Structure of the Human Protein Kinase ZAK in Complex with Vemurafenib.

The mixed lineage kinase ZAK is a key regulator of the MAPK pathway mediating cell survival and inflammatory response. ZAK is targeted by several clinically approved kinase inhibitors, and inhibition of ZAK has been reported to protect from doxorubicin-induced cardiomyopathy. On the other hand, unintended targeting of ZAK has been linked to severe adverse effects such as the development of cutaneous squamous cell carcinoma. Therefore, both specific inhibitors of ZAK, as well as anticancer drugs lacking off-target activity against ZAK, may provide therapeutic benefit. Here, we report the first crystal structure of ZAK in complex with the B-RAF inhibitor vemurafenib. The cocrystal structure displayed a number of ZAK-specific features including a highly distorted P loop conformation enabling rational inhibitor design. Positional scanning peptide library analysis revealed a unique substrate specificity of the ZAK kinase including unprecedented preferences for histidine residues at positions -1 and +2 relative to the phosphoacceptor site. In addition, we screened a library of clinical kinase inhibitors identifying several inhibitors that potently inhibit ZAK, demonstrating that this kinase is commonly mistargeted by currently used anticancer drugs.

[1]  M. Holderfield,et al.  Mechanism and consequences of RAF kinase activation by small-molecule inhibitors , 2014, British Journal of Cancer.

[2]  C. Tournier,et al.  Exploring the function of the JNK (c-Jun N-terminal kinase) signalling pathway in physiological and pathological processes to design novel therapeutic strategies. , 2012, Biochemical Society transactions.

[3]  G. Superti-Furga,et al.  Structural Basis for the Autoinhibition of c-Abl Tyrosine Kinase , 2003, Cell.

[4]  Victor G Prieto,et al.  BRAF inhibitors suppress apoptosis through off-target inhibition of JNK signaling , 2013, eLife.

[5]  Arvin C Dar,et al.  Small molecule recognition of c-Src via the Imatinib-binding conformation. , 2008, Chemistry & biology.

[6]  A. Alonso,et al.  Multiple independent kinase cascades are targeted by hyperosmotic stress but only one activates stress kinase p38. , 2004, Experimental cell research.

[7]  Mindy I. Davis,et al.  Comprehensive analysis of kinase inhibitor selectivity , 2011, Nature Biotechnology.

[8]  H. Saito,et al.  Conserved docking site is essential for activation of mammalian MAP kinase kinases by specific MAP kinase kinase kinases. , 2005, Molecular cell.

[9]  B. Roux,et al.  Explaining why Gleevec is a specific and potent inhibitor of Abl kinase , 2013, Proceedings of the National Academy of Sciences.

[10]  Vincent B. Chen,et al.  Correspondence e-mail: , 2000 .

[11]  W. Kuo,et al.  Transforming growth factor-beta induces the expression of ANF and hypertrophic growth in cultured cardiomyoblast cells through ZAK. , 2004, Biochemical and biophysical research communications.

[12]  E. Tosti,et al.  The Stress Kinase MRK Contributes to Regulation of DNA Damage Checkpoints through a p38γ-independent Pathway* , 2004, Journal of Biological Chemistry.

[13]  E. Jonasch,et al.  Cutaneous squamous cell carcinoma and inflammation of actinic keratoses associated with sorafenib. , 2009, Clinical genitourinary cancer.

[14]  A. Ahluwalia,et al.  ZAK: a MAP3Kinase that transduces Shiga toxin‐ and ricin‐induced proinflammatory cytokine expression , 2008, Cellular microbiology.

[15]  Randy J Read,et al.  Electronic Reprint Biological Crystallography Likelihood-enhanced Fast Translation Functions Biological Crystallography Likelihood-enhanced Fast Translation Functions , 2022 .

[16]  Sridhar Ramaswamy,et al.  Targeting transcription regulation in cancer with a covalent CDK7 inhibitor , 2014, Nature.

[17]  A. Hauschild,et al.  Improved survival with vemurafenib in melanoma with BRAF V600E mutation. , 2011, The New England journal of medicine.

[18]  A. Clerk,et al.  Cardiac protein kinases: the cardiomyocyte kinome and differential kinase expression in human failing hearts. , 2015, Cardiovascular research.

[19]  B. Magun,et al.  Small molecule kinase inhibitors block the ZAK-dependent inflammatory effects of doxorubicin , 2013, Cancer biology & therapy.

[20]  Jaw‐Ji Yang.  Mixed lineage kinase ZAK utilizing MKK7 and not MKK4 to activate the c-Jun N-terminal kinase and playing a role in the cell arrest. , 2002, Biochemical and biophysical research communications.

[21]  J. Kuriyan,et al.  c-Src binds to the cancer drug imatinib with an inactive Abl/c-Kit conformation and a distributed thermodynamic penalty. , 2007, Structure.

[22]  M. Isobe,et al.  ATF3 inhibits doxorubicin-induced apoptosis in cardiac myocytes: a novel cardioprotective role of ATF3. , 2002, Journal of molecular and cellular cardiology.

[23]  S. Jackson,et al.  USP4 Auto-Deubiquitylation Promotes Homologous Recombination , 2015, Molecular cell.

[24]  T. Hunter,et al.  The Protein Kinase Complement of the Human Genome , 2002, Science.

[25]  I. Soubeyran,et al.  The MAP3K ZAK, a novel modulator of ERK-dependent migration, is upregulated in colorectal cancer , 2016, Oncogene.

[26]  G. Murshudov,et al.  Refinement of macromolecular structures by the maximum-likelihood method. , 1997, Acta crystallographica. Section D, Biological crystallography.

[27]  S. Knapp,et al.  A unique inhibitor binding site in ERK1/2 is associated with slow binding kinetics , 2014, Nature chemical biology.

[28]  E. Nigg,et al.  Activity of the Human Centrosomal Kinase, Nek2, Depends on an Unusual Leucine Zipper Dimerization Motif* , 1999, The Journal of Biological Chemistry.

[29]  P. Pai,et al.  ZAK induces cardiomyocyte hypertrophy and brain natriuretic peptide expression via p38/JNK signaling and GATA4/c-Jun transcriptional factor activation , 2015, Molecular and Cellular Biochemistry.

[30]  Kevan M Shokat,et al.  Features of selective kinase inhibitors. , 2005, Chemistry & biology.

[31]  R. Beavis,et al.  Tissue distribution and functional expression of a cDNA encoding a novel mixed lineage kinase. , 2001, Journal of molecular and cellular cardiology.

[32]  Y. Ono,et al.  Regulation of a mitogen-activated protein kinase kinase kinase, MLTK by PKN. , 2003, Journal of biochemistry.

[33]  Mindy I. Davis,et al.  A quantitative analysis of kinase inhibitor selectivity , 2008, Nature Biotechnology.

[34]  John Kuriyan,et al.  Equally potent inhibition of c-Src and Abl by compounds that recognize inactive kinase conformations. , 2009, Cancer research.

[35]  Chad J. Miller,et al.  Rapid Identification of Protein Kinase Phosphorylation Site Motifs Using Combinatorial Peptide Libraries. , 2016, Methods in molecular biology.

[36]  J. Mestan,et al.  Extended kinase profile and properties of the protein kinase inhibitor nilotinib. , 2010, Biochimica et biophysica acta.

[37]  F. Niesen,et al.  The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability , 2007, Nature Protocols.

[38]  Nathanael S Gray,et al.  Developing irreversible inhibitors of the protein kinase cysteinome. , 2013, Chemistry & biology.

[39]  Cristiano R. W. Guimaraes,et al.  Understanding the Impact of the P-loop Conformation on Kinase Selectivity , 2011, J. Chem. Inf. Model..

[40]  Damian Szklarczyk,et al.  Specific CLK Inhibitors from a Novel Chemotype for Regulation of Alternative Splicing , 2011, Chemistry & biology.

[41]  J. Pitha,et al.  A clinicopathologic analysis of adriamycin cardiotoxicity , 1973, Cancer.

[42]  T. C. Liu,et al.  Cloning and expression of ZAK, a mixed lineage kinase-like protein containing a leucine-zipper and a sterile-alpha motif. , 2000, Biochemical and biophysical research communications.

[43]  K. Tsai,et al.  Sorafenib Suppresses JNK-Dependent Apoptosis through Inhibition of ZAK , 2013, Molecular Cancer Therapeutics.

[44]  M. Belvin,et al.  RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth , 2010, Nature.

[45]  A. Hauschild,et al.  Dabrafenib in BRAF-mutated metastatic melanoma: a multicentre, open-label, phase 3 randomised controlled trial , 2012, The Lancet.

[46]  Kwok-Kin Wong,et al.  Development of Selective Covalent Janus Kinase 3 Inhibitors. , 2015, Journal of medicinal chemistry.

[47]  O. Gileadi,et al.  Medium-throughput production of recombinant human proteins: ligation-independent cloning. , 2014, Methods in molecular biology.

[48]  Graeme Winter,et al.  xia2: an expert system for macromolecular crystallography data reduction , 2010 .

[49]  Thomas D. Wu,et al.  Integrated exome and transcriptome sequencing reveals ZAK isoform usage in gastric cancer , 2014, Nature Communications.

[50]  Stefan Knapp,et al.  Kinase inhibitor selectivity profiling using differential scanning fluorimetry. , 2012, Methods in molecular biology.

[51]  Kam Y. J. Zhang,et al.  Clinical efficacy of a RAF inhibitor needs broad target blockade in BRAF-mutant melanoma , 2010, Nature.