The c-Jun N-terminal kinases (JNKs) are members of the mitogen-activated protein kinase (MAPK) family, which includes JNK1–JNK3. Interestingly, JNK1 and JNK2 show opposing functions, with JNK2 activity favoring cell survival and JNK1 stimulating apoptosis. Isoform-selective small molecule inhibitors of JNK1 or JNK2 would be useful as pharmacological probes but have been difficult to develop due to the similarity of their ATP binding pockets. Here, we describe the discovery of a covalent inhibitor YL5084, the first such inhibitor that displays selectivity for JNK2 over JNK1. We demonstrated that YL5084 forms a covalent bond with Cys116 of JNK2, exhibits a 20-fold higher Kinact/KI compared to that of JNK1, and engages JNK2 in cells. However, YL5084 exhibited JNK2-independent antiproliferative effects in multiple myeloma cells, suggesting the existence of additional targets relevant in this context. Thus, although not fully optimized, YL5084 represents a useful chemical starting point for the future development of JNK2-selective chemical probes.
Monday, February 27, 2023
Narek Darabedian, Wenzhi Ji, Mengyang Fan, Shan Lin, Hyuk-Soo Seo, Ekaterina V. Vinogradova, Tomer M. Yaron, Evanna L. Mills, Haopeng Xiao, Kristine Senkane, Emily M. Huntsman, Jared L. Johnson, Jianwei Che, Lewis C. Cantley, Benjamin F. Cravatt, Sirano Dhe-Paganon, Kimberly Stegmaier, Tinghu Zhang, Nathanael S. Gray & Edward T. Chouchan
Nat Chem Biol., 2023
Creatine kinases (CKs) provide local ATP production in periods of elevated energetic demand, such as during rapid anabolism and growth. Thus, creatine energetics has emerged as a major metabolic liability in many rapidly proliferating cancers. Whether CKs can be targeted therapeutically is unknown because no potent or selective CK inhibitors have been developed. Here we leverage an active site cysteine present in all CK isoforms to develop a selective covalent inhibitor of creatine phosphagen energetics, CKi. Using deep chemoproteomics, we discover that CKi selectively engages the active site cysteine of CKs in cells. A co-crystal structure of CKi with creatine kinase B indicates active site inhibition that prevents bidirectional phosphotransfer. In cells, CKi and its analogs rapidly and selectively deplete creatine phosphate, and drive toxicity selectively in CK-dependent acute myeloid leukemia. Finally, we use CKi to uncover an essential role for CKs in the regulation of proinflammatory cytokine production in macrophages.
Wednesday, February 22, 2023
2-Ethynylbenzaldehyde-Based, Lysine-Targeting Irreversible Covalent Inhibitors for Protein Kinases and Nonkinases
Lysine-targeting irreversible covalent inhibitors have attracted growing interests in recent years, especially in the fields of kinase research. Despite encouraging progress, few chemistries are available to develop inhibitors that are exclusively lysine-targeting, selective, and cell-active. We report herein a 2-ethynylbenzaldehyde (EBA)-based, lysine-targeting strategy to generate potent and selective small-molecule inhibitors of ABL kinase by selectively targeting the conserved catalytic lysine in the enzyme. We showed the resulting compounds were cell-active, capable of covalently engaging endogenous ABL kinase in K562 cells with long-residence time and few off-targets. We further validated the generality of this strategy by developing EBA-based irreversible inhibitors against EGFR (a kinase) and Mcl-1 (a nonkinase) that covalently reacted with the catalytic and noncatalytic lysine within each target.
Thursday, February 16, 2023
Template-assisted covalent modification of DCAF16 underlies activity of BRD4 molecular glue degraders
Yen-Der Li, Michelle W Ma, Muhammad Murtaza Hassan, Moritz Hunkeler, Mingxing Teng, Kedar Puvar, Ryan Lumpkin, Brittany Sandoval, Cyrus Y Jin, Scott B Ficarro, Michelle Y Wang, Shawn Xu, Brian J Groendyke, Logan H Sigua, Isidoro Tavares, Charles Zou, Jonathan M Tsai, Paul M C Park, Hojong Yoon, Felix C Majewski, Jarrod A Marto, Jun Qi, Radoslaw P Nowak, Katherine A Donovan, Mikolaj Slabicki, Nathanael S Gray, Eric S Fischer, Benjamin L Ebert
Small molecules that induce protein-protein interactions to exert proximity-driven pharmacology such as targeted protein degradation are a powerful class of therapeutics. Molecular glues are of particular interest given their favorable size and chemical properties and represent the only clinically approved degrader drugs. The discovery and development of molecular glues for novel targets, however, remains challenging. Covalent strategies could in principle facilitate molecular glue discovery by stabilizing the neo-protein interfaces. Here, we present structural and mechanistic studies that define a trans-labeling covalent molecular glue mechanism, which we term "template-assisted covalent modification". We found that a novel series of BRD4 molecular glue degraders act by recruiting the CUL4DCAF16 ligase to the second bromodomain of BRD4 (BRD4BD2). BRD4BD2, in complex with DCAF16, serves as a structural template to facilitate covalent modification of DCAF16, which stabilizes the BRD4-degrader-DCAF16 ternary complex formation and facilitates BRD4 degradation. A 2.2 A cryo-electron microscopy structure of the ternary complex demonstrates that DCAF16 and BRD4BD2 have pre-existing structural complementarity which optimally orients the reactive moiety of the degrader for DCAF16 Cys58 covalent modification. Systematic mutagenesis of both DCAF16 and BRD4BD2 revealed that the loop conformation around BRD4 His437, rather than specific side chains, is critical for stable interaction with DCAF16 and BD2 selectivity. Together our work establishes "template-assisted covalent modification" as a mechanism for covalent molecular glues, which opens a new path to proximity driven pharmacology.
Monday, February 6, 2023
Sulfamate Acetamides as Self-Immolative Electrophiles for Covalent Ligand-Directed Release Chemistry
Electrophiles for covalent inhibitors that are suitable for in vivo administration are rare. While acrylamides are prevalent in FDA-approved covalent drugs, chloroacetamides are considered too reactive for such purposes. We report sulfamate-based electrophiles that maintain chloroacetamide-like geometry with tunable reactivity. In the context of the BTK inhibitor ibrutinib, sulfamate analogues showed low reactivity with comparable potency in protein labeling, in vitro, and cellular kinase activity assays and were effective in a mouse model of CLL. In a second example, we converted a chloroacetamide Pin1 inhibitor to a potent and selective sulfamate acetamide with improved buffer stability. Finally, we show that sulfamate acetamides can be used for covalent ligand-directed release (CoLDR) chemistry, both for the generation of “turn-on” probes as well as for traceless ligand-directed site-specific labeling of proteins. Taken together, this chemistry represents a promising addition to the list of electrophiles suitable for in vivo covalent targeting.
Thursday, February 2, 2023
Wang S, Hadisurya M, Tao WA, Dykhuizen E, Krusemark C.
Targeted covalent inhibitors (TCIs) have increased in popularity among drug candidates and chemical probes. Among current TCIs, the chemistry employed is largely limited to labeling cysteine and lysine side chains. Tyrosine is an attractive residue for TCIs due to its enrichment at protein-protein interfaces. Here, we investigate the utility of cyclic imine Mannich electrophiles as covalent warheads to specifically target a pro-tein tyrosine adjacent to an inhibitor binding pocket. We characterized the intrinsic reaction rates of several cyclic imines to tyrosine and identified the iminolactone to be suitable for a covalent inhibitor (second order rate constant of 0.0029 M-1 s-1). We appended the cyclic imine warheads to a CBX8 chromodomain inhibitor to label a non-conserved tyrosine, which markedly improves both the potency and selectivity of the inhibitor for CBX8 in vitro and in cells. These results indicate that Mannich electrophiles are promising and robust chemical warheads for tyrosine bioconjugation and covalent inhibitors.
Roman C. Sarott, Inchul You, Yen-Der Li, Sean T. Toenjes, Katherine A. Donovan, Pooreum Seo, Martha Ordonez, Woong Sub Byun, Muhammad Murtaz...
Jian Ding, Guo Li, Hejun Liu, Lulu Liu, Ying Lin, Jingyan Gao, Guoqiang Zhou, Lingling Shen, Mengxi Zhao, Yanyan Yu, Weihui Guo, Ulrich Homm...
Ethan S Toriki, James W Papatzimas, Kaila Nishikawa, Dustin Dovala, Lynn M McGregor, Matthew J Hesse, Jeffrey M McKenna, John A Tallarico, M...
Mapping the chemical space of active-site targeted covalent ligands for protein tyrosine phosphatasesHong, S. ho; Xi, S. Y.; Johns, A. C.; Tang, L. C.; Li, A.; Jovanovic, M.; Shah, N. H. ChemRxiv 2022 . https://doi.org/10.26434/chemrxiv-2...