines by base editing of cancer dependency genes [@davidrliu]

Haoxin Li, Tiantai Ma, Jarrett R. Remsberg, Sang Joon Won, Kristen E. DeMeester, Evert Njomen, Daisuke Ogasawara, Kevin T. Zhao, Tony P. Huang, Bingwen Lu, Gabriel M. Simon, Bruno Melillo, Stuart L. Schreiber, Jens Lykke-Andersen, David R. Liu & Benjamin F. Cravatt 

Nat Chem Biol, 2023

https://doi.org/10.1038/s41589-023-01428-w

Covalent chemistry represents an attractive strategy for expanding the ligandability of the proteome, and chemical proteomics has revealed numerous electrophile-reactive cysteines on diverse human proteins. Determining which of these covalent binding events affect protein function, however, remains challenging. Here we describe a base-editing strategy to infer the functionality of cysteines by quantifying the impact of their missense mutation on cancer cell proliferation. The resulting atlas, which covers more than 13,800 cysteines on more than 1,750 cancer dependency proteins, confirms the essentiality of cysteines targeted by covalent drugs and, when integrated with chemical proteomic data, identifies essential, ligandable cysteines in more than 160 cancer dependency proteins. We further show that a stereoselective and site-specific ligand targeting an essential cysteine in TOE1 inhibits the nuclease activity of this protein through an apparent allosteric mechanism. Our findings thus describe a versatile method and valuable resource to prioritize the pursuit of small-molecule probes with high function-perturbing potential.