My research in the area of phosphorylation has made significant impacts in the synthesis of organophosphorus compounds, understanding the substrate recognition by protein kinases and designing their inhibitors, and studying interactions of toxic metals with protein kinases. A summary of my research progress in some of these fields are given here.

1.1. MEDICINAL CHEMISTRY

DESIGNING PROTEIN KINASE INHIBITORS

Protein tyrosine kinases (PTKs) are enzymes that catalyze phosphorylation of tyrosine in many proteins by the transfer of the gama-phosphoryl group from ATP. PTKs can be transiently activated following signals for cell growth or differentiation. The Src family of protein tyrosine kinases, Src, Yes, Lck, Fyn, Lyn, Fgr, Hck, Blk, and Yrk, are non-receptor tyrosine kinases. Enhanced Src tyrosine kinases activity has been directly linked to T-cell activation, mitogenesis, differentiation, cell transformation, and oncogenesis. Src family kinases are involved in the regulation of different cellular processes and in signal transduction pathways. Considerable evidence implicates elevated expression and/or activity of Src in cancer development. Activation of Src is reported in many human cancers, including colon, breast, pancreas, ovarian, lung, gastric, and head and neck. Thus, Src kinase is an important target for anti-cancer drug discovery. The design and evaluation of new compounds against Src tyrosine kinases are important due to the association of Src tyrosine kinases activity with several diseases related to cell signaling, such as cancer, osteoporosis, and inflammation-mediated bone loss. A common strategy for designing Src inhibitors is to target the conserved kinase domain (ATP and substrate binding sites). Although selective inhibitors competitive with ATP have been synthesized for specific protein kinases, the process of PTK inhibitor development is labor intensive due mainly to the presence of a large number of protein kinases that show a conserved ATP binding site. Another major challenge is designing a compound that selectively inhibits one member of the Src family.
The main objective of this research is to design novel Src kinase inhibitors by exploiting the ATP-binding site molecular recognition motif in combination with other recognition motifs. The specific purpose of this project was to design Src kinase inhibitors that incorporate the best features of several successful inhibitor design strategies, including ATP-competitive inhibitors, peptide inhibitors, and structure-guided design.
Different strategies were used in my laboratory for designing inhibitors against Src. Some examples include designing conformationally constrained compounds (Figure 1), bisubstrate analogue inhibitors targeting kinase domain, bisubstrate analogue inhibitors targeting SH2 domain and nucleotide binding site, metal-mediated inhibitors (hydroxamate derivatives), and dendrimer analogs mimicking essential binding sites of protein kinases. The results of these investigations were published in several peer-reviewed manuscripts or as abstracts:

1. Ye, G., Tiwari, R., Parang K. Development of Src tyrosine kinase substrate binding site inhibitors. Curr. Opin. Investig. Drugs (2008) 9, 605-613.

2. Kumar, A., Wang, Y., Lin, X., Sun, G., Parang, K. Synthesis and evaluation of 3-phenylpyrazolopyrimidine-peptide conjugates as Src tyrosine kinase inhibitors. ChemMedChem (2007) 2, 1346-1360.

3. Gu, X., Wang, Y., Kumar, A., Ye, G., Parang, K., Sun, G. Design and evaluation of hydroxamate derivatives as metal-mediated inhibitors of a protein tyrosine kinase. J. Med. Chem. (2006) 49, 7532-7539.

4. Kumar, A., Ye, G., Wang, Y., Lin, X., Sun, G., Parang, K. Synthesis and structure-activity relationships of linear and conformationally constrained peptide analogs of CIYKYY as Src tyrosine kinase inhibitors. J. Med. Chem. (2006) 49, 3395-3401.

5. Ye, G., Ayrapetov, M., Nam, N. H., Sun, G., Parang, K. Solid-phase binding assays of peptides using EGFP-Src SH2 domain fusion protein and biotinylated Src SH2 domain. Bioorg. Med. Chem. Lett. (2005) 15, 4994-4997.

6. Parang, K., Sun, G. Recent advances in the discovery of Src kinases inhibitors. Expert Opin. Ther. Patents (2005), 15, 1183-1207.

7. Parang, K., Sun, G. Protein kinase inhibitors in drug discovery. Drug Discovery Handbook, (2005), Wiley-Interscience, New Jersey, Ed. Gad, S. C. 1191-1257.

8. Parang, K., Sun, G. Design strategies for protein kinases inhibitors. Current Opinions In Drug Discovery (2004) 7, 630-638.

9. Nam, N.-H., Ye, G., Sun, G., Parang, K. Conformationally constrained peptide analogues of pTyr-Glu-Glu-Ile as inhibitors of the Src SH2 domain binding. J. Med. Chem. (2004) 47, 3131-3141.

10. Nam, N. H., Lee, S., Ye, G., Sun, G., Parang, K. ATP-phosphopeptide conjugates as inhibitors of Src tyrosine kinases. Bioorg. Med. Chem. (2004) 12, 5753-5766.

11. Nam, N. H., Pitts, R., Sun, G., Sardari, S., Tiemo, A., Xie, M., Yan, B., Parang, K. Design of tetrapeptide ligands as inhibitors of the Src SH2 domain. Bioorg. Med. Chem. (2004) 12, 779-787.

12. Schmidt, B., Jiricek, J., Titz, A., Ye, G., Parang, K. Copper dipicolinates as peptidomimetic ligands for the Src SH2 domain. Bioorg. Med. Chem. Lett. (2004) 14, 4203-4206.

The figure displays predicted binding mode of a conformationally constrained peptide (ball and stick model) with the Src SH2 domain (surface).

1.2. ORGANIC CHEMISTRY

DEVELOPING SOLID-PHASE REAGENTS FOR THE SYNTHESIS OF ORGANOPHOSPHORUS COMPOUNDS
Organophosphorus compounds are subjects of considerable interest due to their crucial biological roles. Phosphorylated alcohols such as carbohydrate phosphates (e.g., mannose-6-phosphate, glycosyl phosphatidylinositols), nucleosides (e.g., 2',3'-dideoxynucleosides as monophosphates and triphosphates), phosphopeptides composed of phosphoserine, phosphothreonine, and/or phosphotyrosine residues, are involved in several fundamental biological processes and pathways such as molecular recognition and signal transduction. Ready access to organophosphorus compounds, such as carbohydrate and nucleoside phosphates, and phosphopeptides is an important requirement for studying these fundamental biological processes and pathways. Organic chemists investigating these fields are required to prepare many kinds of pure organophosphorus compounds in sufficient quantities. Most of solution- and solid-phase strategies for the synthesis of organophosphorus compounds have been hampered by one or more of the following difficulties: (i) the reactions are not regioselective, involve protection and deprotection reactions for carbohydrates, and lead to low overall yields; (ii) the reactions produce multiple-substituted derivatives; (iii) extensive purification of intermediates and/or final products from the reagents is required; (iv) pure compounds cannot be prepared in sufficient quantities and (v) the current methods cannot be generalized for the synthesis of diverse and large number of compounds. The lack of general strategies and facile synthetic methods has hindered the creation of organophosphorus libraries.
During the past five years, we have designed several solid-phase reagents attached to optimized linkers that can be utilized in the reactions with unprotected alcohols for the regioselective synthesis of variety of organophosphorus compounds. The reagents were linked to a solid phase through several classes of linkers and a variety of resins. Optimal polymer-bound reagents were reacted with several alcohols (e.g., nucleosides and carbohydrates). Oxidation, followed by removal of the cyanoethoxy group with DBU, afforded the corresponding polymer-bound monophosphodiesters, diphosphodiesters, dithiodiphosphodiesters, triphosphodiesters, or trithiotriphosphodiesters. The cleavage of polymer-bound compounds under acidic conditions afforded nucleoside and carbohydrate monophosphates, diphosphates, diphosphodithioates, triphosphates, and triphosphotrithioates. The results of this investigation were published in a number of peer-reviewed manuscripts:

1. Ahmadibeni, Y., Parang, K. Solid-supported reagents for synthesis of nucleoside monothiophosphates, dithiodiphosphates, and trithiotriphosphates. Curr. Protoc. Nucleic Acid Chem., in press.

2. Ahmadibeni, Y., Parang, K. Solid-supported diphosphitylating and triphosphitylating reagents for nucleoside modification. Curr. Protoc. Nucleic Acid Chem. (2008) Chapter 13:Unit13.8.

3.  Ahmadibeni, Y., Parang, K. Solid-phase synthesis of symmetrical 5’,5’-dinucleoside mono-, di-, tri-, and tetraphosphodiesters. Org. Lett. (2007) 9, 4483-4486.

4. Ahmadibeni, Y., Parang, K., Synthesis and evaluation of oligodeoxynucleotides containing diphosphodiester internucleotide linkages. Angew. Chem. Int. Ed. (2007) 46, 4739-4743.

5. Kumar, A., Ye, G., Ahmadibeni, Y., Parang, K. Synthesis of polymer-bound 4-acetoxy-3-phenylbenzaldehyde derivatives: Applications in solid-phase organic synthesis. J. Org. Chem. (2006) 71, 7915-7918.

6. Ahmadibeni, Y., Parang, K. Solid-phase synthesis of dinucleoside and nucleoside-carbohydrate phosphodiesters and thiophosphodiesters. J. Org. Chem. (2006) 71, 6693-6696.

7. Ahmadibeni, Y., Parang, K. Application of a solid-phase β-triphosphitylating reagent in the synthesis of nucleoside β-triphosphates. J. Org. Chem. (2006) 71, 5837-5839.

8. Ahmadibeni, Y., Parang, K. Selective diphosphorylation, dithiodiphosphorylation, triphosphorylation, and trithiotriphosphorylation of unprotected carbohydrates and nucleosides. Org. Lett. (2005) 7, 5589-5592.

9. Ahmadibeni, Y., Parang, K. Polymer-bound oxathiaphospholane: A solid-phase reagent for regioselective monothiophosphorylation and monophosphorylation of unprotected nucleosides and carbohydrates. Org. Lett. (2005) 7, 1955-1958.

10. Ahmadibeni, Y., Parang, K. Solid-phase reagents for selective monophosphorylation of carbohydrates and nucleosides. J. Org. Chem. (2005) 70, 1100-1103.

11. Parang, K. Polymer-supported reagents for methylphosphorylation and phosphorylation of Carbohydrates. Bioorg. Med. Chem. Lett. (2002) 12, 1863-1866.

12. Parang, K., Fournier, E. J.-L., Hindsgaul, O. A solid phase reagent for the capture phosphorylation of carbohydrate and nucleosides. Org. Lett. (2001) 3, 307-309.

Synthesized bifunctional and trifunctional phosphitylating reagents and organophosphorus derivatives of nucleosides and carbohydrates

The long-term objective of this proposal is to develop a general and versatile strategy for the synthesis of novel organophosphorus compounds using solid-phase phosphitylating reagents. The central hypothesis is that several novel solid-phase reagents attached to optimized linkers can be utilized in reactions with unprotected alcohols for the regioselective synthesis of organophosphorus compounds.

1.3. CHEMICAL TOXICOLOGY

PROTEIN TYROSINE KINASES INTERACTIONS WITH METALS

The exposure to toxic metals or metal-containing particles at elevated concentrations is believed to be associated with increased risks of human cancer, neurotoxicity, and immunotoxicity. Cadmium (Cd), arsenite (As), cobalt (Co), lead (Pb), and nickel (Ni) are toxic metals. All of these metals are known as human carcinogens that are believed to play an important role in the development of certain cancers such as skin, lung, and bladder tumors. Cadmium has been shown to inhibit pathways for bone formation. The exact molecular mechanisms of metals-induced toxicities are not well understood. It has been demonstrated that cadmium and arsenite activate cellular Src (c-Src), a protein tyrosine kinase (PTK) that is implicated in the development of cancer and osteoporosis.
The primary focus of our research has centered on the concept that toxic metals bind directly to cellular signaling proteins such as tyrosine kinases through a metal-binding site, thereby inducing conformational changes in those molecules, modulating their activities, and leading to toxic effects. Studying the structural consequences of direct binding of arsenite, cadmium, cobalt, nickel, and lead to a number of protein tyrosine kinases led to the discovery of metal-binding properties of a dicysteine-containing motif in the CT lobe of the kinases.
This study underscored the metal-binding properties of a dicysteine-containing motif located in the CT lobe of several PTKs. CD conformational analyses of peptides, domains, and proteins, site-directed mutagenesis, ICP-MS, NMR and molecular modeling studies, and UV titration analysis provided strong evidence that environmental metals, such as Cd(II), As(III), Co(II), Pb(II), and Ni(II), can selectively bind to the specific motif in PTKs with high affinity and impose a conformational restraint by coordination. An understanding of the binding properties enhanced our knowledge about protein-metal interactions and provided insight into the molecular mechanism(s) by which metals bind to PTKs. Furthermore, these studies provided a model for the future investigations in order to establish how metals may be responsible for toxicity in humans. The results of this investigation were published in one peer-reviewed manuscript:

1. Ahmadibeni, Y., Hanley, M., White, M., Ayrapetov, M., Lin, X., Sun, G., Parang, K. Metal-binding properties of a dicysteine-containing motif in protein tyrosine kinases. ChemBioChem, (2007) 8, 1592-1605.

1.4. BIOCHEMISTRY

DETERMINATION OF MECHAINSM OF KINASE-SUBSTRATE RECOGNITION SITES

This aspect of my research is carried out with collaboration with Dr. Gongqin Sun in Department of Cell and Molecular Biology. The long-term goals of our research are to establish the molecular basis of PTK catalysis and regulation, and use such knowledge to develop potent and specific PTK inhibitors as anti-cancer drugs. Our general approach is to identify catalytically important binding sites on PTKs through structure-function studies, and developing inhibitors to target such binding sites. The results of this investigation were published in several peer-reviewed manuscripts:

1. Bhandari, R., Saiardi, A., Ahmadibeni, Y., Snowman, A. M., Resnick, A. C., Kristiansen, T. Z., Molina, H., Pandey, A., Werner, Jr. J. K., Juluri, K. R., Xu, Y., Prestwich, G. D., Parang, K., Snyder, S. H. Protein pyrophosphorylation by inositol pyrophosphates is a posttranslational event. Proc. Nat. Acad. Sci. U.S.A. (2007) 104, 15305-15310.

2. Ayrapetov, M. K., Wang, Y.-H., Xiaofeng, L., Gu, X., Parang, K., Sun G. Conformational basis for SH2-pTYR527 binding in SRC inactivation. J. Biol. Chem. (2006) 281, 23776-23784.

3. Lee, S., Ayrapetov, M. K., Kemble, D., Parang, K., Sun, G. Docking-based substrate recognition by the catalytic domain of a protein tyrosine kinase, the C-terminal Src kinase. J. Biol. Chem. (2006) 281, 8183-8189.

4. Lin, X., Wang, Y., Ahmadibeni, Y., Parang, K., Sun, G. Structural basis for domain-domain communication in a protein tyrosine kinase, Csk. J. Mol. Biol. (2006) 357, 1263-1273.

5. Ayrapetov, M. K., Nam, N. H., Ye, G., Kumar, A., Parang, K., Sun, G. Functional diversity of Csk, Chk, and Src SH2 domains due to a single residue variation. J. Biol. Chem. (2005) 280, 25780-25787.

6. Lin, X., Ayrapetov, M. K., Lee, S., Parang, K., Sun, G. Probing the communication between the regulatory and catalytic domains of a protein tyrosine kinase, Csk. Biochemistry (2005) 44, 1561-1567.

7. Lee, S., Lin, X., Nam, N. H., Parang, K., Sun, G. Determination of the substrate-docking site of protein tyrosine kinase Csk. Proc. Nat. Acad. Sci. U.S.A. (2003) 100, 14707-14712.