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. Two classes of enzymes are currently being investigated in our laboratory: (i) the enzymes of the mitochondrial β-oxidative pathway, and (ii) the zinc-containing metalloenzymes, viz., carbonic anhydrases, and matrix metalloproteinases. Besides, we are investigating the surface recognition of proteins and enzymes involving differently coated gold nanoparticles and liposomes. Our overall approach involves cloning, expression, and purification of enzymes and proteins, and their characterizations by spectroscopic, kinetic, thermodynamic, and model building methods.

Blockage of Cardiac Preconditioning Effect:
Recently, medium chain acyl-CoA dehydrogenase and other enzymes of the β-oxidative pathways have been found to metabolize 5-hydroxydecanoic acid, the drug that blocks the cardiac preconditioning effect. It appears that after first round of the β-oxidation, the reaction product of 5-hydroxydecanoic acid does not undergo oxidation by the first enzyme of the pathway. Instead, the resultant 3-hydroxy derivative is oxidized by the third enzyme, i.e., L-hydroxyacyl-CoA dehydrogenase, of the β-oxidative pathway. This feature appears to be intrinsic to the inhibition of the fatty acid oxidative pathway, which is manifested in the blockage of the preconditioning effect. We are currently in the process of testing our hypothesis that the origin of precondition and its blockage lies in switching between the fatty acid and carbohydrate metabolism within mitochondria.

Isozyme Selective Inhibition of Carbonic Anhydrases and Matrix Metalloproteinases:
In collaboration with Dr. Sanku Mallik, we are designing isozyme selective inhibitors as potential drugs against carbonic anhydrases and matrix metalloproteinases. We have already cloned, expressed, and purified several isozymes of both carbonic anhydrases and matrix metalloproteinases, and we are currently in the process of developing strategies for selectively inhibiting isozymes of these enzymes. Our approach involves ascertaining the effectiveness of ligands, which not only bind to the active site pockets, but also interacts at the surface exposed residues (the “two-prong” ligands), in inhibiting their cognate enzymes. Since the surface exposed residues are not conserved during the course of evolution, our overall strategy has the potential of producing the isozymes specific inhibitors as potential drugs, herbicides, insecticides etc. By using benzenesulfonamide as the active site directed ligand, and iminodiacetate (IDA) metal conjugates as the peripheral (histidine binding) ligands, we have developed a series of inhibitors, which are specific for one isozyme of carbonic anhydrase versus the other. For example, if a peripheral ligand finds a complementary binding site (e.g., L2, see the cartoon on above-right) on the surface of the protein, the overall binding affinity of the “two-prong” ligand is significantly increased. On the other hand, in the absence of such complementarity (e.g., L1), the binding affinity of the two-prong ligand remains similar to that of the active site directed ligand. Aside from using the active and peripheral site specific ligands, we recently explored the possibility of blocking the accessibility of the active site pockets of matrix metalloproteinases (MMPs) by using the IDA-Cu2+ conjugates as multi-prong ligands (see the cartoon on above-left). Since the surface exposed histidine residues of MMP-9 and MMP-10 are different, we have been able to design an inhibitor which specifically inhibits the former isozyme in preference to the latter.

Surface Recognition of Proteins and Enzymes Involving Gold Nano-particles and Liposomes:
In collaboration of Dr. Sanku Mallik, we are investigating the molecular mechanism underlying the recognition of enzymes and proteins by differently formulated gold nanoparticles and liposomes. The surface recognition is being mediated by incorporating complementary charges, site specific ligands, and a combination of both. The cooperative interplay between different groups of solid surfaces in enhancing the binding affinities of proteins and enzymes are being investigated toward developing diagnostics and drug delivery protocols. For example, by formulating triple-helical peptide coated liposomes, we recently demonstrated their recognition by MMP-9, and subsequently releasing their contents due to “uncorking” of liposomes. This stratagem has the potential of developing the liposome mediated delivery of drugs, contrast agents, etc. to the tumor sites.

Selected Publications

Banerjee, A. L.; Eiler, D.; Roy, B. C.; Jia, X.; Haldar, M. K.; Mallik, S.; Srivastava, D. K. (2005) Spacer based selectivity in the binding of “two-prong” ligands to recombinant human carbonic anhydrase-I. Biochemistry 44, 3673-82.

Banerjee, A. L.; Tobwala, S.; Ganguly, B., Mallik, S.; Srivastava, D. K. (2005) Molecular basis for the origin of differential spectral and binding profiles of dansylamide with human carbonic anhydrase I and II. Biochemistry 44, 3211-24.

Sarkar, N. R.; Rosendahl, T.; Krueger, A. B.; Banerjee, A. L.; Benton, K. A.; Mallik, S.; Srivastava, D. K. (2005) Uncorking of liposomes by matrix metalloproteinase-9. J. Chem. Soc. Chem. Commun. 8, 999-1001.

Banerjee, A. L., Tobwala, S., Haldar, M. K.; Roy, B. C.; Swanson, M.; Mallik, S.; Srivastava, D. K. (2005) Inhibition of Matrix Metalloproteinase-9 by "Multi-Prong" Surface Binding Groups. J. Chem. Soc. Chem. Commun. (ASAP Article: DOI: 10.1039/b501780g)

Hanley, P.J.; Dröse, D.; Brandt, U.; Lareau, R. A.; Banerjee, A. L.; Srivastava, D. K.; Banaszak, L. J.; Barycki, J. J.; Veldhoven, P. P. V.; Daut, J. (2005) 5-hydroxydecanoate is metabolised in mitochondria and creates a rate-limiting bottleneck for beta-oxidation of fatty acids. J. Physiol. 562, 307-318.

Roy, B. C.; Banerjee, A. L.; Kloche, D. L.; Swanson, M.; Jia, X.; Haldar, M. K.; Mallik, S.; Srivastava, D. K. (2004) Two-prong inhibitors for human carbonic anhydrase II. J. Am. Chem. Soc. 126, 13206-7.

Banerjee, A. L.; Swanson, M.; Roy, B. C.; Jia, X.; Haldar, M. K.; Mallik, S.; Srivastava, D. K. (2004) Protein surface-assisted enhancement of the binding affinity of an inhibitor for recombinant human carbonic anhydrase-II. J. Am. Chem. Soc. 126, 10875-83.

Banerjee, A. L., Swanswon, M., Mallik, S., and Srivastava, D. K. (2004) Purification of recombinant human carbonic anhydrase-II without incorporating histidine tags. Protein Expr. Purif. 37, 450-454.

Roy, B. C., Hegge, R., Rosendahl, T., Jia, X., Lareau, R., Mallik, S., and Srivastava, D. K. (2003) Conjugation of poor inhibitors with surface binding groups: a strategy to improve inhibition. J. Chem. Soc. Chem. Comm. 18, 2328-29.

Hanley, P. J., Gopalan, K. V., Lareau, R. A., Srivastava, D. K., Meltzer, M., Daut, J. (2003) beta-oxidation of 5-hydroxydecanoate, a putative blocker of mitochondrial ATP-sensitive potassium channels. J. Physiol. 547, 387-397.

Patents
Mallik, S.; Roy, B. C.; Srivastava, D. K. (2004); Methods and materials for enhancing the effects of protein modulators. Provisional Patent application filed on July 8, 2004 by Regalsky & Weyland, New York ( # 047.00050)

Mallik, S.; Sarkar, N. R.; Srivastava, D. K.; Krueger, A. (2004); Controlled release liposomes and methods of use. U. S. Provisional Patent Application filed on September 16, 2004 by Mueting & Raasch, Minneapolis.

Mallik, S.; Roy, B. C.; Srivastava, D. K. (2004); Methods and materials for enhancing the effects of protein modulators. Full Patent application filed on August 3, 2004 by Regalsky & Weyland, New York (# 047.00052).