Liganding Functional Tyrosine Sites on Proteins Using Sulfur–Triazole Exchange Chemistry [@Hsu_Lab_UVa]

Jeffrey W. Brulet, Adam L. Borne, Kun Yuan, Adam H. Libby, and Ku-Lung Hsu
Journal of the American Chemical Society 2020
DOI: 10.1021/jacs.0c00648

Tuning reactivity of sulfur electrophiles is key for advancing click chemistry and chemical probe discovery. To date, activation of the sulfur electrophile for protein modification has been ascribed principally to stabilization of a fluoride leaving group (LG) in covalent reactions of sulfonyl fluorides and arylfluorosulfates. We recently introduced sulfur–triazole exchange (SuTEx) chemistry to demonstrate the triazole as an effective LG for activating nucleophilic substitution reactions on tyrosine sites of proteins. Here, we probed tunability of SuTEx for fragment-based ligand discovery by modifying the adduct group (AG) and LG with functional groups of differing electron-donating and -withdrawing properties. We discovered the sulfur electrophile is highly sensitive to the position of modification (AG versus LG), which enabled both coarse and fine adjustments in solution and proteome activity. We applied these reactivity principles to identify a large fraction of tyrosine sites (∼30%) on proteins (∼44%) that can be liganded across >1500 probe-modified sites quantified by chemical proteomics. Our proteomic studies identified noncatalytic tyrosine and phosphotyrosine sites that can be liganded by SuTEx fragments with site specificity in lysates and live cells to disrupt protein function. Collectively, we describe SuTEx as a versatile covalent chemistry with broad applications for chemical proteomics and protein ligand discovery.

Cyanopyrrolidine Inhibitors of Ubiquitin Specific Protease 7 Mediate Desulfhydration of the Active-Site Cysteine

Charlene Bashore, Priyadarshini Jaishankar, Nicholas J. Skelton, Jakob Fuhrmann, Brian R. Hearn, Peter S. Liu, Adam R. Renslo, and Erin C. Dueber
ACS Chemical Biology Article ASAP
DOI: 10.1021/acschembio.0c00031

Ubiquitin specific protease 7 (USP7) regulates the protein stability of key cellular regulators in pathways ranging from apoptosis to neuronal development, making it a promising therapeutic target. Here we used an engineered, activated variant of the USP7 catalytic domain to perform structure–activity studies of electrophilic peptidomimetic inhibitors. Employing this USP7 variant, we found that inhibitors with a cyanopyrrolidine warhead unexpectedly promoted a β-elimination reaction of the initial covalent adducts, thereby converting the active-site cysteine residue to dehydroalanine. We determined that this phenomenon is specific for the USP7 catalytic cysteine and that structural features of the inhibitor and protein microenvironment impact elimination rates. Using comprehensive docking studies, we propose that the characteristic conformational dynamics of USP7 allow access to conformations that promote the ligand-induced elimination. Unlike in conventional reversible-covalent inhibition, the compounds described here irreversibly destroy a catalytic residue while simultaneously converting the inhibitor to a nonelectrophilic byproduct. Accordingly, this unexpected finding expands the scope of covalent inhibitor modalities and offers intriguing insights into enzyme–inhibitor dynamics.

2-Sulfonyl pyridines as tunable, cysteine-reactive electrophiles [@mikebollong]

Claudio Zambaldo, Ekaterina V. Vinogradova, Xiaotian Qi, Jonathan Iaconelli, Radu M. Suciu, Minseob Koh, Kristine Senkane, Stormi R Chadwick, Brittany B. Sanchez, Jason S. Chen, Arnab K. Chatterjee, Peng Liu, Peter G Schultz, Benjamin F. Cravatt, and Michael J. Bollong
Journal of the American Chemical Society, 2020
DOI: 10.1021/jacs.0c02721

The emerging use of covalent ligands as chemical probes and drugs would benefit from an expanded repertoire of cysteine-reactive electrophiles for efficient and diverse targeting of the proteome. Here we use the endogenous electrophile sensor of mammalian cells — the KEAP1-NRF2 pathway — to discover cysteine-reactive electrophilic fragments from a reporter-based screen for NRF2 activation. This strategy identified a series of 2-sulfonyl pyridines that selectively react with biological thi-ols via nucleophilic aromatic substitution (SNAr). By tuning the electrophilicity and appended recognition elements, we demonstrate the potential of the 2-sulfonyl pyridine reactive group with the discovery of a selective covalent modifier of adenosine deaminase (ADA). Targeting a cysteine distal to the active site, this molecule attenuates the enzymatic activity of ADA and inhibits proliferation of lymphocytic cells. This study introduces a modular and tunable SNAr-based reactive group for targeting reactive cysteines in the human proteome and illustrates the pharmacological utility of this electrophilic series.

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*
ACS Central Science, 2020

DOI: https://doi.org/10.1021/acscentsci.9b01268

Covalent inhibitors have recently seen a resurgence of interest in drug development. Nevertheless, compounds, which 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.

Identification of the Clinical Development Candidate MRTX849, a Covalent KRASG12C Inhibitor for the Treatment of Cancer

Jay B. Fell, John P. Fischer, Brian R. Baer, James F. Blake, Karyn Bouhana, David M. Briere, Karin D. Brown, Laurence E. Burgess, Aaron C. Burns, Michael R. Burkard, Harrah Chiang, Mark J. Chicarelli, Adam W. Cook, John J. Gaudino, Jill Hallin, Lauren Hanson, Dylan P. Hartley, Erik J. Hicken, Gary P. Hingorani, Ronald J. Hinklin, Macedonio J. Mejia, Peter Olson, Jennifer N. Otten, Susan P. Rhodes, Martha E. Rodriguez, Pavel Savechenkov, Darin J. Smith, Niranjan Sudhakar, Francis X. Sullivan, Tony P. Tang, Guy P. Vigers, Lance Wollenberg, James G. Christensen, Matthew A. Marx

J. Med. Chem., 2020
DOI: https://doi.org/10.1021/acs.jmedchem.9b02052

Capping off an era marred by drug development failures and punctuated by waning interest and presumed intractability toward direct targeting of KRAS, new technologies and strategies are aiding in the target’s resurgence. As previously reported, the tetrahydropyridopyrimidines were identified as irreversible covalent inhibitors of KRASG12C that bind in the switch-II pocket of KRAS and make a covalent bond to cysteine 12. Using structure-based drug design in conjunction with a focused in vitro absorption, distribution, metabolism and excretion screening approach, analogues were synthesized to increase the potency and reduce metabolic liabilities of this series. The discovery of the clinical development candidate MRTX849 as a potent, selective covalent inhibitor of KRASG12C is described.

Targeted Degradation of Oncogenic KRASG12C by VHL-recruiting PROTACs [@CraigMCrews]

Bond, Michael J.; Chu, Ling; Nalawansha, Dhanusha A.; Li, Ke; Crews, Craig

ChemRxiv, 2020
DOI: https://doi.org/10.26434/chemrxiv.12091176.v1

We report the development of LC-2, the first PROTAC capable of degrading endogenous KRAS^G12C. LC-2 covalently binds KRAS^G12C with a MRTX849 warhead and recruits the E3 ligase VHL, inducing rapid and sustained KRAS^G12C degradation leading to suppression of MAPK signaling in both homozygous and heterozygous KRAS^G12C cell lines. LC-2 demonstrates that PROTAC-mediated degradation is a viable option for attenuating oncogenic KRAS levels and downstream signaling in cancer cells.

Structure of Mpro from COVID-19 virus and discovery of its inhibitors.

Zhenming Jin, Xiaoyu Du, Yechun Xu, Yongqiang Deng, Meiqin Liu, Yao Zhao, Bing Zhang, Xiaofeng Li, Leike Zhang, Chao Peng, Yinkai Duan, Jing Yu, Lin Wang, Kailin Yang, Fengjiang Liu, Rendi Jiang, Xinglou Yang, Tian You, Xiaoce Liu, Xiuna Yang, Fang Bai, Hong Liu, Xiang Liu, Luke W. Guddat, Wenqing Xu, Gengfu Xiao, Chengfeng Qin, Zhengli Shi, Hualiang Jiang, Zihe Rao & Haitao Yang

Nature, 2020
doi https://doi.org/10.1038/s41586-020-2223-y

A new coronavirus (CoV) identified as COVID-19 virus is the etiological agent responsible for the 2019-2020 viral pneumonia outbreak that commenced in Wuhan1–4. Currently there are no targeted therapeutics and effective treatment options remain very limited. In order to rapidly discover lead compounds for clinical use, we initiated a program of combined structure-assisted drug design, virtual drug screening and high-throughput screening to identify new drug leads that target the COVID-19 virus main protease (Mpro). Mpro is a key CoV enzyme, which plays a pivotal role in mediating viral replication and transcription, making it an attractive drug target for this virus5,6. Here, we identified a mechanism-based inhibitor, N3, by computer-aided drug design and subsequently determined the crystal structure of COVID-19 virus Mpro in complex with this compound. Next, through a combination of structure-based virtual and high-throughput screening, we assayed over 10,000 compounds including approved drugs, drug candidates in clinical trials, and other pharmacologically active compounds as inhibitors of Mpro. Six of these compounds inhibited Mpro with IC50 values ranging from 0.67 to 21.4 μM. Ebselen also exhibited promising antiviral activity in cell-based assays. Our results demonstrate the efficacy of this screening strategy, which can lead to the rapid discovery of drug leads with clinical potential in response to new infectious diseases for which no specific drugs or vaccines are available.

Rationally Designed Covalent BCL6 Inhibitor That Targets a Tyrosine Residue in the Homodimer Interface

Mingxing Teng, Scott B. Ficarro, Hojong Yoon, Jianwei Che, Jing Zhou, Eric S. Fischer, Jarrod A. Marto, Tinghu Zhang, and Nathanael S. Gray

ACS Medicinal Chemistry Letters Article 2020
DOI: 10.1021/acsmedchemlett.0c00111

B-cell lymphoma 6 (BCL6) is a transcriptional repressor frequently deregulated in lymphoid malignancies. BCL6 engages with number of corepressors, and these protein–protein interactions are being explored as a strategy for drug development. Here, we report the development of an irreversible BCL6 inhibitor TMX-2164 that uses a sulfonyl fluoride to covalently react with the hydroxyl group of Tyrosine 58 located in the lateral groove. TMX-2164 exhibits significantly improved inhibitory activity compared to that of its reversible parental compound and displays sustained target engagement and antiproliferative activity in cells. TMX-2164 therefore represents an example of a tyrosine-directed covalent inhibitor of BCL6 which demonstrates advantages relative to reversible targeting.

A Nimbolide-Based Kinase Degrader Preferentially Degrades Oncogenic BCR-ABL [@DanNomura]

Bingqi Tong, Jessica N. Spradlin, Luiz F.T. Novaes, Erika Zhang, Xirui Hu Malte Moeller,
Scott M. Brittain, Lynn M. McGregor, Jeffrey M. McKenna, John A. Tallarico, Markus Schirle,
Thomas J. Maimone, and Daniel K. Nomura

BioRXiv, 2020
doi: https://doi.org/10.1101/2020.04.02.022541

Targeted protein degradation (TPD) and proteolysis-targeting chimeras (PROTACs) have arisen as powerful therapeutic modalities for degrading specific protein targets in a proteasome-dependent manner. However, a major limitation to broader TPD applications is the lack of E3 ligase recruiters. Recently, we discovered the natural product nimbolide as a covalent ligand for the E3 ligase RNF114. When linked to the BET family inhibitor JQ1, the resulting heterobifunctional PROTAC molecule was capable of selectively degrading BRD4 in cancer cells. Here, we show the broader utility of nimbolide as an E3 ligase recruiter for TPD applications. We demonstrate that a PROTAC linking nimbolide to the kinase and BCR-ABL fusion oncogene inhibitor dasatinib, BT1, selectively degrades BCR-ABL over c-ABL in leukemia cancer cells, compared to previously reported cereblon or VHL-recruiting BCR-ABL degraders that show opposite selectivity or in some cases inactivity. Further contrasting from cereblon or VHL-recruiting degradation, we show that BT1 treatment not only leads to BCR-ABL degradation, but also stabilizes the endogenous RNF114 substrate and tumor suppressor substrate p21. This leads to additional anti-proliferative effects in leukemia cancer cells beyond those observed with cereblon or VHL-recruiting BCR-ABL PROTACs. Thus, we further establish nimbolide as an additional general E3 ligase recruiter for PROTACs with unique additional benefits for oncology applications. We also further demonstrate the importance of expanding upon the arsenal of E3 ligase recruiters, as such molecules confer differing and unpredictable selectivity for the degradation of neo-substrate proteins.

Targeted Protein Degradation via a Covalent Reversible Degrader Based on Bardoxolone [@DanNomura]

Tong, Bingqi; Luo, Mai; Xie, Yi; Spradlin, Jessica; Tallarico, John A.; McKenna, Jeffrey M. McKenna, Markus Schirle, Thomas J. Maimone, and Daniel K. Nomura

ChemRxiv. 2020
https://doi.org/10.26434/chemrxiv.12055935.v1

Targeted protein degradation (TPD) has emerged as a powerful tool in drug discovery for the perturbation of protein levels using heterobifunctional small molecules (i.e. PROTACs). E3 ligase recruiters remain central to this process yet relatively few have been identified relative to the >500 predicted human E3 ligases. While, initial recruiters have utilized non-covalent chemistry for protein binding, very recently covalent engagement to novel E3’s has proven fruitful in TPD application. Herein we demonstrate efficient proteasome-mediated degradation of BRD4 by a bifunctional small molecule linking the KEAP1-NRF2 activator bardoxolone to a BRD4 inhibitor JQ1. Notably, this work reports the first covalent, reversible E3 ligase recruiter for TPD applications.

Advances in covalent kinase inhibitors [@GunningLabUofT]

Ayah Abdeldayem, Yasir S. Raouf,  Stefan N. Constantinescu,  Richard Moriggl, and Patrick T. Gunning

Chem. Soc. Rev., 2020
https://doi.org/10.1039/C9CS00720B

Over the past decade, covalent kinase inhibitors (CKI) have seen a resurgence in drug discovery. Covalency affords a unique set of advantages as well as challenges relative to their non-covalent counterpart. After reversible protein target recognition and binding, covalent inhibitors irreversibly modify a proximal nucleophilic residue on the protein via reaction with an electrophile. To date, the acrylamide group remains the predominantly employed electrophile in CKI development, with its incorporation in the majority of clinical candidates and FDA approved covalent therapies. Nonetheless, in recent years considerable efforts have ensued to characterize alternative electrophiles that exhibit irreversible or reversibly covalent binding mechanisms towards cysteine thiols and other amino acids. This review article provides a comprehensive overview of CKIs reported in the literature over a decade period, 2007–2018. Emphasis is placed on the rationale behind warhead choice, optimization approach, and inhibitor design. Current FDA approved CKIs are also highlighted, in addition to a detailed analysis of the common trends and themes observed within the listed data set.