Structure-based virtual screening of Src kinase inhibitors.

Src is an important target in multiple processes associated with tumor growth and development, including proliferation, neovascularization, and metastasis. In this study, hit identification was performed by virtual screening of commercial and in-house compound libraries. Docking studies for the hits were performed, and scoring functions were used to evaluate the docking results and to rank ligand-binding affinities. Subsequently, hit optimization for potent and selective candidate Src inhibitors was performed through focused library design and docking analyses. Consequently, we report that a novel compound '43' with an IC(50) value of 89 nM, representing (S)-N-(4-(5-chlorobenzo[d][1,3]dioxol-4-ylamino)-7-(2-methoxyethoxy)quinazolin-6-yl)pyrrolidine-2-carboxamide, is highly selective for Src in comparison to EGFR (IC(50) ratio>80-fold) and VEGFR-2 (IC(50) ratio>110-fold). Compound 43 exerted anti-proliferative effects on Src-expressing PC3 human prostate cancer and A431 human epidermoid carcinoma cells, with calculated IC(50) values of 1.52 and 0.78 microM, respectively. Moreover, compound 43 (0.1 microM) suppressed the phosphorylation of extracellular signal-regulated kinases and p90 ribosomal S6 kinase, downstream molecules of Src, in a time-dependent manner, in both PC3 and A431 cell lines. The docking structure of compound 43 with Src disclosed that the chlorobenzodioxole moiety and pyrrolidine ring of C-6 quinazoline appeared to fit tightly into the hydrophobic pocket of Src. Additionally, the pyrrolidine NH forms a hydrogen bond with the carboxyl group of Asp348. These results confirm the successful application of virtual screening studies in the lead discovery process, and suggest that our novel compound 43 can be an effective Src inhibitor candidate for further lead optimization.

[1]  Ping Chen,et al.  Discovery of N-(2-chloro-6-methyl- phenyl)-2-(6-(4-(2-hydroxyethyl)- piperazin-1-yl)-2-methylpyrimidin-4- ylamino)thiazole-5-carboxamide (BMS-354825), a dual Src/Abl kinase inhibitor with potent antitumor activity in preclinical assays. , 2004, Journal of medicinal chemistry.

[2]  T. Boggon,et al.  Structure and regulation of Src family kinases , 2004, Oncogene.

[3]  Sheila M. Thomas,et al.  Cellular functions regulated by Src family kinases. , 1997, Annual review of cell and developmental biology.

[4]  Brian K. Shoichet,et al.  Virtual screening of chemical libraries , 2004, Nature.

[5]  G. Gallick,et al.  Src family kinases in tumor progression and metastasis , 2003, Cancer and Metastasis Reviews.

[6]  Jürgen Bajorath,et al.  Integration of virtual and high-throughput screening , 2002, Nature Reviews Drug Discovery.

[7]  E. Avizienyte,et al.  Identification of Src-specific phosphorylation site on focal adhesion kinase: dissection of the role of Src SH2 and catalytic functions and their consequences for tumor cell behavior. , 2005, Cancer research.

[8]  T. Hunter,et al.  Transforming gene product of Rous sarcoma virus phosphorylates tyrosine , 1980, Proceedings of the National Academy of Sciences.

[9]  J. Parsons,et al.  Src family kinases, key regulators of signal transduction , 2004, Oncogene.

[10]  D. Boschelli,et al.  Synthesis and Src kinase inhibitory activity of 2-phenyl- and 2-thienyl-7-phenylaminothieno[3,2-b]pyridine-6-carbonitriles. , 2005, Journal of medicinal chemistry.

[11]  Ajay,et al.  Computational methods to predict binding free energy in ligand-receptor complexes. , 1995, Journal of medicinal chemistry.

[12]  J. Mestan,et al.  Advances in the structural biology, design and clinical development of Bcr-Abl kinase inhibitors for the treatment of chronic myeloid leukaemia. , 2005, Biochimica et biophysica acta.

[13]  Qian Huang,et al.  The Discovery of Novel Vascular Endothelial Growth Factor Receptor Tyrosine Kinases Inhibitors: Pharmacophore Modeling, Virtual Screening and Docking Studies , 2007, Chemical biology & drug design.

[14]  L. Otterbein,et al.  N-(5-chloro-1,3-benzodioxol-4-yl)-7-[2-(4-methylpiperazin-1-yl)ethoxy]-5- (tetrahydro-2H-pyran-4-yloxy)quinazolin-4-amine, a novel, highly selective, orally available, dual-specific c-Src/Abl kinase inhibitor. , 2006, Journal of medicinal chemistry.

[15]  Fei Ye,et al.  SKI-606, a Src/Abl inhibitor with in vivo activity in colon tumor xenograft models. , 2005, Cancer research.

[16]  Susan Adams,et al.  Structural Basis of Src Tyrosine Kinase Inhibition with a New Class of Potent and Selective Trisubstituted Purine‐based Compounds , 2006, Chemical biology & drug design.

[17]  Robert Huber,et al.  Crystal structures of active SRC kinase domain complexes. , 2005, Journal of molecular biology.

[18]  P. Schwartzberg,et al.  The many faces of Src: multiple functions of a prototypical tyrosine kinase , 1998, Oncogene.

[19]  Yuzhu Chen,et al.  N2-substituted O6-cyclohexylmethylguanine derivatives: potent inhibitors of cyclin-dependent kinases 1 and 2. , 2004, Journal of medicinal chemistry.

[20]  Renxiao Wang,et al.  Comparative evaluation of 11 scoring functions for molecular docking. , 2003, Journal of medicinal chemistry.

[21]  D. Boschelli,et al.  Optimization of 4-phenylamino-3-quinolinecarbonitriles as potent inhibitors of Src kinase activity. , 2001, Journal of medicinal chemistry.

[22]  Thierry Langer,et al.  Impact of Scoring Functions on Enrichment in Docking-Based Virtual Screening: An Application Study on Renin Inhibitors , 2004, J. Chem. Inf. Model..

[23]  P. Plé,et al.  Discovery of a new class of anilinoquinazoline inhibitors with high affinity and specificity for the tyrosine kinase domain of c-Src. , 2004, Journal of medicinal chemistry.

[24]  J. Siegfried,et al.  Src Family Kinases Mediate Epidermal Growth Factor Receptor Ligand Cleavage, Proliferation, and Invasion of Head and Neck Cancer Cells , 2004, Cancer Research.

[25]  D. Fabbro,et al.  The crystal structure of a c-Src complex in an active conformation suggests possible steps in c-Src activation. , 2005, Structure.

[26]  Tudor I. Oprea,et al.  Integrating virtual screening in lead discovery. , 2004, Current opinion in chemical biology.

[27]  D. Rognan,et al.  Protein-based virtual screening of chemical databases. 1. Evaluation of different docking/scoring combinations. , 2000, Journal of medicinal chemistry.

[28]  S. Wedge,et al.  Novel 4-anilinoquinazolines with C-7 basic side chains: design and structure activity relationship of a series of potent, orally active, VEGF receptor tyrosine kinase inhibitors. , 2002, Journal of medicinal chemistry.