|
1. CHEMISTRY AND BIOLOGY OF
PHOSPHORYLATION
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 (see Figure below), 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 Figure displays predicted binding mode of a conformationally constrained peptide (ball and stick
model) with the Src SH2 domain (surface).
The results of these investigations were published in
several peer-reviewed manuscripts:
1. Rao, V. K., Chhikara, B. S., Nasrolahi Shirazi, A. N., Tiwari, R., Parang, K., Kumar, A. 3-Substitued indoles:
One pot synthesis and evaluation of anticancer and Src
kinase inhibitory activities. Bioorg.
Med. Chem. Lett., (2011)
doi:10.1016/j.bmcl.2011.05.010.
2. Fallah-Tafti, A., Tiwari, R., Shirazi, A. N., Akbarzadeh, T., Mandal, D., Shafiee, A., Parang, K., Foroumadi, A. 4-Aryl-4H-chromene-3-carbonitrile
derivatives: Evaluation of Src kinase inhibitory and anticancer activities. Med.
Chem. In press.
3. Kumar, A., Ahmad, I., Chhikara,
B. S., Tiwari, R., Mandal,
D., Parang, K. Synthesis of 3-phenylpyrazolopyrimidine-1,2,3-triazole
conjugates and evaluation of their Src kinase inhibitory and anticancer activities. Bioorg. Med. Chem. Lett.
(2011) 21, 1342-1346.
4. Kumar, D., Buchi Reddy, V.,
Kumar, A., Mandal, D., Tiwari,
R., Parang, K. Click chemistry inspired one-pot
synthesis of 1,4-disubstituted 1,2,3-triazoles and their Src kinase inhibitory
activity. Bioorg. Med. Chem. Lett. (2011) 2, 449-452.
5. Sharma, D., Bhatia, S., Sharma, R. K., Tiwari, R., Olsen, C. E., Mandal,
D., Lehmann, J., Parang, K., Parmar,
V. S., Prasad, A. S. Synthesis, Src kinase inhibitory and anticancer activities of
1-substituted 3-(N-alkyl-N-phenylamino)propane-2-ols.
Biochimie (2010) 92,
1164-1172.
6. Tiwari, R., Brown, A., Narramaneni, S., Sub, G., Parang,
K. Synthesis and evaluation of conformationally
constrained peptide analogues as the Src SH3
domain binding ligands. Biochimie
(2010) 92, 1153-1163.
7. Ye, G., Schuler, A., Ahmadibeni, Y., Morgan, J. R., Faruqui,
A., Huang, K., Sun, G., Zebala, J. A., Parang, K. Synthesis and evaluation of peptides
containing iminodiacetate groups as binding ligands of the Src SH2
domain. Bioorg. Chem. (2009)
37, 133-142.
8. Ye, G., Tiwari,
R., Parang K. Development of Src
tyrosine kinasesubstrate binding site inhibitors.
Curr. Opin. Investig. Drugs (2008) 9, 605-613.
9. 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.
10. 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.
11. 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.
12. 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.
13. Parang, K., Sun, G. Recent
advances in the discovery of Src kinases inhibitors. Expert Opin.
Ther. Patents (2005), 15,
1183-1207.
14. Parang, K., Sun, G. Protein kinase inhibitors in drug discovery. Drug Discovery
Handbook, (2005),
Wiley-Interscience, New Jersey, Ed. Gad, S. C.
1191-1257.
15. Parang, K., Sun, G. Design
strategies for protein kinases inhibitors.
Current Opinions In Drug Discovery (2004) 7, 630-638.
16. 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.
17. 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.
18. 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.
19. 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.
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 eight 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.
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.
The
results of this investigation were published in a number of peer-reviewed
manuscripts:
1. Ahmadibeni, Y., Tiwari, R., Swepson, C., Pandhare, J., Dash, C., Doncel,
G. F., Parang, K. Synthesis and anti-HIV
activities of bis-(cycloSaligenyl)
pronucleotides derivatives of
3'-fluoro-3'-deoxythymidine and 3'-azido-3'-deoxythymidine. Tetrahedron Lett. (2011) 52, 802-805.
2. Ahmadibeni, Y., Dash, C., Le
Grice, S. F. J., Parang K. Solid-phase synthesis
of 5'-O-?,?-methylenetriphosphate derivatives of
nucleosides and evaluation of their inhibitory activity against HIV-1
reverse transcriptase. Tetrahedron Lett. (2010)
51, 3010-3013.
3. Ahmadibeni, Y., Dash, C.,
Hanley, M. J., Le Grice, S. F. J., Agarwal, H.
K., Parang K. Synthesis of nucleoside
5'-O-alpha,beta-methylene-beta-triphosphates and evaluation of their
potency towards inhibition of HIV-1 reverse transcriptase. Org. Biomol. Chem. (2010) 8, 1271-1274.
4. Ahamadibeni, Y., Tiwari, R., Sun, G., Parang,
K. Synthesis of nucleoside mono-, di-, and triphosphoramidates from solid-phase cycloSaligenyl phosphitylating
reagents. Org. Lett. (2009) 11,
2157-2160.
5. Ahmadibeni, Y., Parang, K. Solid-supported reagents for synthesis of
nucleoside monothiophosphates, dithiodiphosphates, and trithiotriphosphates.
Curr. Protoc. Nucleic
Acid Chem., Chapter 13:Unit13.9.
6. Ahmadibeni, Y., Parang, K. Solid-supported diphosphitylating
and triphosphitylating reagents for nucleoside
modification. Curr. Protoc.
Nucleic Acid Chem. (2008)
Chapter 13:Unit13.8.
7. Ahmadibeni, Y., Parang, K. Solid-phase synthesis of symmetrical
5’,5’-dinucleoside mono-, di-, tri-, and tetraphosphodiesters. Org. Lett.
(2007)
9, 4483-4486.
8. Ahmadibeni, Y., Parang, K.,
Synthesis and evaluation of oligodeoxynucleotides
containing diphosphodiester internucleotide
linkages. Angew. Chem. Int. Ed. (2007) 46, 4739-4743.
9.
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.
10. Ahmadibeni, Y., Parang, K. Solid-phase synthesis of dinucleoside
and nucleoside-carbohydrate phosphodiesters and thiophosphodiesters. J. Org. Chem. (2006) 71,
6693-6696.
11. 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.
12. Ahmadibeni, Y., Parang, K.
Selective diphosphorylation, dithiodiphosphorylation,
triphosphorylation, and trithiotriphosphorylation
of unprotected carbohydrates and nucleosides. Org. Lett.
(2005) 7, 5589-5592.
13. 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.
14. Ahmadibeni, Y., Parang, K.
Solid-phase reagents for selective monophosphorylation
of carbohydrates and nucleosides. J. Org. Chem. (2005) 70, 1100-1103.
15. Parang, K. Polymer-supported reagents for methylphosphorylation and phosphorylation
of Carbohydrates. Bioorg. Med. Chem. Lett. (2002) 12, 1863-1866.
16. 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.
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.
Tiwari, R., Parang K.
Protein conjugates of SH3 domain ligands and
ATP-competitive inhibitors as bivalent inhibitors of protein kinases. ChemBioChem (2009)
10, 2445-2448.
2. 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.
3. 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.
4. 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.
5. 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.
6. 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.
7. 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.
8. 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.
|
