John S. Porter

A Novel Post-translational Modification Involving Bromination of Tryptophan IDENTIFICATION OF THE RESIDUE, L-6-BROMOTRYPTOPHAN, IN PEPTIDES FROM Conus imperialis AND Conus radiatus VENOM

We report a novel post-translational modification involving halogenation of tryptophan in peptides recovered from the venom of carnivorous marine cone snails (Conus). The residue, L-6-bromotryptophan, was identified in the sequence of a heptapeptide, isolated from Conus imperialis, a worm-hunting cone. This peptide does not elicit gross behavioral symptoms when injected centrally or peripherally in mice. L-6-Bromotryptophan was also identified in a 33-amino acid peptide from Conus radiatus; this peptide has been shown to induce a sleep-like state in mice of all ages and is referred to as bromosleeper peptide. The sequences of the two peptides and were determined using a combination of mass spectrometry, amino acid, and chemical sequence analyses, where Pca = pyroglutamic acid, Hyp = hydroxyproline, Gla = γ-carboxyglutamate, and Trp* = L-6-bromotryptophan. The precise structure and stereochemistry of the modified residue were determined as L-6-bromotryptophan by synthesis, co-elution, and enzymatic hydrolysis experiments. To our knowledge this is the first documentation of tryptophan residues in peptides/proteins being modified in a eukaryotic system and the first report of halogenation of tryptophan in vivo.

Structural modifications of non-mammalian gonadotropin-releasing hormone (GnRH) isoforms: design of novel GnRH analogues.

Three natural forms of vertebrate gonadotropin-releasing hormone (GnRH) provided the structural basis upon which to design new GnRH agonists: [His5,Trp7,Leu8]-GnRH, dogfish (df) GnRH; [His5,Asn8]-GnRH, catfish (cf) GnRH; and [His5,Trp7,Tyr8]-GnRH, chicken (c) GnRH-II. The synthetic peptides incorporated the position 6 dextro (D)-isomers D-arginine (D-Arg) or D-naphthylalanine (D-Nal) in combination with an ethylamide substitution of position 10. The in vitro potencies for LH and FSH release of these analogues were assessed using static cultures of rat anterior pituitary cells. Efficacious peptides were examined for their gonadotropin-II and growth hormone releasing abilities from perifused goldfish pituitary fragments. Rat LH and FSH release was measured using homologous radioimmunoassays, whereas goldfish growth hormone and gonadotropin-II release were determined using heterologous carp hormone radioimmunoassays. The receptor binding of the most potent analogues was determined in bovine pituitary membrane preparations. Substitution of D-Nal6 into [His5,Asn8]-GnRH increased the potency over 2200-fold compared with the native ligand (cfGnRH) in cultured rat pituitary cells. This was equivalent to a 55-fold greater potency than that of the native mammal (m) GnRH peptide. Substitution of D-Nal6 or D-Arg6 into dfGnRH or cGnRH-II resulted in potencies that were related to the overall hydrophobicity of the analogues. The [D-Nal6,Pro9NEt]-cfGnRH bound to the bovine membrane preparation with an affinity statistically similar to that of [D-Nal6,Pro9NEt]-mGnRH (kd = 0.40 +/- 0.04 and 0.55 +/- 0.10 nM, respectively) in cultured rat pituitary cells. All analogues tested released the same ratio of FSH to LH. In goldfish, the analogues did not possess superagonistic activity but instead desensitized the pituitary fragments at lower analogue doses than that of the sGnRH standard suggesting differences in receptor affinity or signal transduction.

DESIGN, COMPUTER DERIVED STRUCTURE AND BIOLOGICAL ACTIVITY OF THREE BICYCLIC GONADOTROPIN RELEASING HORMONE (GnRH) ANTAGONISTS

In order to determine the bioactive conformation of GnRH we decided to synthesize fully rigid and biologically active analogs which could be studied ultimately by NMR spectroscopy or X-ray crystallography. Because we had found that bridging GnRH sequences at positions 1 and 10 or 4 and 10 in fact lead to partial agonists and antagonists resp., and because we have more latitude in the selection of amino acid substitutions in the design of antagonists, we designed, synthesized and tested cyclic GnRH antagonists. These antagonists are characterized by specific substitutions at positions 1, 2 and 3. Here, we show that some antagonists of GnRH which had been bridged at positions 4 and 10 and had been found to be equipotent to the most potent linear analogs, could be further constrained by the introduction of a second bridge between residues 5 and 8 with retention of high potency.

Characterization of Gonadotropin Hormone-Releasing Hormone Analogs

Characterization, a word often used in the chemical/biochemical and biological sciences, refers in each of these disciplines to distinct but certainly complementary analytical approaches to defining intrinsic properties of a particular compound or activity (including bio-, immuno-, radio- etc. activities). Here we describe some aspects of our research efforts at characterizing the decapeptide GnRH (gonadotropin hormone releasing hormone) using analogs as probes for the study of receptor binding and activation.