Two dipeptidyl peptidase IV (DPPIV, DPP4)-related proteins, DPP8 and DPP9, have been identified recently [Abbott, Yu, Woollatt, Sutherland, McCaughan, and Gorrell (2000) Eur. J. Biochem. 267, 6140-6150; Olsen and Wagtmann (2002) Gene 299, 185-193; Qi, Akinsanya, Riviere, and Junien (2002) Patent application WO0231134]. In the present study, we describe the cloning of DPP10, a novel 796-amino-acid protein, with significant sequence identity to DPP4 (32%) and DPP6 (51%) respectively. We propose that DPP10 is a new member of the S9B serine proteases subfamily. The DPP10 gene is located on the long arm of chromosome 2 (2q12.3-2q14.2), close to the DPP4 (2q24.3) and FAP (2q23) genes. The active-site serine residue is replaced by a glycine residue in DPP10, resulting in the loss of DPP activity. The serine residue is also replaced in DPP6, which lacks peptidase activity. DPP8 and DPP9 share an identical active site with DPP4 (Gly-Trp-Ser-Tyr-Gly). In contrast with the previous results suggesting that DPP9 is inactive, we show that DPP9 is a DPP, hydrolysing Ala-Pro-(7-amino-4-methyl-coumarin) with similar pH-specificity and protease-inhibitor-sensitivity to those of DPP4 and DPP8. Northern-blot analysis shows that whereas DPP8 and DPP9 are widely expressed, DPP10 is expressed mainly in the brain and pancreas. DPP6, which has the highest amino acid identity with DPP10, has been shown previously [Nadal, Ozaita, Amarillo, de Miera, Ma, Mo, Goldberg, Misumi, Ikehara, Neubert et al. (2003) Neuron 37, 449-461] to associate with A-type K(+) channel subunits, modulating their transport and function in somatodendritic compartments of neurons. It is possible that DPP10 is involved in similar functions in the brain. Elucidation of the physiological or pathophysiological role of DPP8, DPP9 and DPP10 and characterization of their structure-function relationships will add impetus to the development of inhibitor molecules for pharmacological or therapeutic use.

Cysteine proteinases are important not only in the intracellular catabolism of peptides and proteins and in the processing of prohormones and proenzymes, but also in the penetration of normal human tissue by malignant cells and possibly microorganisms, including viruses. Cystatin C is a human cysteine proteinase inhibitor present in extracellular fluids. We have synthesized peptide derivatives mimicking the proposed proteinase-binding centre of cystatin C and find that they irreversibly inhibit cysteine proteinases. Several bacteria produce proteinases, so we tested a tripeptide derivative (Z-LVG-CHN2) for in vitro anti-bacterial activity against a large number of bacterial strains belonging to thirteen different species. It was found to inhibit specifically the growth of all strains of group A streptococci. The susceptibility of these human pathogens to the peptide was compared with that to well-established anti-streptococcal antibiotics such as tetracycline and bacitracin. Moreover, the peptide was active in vivo against group A streptococci: mice injected with lethal doses of these bacteria were cured by a single injection of Z-LVG-CHN2. The cysteine proteinase produced by group A streptococci was isolated and found to be inhibited by Z-LVG-CHN2; moreover, excess proteinase relieved the growth inhibition caused by the peptide derivative, suggesting that the antibacterial activity of Z-LVG-CHN2 is due to inhibition of this cysteine proteinase. This strategy of blocking proteinases with peptide derivatives that mimic naturally occurring inhibitors could be useful in the construction of new agents against other microorganisms, including viruses.

A series of antagonists of gonadotropin-releasing hormone (GnRH) of the general formula Ac-D2Nal-D4Cpa-D3Pal-Ser-4Aph/4Amf(P)-D4Aph/D4Amf(Q)-Leu-ILys-Pro-DAla-NH2 was synthesized, characterized, and screened for duration of inhibition of luteinizing hormone release in a castrated male rat assay. Selected analogues were tested in a reporter gene assay (IC50 and pA2) and an in vitro histamine release assay. P and Q contain urea/carbamoyl functionalities designed to increase potential intra- and intermolecular hydrogen bonding opportunities for structural stabilization and peptide/receptor interactions, respectively. These substitutions resulted in analogues with increased hydrophilicity and a lesser propensity to form gels in aqueous solution than azaline B [Ac-D2Nal-D4Cpa-D3Pal-Ser-4Aph(Atz)-D4Aph(Atz)-Leu-ILys-Pro-DAla-NH2 with Atz = 3'-amino-1H-1',2',4'-triazol-5'-yl, 5], and in some cases they resulted in a significant increase in duration of action after subcutaneous (s.c.) administration. Ac-D2Nal-D4Cpa-D3Pal-Ser-4Aph(L-hydroorotyl)-D4Aph(carbamoyl)-Leu-ILys-Pro-DAla-NH2 (acetate salt is FE200486) (31) and eight other congeners (20, 35, 37, 39, 41, 45-47) were identified that exhibited significantly longer duration of action than acyline [Ac-D2Nal-D4Cpa-D3Pal-Ser-4Aph(Ac)-D4Aph(Ac)-Leu-ILys-Pro-DAla-NH2] (6) when administered subcutaneously in castrated male rats at a dose of 50 microg in 100 microL of phosphate buffer. No correlation was found between retention times on a C18 reverse phase column using a triethylammonium phosphate buffer at pH 7.0 (a measure of hydrophilicity) or affinity in an in vitro human GnRH report gene assay (pA2) and duration of action. FE200486 was selected for preclinical studies, and some of its properties were compared to those of other clinical candidates. In the intact rat, ganirelix, abarelix, azaline B, and FE200486 inhibited plasma testosterone for 1, 1, 14, and 57 days, respectively, at 2 mg/kg s.c. in 5% mannitol (injection volume = 20 microL). Based on the information that 31, 33, 35 and 37 were significantly shorter acting than acyline or azaline B after intravenous administration (100 microg/rat), we surmised that the very long duration of action of the related FE200486 (for example) was likely due to unique physicochemical properties such as solubility in aqueous milieu, comparatively low propensity to form gels, and ability to diffuse at high concentrations in a manner similar to that described for slow release formulations of peptides. Indeed, in rats injected s.c. with FE200486 (2 mg/kg), plasmatic concentrations of FE200486 remained above 5 ng/mL until day 41, and the time after which they dropped below 3 ng/mL and plasma LH levels started rising until full recovery was reached at day 84 with levels of FE200486 hovering around 1 ng/mL. Additionally, FE200486 was less potent at releasing histamine from isolated rat mast cells than any of the GnRH antagonists presently described in preclinical reports.

No abstract