Tuesday, December 11, 2018

A Chemoproteomic Strategy for Direct and Proteome-wide Covalent Inhibitor Target-site Identification

Christopher Michael BrowneBaishan JiangScott B FicarroZainab M DoctorJared Lee JohnsonJoseph D CardSindhu Carmen SivakumarenWilliam M AlexanderTomer YaronCharles Joseph MurphyNicholas P KwiatkowskiTinghu ZhangLewis C. CantleyNathanael S Gray, and Jarrod A. Marto

Journal of the American Chemical Society 2018
DOI: 10.1021/jacs.8b07911

Despite recent clinical successes for irreversible drugs, potential toxicities mediated by unpredictable modification of off-target cysteines represents a major hurdle for expansion of covalent drug programs. Understanding the proteome-wide binding profile of covalent inhibitors can significantly accelerate their development; however, current mass spectrometry strategies typically do not provide a direct, amino acid level readout of covalent activity for complex, selective inhibitors. Here we report the development of CITe-Id, a novel chemoproteomic approach that employs covalent pharmacologic inhibitors as enrichment reagents in combination with an optimized proteomic platform to directly quantify dose-dependent binding at cysteine-thiols across the proteome. CITe-Id analysis of our irreversible CDK inhibitor THZ1 identified dose-dependent covalent modification of several unexpected kinases, including a previously unannotated cysteine (C840) on the understudied kinase PKN3. These data streamlined our development of JZ128 as a new selective covalent inhibitor of PKN3. Using JZ128 as a probe compound, we identified novel potential PKN3 substrates, thus offering an initial molecular view of PKN3 cellular activity. CITe-Id provides a powerful complement to current chemoproteomic platforms to characterize the selectivity of covalent inhibitors, identify new, pharmacologically-addressable cysteine-thiols, and inform structure-based drug design programs.

Monday, December 3, 2018

Overview of Current Type I/II Kinase Inhibitors

Zheng Zhao, Philip E. Bourne


Research on kinase-targeting drugs has made great strides over the last 30 years and is attracting greater attention for the treatment of yet more kinase-related diseases. Currently, 42 kinase drugs have been approved by the FDA, most of which (39) are Type I/II inhibitors. Notwithstanding these advances, it is desirable to target additional kinases for drug development as more than 200 diseases, particularly cancers, are directly associated with aberrant kinase regulation and signaling. Here, we review the extant Type I/II drugs systematically to obtain insights into the binding pocket characteristics, the associated features of Type I/II drugs, and the mechanism of action to facilitate future kinase drug design and discovery. We conclude by summarizing the main successes and limitations of targeting kinase for the development of drugs.

Tuesday, November 20, 2018

Covalent Docking Identifies a Potent and Selective MKK7 Inhibitor [@london_lab]

Amit Shraga, Evgenia Olshvang, Natalia Davidzohn, Payam Khoshkenar, Nicolas Germain, Khriesto Shurrush, Silvia Carvalho, Liat Avram, Shira Albeck, Tamar Unger, Bruce Lefker, Chakrapani Subramanyam, Robert L. Hudkins, Amir Mitchell, Ziv Shulman, Takayoshi Kinoshita, Nir London,
Covalent Docking Identifies a Potent and Selective MKK7 Inhibitor,
Cell Chemical Biology, 2018

DOI: 10.1016/j.chembiol.2018.10.011

The c-Jun NH2-terminal kinase (JNK) signaling pathway is central to the cell response to stress, inflammatory signals, and toxins. While selective inhibitors are known for JNKs and for various upstream MAP3Ks, no selective inhibitor is reported for MKK7––one of two direct MAP2Ks that activate JNK. Here, using covalent virtual screening, we identify selective MKK7 covalent inhibitors. We optimized these compounds to low-micromolar inhibitors of JNK phosphorylation in cells. The crystal structure of a lead compound bound to MKK7 demonstrated that the binding mode was correctly predicted by docking. We asserted the selectivity of our inhibitors on a proteomic level and against a panel of 76 kinases, and validated an on-target effect using knockout cell lines. Lastly, we show that the inhibitors block activation of primary mouse B cells by lipopolysaccharide. These MKK7 tool compounds will enable better investigation of JNK signaling and may serve as starting points for therapeutics.

Saturday, November 17, 2018

Balancing reactivity and antitumor activity: Heteroarylthio acetamide derivatives as potent and time-dependent inhibitors of EGFR

Riccardo Castelli, Nicole Bozza, Andrea Cavazzoni, Mara Bonelli, Federica Vacondio, Francesca Ferlenghi, Donatella Callegari, Claudia Silva, Silvia Rivara, Alessio Lodola, Graziana Digiacomo, Claudia Fumarola, Roberta Alfieri, Pier Giorgio Petronini, Marco Mor

European Journal of Medicinal Chemistry, 2018
DOI: 10.1016/j.ejmech.2018.11.029

Second- and third-generation inhibitors of EGFR possess an acrylamide group which alkylates Cys797, allowing to overcome resistance due to insurgence of T790M mutation. Less reactive warheads, yet capable to bind the target cysteine, may be useful to design newer and safer inhibitors. In the present work, we synthesized a 2-chloro-N-(4-(phenylamino)quinazolin-6-yl)acetamide (8) derivative as a prototype of EGFR inhibitor potentially able to react with Cys797 by nucleophilic substitution. We then tuned the reactivity of the acetamide fragment by replacing the chlorine leaving group with (hetero)-aromatic thiols or carboxylate esters. Among the synthesized derivatives, the 2-((1H-imidazol-2-yl)thio)acetamide 16, while showing negligible reactivity with cysteine in solution, caused long-lasting inhibition of wild-type EGFR autophosphorylation in A549 cells, resulted able to bind recombinant EGFR L858R/T790M in a time-dependent manner, and inhibited both EGFR autophosphorylation and proliferation in gefitinib-resistant H1975 (EGFR L858R/T790M) lung cancer cells at low micromolar concentration.

Tuesday, November 6, 2018

Neolymphostin A is a Covalent Phosphoinositide-3-kinase (PI3-K)/Mammalian Target of Rapamycin (mTOR) Dual Inhibitor that Employs an Unusual Electrophilic Vinylogous Ester

Gabriel Castro-Falcón, Grant Seiler, Ozlem Demir, Manoj Rathinaswamy, David Hamelin, Reece M. Hoffmann, Stefanie Makowski, Anne-Catrin Letzel, Seth Field, John Burke, Rommie E. Amaro, and Chambers C. Hughes

Journal of Medicinal Chemistry 2018
DOI: 10.1021/acs.jmedchem.8b00975

Using a novel chemistry-based assay for identifying electrophilic natural products from unprocessed extracts, we identified the PI3-kinase/mTOR dual inhibitor neolymphostin A from Salinispora arenicola CNY-486. The method further showed that the vinylogous ester substituent on the neolymphostin core was the exact site for enzyme conjugation. Tandem MS/MS experiments on PI3Kα treated with the inhibitor revealed that neolymphostin covalently modified Lys802 with a shift in mass of +306 amu, corresponding to addition of the inhibitor and elimination of methanol. The binding pose of the inhibitor bound to PI3Kα was modelled, and hydrogen-deuterium exchange mass spectrometry experiments supported this model. Against a panel of kinases, neolymphostin showed good selectivity for PI3-kinase and mTOR. In addition, the natural product blocked AKT phosphorylation in live cells with an IC50 of ~3 nM. Taken together, neolymphostin is the first reported example of a covalent kinase inhibitor from the bacterial domain of life.

Saturday, November 3, 2018

Structure-based engineering of irreversible inhibitors against histone lysine demethylase KDM5A

John R Horton, Clayton B Woodcock, Qin Chen, Xu Liu, Xing Zhang, John Shanks, Ganesha Rai, Bryan T Mott, Daniel J Jansen, Stephen C Kales, Mark J Henderson, Matthew Cyr, Katherine Pohida, Xin Hu, Pranav Shah, Xin Xu, Ajit Jadhav, David J. Maloney, Matthew D. Hall, Anton Simeonov, Haian Fu, Paula M. Vertino, and Xiaodong Cheng

J. Med. Chem., 2018
DOI: 10.1021/acs.jmedchem.8b01219

The active sites of hundreds of human α-ketoglutarate (αKG) and Fe(II)-dependent dioxygenases are exceedingly well preserved, which challenges the design of selective inhibitors. We identified a non-catalytic cysteine (Cys481 in KDM5A) near the active sites of KDM5 histone H3 lysine 4 demethylases – which is absent in other histone demethylase families - that could be explored for interaction with the cysteine-reactive electrophile acrylamide. We synthesized analogs of a thienopyridine-based inhibitor chemotype, namely 2-((3-aminophenyl)(2-(piperidin-1-yl)ethoxy)methyl)thieno[3,2-b]pyridine-7-carboxylic acid (N70) and a derivative containing a (dimethylamino)but-2-enamido)phenyl moiety (N71) designed to form a covalent interaction with Cys481. We characterized the inhibitory and binding activities against KDM5A and determined the co-crystal structures of the catalytic domain of KDM5A in complex with N70 and N71. Whereas the non-covalent inhibitor N70 displayed αKG-competitive inhibition that could be reversed after dialysis, inhibition by N71 was dependent on enzyme concentration and persisted even after dialysis, consistent with covalent modification.

Tuesday, October 30, 2018

Genetic Incorporation of Olefin Cross-Metathesis Reaction Tags for Protein Modification

Bhaskar Bhushan, Yuya A. Lin, Martin Bak, Anuchit Phanumartwiwath, Nan Yang, Matthew K. Bilyard, Tomonari Tanaka, Kieran L. Hudson, Lukas Lercher, Monika Stegmann, Shabaz Mohammed, and Benjamin G. Davis

J. Am. Chem. Soc., 2018
DOI: 10.1021/jacs.8b09433

Olefin cross-metathesis (CM) is a viable reaction for the modification of alkene-containing proteins. Although allyl sulfide or selenide side-chain motifs in proteins can critically enhance the rate of CM reactions, no efficient method for their site-selective genetic incorporation into proteins has been reported to date. Here, through the systematic evaluation of olefin-bearing unnatural amino acids for their metabolic incorporation, we have discovered S-allylhomocysteine (Ahc) as a genetically encodable Met analogue that is not only processed by translational cellular machinery but also a privileged CM substrate residue in proteins. In this way, Ahc was used for efficient Met codon reassignment in a Met-auxotrophic strain of E. coli (B834 (DE3)) as well as metabolic labeling of protein in human cells and was reactive toward CM in several representative proteins. This expands the use of CM in the toolkit for “tag-and-modify” functionalization of proteins.

A Chemoproteomic Strategy for Direct and Proteome-wide Covalent Inhibitor Target-site Identification

Christopher Michael Browne ,  Baishan Jiang ,  Scott B Ficarro ,  Zainab M Doctor ,  Jared Lee Johnson ,  Joseph D Card ,  Sindhu Carmen Si...