Protein kinase D (PKD) has emerged as a potential therapeutic target in multiple pathological conditions, including cancer and heart diseases. Potent and selective small molecule inhibitors of PKD are valuable for dissecting PKD-mediated cellular signaling pathways and for therapeutic application. In this study, we evaluated a targeted library of 235 small organic kinase inhibitors for PKD1 inhibitory activity at a single concentration. Twenty-eight PKD inhibitory chemotypes were identified and six exhibited excellent PKD1 selectivity. Five of the six lead structures share a common scaffold, with compound 139 being the most potent and selective for PKD vs PKC and CAMK. Compound 139 was an ATP-competitive PKD1 inhibitor with a low double-digit nanomolar potency and was also cell-active. Kinase profiling analysis identified this class of small molecules as pan-PKD inhibitors, confirmed their selectivity again PKC and CAMK, and demonstrated an overall favorable selectivity profile that could be further enhanced through structural modification. Furthermore, using a PKD homology model based on similar protein kinase structures, docking modes for compound 139 were explored and compared to literature examples of PKD inhibition. Modeling of these compounds at the ATP-binding site of PKD was used to rationalize its high potency and provide the foundation for future further optimization. Accordingly, using biochemical screening of a small number of privileged scaffolds and computational modeling, we have identified a new core structure for highly potent PKD inhibition with promising selectivity against closely related kinases. These lead structures represent an excellent starting point for the further optimization and the design of selective and therapeutically effective small molecule inhibitors of PKD.
References
[1]
Manning G, Whyte DB, Martinez R, Hunter T, Sudarsanam S (2002) The protein kinase complement of the human genome. Science 298: 1912–1934.
[2]
Wang QJ (2006) PKD at the crossroads of DAG and PKC signaling. Trends Pharmacol Sci 27: 317–323.
[3]
Rozengurt E (2011) Protein kinase D signaling: multiple biological functions in health and disease. Physiology (Bethesda) 26: 23–33.
[4]
LaValle CR, George KM, Sharlow ER, Lazo JS, Wipf P, et al. (2010) Protein kinase D as a potential new target for cancer therapy. Biochim Biophys Acta 1806: 183–192.
[5]
Ochi N, Tanasanvimon S, Matsuo Y, Tong Z, Sung B, et al. (2011) Protein kinase D1 promotes anchorage-independent growth, invasion, and angiogenesis by human pancreatic cancer cells. J Cell Physiol 226: 1074–1081.
[6]
Rennecke J, Rehberger PA, Furstenberger G, Johannes FJ, Stohr M, et al. (1999) Protein-kinase-Cmu expression correlates with enhanced keratinocyte proliferation in normal and neoplastic mouse epidermis and in cell culture. Int J Cancer 80: 98–103.
[7]
Ristich VL, Bowman PH, Dodd ME, Bollag WB (2006) Protein kinase D distribution in normal human epidermis, basal cell carcinoma and psoriasis. Br J Dermatol 154: 586–593.
[8]
Biswas MH, Du C, Zhang C, Straubhaar J, Languino LR, et al. (2010) Protein kinase D1 inhibits cell proliferation through matrix metalloproteinase-2 and matrix metalloproteinase-9 secretion in prostate cancer. Cancer Res 70: 2095–2104.
[9]
Chen J, Deng F, Singh SV, Wang QJ (2008) Protein kinase D3 (PKD3) contributes to prostate cancer cell growth and survival through a PKCepsilon/PKD3 pathway downstream of Akt and ERK 1/2. Cancer Res 68: 3844–3853.
[10]
Eiseler T, Doppler H, Yan IK, Goodison S, Storz P (2009) Protein kinase D1 regulates matrix metalloproteinase expression and inhibits breast cancer cell invasion. Breast Cancer Res 11: R13.
[11]
Kim M, Jang HR, Kim JH, Noh SM, Song KS, et al. (2008) Epigenetic inactivation of protein kinase D1 in gastric cancer and its role in gastric cancer cell migration and invasion. Carcinogenesis 29: 629–637.
[12]
Azoitei N, Kleger A, Schoo N, Thal DR, Brunner C, et al. (2011) Protein kinase D2 is a novel regulator of glioblastoma growth and tumor formation. Neuro Oncol 13: 710–724.
[13]
Fielitz J, Kim MS, Shelton JM, Qi X, Hill JA, et al. (2008) Requirement of protein kinase D1 for pathological cardiac remodeling. Proc Natl Acad Sci U S A 105: 3059–3063.
[14]
Harrison BC, Kim MS, van Rooij E, Plato CF, Papst PJ, et al. (2006) Regulation of cardiac stress signaling by protein kinase d1. Mol Cell Biol 26: 3875–3888.
[15]
Vega RB, Harrison BC, Meadows E, Roberts CR, Papst PJ, et al. (2004) Protein kinases C and D mediate agonist-dependent cardiac hypertrophy through nuclear export of histone deacetylase 5. Mol Cell Biol 24: 8374–8385.
[16]
Bossuyt J, Helmstadter K, Wu X, Clements-Jewery H, Haworth RS, et al. (2008) Ca2+/Calmodulin-Dependent Protein Kinase II{delta} and Protein Kinase D Overexpression Reinforce the Histone Deacetylase 5 Redistribution in Heart Failure. Circ Res
[17]
Sharlow ER, Giridhar KV, Lavalle CR, Chen J, Leimgruber S, et al. (2008) Potent and selective disruption of protein kinase d functionality by a benzoxoloazepinolone. J Biol Chem 283: 33516–33526.
[18]
George KM, Frantz MC, Bravo-Altamirano K, Lavalle CR, Tandon M, et al. (2011) Design, Synthesis, and Biological Evaluation of PKD Inhibitors. Pharmaceutics 3: 186–228.
[19]
Monovich L, Vega RB, Meredith E, Miranda K, Rao C, et al. (2010) A novel kinase inhibitor establishes a predominant role for protein kinase D as a cardiac class IIa histone deacetylase kinase. FEBS Lett 584: 631–637.
[20]
Meredith EL, Beattie K, Burgis R, Capparelli M, Chapo J, et al. (2010) Identification of potent and selective amidobipyridyl inhibitors of protein kinase D. J Med Chem 53: 5422–5438.
[21]
Meredith EL, Ardayfio O, Beattie K, Dobler MR, Enyedy I, et al. (2010) Identification of orally available naphthyridine protein kinase D inhibitors. J Med Chem 53: 5400–5421.
[22]
Gamber GG, Meredith E, Zhu Q, Yan W, Rao C, et al. (2011) 3,5-diarylazoles as novel and selective inhibitors of protein kinase D. Bioorg Med Chem Lett 21: 1447–1451.
[23]
Harikumar KB, Kunnumakkara AB, Ochi N, Tong Z, Deorukhkar A, et al. (2010) A novel small-molecule inhibitor of protein kinase D blocks pancreatic cancer growth in vitro and in vivo. Mol Cancer Ther 9: 1136–1146.
[24]
Evans IM, Bagherzadeh A, Charles M, Raynham T, Ireson C, et al. (2010) Characterization of the biological effects of a novel protein kinase D inhibitor in endothelial cells. Biochem J 429: 565–572.
[25]
Bravo-Altamirano K, George KM, Frantz MC, Lavalle CR, Tandon M, et al. (2011) Synthesis and Structure-Activity Relationships of Benzothienothiazepinone Inhibitors of Protein Kinase D. ACS Med Chem Lett 2: 154–159.
[26]
Lavalle CR, Bravo-Altamirano K, Giridhar KV, Chen J, Sharlow E, et al. (2010) Novel protein kinase D inhibitors cause potent arrest in prostate cancer cell growth and motility. BMC Chem Biol 10: 5.
[27]
Sharlow ER, Mustata Wilson G, Close D, Leimgruber S, Tandon M, et al. (2011) Discovery of diverse small molecule chemotypes with cell-based PKD1 inhibitory activity. PLoS One 6: e25134.
[28]
Zugaza JL, Sinnett-Smith J, Van Lint J, Rozengurt E (1996) Protein kinase D (PKD) activation in intact cells through a protein kinase C-dependent signal transduction pathway. Embo J 15: 6220–6230.
[29]
Rozengurt E, Rey O, Waldron RT (2005) Protein kinase D signaling. J Biol Chem 280: 13205–13208.
[30]
Waldron RT, Rey O, Iglesias T, Tugal T, Cantrell D, et al. (2001) Activation loop Ser744 and Ser748 in protein kinase D are transphosphorylated in vivo. J Biol Chem 276: 32606–32615.
[31]
Matthews SA, Rozengurt E, Cantrell D (1999) Characterization of serine 916 as an in vivo autophosphorylation site for protein kinase D/Protein kinase Cmu. J Biol Chem 274: 26543–26549.
[32]
Trejo A, Arzeno H, Browner M, Chanda S, Cheng S, et al. (2003) Design and synthesis of 4-azaindoles as inhibitors of p38 MAP kinase. J Med Chem 46: 4702–4713.
[33]
Chen S, Chen L, Le NT, Zhao C, Sidduri A, et al. (2007) Synthesis and activity of quinolinyl-methylene-thiazolinones as potent and selective cyclin-dependent kinase 1 inhibitors. Bioorg Med Chem Lett 17: 2134–2138.
[34]
Chen JZ, Wang J, Xie XQ (2007) GPCR structure-based virtual screening approach for CB2 antagonist search. J Chem Inf Model 47: 1626–1637.
[35]
Xie XQ, Chen JZ, Billings EM (2003) 3D structural model of the G-protein-coupled cannabinoid CB2 receptor. Proteins: Structure, Function, and Bioinformatics 53: 307–319.
[36]
Meurice N, Wang L, Lipinski CA, Yang Z, Hulme C, et al. (2010) Structural conservation in band 4.1, ezrin, radixin, moesin (FERM) domains as a guide to identify inhibitors of the proline-rich tyrosine kinase 2. J Med Chem 53: 669–677.
[37]
Kim YI, Park JE, Brand DD, Fitzpatrick EA, Yi AK (2010) Protein kinase D1 is essential for the proinflammatory response induced by hypersensitivity pneumonitis-causing thermophilic actinomycetes Saccharopolyspora rectivirgula. J Immunol 184: 3145–3156.
[38]
Yamashita K, Gon Y, Shimokawa T, Nunomura S, Endo D, et al. (2010) High affinity receptor for IgE stimulation activates protein kinase D augmenting activator protein-1 activity for cytokine producing in mast cells. Int Immunopharmacol 10: 277–283.
[39]
Tan M, Hao F, Xu X, Chisolm GM, Cui MZ (2009) Lysophosphatidylcholine activates a novel PKD2-mediated signaling pathway that controls monocyte migration. Arterioscler Thromb Vasc Biol 29: 1376–1382.
[40]
Park JE, Kim YI, Yi AK (2009) Protein kinase D1 is essential for MyD88-dependent TLR signaling pathway. J Immunol 182: 6316–6327.
[41]
Ivison SM, Graham NR, Bernales CQ, Kifayet A, Ng N, et al. (2007) Protein kinase D interaction with TLR5 is required for inflammatory signaling in response to bacterial flagellin. J Immunol 178: 5735–5743.
[42]
Sundram V, Chauhan SC, Jaggi M (2011) Emerging Roles of Protein Kinase D1 in Cancer. Mol Cancer Res
[43]
Zhu H, Yang Y, Zhang H, Han Y, Li Y, et al. (2008) Interaction between protein kinase D1 and transient receptor potential V1 in primary sensory neurons is involved in heat hypersensitivity. Pain 137: 574–588.
[44]
Zhang Y (2008) I-TASSER server for protein 3D structure prediction. BMC Bioinformatics 9: 40.
[45]
Jain AN (2003) Surflex: fully automatic flexible molecular docking using a molecular similarity-based search engine. J Med Chem 46: 499–511.
[46]
Yera ER, Cleves AE, Jain AN (2011) Chemical structural novelty: on-targets and off-targets. J Med Chem 54: 6771–6785.
[47]
Fabian MA, Biggs WH 3rd, Treiber DK, Atteridge CE, Azimioara MD, et al. (2005) A small molecule-kinase interaction map for clinical kinase inhibitors. Nat Biotechnol 23: 329–336.
