Novel 3-amino-2-hydroxy acids containing protease inhibitors. Part 1: Synthesis and kinetic characterization as aminopeptidase P inhibitors.

Novel, potent inhibitors of aminopeptidase P, containing a 3-amino-2-hydroxy acid and a proline or a proline analogues, have been prepared. One part of the bestatin-derived inhibitors was found to inhibit APP from Escherichia coli and from rat intestine according to a mixed-type mechanism, with Ki values up to 1.26 microM. The other compounds, 3-amino-2-hydroxy acyl prolines of a different configuration, inhibit APP competitively, according to a slow-binding mechanism, with Ki values in the nanomolar up to the micromolar range.

Different modes of dipeptidyl peptidase IV (CD26) inhibition by oligopeptides derived from the N-terminus of HIV-1 Tat indicate at least two inhibitor binding sites.

Dipeptidyl peptidase IV (DP IV, CD26) plays an essential role in the activation and proliferation of lymphocytes, which is shown by the immunosuppressive effects of synthetic DP IV inhibitors. Similarly, both human immunodeficiency virus-1 (HIV-1) Tat protein and the N-terminal peptide Tat(1-9) inhibit DP IV activity and T cell proliferation. Therefore, the N-terminal amino acid sequence of HIV-1 Tat is important for the inhibition of DP IV. Recently, we characterized the thromboxane A2 receptor peptide TXA2-R(1-9), bearing the N-terminal MWP sequence motif, as a potent DP IV inhibitor possibly playing a functional role during antigen presentation by inhibiting T cell-expressed DP IV [Wrenger, S., Faust, J., Mrestani-Klaus, C., Fengler, A., Stöckel-Maschek, A., Lorey, S., Kähne, T., Brandt, W., Neubert, K., Ansorge, S. & Reinhold, D. (2000) J. Biol. Chem.275, 22180-22186]. Here, we demonstrate that amino acid substitutions at different positions of Tat(1-9) can result in a change of the inhibition type. Certain Tat(1-9)-related peptides are found to be competitive, and others linear mixed-type or parabolic mixed-type inhibitors indicating different inhibitor binding sites on DP IV, at the active site and out of the active site. The parabolic mixed-type mechanism, attributed to both non-mutually exclusive inhibitor binding sites of the enzyme, is described in detail. From the kinetic investigations and molecular modeling experiments, possible interactions of the oligopeptides with specified amino acids of DP IV are suggested. These findings give new insights for the development of more potent and specific peptide-based DP IV inhibitors. Such inhibitors could be useful for the treatment of autoimmune and inflammatory diseases.

A continuous fluorimetric assay for aminopeptidase P detailed analysis of product inhibition.

Aminopeptidase P (APP; EC 3.4.11.9) is a proline-specific peptidase hydrolyzing N-terminal Xaa-Pro peptide bonds. On the basis of its unique substrate specificity it is difficult to determine enzyme activity and to estimate potential enzyme inhibitors. In this report, we describe the synthesis of a new fluorogenic substrate to assay APP. The substrate was characterized using Escherichia coli APP and rat intestine APP. The compound contains the fluorogenic 2-aminobenzoyl residue and 4-nitroanilide as internal quencher. Both enzymes hydrolyze the substrate Lys(N(epsilon)-2-aminobenzoyl)-Pro-Pro-4-nitroanilide at the Lys-Pro peptide bond with Km values in the micromolar range. Lys(N(epsilon)-2-aminobenzoyl)-Pro-Pro-4-nitroanilide is the best substrate of APP from rat intestine that is known with a Km value of 3.54 microM and a second-order rate constant of 1,142,000 M(-1) s(-1). Unfortunately, product inhibition occurs. Inhibition studies using the hydrolysis product Pro-Pro-4-nitroanilide demonstrate a linear mixed-type mechanism with a K(i) value of 20.8 microM and an alpha value of 5.1 for rat APP and a K(i) value of 76.1 microM and an alpha value of 0.4 for E. coli APP, respectively. Furthermore, the hydrolysis of the fluorogenic APP substrate catalyzed by prolyl oligopeptidase was investigated.