Proteome-wide Map of Targets of T790M-EGFR-Directed Covalent Inhibitors

Sherry Niessen,, Melissa M. Dix, Sabrina Barbas, Zachary E. Potter, Shuyan Lu, Oleg Brodsky, Simon Planken, Douglas Behenna, Chau Almaden, Ketan S. Gajiwala, Kevin Ryan, RoseAnn Ferre, Michael R. Lazear, Matthew M. Hayward, John C. Kath, Benjamin F. Cravatt

Cell Chemical Biology, 2017 doi: 10.1016/j.chembiol.2017.08.017

Abstract

Patients with non-small cell lung cancers that have kinase-activating epidermal growth factor receptor (EGFR) mutations are highly responsive to first- and second-generation EGFR inhibitors. However, these patients often relapse due to a secondary, drug-resistant mutation in EGFR whereby the gatekeeper threonine is converted to methionine (T790M). Several third-generation EGFR inhibitors have been developed that irreversibly inactivate T790M-EGFR while sparing wild-type EGFR, thus reducing epithelium-based toxicities. Using chemical proteomics, we show here that individual T790M-EGFR inhibitors exhibit strikingly distinct off-target profiles in human cells. The FDA-approved drug osimertinib (AZD9291), in particular, was found to covalently modify cathepsins in cell and animal models, which correlated with lysosomal accumulation of the drug. Our findings thus show how chemical proteomics can be used to differentiate covalent kinase inhibitors based on global selectivity profiles in living systems and identify specific off-targets of these inhibitors that may affect drug activity and safety.

Sequence-Based Prediction of Cysteine Reactivity Using Machine Learning

Haobo Wang, Xuemin Chen, Can Li, Yuan Liu, Fan Yang, and Chu Wang

Biochemistry, 2017 DOI: 10.1021/acs.biochem.7b00897

As one of the most intrinsically reactive amino acids, cysteine carries a variety of important biochemical functions, including catalysis and redox regulation. Discovery and characterization of cysteines with heightened reactivity will help annotate protein functions. Chemical proteomic methods have been used to quantitatively profile cysteine reactivity in native proteomes, showing a strong correlation between the chemical reactivity of a cysteine and its functionality; however, the relationship between the cysteine reactivity and its local sequence has not yet been systematically explored. Herein, we report a machine learning method, sbPCR (sequence-based prediction of cysteine reactivity), which combines the basic local alignment search tool, truncated composition of k-spaced amino acid pair analysis, and support vector machine to predict cysteines with hyper-reactivity based on only local sequence features. Using a benchmark set compiled from hyper-reactive cysteines in human proteomes, our method can achieve a prediction accuracy of 98%, a precision of 95%, and a recall ratio of 89%. We utilized these governing features of local sequence motifs to expand the prediction to potential hyper-reactive cysteines in other proteomes deposited in the UniProt database. We validated our predictions in Escherichia coli by activity-based protein profiling and discovered a hyper-reactive cysteine from a functionally uncharacterized protein, YecH. Biochemical analysis suggests that the hyper-reactive cysteine might be involved in metal binding. Our computational method provides a large inventory of potential hyper-reactive cysteines in proteomes and is highly complementary to other experimental approaches to guide systematic annotation of protein functions in the postgenome era.

Targeting the Protein Kinases Cysteinome

Apirat Chaikuad, Pierre Koch, Stefan Laufer, and Stefan Knapp

Angew. Chem. Int. Ed. 2017

doi: 10.1002/anie.201707875

Drugs that function by covalent bond formation represent a considerable fraction of our repository of effective medicines but safety concerns and the complexity of developing covalent inhibitors has rendered covalent targeting a less attractive strategy for rational drug design. The recent approval of four covalent kinase inhibitors and the development of highly potent covalent kinase probes with exceptional selectivity has raised significant interest in industry and academic research and validated the concept of covalent kinase targeting for clinical applications. The abundance of cysteines at diverse positions in and around the kinase active site suggests that a large fraction of kinases can be targeted by covalent inhibitors. Here we review recent developments of this rapidly growing area in kinase drug development and highlight the unique opportunities and challenges of this strategy.

Covalent lectin inhibition and application in bacterial biofilm imaging

Stefanie Wagner, Dirk Hauck, Michael Hoffmann, Roman Sommer, Ines Joachim, Rolf Müller, Anne Imberty, Annabelle Varrot, Alexander Titz
Angew. Chem. 2017
DOI: 10.1002/ange.201709368

Biofilm formation by pathogenic bacteria is a hallmark of chronic infections. In many cases, lectins play key roles in establishing biofilms. The pathogen Pseudomonas aeruginosa often exhibiting various drug resistances employs its lectins LecA and LecB as virulence factors and biofilm building blocks. Therefore, inhibition of the function of these proteins is thought to have potential in developing 'pathoblockers' preventing biofilm formation and virulence. Here, we describe for the first time a covalent lectin inhibitor specific to a carbohydrate binding site. In addition we report its application in the LecA-specific in vitro imaging of biofilms formed by P. aeruginosa.

Cytosolic Delivery of Proteins by Bioreversible Esterification

Kalie A. Mix, Jo E. Lomax, and Ronald T. Raines



J. Am. Chem. Soc., Article ASAP

DOI: 10.1021/jacs.7b06597



Cloaking its carboxyl groups with a hydrophobic moiety is shown to enable a protein to enter the cytosol of a mammalian cell. Diazo compounds derived from (p-methylphenyl)glycine were screened for the ability to esterify the green fluorescent protein (GFP) in an aqueous environment. Esterification of GFP with 2-diazo-2-(p-methylphenyl)-N,N-dimethylacetamide was efficient. The esterified protein entered the cytosol by traversing the plasma membrane directly, like a small-molecule prodrug. As with prodrugs, the nascent esters are substrates for endogenous esterases, which regenerate native protein. Thus, esterification could provide a general means to deliver native proteins to the cytosol.

Proteome-wide Map of Targets of T790M-EGFR-Directed Covalent Inhibitors

Sherry Niessen, Melissa M.Dix, Sabrina Barbas, Zachary Potter. Shuyan Lu, Oleg Brodsky, Simon Planken, Douglas Behenna, Chau Almaden, Ketan S. Gajiwala, Kevin Ryan, Rose Ann Ferre, Michael R. Lazear. Matthew M.Hayward, John C. Kath, Benjamin F. Cravatt

Cell Chemical Biology, 2017
doi: 10.1016/j.chembiol.2017.08.017

Patients with non-small cell lung cancers that have kinase-activating epidermal growth factor receptor (EGFR) mutations are highly responsive to first- and second-generation EGFR inhibitors. However, these patients often relapse due to a secondary, drug-resistant mutation in EGFR whereby the gatekeeper threonine is converted to methionine (T790M). Several third-generation EGFR inhibitors have been developed that irreversibly inactivate T790M-EGFR while sparing wild-type EGFR, thus reducing epithelium-based toxicities. Using chemical proteomics, we show here that individual T790M-EGFR inhibitors exhibit strikingly distinct off-target profiles in human cells. The FDA-approved drug osimertinib (AZD9291), in particular, was found to covalently modify cathepsins in cell and animal models, which correlated with lysosomal accumulation of the drug. Our findings thus show how chemical proteomics can be used to differentiate covalent kinase inhibitors based on global selectivity profiles in living systems and identify specific off-targets of these inhibitors that may affect drug activity and safety.