Type A allatostatins from Drosophila melanogaster and Diplotera puncata activate two Drosophila allatostatin receptors, DAR-1 and DAR-2, expressed in CHO cells.

The type-A allatostatins A (AST-A) are a group of insect peptides with a common C-terminal motif Y/FXFGL-NH(2). The existence of at least four putative type A Drosophila melanogaster ASTs (called type A drostatins or DST-As) has been predicted from the sequence of a recently cloned DST-A preprohormone [C. Lenz et al. (2000) Biochem. Biophys. Res. Commun. 273, 126-1131]. SRPYSFGL-NH(2), (DST-3A), the only DST isolated from Drosophila so far, activated the first cloned DST-A GPCR (DAR-1) [N. Birgül et al. (1999) EMBO J. 18, 5892-5900]. A newly cloned orphan Dm GPCR, which shares 47% overall and 60% transmembrane region sequence identity with DAR-1, was classified as a second putative Dm DST-A receptor (DAR-2) [C. Lenz et al. (2000) Biochem. Biophys. Res. Commun. 273, 571-577]. Although activation of DAR-2 by DSTs has been postulated, no experimental evidence for that has been presented to date. In this study, we expressed both DAR-1 and DAR-2 in CHO cells and used a GTPgammaS and a Ca(2+) mobilization assay for pharmacological evaluation of the receptors. Synthetically prepared DST-As, as well as selected Diplotera punctata (cockroach) ASTs, activated DAR-1 and DAR-2 in both functional assays indicating ligand redundancy and cross species activity. Cell pretreatment with pertussis toxin led to some differences in the nature and magnitude of signaling pathways at the DAR-1 and DAR-2 receptors, suggesting possible differential coupling to cellular effector system(s) and distinct biological functions of each receptor in vivo.

Synthesis and biological activity of anthelmintic thiadiazoles using an AF-2 receptor binding assay.

Following our discovery of the strong binding of thiadiazole 1 to the AF-2 neuropeptide receptor of gastrointestinal nematodes (e.g., Ascaris suum), we prepared two series of analogs. Only the series containing the thiadiazole ring had potencies comparable to that of compound 1. Analog 50 exhibited an apparent potency in the AF-2 binding assay 300 times that of compound 1.

Haemonchus contortus: cloning and functional expression of a cDNA encoding ornithine decarboxylase and development of a screen for inhibitors.

Polyamines (PA) are essential for viability and replication of all cells; organisms either synthesize PA or acquire them from the environment. How nematodes that parasitize the gut satisfy their PA requirement has not been resolved. The primary regulatory enzyme in PA biosynthesis in most animals is ornithine decarboxylase (ODC). This enzyme has recently been characterized in free-living nematodes and in the parasitic species. Haemonchus contortus. Nematode and mammalian ODC are reported to differ in subcellular localization, kinetics, and sensitivity to inhibitors. We cloned an H. contortus cDNA that encodes a full-length ODC (sequence data from this article have been deposited with the GenBank Data Library under Accession Nos. AF016538 and AF016891). This cDNA was functionally expressed in strains of Escherichia coli and Saccharomyces cerevisiae that lack ODC and are dependent upon exogenous PA for survival. Expression of nematode ODC reversed the PA-dependence phenotype of both microorganisms. The complemented yeast strain was used to develop a nutrient-dependent viability screen for selective inhibitors of nematode ODC. The antiprotozoal drug stilbamidine isethionate was identified as active in this screen, but biochemical characterization revealed that this compound did not inhibit ODC. Instead, like other cationic diamidines, stilbamidine probably inhibits yeast S-adenosylmethionine decarboxylase. Nonetheless, the activity in the screen of the known ODC inhibitor difluoromethylornithine (DFMO) validates the concept that specific recombinant microorganisms can serve as the basis for extremely selective and facile screens.