Light-Activatable, 2,5-Disubstituted Tetrazoles for the Proteome-Wide Profiling of Aspartates and Glutamates in Living Bacteria

Kathrin Bach Bert L. H. Beerkens Patrick R. A. Zanon Stephan M. Hacker
ChemRxiv, 2019
doi: 10.26434/chemrxiv.11352101.v1

Covalent inhibitors have recently seen a resurgence of interest in drug development. Nevertheless, compounds, that do not rely on an enzymatic activity, have almost exclusively been developed to target cysteines. Expanding the scope to other amino acids would be largely facilitated by the ability to globally monitor their engagement by covalent inhibitors. Here, we present the use of light-activatable 2,5-disubstituted tetrazoles that allow quantifying 8971 aspartates and glutamates in the bacterial proteome with excellent selectivity. Using these probes, we competitively map the binding sites of two isoxazolium salts and introduce hydrazonyl chlorides as a new class of carboxylic acid-directed covalent protein ligands. As the probes are unreactive prior to activation, they allow global profiling even in living Gram-positive and Gram-negative bacteria. Taken together, this method to monitor aspartates and glutamates proteome-wide will lay the foundation to efficiently develop covalent inhibitors targeting these amino acids

Kinetic Optimization of Lysine-Targeting Covalent Inhibitors of HSP72

Jonathan Pettinger, Michael Carter, Keith Jones, and Matthew D. Cheeseman

Journal of Medicinal Chemistry 2019

DOI: 10.1021/acs.jmedchem.9b01709

The covalent inhibition mechanism of action, which overcomes competition with high-affinity, high-abundance substrates of challenging protein targets, can deliver effective chemical probes and drugs. The success of this strategy has centered on exposed cysteine residues as nucleophiles but the low abundance of cysteine in the proteome has limited its application. We have recently reported our discovery that lysine-56 in the difficult-to-drug target HSP72 could form a covalent bond with a small-molecule inhibitor. We now disclose the optimization of these targeted covalent inhibitors using rational design. Essential to our optimization was the development of a new covalent fluorescence polarization assay, which allows for the direct measurement of the key kinetic parameter in covalent inhibitor design, kinact/KI, extrapolation of the underlying parameters, kinact and Ki, and direct comparison to reversible analogues. Using our approach, we demonstrate a >100-fold enhancement in covalent efficiency and key learnings in lysine-selective electrophile optimization.

Discovery of a covalent inhibitor of KRASG12C (AMG 510) for the treatment of solid tumors

Brian A Lanman, Jennifer R. Allen, John G. Allen, Albert K Amegadzie, Kate S. Ashton, Shon K. Booker, Jian Jeffrey Chen, Ning Chen, Michael J Frohn, Guy Goodman, David J Kopecky, Longbin Liu, Patricia Lopez, Jonathan D Low, Vu Ma, Ana Elena Minatti, Thomas T Nguyen, Nobuko Nishimura, Alexander J. Pickrell, Anthony B. Reed, Youngsook Shin, Aaron Siegmund, Nuria A. Tamayo, Christopher M Tegley, Mary C Walton, Hui-Ling Wang, Ryan P. Wurz, May Xue, Kevin C Yang, Pragathi Achanta, Michael D. Bartberger, Jude Canon, L Steven Hollis, John D McCarter, Christopher Mohr, Karen Rex, Anne Y Saiki, Tisha San Miguel, Laurie Volak, Kevin H Wang, Douglas A. Whittington, Stephan G Zech, J. Russell Lipford, and Victor J. Cee

Journal of Medicinal Chemistry 2019

DOI: 10.1021/acs.jmedchem.9b01180

KRASG12C has emerged as a promising target in the treatment of solid tumors. Covalent inhibitors targeting the mutant cysteine-12 residue have been shown to disrupt signaling by this long-“undruggable” target, however clinically viable inhibitors have yet to be identified. Here, we report efforts to exploit a cryptic pocket (H95/Y96/Q99) we identified in KRASG12C to identify inhibitors suitable for clinical development. Structure-based design efforts leading to the identification of a novel quinazolinone scaffold are described, along with optimization efforts that overcame a configurational stability issue arising from restricted rotation about an axially chiral biaryl bond. Biopharmaceutical optimization of the resulting leads culminated in the identification of AMG 510, a highly potent, selective, and well-tolerated KRASG12C inhibitor currently in Phase I clinical trials (NCT03600883).

Global targeting of functional tyrosines using sulfur-triazole exchange chemistry

Heung Sik Hahm, Emmanuel K. Toroitich, Adam L. Borne, Jeffrey W. Brulet, Adam H. Libby, Kun Yuan, Timothy B. Ware, Rebecca L. McCloud, Anthony M. Ciancone & Ku-Lung Hsu

Nat Chem Biol (2019) doi:10.1038/s41589-019-0404-5

Covalent probes serve as valuable tools for global investigation of protein function and ligand binding capacity. Despite efforts to expand coverage of residues available for chemical proteomics (e.g., cysteine and lysine), a large fraction of the proteome remains inaccessible with current activity-based probes. Here, we introduce sulfur-triazole exchange (SuTEx) chemistry as a tunable platform for developing covalent probes with broad applications for chemical proteomics. We show modifications to the triazole leaving group can furnish sulfonyl probes with ~5-fold enhanced chemoselectivity for tyrosines over other nucleophilic amino acids to investigate more than 10,000 tyrosine sites in lysates and live cells. We discover that tyrosines with enhanced nucleophilicity are enriched in enzymatic, protein–protein interaction and nucleotide recognition domains. We apply SuTEx as a chemical phosphoproteomics strategy to monitor activation of phosphotyrosine sites. Collectively, we describe SuTEx as a biocompatible chemistry for chemical biology investigations of the human proteome.

Use of Pyridazinediones as Extracellular Cleavable Linkers Through Reversible Cysteine Conjugation

Calise Bahou , Richard Spears , Abil Aliev , Antoine Maruani , Marcos Fernandez , Faiza Javaid , Peter Szijj , James Baker and Vijay Chudasama

Chem. Commun. 2019
DOI: 10.1039/C9CC08362F

Herein we report a retro-Michael deconjugation pathway of thiol-pyridazinedione linked protein bioconjugates to provide a novel cleavable linker technology. We demonstrate that the novel pyridazinedione linker does not suffer from off-target modification with blood thiols (e.g. glutathione, human serum albumin (HSA)), which is in sharp contrast to an analogous maleimide linker.

Modulating multi-functional ERK complexes by covalent targeting of a recruitment site in vivo

Tamer S. Kaoud, William H. Johnson, Nancy D. Ebelt, Andrea Piserchio, Diana Zamora-Olivares, Sabrina X. Van Ravenstein, Jacey R. Pridgen, Ramakrishna Edupuganti, Rachel Sammons, Micael Cano, Mangalika Warthaka, Matthew Harger, Clint D. J. Tavares, Jihyun Park, Mohamed F. Radwan, Pengyu Ren, Eric V. Anslyn, Kenneth Y. Tsai, Ranajeet Ghose & Kevin N. Dalby

Nat. Commun. 2019, 10, 5232

DOI: https://doi.org/10.1038/s41467-019-12996-8

Recently, the targeting of ERK with ATP-competitive inhibitors has emerged as a potential clinical strategy to overcome acquired resistance to BRAF and MEK inhibitor combination therapies. In this study, we investigate an alternative strategy of targeting the D-recruitment site (DRS) of ERK. The DRS is a conserved region that lies distal to the active site and mediates ERK–protein interactions. We demonstrate that the small molecule BI-78D3 binds to the DRS of ERK2 and forms a covalent adduct with a conserved cysteine residue (C159) within the pocket and disrupts signaling in vivo. BI-78D3 does not covalently modify p38MAPK, JNK or ERK5. BI-78D3 promotes apoptosis in BRAF inhibitor-naive and resistant melanoma cells containing a BRAF V600E mutation. These studies provide the basis for designing modulators of protein–protein interactions involving ERK, with the potential to impact ERK signaling dynamics and to induce cell cycle arrest and apoptosis in ERK-dependent cancers.

Structure–Activity Relationship Study of Covalent Pan-phosphatidylinositol 5-Phosphate 4-Kinase Inhibitors

Theresa D. Manz, Sindhu C. Sivakumaren, Adam Yasgar, Matthew D. Hall, Mindy I. Davis, Hyuk-Soo Seo, Joseph D. Card, Scott B. Ficarro, Hyeseok Shim, Jarrod A. Marto, Sirano Dhe-Paganon, Atsuo T. Sasaki, Matthew B. Boxer, Anton Simeonov, Lewis C. Cantley, Min Shen, Tinghu Zhang, Fleur M. Ferguson, and Nathanael S. Gray

ACS Medicinal Chemistry Letters  2019, DOI: 10.1021/acsmedchemlett.9b00402

Phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) are important molecular players in a variety of diseases, such as cancer. Currently available PI5P4K inhibitors are reversible small molecules, which may lack selectivity and sufficient cellular on-target activity. In this study, we present a new class of covalent pan-PI5P4K inhibitors with potent biochemical and cellular activity. Our designs are based on THZ-P1-2, a covalent PI5P4K inhibitor previously developed in our lab. Here, we report further structure-guided optimization and structure–activity relationship (SAR) study of this scaffold, resulting in compound 30, which retained biochemical and cellular potency, while demonstrating a significantly improved selectivity profile. Furthermore, we confirm that the inhibitors show efficient binding affinity in the context of HEK 293T cells using isothermal CETSA methods. Taken together, compound 30 represents a highly selective pan-PI5P4K covalent lead molecule.