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.

Selective covalent targeting of GPX4 using masked nitrile-oxide electrophiles

John K. Eaton, Laura Furst, Richard A. Ruberto, Dieter Moosmayer, André Hilpmann, Matthew J. Ryan, Katja Zimmermann, Luke L. Cai, Michael Niehues, Volker Badock, Anneke Kramm, Sixun Chen, Roman C. Hillig, Paul A. Clemons, Stefan Gradl, Claire Montagnon, Kiel E. Lazarski, Sven Christian, Besnik Bajrami, Roland Neuhaus, Ashley L. Eheim, Vasanthi S. Viswanathan & Stuart L. Schreiber

Nature Chemical Biology,  2020

DOI: https://doi.org/10.1038/s41589-020-0501-5

We recently described glutathione peroxidase 4 (GPX4) as a promising target for killing therapy-resistant cancer cells via ferroptosis. The onset of therapy resistance by multiple types of treatment results in a stable cell state marked by high levels of polyunsaturated lipids and an acquired dependency on GPX4. Unfortunately, all existing inhibitors of GPX4 act covalently via a reactive alkyl chloride moiety that confers poor selectivity and pharmacokinetic properties. Here, we report our discovery that masked nitrile-oxide electrophiles, which have not been explored previously as covalent cellular probes, undergo remarkable chemical transformations in cells and provide an effective strategy for selective targeting of GPX4. The new GPX4-inhibiting compounds we describe exhibit unexpected proteome-wide selectivity and, in some instances, vastly improved physiochemical and pharmacokinetic properties compared to existing chloroacetamide-based GPX4 inhibitors. These features make them superior tool compounds for biological interrogation of ferroptosis and constitute starting points for development of improved inhibitors of GPX4.

Insight Into the Therapeutic Selectivity of the Irreversible EGFR Tyrosine Kinase Inhibitor Osimertinib Through Enzyme Kinetic Studies

Xiang Zhai, Richard A Ward, Peter Doig, and Argyrides Argyrou
Biochemistry 2020
DOI: 10.1021/acs.biochem.0c00104

Osimertinib is a covalent, third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) approved for treating non-small cell lung cancer (NSCLC) patients with activating EGFR mutations (Exon19del or L858R), or with T790M resistance mutation following disease progression on first- or second-generation EGFR TKIs. The aim of this work was to understand how osimertinib achieves selective inhibition of mutant EGFR relative to wildtype through evaluating its kinetic mechanism of action. In doing so, we developed methodologies combining steady-state and pre-steady-state kinetics to determine the covalent inactivation rates (kinact) and reversible binding affinities (Ki) of osimertinib for WT, L858R and L858R/T790M EGFR, and compared these data to the inhibition kinetics of earlier generations of EGFR TKIs. The kinact/KI values indicate that osimertinib inactivates L858R and L858R/T790M with 20-fold and 50-fold higher overall efficiencies compared to WT, respectively. The Ki values reveal that osimertinib binds 3-fold and 17-fold tighter to L858R and L858R/T790M than to WT, respectively, while the kinact values show that osimertinib reacts 3-fold faster with L858R and L858R/T790M than with WT EGFR. We conclude that osimertinib achieves selective inhibition of L858R and L858R/T790M through improved affinities and improved rates of covalent bond formation via better positioning of the acrylamide warhead. This work highlights the importance of optimizing both reversible drug-target interactions and the inactivation rates for covalent inhibitors to achieve selectivity targeting mutant EGFR.

Sulfopin, a selective covalent inhibitor of Pin1, blocks Myc-driven tumor initiation and growth in vivo

Christian Dubiella, Benika J. Pinch, Daniel Zaidman, Theresa D. Manz, Evon Poon, Shuning He, Efrat Resnick, Ellen M. Langer, Colin J. Daniel, Hyuk-Soo Seo, Ying Chen, Scott B. Ficarro, Yann Jamin, Xiaolan Lian, Shin Kibe, Shingo Kozono, Kazuhiro Koikawa, Zainab M. Doctor, Behnam Nabet, Christopher M. Browne, Annan Yang, Liat Stoler-Barak, Richa B. Shah, Nick E. Vangos, Ezekiel A. Geffken, Roni Oren, Samuel Sidi, Ziv Shulman, Chu Wang, Jarrod A. Marto, Sirano Dhe-Paganon, Thomas Look, Xiao Zhen Zhou, Kun Ping Lu, Rosalie C. Sears, Louis Chesler, Nathanael S. Gray, Nir London

BioRXiv, 2020
doi: https://doi.org/10.1101/2020.03.20.998443

The peptidyl-prolyl cis-trans isomerase, Pin1, acts as a unified signaling hub that is exploited in cancer to activate oncogenes and inactivate tumor suppressors, in particular through up-regulation of c-Myc target genes. However, despite considerable efforts, Pin1 has remained an elusive drug target. Here, we screened an electrophilic fragment library to discover covalent inhibitors targeting Pin1’s active site nucleophile - Cys113, leading to the development of Sulfopin, a double-digit nanomolar Pin1 inhibitor. Sulfopin is highly selective for Pin1, as validated by two independent chemoproteomics methods, achieves potent cellular and in vivo target engagement, and phenocopies genetic knockout of Pin1. Although Pin1 inhibition had a modest effect on viability in cancer cell cultures, Sulfopin induced downregulation of c-Myc target genes and reduced tumor initiation and tumor progression in murine and zebrafish models of MYCN-driven neuroblastoma. Our results suggest that Sulfopin is a suitable chemical probe for assessing Pin1-dependent pharmacology in cells and in vivo. Moreover, these studies indicate that Pin1 should be further investigated as a potential cancer target.

Crystal structure of SARS-CoV-2 main protease provides a basis for design of improved α-ketoamide inhibitors

Linlin Zhang, Daizong Lin, Xinyuanyuan Sun, Ute Curth, Christian Drosten, Lucie Sauerhering, Stephan Becker, Katharina Rox, Rolf Hilgenfeld

Science, 2020: eabb3405

DOI: 10.1126/science.abb3405

The COVID-19 pandemic caused by SARS-CoV-2 is a global health emergency. An attractive drug target among coronaviruses is the main protease (Mpro, 3CLpro), due to its essential role in processing the polyproteins that are translated from the viral RNA. We report the X-ray structures of the unliganded SARS-CoV-2 Mpro and its complex with an α-ketoamide inhibitor. This was derived from a previously designed inhibitor but with the P3-P2 amide bond incorporated into a pyridone ring to enhance the half-life of the compound in plasma. Based on the structure, we developed the lead compound into a potent inhibitor of the SARS-CoV-2 Mpro. The pharmacokinetic characterization of the optimized inhibitor reveals a pronounced lung tropism and suitability for administration by the inhalative route.