Potent and Selective Inhibitors of Long Chain l-2-Hydroxy Acid Oxidase Reduced Blood Pressure in DOCA Salt-Treated Rats.

l-2-Hydroxy acid oxidase (Hao2) is a peroxisomal enzyme with predominant expression in the liver and kidney. Hao2 was recently identified as a candidate gene for blood pressure quantitative trait locus in rats. To investigate a pharmacological role of Hao2 in the management of blood pressure, selective Hao2 inhibitors were developed.

Matrix metalloproteinase-assisted triggered release of liposomal contents.

We offer a novel methodology for formulating liposomes by incorporating sequence-specific collagen-mimetic peptides such that they are specifically "uncorked" by a matrix metalloproteinase, MMP-9. By encapsulating carboxyfluorescein (as a self-quenching fluorescent dye), we demonstrate that the time-dependent release of the dye from liposomes is due to the specific enzymatic cleavage of the surface-exposed collagen-mimetic peptides. The specificity of such cleavage is attested by the fact that the liposomal "uncorking" and their content release occur only by MMP-9 and not by a general proteolytic enzyme, trypsin, despite the fact that the collagen mimetic peptides contain the trypsin cleavage site.

Structural analysis of charge discrimination in the binding of inhibitors to human carbonic anhydrases I and II.

Despite the similarity in the active site pockets of carbonic anhydrase (CA) isozymes I and II, the binding affinities of benzenesulfonamide inhibitors are invariably higher with CA II as compared to CA I. To explore the structural basis of this molecular recognition phenomenon, we have designed and synthesized simple benzenesulfonamide inhibitors substituted at the para position with positively charged, negatively charged, and neutral functional groups, and we have determined the affinities and X-ray crystal structures of their enzyme complexes. The para-substituents are designed to bind in the midsection of the 15 A deep active site cleft, where interactions with enzyme residues and solvent molecules are possible.

Ultrahigh resolution crystal structures of human carbonic anhydrases I and II complexed with "two-prong" inhibitors reveal the molecular basis of high affinity.

The atomic-resolution crystal structures of human carbonic anhydrases I and II complexed with "two-prong" inhibitors are reported. Each inhibitor contains a benzenesulfonamide prong and a cupric iminodiacetate (IDA-Cu(2+)) prong separated by linkers of different lengths and compositions. The ionized NH(-) group of each benzenesulfonamide coordinates to the active site Zn(2+) ion; the IDA-Cu(2+) prong of the tightest-binding inhibitor, BR30, binds to H64 of CAII and H200 of CAI.

Inhibition of matrix metalloproteinase-9 by "multi-prong" surface binding groups.

A novel strategy of blocking the active site accessibility of MMP-9 by "multi-prong" surface binding groups is described.

Molecular basis for the origin of differential spectral and binding profiles of dansylamide with human carbonic anhydrase I and II.

Sulfonamide derivatives serve as potent inhibitors of carbonic anhydrases (CAs), and a few such inhibitors have been currently used as drugs for the treatment of different pathogenic conditions in humans. In pursuit of designing the isozyme-specific inhibitors of human CAs, we observed that the fluorescence spectral properties and binding profiles of a fluorogenic sulfonamide derivative, 5-(dimethylamino)-1-naphthalenesulfonamide (dansylamide, DNSA), were markedly different between the recombinant forms of human carbonic anhydrase I (hCA I) and II (hCA II). The kinetic evaluation of the overall microscopic pathways for the binding of DNSA to hCA I versus hCA II revealed that the protein isomerization step served as a major determinant of the above discrepancy.

Spacer-based selectivity in the binding of "two-prong" ligands to recombinant human carbonic anhydrase I.

Benzenesulfonamide and iminodiacetate (IDA)-conjugated Cu(2+) independently interact at the active site and a peripheral site of carbonic anhydrases, respectively [Banerjee, A. L., Swanson, M.

"Uncorking" of liposomes by matrix metalloproteinase-9.

A triggered release methodology of liposomal contents via the enzyme MMP-9 is described.

5-Hydroxydecanoate is metabolised in mitochondria and creates a rate-limiting bottleneck for beta-oxidation of fatty acids.

5-Hydroxydecanoate (5-HD) blocks pharmacological and ischaemic preconditioning, and has been postulated to be a specific inhibitor of mitochondrial ATP-sensitive K(+) (K(ATP)) channels. However, recent work has shown that 5-HD is activated to 5-hydroxydecanoyl-CoA (5-HD-CoA), which is a substrate for the first step of beta-oxidation. We have now analysed the complete beta-oxidation of 5-HD-CoA using specially synthesised (and purified) substrates and enzymes, as well as isolated rat liver and heart mitochondria, and compared it with the metabolism of the physiological substrate decanoyl-CoA.

Two-prong inhibitors for human carbonic anhydrase II.

The enzyme inhibitors are usually designed by taking into consideration the overall dimensions of the enzyme's active site pockets. This conventional approach often fails to produce desirable affinities of inhibitors for their cognate enzymes. To circumvent such constraints, we contemplated enhancing the binding affinities of inhibitors by attaching tether groups, which would interact with the surface exposed amino acid residues.

Purification of recombinant human carbonic anhydrase-II by metal affinity chromatography without incorporating histidine tags.

Due to their involvement in diverse pathological conditions, carbonic anhydrases have been the targets of drug developments for the treatments of glaucoma, epilepsy, high altitude sickness, as well as cancer. Of about 14 isozymes of carbonic anhydrases, carbonic anhydrase-II (hCA-II) has been most extensively investigated from the structural, functional, and inhibitor design point of view. We discovered that hCA-II preferentially binds to the Sepharose-iminodiacetate (IDA)-Zn(2+) column, and such binding does not require incorporation of either N- or C-terminal histidine tags in the protein structure.

Protein surface-assisted enhancement in the binding affinity of an inhibitor for recombinant human carbonic anhydrase-II.

We elaborate on a novel strategy for enhancing the binding affinity of an active-site directed inhibitor by attaching a tether group, designed to interact with the surface-exposed histidine residue(s) of enzymes. In this approach, we have utilized the recombinant form of human carbonic anhydrase-II (hCA-II) as the enzyme source and benzenesulfonamide and its derivatives as inhibitors. The steady-state kinetic and the ligand binding data revealed that the attachment of iminodiacetate (IDA)-Cu(2+) to benzenesulfonamide (via a triethylene glycol spacer) enhanced its binding affinity for hCA-II by about 40-fold.