D.K. Srivastava

Mechanistic studies of the triggered release of liposomal contents by matrix metalloproteinase-9.

Matrix metalloproteinases (MMPs) constitute a class of extracellular-matrix-degrading enzymes overexpressed in many cancers and contribute to the metastatic ability of the cancer cells. We have recently demonstrated that liposomal contents can be released when triggered by the enzyme MMP-9. Herein, we report the results of our mechanistic studies of the MMP-9-triggered release of liposomal contents. We synthesized peptides containing the cleavage site for MMP-9 and conjugated them with fatty acids to prepare the corresponding lipopeptides. By employing circular dichroism (CD) spectroscopy, we demonstrated that the lipopeptides, when incorporated into liposomes, are demixed in the lipid bilayers and generate triple-helical structures. MMP-9 cleaves the triple-helical peptides, leading to the release of the liposomal contents. Other MMPs, which cannot hydrolyze triple-helical peptides, fail to release the contents from the liposomes. We also observed that the rate and extent of release of the liposomal contents depend on the mismatch between the acyl chains of the synthesized lipopeptide and phospholipid components of the liposomes. CD spectroscopic studies imply that the observed differences in the release reflect the ability of the liposomal membrane to anneal the defects following the enzymatic cleavage of the liposome-incorporated lipopeptides.

Synthesis of barbiturate-based methionine aminopeptidase-1 inhibitors

The syntheses of a new class of barbiturate-based inhibitors for human and Escherichia Coli methionine aminopeptidase-1 (MetAP-1) are described. Some of the synthesized inhibitors show selective inhibition of the human enzyme with high potency.

Intrinsic selectivity in binding of matrix metalloproteinase-7 to differently charged lipid membranes

We provide evidence that matrix metalloproteinase‐7 (MMP‐7) interacts with anionic, cationic and neutral lipid membranes, although it interacts strongest with anionic membranes. While the catalytic activity of the enzyme remains unaffected upon binding to neutral and negatively charged membranes, it is drastically impaired upon binding to the positively charged membranes. The structural data reveal that the origin of these features lies in the “bipolar” distribution of the electrostatic surface potentials on the crystallographic structure of MMP‐7.

Novel bis-(arylsulfonamide) hydroxamate-based selective MMP inhibitors

A series of bis-(arylsulfonamide) hydroxamate inhibitors were synthesized. These compounds exhibit good potency against MMP-7 and MMP-9 depending on the nature, steric bulk, and substitution pattern of the substituents in the benzene ring. In general, the preliminary structure-activity relationships (SAR) suggest that among the DAPA hydroxamates (i) electron-rich benzene rings of the sulfonamides may produce better inhibitors than electron-poor analogs. However, potential H-bond acceptors can reverse the trend depending on the isozyme; (ii) isozyme selectivity between MMP-7 and -9 can be conferred through steric bulk and substitution pattern of the substituents in the benzene ring, and (iii) the MMP-10 inhibition pattern of the compounds paralleled that for MMP-9.

Influence of Glu-376 → Gln mutation on enthalpy and heat capacity changes for the binding of slightly altered ligands to medium chain acyl-CoA dehydrogenase

We showed that the α‐CH2 → NH substitution in octanoyl‐CoA alters the ground and transition state energies for the binding of the CoA ligands to medium‐chain acyl‐CoA dehydrogenase (MCAD), and such an effect is caused by a small electrostatic difference between the ligands. To ascertain the extent that the electrostatic contribution of the ligand structure and/or the enzyme site environment modulates the thermodynamics of the enzyme–ligand interaction, we undertook comparative microcalorimetric studies for the binding of 2‐azaoctanoyl‐CoA (α‐CH2 → NH substituted octanoyl‐CoA) and octenoyl‐CoA to the wild‐type and Glu‐376 → Gln mutant enzymes. The experimental data revealed that both enthalpy (ΔH°) and heat capacity changes (ΔCp°) for the binding of 2‐azaoctanoyl‐CoA (ΔH°298 = −21.7 ± 0.8 kcal/mole, ΔCp° = −0.627 ± 0.04 kcal/mole/K) to the wild‐type MCAD were more negative than those obtained for the binding of octenoyl‐CoA (ΔH°298 = −17.2 ± 1.6 kcal/mole, ΔCp° = −0.526 ± 0.03 kcal/mole/K). Of these, the decrease in the magnitude of ΔCp° for the binding of 2‐azaoctanoyl‐CoA (vis‐à‐vis octenoyl‐CoA) to the enzyme was unexpected, because the former ligand could be envisaged to be more polar than the latter. To our further surprise, the ligand‐dependent discrimination in the above parameters was completely abolished on Glu‐376 → Gln mutation of the enzyme. Both ΔH° and ΔCp° values for the binding of 2‐azaoctanoyl‐CoA (ΔH°298 = −13.3 ± 0.6 kcal/mole, ΔCp° = −0.511 ± 0.03 kcal/mole/K) to the E376Q mutant enzyme were found to be correspondingly identical to those obtained for the binding of octenoyl‐CoA (ΔH°298 = −13.2 ± 0.6 kcal/mole, ΔCp° = −0.520 ± 0.02 kcal/mole/K). However, in neither case could the experimentally determined ΔCp° values be predicted on the basis of the changes in the water accessible surface areas of the enzyme and ligand species. Arguments are presented that the origin of the above thermodynamic differences lies in solvent reorganization and water‐mediated electrostatic interaction between ligands and enzyme site groups, and such interactions are intrinsic to the molecular basis of the enzyme–ligand complementarity.

Differential modulation of the active site environment of human carbonic anhydrase XII by cationic quantum dots and polylysine

Due to prevalence of negative charges on the protein surface, opposite to the active site pocket of human carbonic anhydrase XII (hCA XII), both positively charged CdTe quantum dots (Qds(+)) and polylysine electrostatically interact with the enzyme, and such interaction does not influence the catalytic activity of the enzyme. However, both these cationic macromolecules differently modulate the active site environment of the enzyme. The steady-state kinetic data revealed that whereas polylysine exhibited no influence on dansylamide (DNSA) dependent inhibition of the enzyme, Qds(+) overcame such an inhibitory effect, leading to almost 70% restoration of the catalytic activity of the enzyme. We provide evidence that DNSA remains bound to the enzyme upon interaction with both polylysine and Qds(+). Arguments are presented that the above differential feature of polylysine and Qds(+) on hCA XII is encoded in the rigidity versus flexibility of these cationic macromolecules.

Energetic rationale for an unexpected and abrupt reversal of guanidinium chloride-induced unfolding of peptide deformylase.

Peptide deformylase (PDF) catalyzes the removal of formyl group from the N‐terminal methionine residues of nascent proteins in prokaryotes, and this enzyme is a high priority target for antibiotic design. In pursuit of delineating the structural–functional features of Escherichia coli PDF (EcPDF), we investigated the mechanistic pathway for the guanidinium chloride (GdmCl)‐induced unfolding of the enzyme by monitoring the secondary structural changes via CD spectroscopy. The experimental data revealed that EcPDF is a highly stable enzyme, and it undergoes slow denaturation in the presence of varying concentrations of GdmCl. The most interesting aspect of these studies has been the abrupt reversal of the unfolding pathway at low to moderate concentrations of the denaturant, but not at high concentration. An energetic rationale for such an unprecedented feature in protein chemistry is offered.

Recognition of isozymes via lanthanide ion incorporated polymerized liposomes

We report the selective recognition of carbonic anhydrase isozymes based on the excited-state lifetimes of chelated Eu(3+) ions incorporated in polymerized liposomes.