Kerstin Kühn-Wache

Dipeptidyl peptidase IV (DPIV/CD26) degradation of glucagon. Characterization of glucagon degradation products and DPIV-resistant analogs.

Over the past decade, numerous studies have been targeted at defining structure-activity relationships of glucagon. Recently, we have found that glucagon1–29 is hydrolyzed by dipeptidyl peptidase IV (DPIV) to produce glucagon3–29 and glucagon5–29; in human serum, [pyroglutamyl (pGlu)3]glucagon3–29 is formed from glucagon3–29, and this prevents further hydrolysis of glucagon by DPIV (H.-U. Demuth, K. Glund, U. Heiser, J. Pospisilik, S. Hinke, T. Hoffmann, F. Rosche, D. Schlenzig, M. Wermann, C. McIntosh, and R. Pederson, manuscript in preparation). In the current study, the biological activity of these peptides was examined in vitro. The amino-terminally truncated peptides all behaved as partial agonists in cyclic AMP stimulation assays, with Chinese hamster ovary K1 cells overexpressing the human glucagon receptor (potency: glucagon1–29 > [pGlu3]glu- cagon3–29 > glucagon3–29 > glucagon5–29 > [Glu9]glu- cagon2–29). In competition binding experiments, [pGlu3]glucagon3–29 and glucagon5–29 both demonstrated 5-fold lower affinity for the receptor than glucagon1–29, whereas glucagon3–29 exhibited 18-fold lower affinity. Of the peptides tested, only glucagon5–29 showed antagonist activity, and this was weak compared with the classical glucagon antagonist, [Glu9]glucagon2–29. Hence, DPIV hydrolysis of glucagon yields low affinity agonists of the glucagon receptor. As a corollary to evidence indicating that DPIV degrades glucagon (Demuth, et al., manuscript in preparation), DPIV-resistant analogs were synthesized. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry was used to assess DPIV resistance, and it allowed kinetic analysis of degradation. Of several analogs generated, only [d-Ser2] and [Gly2]glucagon retained high affinity binding and biological potency, similar to native glucagon in vitro. [d-Ser2]Glucagon exhibited enhanced hyperglycemic activity in a bioassay, whereas [Gly2]glucagon was not completely resistant to DPIV degradation.

Analogs of Glucose-Dependent Insulinotropic Polypeptide With Increased Dipeptidyl Peptidase IV Resistance

Fully and partially DPIV-resistant analogs of GIP1–30 could be synthesized. The introduction of D-amino acids in P1- and P1’-position resulted in a slight reduction in binding and bioactivity. The examined C-terminal truncated fragments (with exception of the GIP1–30 fragment) showed no binding affinity, whereas the antagonistic N-terminal truncated fragments were able to bind to transfected rat GIP receptor. These results emphasize the hypothesis of an existing one-receptor-two-interaction-sites-model which was shown for peptides of the GRF-family.

Selective inhibition of dipeptidyl peptidase 4 by targeting a substrate-specific secondary binding site.

Abstract Dipeptidyl peptidase 4/CD26 (DP4) is a multifunctional serine protease liberating dipeptide from the N-terminus of (oligo)peptides which can modulate the activity of these peptides. The enzyme is involved in physiological processes such as blood glucose homeostasis and immune response. DP4 substrate specificity is characterized in detail using synthetic dipeptide derivatives. The specificity constant k cat/K m strongly depends on the amino acid in P1-position for proline, alanine, glycine and serine with 5.0×105 m -1s-1, 1.8×104 m -1s-1, 3.6×102 m -1s-1, 1.1×102 m -1s-1, respectively. By contrast, kinetic investigation of larger peptide substrates yields a different pattern. The specific activity of DP4 for neuropeptide Y (NPY) cleavage comprising a proline in P1-position is the same range as the k cat/K m values of NPY derivatives containing alanine or serine in P1-position with 4×105 m -1s-1, 9.5×105 m -1s-1 and 2.1×105 m -1s-1, respectively. The proposed existence of an additional binding region outside the catalytic center is supported by measurements of peptide substrates with extended chain length. This ‘secondary’ binding site interaction depends on the amino acid sequence in P4′–P8′-position. Interactions with this binding site could be specifically blocked for substrates of the GRF/glucagon peptide family. By contrast, substrates not belonging to this peptide family and dipeptide derivative substrates that only bind to the catalytic center of DP4 were not inhibited. This more selective inhibition approach allows, for the first time, to distinguish between substrate families by substrate-discriminating inhibitors.

Structure-activity relationships of glucose-dependent insulinotropic polypeptide (GIP).

Abstract Six GIP1-30NH2 analogs were synthesized with modifications (de-protonation, N-methylation, reversed chirality, and substitution) at positions 1, 3, and 4 of the N-terminus, and additionally, a cyclized GIP derivative was synthesized. The relationship between altered structure to biological activity was assessed by measuring receptor binding affinity and ability to stimulate adenylyl cyclase in CHO-K1 cells transfected with the wild-type GIP receptor (wtGIPR). These structureactivity relationship studies demonstrate the importance of the GIP N-terminus and highlight structural constraints that can be introduced in GIP analogs. These analogs may be useful starting points for design of peptides with enhanced in vivo bioactivity.

The Specificity of DP IV for Natural Substrates is Peptide Structure Determined

Our results indicate that the substrate properties of peptides are encoded by their own structure. That means, that substrate characteristics depend not only on the primary structure around the catalytic site rather C-terminal located secondary interactions strongly influence the binding and catalysis of the substrates. Such interaction sites seem to force the ligand in a proper orientation to the active site of DP IV. As result of these relations the hydrolysis of peptides with non-proline and non-alanine residues in P1-position (Ser, Val, Gly) becomes possible in longer peptides.

Glucose-dependent insulinotropic polypeptide (GIP): development of DP IV-resistant analogues with therapeutic potential.

Although type 2 diabetic patients exhibit resistance to GIP when the peptide is administered in doses that result in circulating levels approximating those found physiologically, it is likely that DP IV-resistant forms of the peptide administered in pharmacological doses will prove to be effective in improving glucose tolerance. Additionally, in view of recent studies showing that GIP receptor knockout mice are resistant to diet induced obesity25, it is possible that GIP-antagonists will prove useful in obesity treatment.

Isolation and Characterization of Attractin-2

Attractin 2/4 has been isolated to homogeneity from human plasma. Based on the native molecular weight of 178 kDa and pI-value around pH 3.5, a new reproducible purification procedure has been developed. N-terminal sequencing of attractin confirmed the predicted signal peptidase cleavage site in the insertion of the isoforms 1, 2, 4 and 5 and questioned the predicted Ser 26 as an active site residue.

A phase 1 study to evaluate the safety and pharmacokinetics of PQ912, a glutaminyl cyclase inhibitor, in healthy subjects

Pyroglutamate‐amyloid‐β (pE‐Aβ) peptides are major components of Aβ‐oligomers and Aβ‐plaques, which are regarded as key culprits of Alzheimers disease (AD) pathology. PQ912 is a competitive inhibitor of the enzyme glutaminyl cyclase (QC), essential for the formation of pE‐Aβ peptides.

Acylated Hydroxamates as Selective and Highly Potent Inhibitors of Dipeptidyl Peptidase I

The compounds 10–14 which are N-dipeptidyl derivatives of O-acyl hydroxamates proved to be potent, selective and irreversible inhibitors of DP I.