C797S Resistance: The Undruggable EGFR Mutation in Non-Small Cell Lung Cancer?

Tobias Grabe, Jonas Lategahn, and Daniel Rauh

ACS Med. Chem. Lett., 2018
DOI: 10.1021/acsmedchemlett.8b00314

The first evidence of osimertinib resistance mediated by the epidermal growth factor receptor (EGFR) mutation C797S was reported three years ago. Since then, no major breakthroughs have been achieved to target the clinically relevant mutant variant that impedes covalent bond formation with irreversible EGFR inhibitors. Although several biochemically active compounds have been described, only a few inhibitors that potently act on the cellular level or in vivo have been introduced so far. Herein, we give an overview of current approaches in the field and highlight the challenges that need to be addressed in future research projects to overcome the C797S-mediated drug resistance.

Structural basis of substrate recognition and covalent inhibition of Cdu1 from Chlamydia trachomatis

Yesid Andres Ramirez  Thomas Adler  Eva Altmann  Christian Tiesmeyer  Theresa Klemm Florian Sauer  Stefan Kathman  Alexander Statsyuk  Christoph Sotriffer  Caroline Kisker

ChemMedChem, 2018

doi: 10.1002/cmdc.201800364

Based on the similarity between the active sites of the deubiquitylating and deneddylating enzyme ChlaDub1 (Cdu1) and the evolutionary related protease adenain a target‐hopping approach screening on a focused set of adenain inhibitors has been pursued. The thereby identified cyano‐pyrimidine based inhibitors represent the first active‐site directed small molecule inhibitors for Cdu1. High‐resolution crystal structures of Cdu1 in complex with two covalently bound cyano‐pyrimidines as well as with its substrate ubiquitin have been obtained. These structural data were complemented by enzymatic assays and covalent docking studies to provide insight into Cdu1s substrate recognition, active site pocket flexibility and potential hotspots for ligand interaction. Combined, these data provide a strong foundation for future structure‐guided medicinal chemistry optimization of this cyano‐pyrimidine based scaffold towards more potent and specific Cdu1 inhibitors.

Mechanistic Insight through Irreversible Inhibition: DNA Polymerase θ Uses a Common Active Site for Polymerase and Lyase Activities

Daniel J. Laverty, Ifor P. Mortimer, and Marc M. Greenberg
J. Am. Chem. Soc. 2018 140 (29), 9034–9037
doi: 10.1021/jacs.8b04158

DNA polymerase θ (Pol θ) is a multifunctional enzyme. It is nonessential in normal cells, but its upregulation in cancer cells correlates with cellular resistance to oxidative damage and poor prognosis. Pol θ possesses polymerase activity and poorly characterized lyase activity. We examined the Pol θ lyase activity on various abasic sites and determined that the enzyme is inactivated upon attempted removal of the oxidized abasic site commonly associated with C4′-oxidation (pC4-AP). Covalent modification of Pol θ by the DNA lesion enabled determination of the primary nucleophile (Lys2383) responsible for Schiff base formation in the lyase reaction. Unlike some other base excision repair polymerases, Pol θ uses a single active site for polymerase and lyase activity. Mutation of Lys2383 significantly reduces both enzyme activities but not DNA binding. Demonstration that Lys2383 is required for polymerase and lyase activities indicates that this residue is an Achilles heel for Pol θ and suggests a path forward for designing inhibitors of this attractive anticancer target.

Arylfluorosulfate‐Based Warheads for Covalent Protein Labeling: A New Addition to the Arsenal

Pablo Martin-Gago, Christian Adam Olsen

Angewandte Chemie, 2018

doi: 10.1002/anie.201806037

Selective covalent modification of a targeted protein is a powerful tool in chemical biology and drug discovery, with applications ranging from identification and characterization of proteins and their functions to the development of targeted covalent inhibitors. Most covalent ligands contain an "affinity motif" and an electrophilic warhead that reacts with a nucleophilic residue of the targeted protein. Because the electrophilic warhead is prone to react and modify off‐target nucleophiles, its reactivity should be balanced carefully to maximize target selectivity. Arylfluorosulfates have recently emerged as latent electrophiles for selective labeling of context‐specific tyrosine and lysine residues in protein pockets. Here, we review the recent but intense introduction of arylfluorosulfates into the arsenal of available warheads for selective covalent modification of proteins. We highlight the untapped potential of this functional group for use in chemical biology and drug discovery.

The Meisenheimer Complex as a Paradigm in Drug Discovery: Reversible Covalent Inhibition through C67 of the ATP Binding Site of PLK1

Russell J.Pearson, David G. Blake, Mokdad Mezna, Peter M.Fischer, Nicholas J.Westwood, Campbell McInnes

Cell Chem. Biol. 2018

DOI: 10.1016/j.chembiol.2018.06.001

The polo kinase family are important oncology targets that act in regulating entry into and progression through mitosis. Structure-guided discovery of a new class of inhibitors of Polo-like kinase 1 (PLK1) catalytic activity that interact with Cys67 of the ATP binding site is described. Compounds containing the benzothiazole N-oxide scaffold not only bind covalently to this residue, but are reversible inhibitors through the formation of Meisenheimer complexes. This mechanism of kinase inhibition results in compounds that can target PLK1 with high selectivity, while avoiding issues with irreversible covalent binding and interaction with other thiol-containing molecules in the cell. Due to renewed interest in covalent drugs and the plethora of potential drug targets, these represent prototypes for the design of kinase inhibitory compounds that achieve high specificity through covalent interaction and yet still bind reversibly to the ATP cleft, a strategy that could be applied to avoid issues with conventional covalent binders.

Merits and Pitfalls in the Characterization of Covalent Inhibitors of Bruton’s Tyrosine Kinase

Christopher M. Harris, Sage E. Foley, Eric R. Goedken, Mark Michalak, Sara Murdock, and Noel S. Wilson

SLAS Discovery, 2018, 1–11

DOI: 10.1177/2472555218787445

In vitro analysis of covalent inhibitors requires special consideration, due to the time-dependent and typically irreversible nature of their target interaction. While many analyses are reported for the characterization of a final candidate, it is less clear which are most useful in the lead optimization phase of drug discovery. In the context of identifying covalent inhibitors of Bruton’s tyrosine kinase (BTK), we evaluated multiple techniques for characterizing covalent inhibitors. Several methods qualitatively support the covalent mechanism of action or support a particular aspect of interaction but were not otherwise informative to differentiate inhibitors. These include the time dependence of IC₅₀, substrate competition, mass spectrometry, and recovery of function after inhibitor removal at the biochemical and cellular level. A change in IC₅₀ upon mutation of the targeted BTK C481 nucleophile or upon removal of the electrophilic moiety of the inhibitor was not always a reliable indicator of covalent inhibition. Determination of k_inact and K_I provides a quantitative description of covalent interactions but was challenging at scale and frequently failed to provide more than the ratio of the two values, k_inact/K_I. Overall, a combination of approaches is required to assess time-dependent, covalent, and irreversible inhibitors in a manner suitable to reliably advance drug candidates.

Structure-Based Design, Synthesis, and Characterization of the First Irreversible Inhibitor of Focal Adhesion Kinase

Acebrón-Garcia-de-Eulate, Marta; Tomkiewicz-Raulet, Céline; Dawson, John; Lietha, Daniel; Frame, Margaret C.; Coumoul, Xavier; Garbay, Christiane; Etheve-Quelquejeu, Mélanie Chen, Huixiong

ACS Chem. Biol., Article ASAP

DOI: 10.1021/acschembio.8b00250

Focal Adhesion Kinase signaling pathway and its functions have been involved in the development and aggressiveness of tumor malignancy, it then presents a promising cancer therapeutic target. Several reversible FAK inhibitors have been developed and are being conducted in clinical trials. On the other hand, irreversible covalent inhibitors would bring many desirable pharmacological features including high potency and increased duration of action. Herein we report the structure-guided development of the first highly potent and irreversible inhibitor of the FAK kinase. This inhibitor showed a very potent decrease of autophosphorylation of FAK in squamous cell carcinoma. A cocrystal structure of the FAK kinase domain in complex with this compound revealed the inhibitor binding mode within the ATP binding site and confirmed the covalent linkage between the targeted Cys427 of the protein and the inhibitor.

Oridonin is a covalent NLRP3 inhibitor with strong anti-inflammasome activity

Hongbin He, Hua Jiang, Yun Chen, Jin Ye, Aoli Wang, Chao Wang, Qingsong Liu, Gaolin Liang, Xianming Deng, Wei Jiang & Rongbin Zhou
Nature Communications, 2018, 9, 2550, doi: 10.1038/s41467-018-04947-6

Oridonin (Ori) is the major active ingredient of the traditional Chinese medicinal herb Rabdosia rubescens and has anti-inflammatory activity, but the target of Ori remains unknown. NLRP3 is a central component of NLRP3 inflammasome and has been involved in a wide variety of chronic inflammation-driven human diseases. Here, we show that Ori is a specific and covalent inhibitor for NLRP3 inflammasome. Ori forms a covalent bond with the cysteine 279 of NLRP3 in NACHT domain to block the interaction between NLRP3 and NEK7, thereby inhibiting NLRP3 inflammasome assembly and activation. Importantly, Ori has both preventive or therapeutic effects on mouse models of peritonitis, gouty arthritis and type 2 diabetes, via inhibition of NLRP3 activation. Our results thus identify NLRP3 as the direct target of Ori for mediating Ori’s anti-inflammatory activity. Ori could serve as a lead for developing new therapeutics against NLRP3-driven diseases.

Identification of the histidine residue in vitamin D receptor that covalently binds to electrophilic ligands

Mami Yoshizawa, Toshimasa Itoh, Tatsuya Hori, Akira Kato, Yasuaki Anami, Nobuko Yoshimoto, and Keiko Yamamoto

J. Med. Chem., 2018

DOI: 10.1021/acs.jmedchem.8b00774

We designed and synthesized vitamin D analogues with an electrophile as covalent modifiers for the vitamin D receptor (VDR). Novel vitamin D analogues 1-4 have an electrophilic enone group at the side chain for conjugate addition to His301 or His393 in the VDR. All compounds showed specific VDR-binding potency and agonistic activity. Covalent bond formations of 1-4 with the ligand-binding domain (LBD) of VDR was evaluated by electrospray ionization mass spectrometry. All compounds were shown to covalently bind to the VDR-LBD, and the abundance of VDR-LBD corresponding conjugate adducts of 1-4 increased with incubation time. Enone compounds 1 and 2 showed higher reactivity than the ene-ynone 3 and dienone 4 compounds. Furthermore, we successfully obtained co-crystals of VDR-LBD with analogues 1-4. X-ray crystallographic analysis showed a covalent bond with His301 in VDR-LBD. We successfully synthesized vitamin D analogues that form a covalent bond with VDR-LBD.