We have purified contulakin-G, a 16-amino acid Olinked glycopeptide (pGlu-Ser-Glu-Glu-Gly-Gly-SerAsn-Ala-Thr-Lys-Lys-Pro-Tyr-Ile-Leu-OH, pGlu is pyroglutamate) from Conus geographus venom. The major glycosylated form of contulakin-G was found to incorporate the disaccharide ␤-D-Galp-(133)-␣-D-GalpNAc-(13) attached to Thr 10 . The C-terminal sequence of contulakin-G shows a high degree of similarity to the neurotensin family of peptides. Synthetic peptide replicates of Gal(␤33) GalNAc(␣3)Thr 10 contulakin-G and its nonglycosylated analog were prepared using an Fmoc (9-fluorenylmethoxycarbonyl) protected solid phase synthesis strategy. The synthetic glycosylated contulakin-G, when administered intracerebroventricular into mice, was found to result in motor control-associated dysfunction observed for the native peptide. Contulakín-G was found to be active at 10-fold lower doses than the nonglycosylated Thr 10 contulakin-G analog. The binding affinities of contulakin-G and the nonglycosylated Thr 10 contulakin-G for a number of neurotensin receptor types including the human neurotensin type 1 receptor (hNTR1), the rat neurotensin type 1 and type 2 receptors, and the mouse neurotensin type 3 receptor were determined. The binding affinity of the nonglycosylated Thr 10 contulakin-G was approximately an order of magnitude lower than that of neurotensin [1][2][3][4][5][6][7][8][9][10][11][12][13] for all the receptor types tested. In contrast, the glycosylated form of contulakin-G exhibited significantly weaker binding affinity for all of the receptors tested. However, both contulakin-G and nonglycosylated Thr 10 contulakin-G were found to be potent agonists of rat neurotensin receptor type 1. Based on these results, we conclude that O-linked glycosylation appears to be a highly unusual strategy for increasing the efficacy of toxins directed against neurotransmitter receptors.

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.

In this work, a new family of Conus peptides, the ␣A-conotoxins, which target the nicotinic acetylcholine receptor, is defined. The first members of this family have been characterized from the eastern Pacific species, Conus purpurascens (the purple cone); three peptides that cause paralysis in fish were purified and characterized from milked venom. The sequence and disulfide bonding pattern of one of these, ␣A-conotoxin PIVA, is as follows:

We purified and characterized a novel peptide from the venom of the fish-hunting cone snail Conus striatus that inhibits voltage-gated K+ channels. The peptide, kappaA-conotoxin SIVA, causes characteristic spastic paralytic symptoms when injected into fish, and in frog nerve-muscle preparations exposed to the toxin, repetitive action potentials are seen in response to a single stimulus applied to the motor nerve. Other electrophysiological tests on diverse preparations provide evidence that is consistent with the peptide blocking K+ channels. The peptide has three disulfide bonds; the locations of Cys residues indicate that the spastic peptide may be the first and defining member of a new family of Conus peptides, the kappaA-conotoxins, which are structurally related to, but pharmacologically distinct from, the alphaA-conotoxins. This 30 AA tricyclic toxin has several characteristics not previously observed in Conus peptides. In addition to the distinctive biological and physiological activity, a novel biochemical feature is the unusually long linear N-terminal tail (11 residues) which contains one O-glycosylated serine at position 7. This is the first evidence for O-glycosylation as a posttranslational modification in a biologically active Conus peptide.