Identification of small molecular weight inhibitors of Src homology 2 domain-containing tyrosine phosphatase 2 (SHP-2) via in silico database screening combined with experimental assay.

Virtual screening methods combined with experimental assays were used to identify low molecular weight inhibitors for Src homology 2 domain-containing phosphatase 2 (SHP-2) that is mutated and hyperactivated in Noonan syndrome and a significant portion of childhood leukemias. Virtual screening included multiple conformations of the protein, score normalization procedures, and chemical similarity considerations. As the catalytic core of SHP-2 shares extremely high homology to those of the related SHP-1 phosphatase and other tyrosine phosphatases, in order to identify selective inhibitors, we chose to target an adjacent protein surface pocket that is predicted to be important for binding to phosphopeptides and that has structural features unique to SHP-2. From a database of 1.3 million compounds, 9 out of 165 computationally selected compounds were shown to inhibit SHP-2 activity with IC(50) values of approximately 100 microM. Two of the active compounds were further verified for their ability to inhibit SHP-2-mediated cellular functions. Fluorescence titration experiments confirmed their direct binding to SHP-2. Because of their simple chemical structures, these small organic compounds have the potential to act as lead compounds for the development of novel anti-SHP-2 drugs.

[1]  Alexander D. MacKerell,et al.  Extending the treatment of backbone energetics in protein force fields: Limitations of gas‐phase quantum mechanics in reproducing protein conformational distributions in molecular dynamics simulations , 2004, J. Comput. Chem..

[2]  S. Harrison,et al.  Spatial constraints on the recognition of phosphoproteins by the tandem SH2 domains of the phosphatase SH-PTP2 , 1996, Nature.

[3]  F. Lombardo,et al.  Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. , 2001, Advanced drug delivery reviews.

[4]  M. L. Connolly.  Solvent-accessible surfaces of proteins and nucleic acids. , 1983, Science.

[5]  Conrad C. Huang,et al.  The MIDAS display system , 1988 .

[6]  J. Licht,et al.  Somatic mutations in PTPN11 in juvenile myelomonocytic leukemia, myelodysplastic syndromes and acute myeloid leukemia , 2003, Nature Genetics.

[7]  I. Kuntz,et al.  Conformational analysis of flexible ligands in macromolecular receptor sites , 1992 .

[8]  P. Goodford.  A computational procedure for determining energetically favorable binding sites on biologically important macromolecules. , 1985, Journal of medicinal chemistry.

[9]  Martin Zacharias,et al.  Sequence Specificity of SHP-1 and SHP-2 Src Homology 2 Domains , 2006, Journal of Biological Chemistry.

[10]  M. Loh,et al.  Functional analysis of leukemia-associated PTPN11 mutations in primary hematopoietic cells. , 2004, Blood.

[11]  Darko Butina,et al.  Unsupervised Data Base Clustering Based on Daylight's Fingerprint and Tanimoto Similarity: A Fast and Automated Way To Cluster Small and Large Data Sets , 1999, J. Chem. Inf. Comput. Sci..

[12]  J M Blaney,et al.  A geometric approach to macromolecule-ligand interactions. , 1982, Journal of molecular biology.

[13]  Michael A. Patton,et al.  Mutations in PTPN11, encoding the protein tyrosine phosphatase SHP-2, cause Noonan syndrome , 2001, Nature Genetics.

[14]  Z. Zhao,et al.  Crystal Structure of Human Protein-tyrosine Phosphatase SHP-1* , 2003, The Journal of Biological Chemistry.

[15]  J. Richardson,et al.  Asparagine and glutamine: using hydrogen atom contacts in the choice of side-chain amide orientation. , 1999, Journal of molecular biology.

[16]  John D. Minna,et al.  Activating Mutations of the Noonan Syndrome-Associated SHP2/PTPN11 Gene in Human Solid Tumors and Adult Acute Myelogenous Leukemia , 2004, Cancer Research.

[17]  Farhi Dc.  Myelodysplastic syndromes and acute myeloid leukemia. Diagnostic criteria and pitfalls. , 1995 .

[18]  G J Williams,et al.  The Protein Data Bank: a computer-based archival file for macromolecular structures. , 1978, Archives of biochemistry and biophysics.

[19]  Robert G Hawley,et al.  Catalytic-dependent and -independent roles of SHP-2 tyrosine phosphatase in interleukin-3 signaling , 2003, Oncogene.

[20]  B. Neel,et al.  The 'Shp'ing news: SH2 domain-containing tyrosine phosphatases in cell signaling. , 2003, Trends in biochemical sciences.

[21]  Menggang Yu,et al.  Human somatic PTPN11 mutations induce hematopoietic-cell hypersensitivity to granulocyte-macrophage colony-stimulating factor. , 2005, Blood.

[22]  Alexander D. MacKerell,et al.  Identification of non-phosphate-containing small molecular weight inhibitors of the tyrosine kinase p56 Lck SH2 domain via in silico screening against the pY + 3 binding site. , 2004, Journal of medicinal chemistry.

[23]  W. Guida,et al.  Discovery of a Novel Shp2 Protein Tyrosine Phosphatase Inhibitor , 2006, Molecular Pharmacology.

[24]  Todd J. A. Ewing,et al.  Critical evaluation of search algorithms for automated molecular docking and database screening , 1997, J. Comput. Chem..

[25]  C. Qu,et al.  Effects of a Leukemia-associated Gain-of-Function Mutation of SHP-2 Phosphatase on Interleukin-3 Signaling* , 2006, Journal of Biological Chemistry.

[26]  I. Kuntz.  Structure-Based Strategies for Drug Design and Discovery , 1992, Science.

[27]  Tudor I. Oprea,et al.  Is There a Difference Between Leads and Drugs? A Historical Perspective. , 2001 .

[28]  D. Gilliland,et al.  Mouse model of Noonan syndrome reveals cell type– and gene dosage–dependent effects of Ptpn11 mutation , 2004, Nature Medicine.

[29]  Ray A. Jarvis,et al.  Clustering Using a Similarity Measure Based on Shared Near Neighbors , 1973, IEEE Transactions on Computers.

[30]  N. Tonks,et al.  Protein tyrosine phosphatases: from genes, to function, to disease , 2006, Nature Reviews Molecular Cell Biology.

[31]  Malcolm McGregor,et al.  Diverse Biochemical Properties of Shp2 Mutants , 2005, Journal of Biological Chemistry.

[32]  M. Loh,et al.  Mutations in PTPN11 implicate the SHP-2 phosphatase in leukemogenesis. , 2004, Blood.

[33]  D. Barford,et al.  Revealing mechanisms for SH2 domain mediated regulation of the protein tyrosine phosphatase SHP-2. , 1998, Structure.

[34]  K Schulten,et al.  VMD: visual molecular dynamics. , 1996, Journal of molecular graphics.

[35]  Z. Zhao,et al.  Crystal Structure of the Catalytic Domain of Protein-tyrosine Phosphatase SHP-1* , 1998, The Journal of Biological Chemistry.

[36]  S. Shoelson,et al.  Crystal Structure of the Tyrosine Phosphatase SHP-2 , 1998, Cell.

[37]  Z. Zhao,et al.  Structural Basis for Substrate Specificity of Protein-tyrosine Phosphatase SHP-1* , 2000, The Journal of Biological Chemistry.

[38]  C. Qu.  Role of the SHP-2 tyrosine phosphatase in cytokine-induced signaling and cellular response. , 2002, Biochimica et biophysica acta.

[39]  L. Kelley,et al.  An automated approach for clustering an ensemble of NMR-derived protein structures into conformationally related subfamilies. , 1996, Protein engineering.

[40]  J. Kutok,et al.  Prognostic, therapeutic, and mechanistic implications of a mouse model of leukemia evoked by Shp2 (PTPN11) mutations. , 2005, Cancer cell.

[41]  Alexander D. MacKerell,et al.  All-atom empirical potential for molecular modeling and dynamics studies of proteins. , 1998, The journal of physical chemistry. B.