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D. K. Srivastava

Research Interests - Enzymological Studies of Human Diseases

Our research group is currently interested in investigating the mechanistic and structural-functional aspects of selected enzymes, which are responsible for causing human diseases. These studies are geared toward identifying and characterizing enzyme inhibitors as potential therapeutic agents (Drug Design), developing nanoparticles based approaches for selective detection of pathogenic enzymes (Diagnostics), and investigating the enzyme assisted cleavage of lipid conjugates as well as protein-lipid interactions from the point of view of releasing liposomal contents (Drug Delivery). These research objectives are being pursued using different isoforms of Matrix Metalloproteinases, Carbonic Anhydrases, Histone Deacetylases, Sirtuins, Methionine Aminopeptidases and Peptide deformylases. Our overall approach involves cloning, expression, and purification of enzymes and proteins, and their characterizations by a variety of spectroscopic, kinetic, thermodynamic, and model building techniques.

Drug Design

We are employing rationale approaches in designing enzyme inhibitors as potential drugs. In this endeavor, we investigate a detailed account of structural-functional and mechanistic features of the above noted pathogenic enzymes and design their potent inhibitors in collaboration with Dr. Sanku Mallik. In effort of further fine tuning the inhibitor designs, particularly for achieving isozyme selectivity of pathogenic enzymes, we have been employing our recently developed “multi-prong” approach. This approach involves designing inhibitors, which not only bind at the active site of enzymes but also bind at the peripheral sites. Since the surface exposed residues are not conserved during the course of evolution, our overall strategy has potential to find applications in producing highly potent and selective therapeutic agents. In a few cases we have demonstrated that by attaching iminodiacetate (IDA) metal conjugates to the active site directed (albeit weak) inhibitors, we could block the accessibility of the enzymes’ active site pockets due to the multi-prong effect as depicted by the cartoon of the Figure. The multi-prong inhibitor designing approach has been expanded by fabricating liposomes which contain both active site as well as peripheral site affine ligands. A marked advantage in using the liposome resident ligands is their ability to find (due to mobility of the lipid components within the liposomes) complementary binding surfaces on the target enzymes, and resulting in highly potent and selective inhibitors for their cognate enzymes. Aside from the multi-prong approaches, we have also been assessing the efficacies of several metal-chelating as well as mechanism based inhibitors toward desensitizing our selected pathogenic enzymes.

Diagnostics

By using different types of nanoparticles, we are developing fluorescence based methodologies for selective detection of pathogenic enzymes involved in human diseases. Our overall approach involves using intrinsic fluorescence signals associated with proteins, protein bound ligands and/or nanoparticles themselves, and probe the unique changes in such signals upon binding of protein to the nanoparticles. For example, by using cationic quantum dots (QD), we could demonstrate that dansylamide (DNSA) bound to the active site of carbonic anhydrase XII (CA) uniquely transfers its excitation energy to the quantum dots upon formation of the electrostatic complex between the enzyme and the nanoparticles. The latter interaction is further manifested in the weakening of the enzyme-ligand complex due to nanoparticles mediated changes in the protein conformation as depicted in the cartoon of the Figure. This approach is being extended using differently formulated lipid nanoparticles containing fluorescence reporter groups for selective detection of pathogenic enzymes and proteins.

Drug Delivery

We are developing methodologies for delivering both small and large molecular weight compounds to cellular systems using nanoparticles as well as liposomes as drug delivery vehicles. In collaboration with Dr. Sanku Mallik, we are formulating liposomes, which are selectively “uncorked” by Matrix Metalloproteinases leading to the release of their encapsulated materials. The effectiveness of these as well as the nanoparticles based drug delivery system is being evaluated at the cellular level.

Research Support: These research projects are being supported by the funds from two NIH-R01 and one NSF collaborative grants.

Selected Publications

Elegebede, A. I., Banerjee, J., Hanson, A. J., Tobwala, S., Ganguly, B., Wang, R., Lu, X., Srivastava, D. K., and Mallik, S. (2008) Mechanistic Studies of the Triggered Release of Liposomal Contents by Matrix Metalloproteinase-9 J. Am. Chem. Soc. (ASAP article, doi:ja801548g)

Subramaniam R, Haldar MK, Tobwala S, Ganguly B, Srivastava DK, Mallik S. Novel bis-(arylsulfonamide) hydroxamate-based selective MMP inhibitors (2008)
Bioorg Med Chem Lett. 18, 3333-7.

Haldar MK, Scott MD, Sule N, Srivastava DK, Mallik S. (2008) Synthesis of barbiturate-based methionine aminopeptidase-1 inhibitors. Bioorg Med Chem Lett. 18, 2373-6.

Sarkar N, Banerjee J, Hanson AJ, Elegbede AI, Rosendahl T, Krueger AB, Banerjee AL, Tobwala S, Wang R, Lu X, Mallik S, Srivastava DK. (2008) Matrix metalloproteinase-assisted triggered release of liposomal contents. Bioconjug Chem. 19, 57-64.

Berg, A., K., Manokaran, S., Eiler, D., Kooren, J., Mallik, S., and Srivastava, D. K. (2008) Energetic rationale for an unexpected and abrupt reversal of guanidinium chloride-induced unfolding of peptide deformylase. Prot. Sci. 17, 11-5

Elegbede, A. I., Haldar, M. K., Manokaran, S., Mallik, S., and Srivastava, D. K. (2007) Recognition of isozymes via lanthanide ion incorporated polymerized liposomes. Chem. Comm. 43, 4495-4497.

Ganguly, B., Banerjee, J., Elegbede, A. I., Klocke, D. J., Mallik, S., and Srivastava, D. K. (2007) Intrinsic selectivity in binding of matrix metalloproteinase-7 to differently charged lipid membranes. FEBS Lett 581, 5723-5726.

Srivastava, D. K., Jude, K. M., Banerjee, A. L., Haldar, M., Manokaran, S., Kooren, J., Mallik, S., Christianson, D. W. (2007) Structural analysis of charge discrimination in the binding of inhibitors to human carbonic anhydrases I and II. J. Am. Chem. Soc. 129, 5528-37.

Banerjee, J., Haldar, M. K., Manokaran, S., Mallik, S., and Srivastava, D. K. (2007) New Fluorescent probes for carbonic anhydrases. Chem. Comm. 26, 2723-2725.

Subramaniam, R., Mallik, S., and Srivastava, D. K. (2007) Natural Products as Inhibitors of Matrix Metalloproteinases and Histone deacetylases. In “Natural Products: Chemistry, Biochemistry, and Pharmacology” Chapter 15, (Editor: G. Brahmachari), Alpha Science Int., Oxford.

Elegbede, A. I., Haldar, M. K., Manokaran, S., Kooren, J., Roy, B., Mallik, S., and Srivastava, D. K. (2007) A Strategy of designing multi-prong enzyme inhibitors by incorporating selective ligands to the liposomal surface. Chem. Comm. 32, 3377-3379.

Elegbede, A. I., Srivastava, D. K., and Hinderliter, A. (2006) Purification of recombinant annexins without the use of phospholipids. Prot. Expr. Purif. 50, 157-162.

Jude, K.M., Banerjee, A. L.; Haldar, M. K., Manokaran, S.; Roy, B. C.; Mallik, S.; Srivastava, D. K., Christianson, D. W. (2006) Ultrahigh resolution crystal structure of human carbonic anhydrase I and II complexed with “two-prong” inhibitors reveal the structural basis for potent inhibition. J. Am. Chem. Soc. 128, 3011-3018.

Ngwendson, J. N.; Amiot, C. L., Srivastava, D. K.,and Banerjee, A. (2006) Design of a Zinc(II) ion Specific Fluorescence Sensor. Tett. Lett. 47, 2327-2330.

Jude, K.M., Banerjee, A. L.; Haldar, M. K., Manokaran, S.; Roy, B. C.; Mallik, S.; Srivastava, D. K., Christianson, D. W. (2006) Ultrahigh resolution crystal structure of human carbonic anhydrase I and II complexed with “two-prong” inhibitors reveal the structural basis for potent inhibition. J. Am. Chem. Soc. 128, 3011-3018.

Chandra, B., Subramaniam, R., Mallik, S. and Srivastava, D. K. (2006) Formulation of Photo-cleavable Liposomes and the Mechanism of Their Content Release. Org. Biomol. Chem. 4, 1730-404.

 

 

James A. Meir Professor

MS, Banaras University, Varanasi, India, 1975
PhD, Banaras University, Varanasi, India, 1982

Office: Quentin Burdick Building 312

tel 701-231-7831
fax 701-231-8342

DK.Srivastava@
ndsu.edu