CysDB: a human cysteine database based on experimental quantitative chemoproteomic [@Keribackus]

Lisa M. Boatner , Maria F. Palafox, Devin K. Schwepp, Keriann M. Backus 

Cell Chem. Bio. 2023

https://doi.org/10.1016/j.chembiol.2023.04.004

Cysteine chemoproteomics provides proteome-wide portraits of the ligandability or potential “druggability” for thousands of cysteine residues. Consequently, these studies are facilitating resources for closing the druggability gap, namely, achieving pharmacological manipulation of ∼96% of the human proteome that remains untargeted by U.S. Food and Drug Administration (FDA) approved small molecules. Recent interactive datasets have enabled users to interface more readily with cysteine chemoproteomics datasets. However, these resources remain limited to single studies and therefore do not provide a mechanism to perform cross-study analyses. Here we report CysDB as a curated community-wide repository of human cysteine chemoproteomics data derived from nine high-coverage studies. CysDB is publicly available at https://backuslab.shinyapps.io/cysdb/ and features measures of identification for 62,888 cysteines (24% of the cysteinome), as well as annotations of functionality, druggability, disease relevance, genetic variation, and structural features. Most importantly, we have designed CysDB to incorporate new datasets to further support the continued growth of the druggable cysteinome.

Hydrazonyl Sultones as Stable Tautomers of Highly Reactive Nitrile Imines for Fast Bioorthogonal Ligation Reaction

Ming Fang, Gangam Srikanth Kumar, Stefano Racioppi, Heyang Zhang, Johnathan D. Rabb, Eva Zurek, and Qing Lin

Journal of the American Chemical Society 2023

DOI: 10.1021/jacs.2c12325

Here we report the design and synthesis of a new class of bioorthogonal reagents called hydrazonyl sultones (HS) that serve as stable tautomers of highly reactive nitrile imines (NI). Compared to the photogenerated NI, HS display a broad range of aqueous stability and tunable reactivity in a 1,3-dipolar cycloaddition reaction, depending on substituents, sultone ring structure, and solvent conditions. DFT calculations have provided vital insights into the HS → NI tautomerism, including a base-mediated anionic tautomerization pathway and a small activation barrier. Comparative kinetic analysis of tetrazole vs HS-mediated cycloadditions reveals that a tiny fraction of the reactive NI (∼15 ppm) is present in the tautomeric mixture, underpinning the extraordinary stability of the six-membered HS. We further demonstrate the utilities of HS in selective modification of bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN)-lysine-containing nanobodies in phosphate buffered saline and fluorescent labeling of a BCN-lysine-encoded transmembrane glucagon receptor on live cells.

Rational Design of Highly Potent and Selective Covalent MAP2K7 Inhibitors

Dalton R. Kim, Meghan J. Orr, Ada J. Kwong, Kristine K. Deibler, Hasan H. Munshi, Cory Seth Bridges, Taylor Jie Chen, Xiaoyu Zhang, H. Daniel Lacorazza, and Karl A. Scheidt

ACS Medicinal Chemistry Letters 2023

DOI: 10.1021/acsmedchemlett.3c00029

The mitogen-activated protein kinase signaling cascade is conserved across eukaryotes, where it plays a critical role in the regulation of activities including proliferation, differentiation, and stress responses. This pathway propagates external stimuli through a series of phosphorylation events, which allows external signals to influence metabolic and transcriptional activities. Within the cascade, MEK, or MAP2K, enzymes occupy a molecular crossroads immediately upstream to significant signal divergence and cross-talk. One such kinase, MAP2K7, also known as MEK7 and MKK7, is a protein of great interest in the molecular pathophysiology underlying pediatric T cell acute lymphoblastic leukemia (T-ALL). Herein, we describe the rational design, synthesis, evaluation, and optimization of a novel class of irreversible MAP2K7 inhibitors. With a streamlined one-pot synthesis, favorable in vitro potency and selectivity, and promising cellular activity, this novel class of compounds wields promise as a powerful tool in the study of pediatric T-ALL.

Discovery of Futibatinib: The First Covalent FGFR Kinase Inhibitor in Clinical Use

Satoru Ito, Sachie Otsuki, Hirokazu Ohsawa, Atsushi Hirano, Hideki Kazuno, Satoshi Yamashita, Kosuke Egami, Yoshihiro Shibata, Ikuo Yamamiya, Fumiaki Yamashita, Yasuo Kodama, Kaoru Funabashi, Hiromi Kazuno, Toshiharu Komori, Satoshi Suzuki, Hiroshi Sootome, Hiroshi Hirai, and Takeshi Sagara

ACS Medicinal Chemistry Letters 2023 14 (4), 396-404

DOI: 10.1021/acsmedchemlett.3c00006

Deregulating fibroblast growth factor receptor (FGFR) signaling is a promising strategy for cancer therapy. Herein, we report the discovery of compound 5 (TAS-120, futibatinib), a potent and selective covalent inhibitor of FGFR1–4, starting from a unique dual inhibitor of mutant epidermal growth factor receptor and FGFR (compound 1). Compound 5 inhibited all four families of FGFRs in the single-digit nanomolar range and showed high selectivity for over 387 kinases. Binding site analysis revealed that compound 5 covalently bound to the cysteine 491 highly flexible glycine-rich loop region of the FGFR2 adenosine triphosphate pocket. Futibatinib is currently in Phase I–III trials for patients with oncogenically driven FGFR genomic aberrations. In September 2022, the U.S. Food & Drug Administration granted accelerated approval for futibatinib in the treatment of previously treated, unresectable, locally advanced, or metastatic intrahepatic cholangiocarcinoma harboring an FGFR2 gene fusion or other rearrangement.

Discovery of a spirocyclic 3-bromo-4,5-dihydroisoxazole covalent inhibitor of hGAPDH with antiproliferative activity against pancreatic cancer cells,

Andrea Galbiati, Stefania Bova, Raffaella Pacchiana, Chiara Borsari, Marco Persico, Aureliano Zana, Stefano Bruno, Massimo Donadelli, Caterina Fattorusso, Paola Conti,

European Journal of Medicinal Chemistry, 2023

https://doi.org/10.1016/j.ejmech.2023.115286

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key glycolytic enzyme, plays a crucial role in the energy metabolism of cancer cells and has been proposed as a valuable target for the development of anticancer agents. Among a series of 5-substituted 3-bromo-4,5-dihydroisoxazole (BDHI) derivatives, we identified the spirocyclic compound 11, which is able to covalently inactivate recombinant human GAPDH (hGAPDH) with a faster reactivity than koningic acid, one of the most potent hGAPDH inhibitors known to date. Computational studies confirmed that conformational rigidification is crucial to stabilize the interaction of the inhibitor with the binding site, thus favoring the subsequent covalent bond formation. Investigation of intrinsic warhead reactivity at different pH disclosed the negligible reactivity of 11 with free thiols, highlighting its ability to selectively react with the activated cysteine of hGAPDH with respect to other sulfhydryl groups. Compound 11 strongly reduced cancer cell growth in four different pancreatic cancer cell lines and its antiproliferative activity correlated well with the intracellular inhibition of hGAPDH. Overall, our results qualify 11 as a potent hGAPDH covalent inhibitor with a moderate drug-like reactivity that could be further exploited to develop anticancer agents.

Reversible Dual-Covalent Molecular Locking of the 14-3-3/ERRγ Protein–Protein Interaction as a Molecular Glue Drug Discovery Approach

Bente A. Somsen, Rick J.C. Schellekens, Carlo J.A. Verhoef, Michelle R. Arkin, Christian Ottmann, Peter J. Cossar, and Luc Brunsveld

Journal of the American Chemical Society 2023

DOI: 10.1021/jacs.2c12781

Molecules that stabilize protein–protein interactions (PPIs) are invaluable as tool compounds for biophysics and (structural) biology, and as starting points for molecular glue drug discovery. However, identifying initial starting points for PPI stabilizing matter is highly challenging, and chemical optimization is labor-intensive. Inspired by chemical crosslinking and reversible covalent fragment-based drug discovery, we developed an approach that we term “molecular locks” to rapidly access molecular glue-like tool compounds. These dual-covalent small molecules reversibly react with a nucleophilic amino acid on each of the partner proteins to dynamically crosslink the protein complex. The PPI between the hub protein 14-3-3 and estrogen-related receptor γ (ERRγ) was used as a pharmacologically relevant case study. Based on a focused library of dual-reactive small molecules, a molecular glue tool compound was rapidly developed. Biochemical assays and X-ray crystallographic studies validated the ternary covalent complex formation and overall PPI stabilization via dynamic covalent crosslinking. The molecular lock approach is highly selective for the specific 14-3-3/ERRγ complex, over other 14-3-3 complexes. This selectivity is driven by the interplay of molecular reactivity and molecular recognition of the composite PPI binding interface. The long lifetime of the dual-covalent locks enabled the selective stabilization of the 14-3-3/ERRγ complex even in the presence of several other competing 14-3-3 clients with higher intrinsic binding affinities. The molecular lock approach enables systematic, selective, and potent stabilization of protein complexes to support molecular glue drug discovery.

Quantifying KRAS G12C Covalent Drug Inhibitor Activity in Mouse Tumors Using Mass Spectrometry

John C. Tran, Thomas Hunsaker, Christina Bell, Taylur P. Ma, Emily Chan, Pablo Saenz-Lopez Larrocha, Kelsey Homyk, Liling Liu, Hank La, Jialin Mao, Cecile C. de la Cruz, Kebing Yu, Maureen Beresini, William F. Forrest, Yang Xiao, Anne Jang, Natalia Samus, Nicholas Dupuis Stesco, Marija Mentinova, Stephane Parent, Gwenael Pottiez, Michael Schirm, Hans E. Purkey, Yichin Liu, and Mark Merchant

Analytical Chemistry 2023

DOI: 10.1021/acs.analchem.2c04417

The growing opportunities recognized for covalent drug inhibitors, like KRAS G12C inhibitors, are driving the need for mass spectrometry methods that can quickly and robustly measure therapeutic drug activity in vivo for drug discovery research and development. Effective front-end sample preparation is critical for proteins extracted from tumors but is generally labor intensive and impractical for large sample numbers typical in pharmacodynamic (PD) studies. Herein, we describe an automated and integrated sample preparation method for the measurement of activity levels of KRAS G12C drug inhibitor alkylation from complex tumor samples involving high throughput detergent removal and preconcentration followed by quantitation using mass spectrometry. We introduce a robust assay with an average intra-assay coefficient of variation (CV) of 4% and an interassay CV of 6% obtained from seven studies, enabling us to understand the relationship between KRAS G12C target occupancy and the therapeutic PD effect from mouse tumor samples. Further, the data demonstrated that the drug candidate GDC-6036, a KRAS G12C covalent inhibitor, shows dose-dependent target inhibition (KRAS G12C alkylation) and MAPK pathway inhibition, which correlate with high antitumor potency in the MIA PaCa-2 pancreatic xenograft model.