Abstract
ABSTRACT|Human-a-calcitonin gene-related peptide (CGRP) is a 37 amino acid residue neuropeptide produced from tissue-specific alternative splicing of the primary RNA transcript of the calcitonin gene. CGRP is widely distributed throughout the central and peripheral nervous systems and is involved in wound healing, inflammatory responses, nociception, appetite suppression, gastric emptying, regulation of vascular tone and regional organ blood flow.|The conformation of CGRP, defined in CD and two-dimensional 1H-NMR studies, includes a disulfide bridged N-terminal ring structure between residues 2 and 7, an amphipathic a-helical segment between residues 8 through 18 which ends in a ß-turn centered on residues 19 through 22 and a conformationally disordered C-terminus. Molecular modeling studies suggest a turn structure centered on residue 33 exists. Previous studies have shown that the N-terminal loop of CGRP is required for the activation of signal transduction by CGRP receptors. Truncation of the N-terminal ring, resulting in CGRP(8-37), leads to loss of agonist activity while retaining the ability to bind to the receptor and CGRP(8-37) became the primary research tool used to investigate CGRP receptors. The development of CGRP receptor antagonists has progressed slowly since the inception of the CGRP field. Further truncation of the N-terminus of CGRP(8-37) has resulted in antagonists at CGRP receptors with marked reductions in antagonist potency, suggesting that N-terminal structural elements of CGRP(8-37) are important for high affinity binding to CGRP receptors.|Previous studies in my lab described a series of N-termina!ly modified CGRP(8-37) analogs that were high affinity antagonists of calcitonin gene-related peptide receptors. These analogues, N-a-benzoyl-h-a-CGRP(8-37), N-a-benzyl- h-a-CGRP(8-37) and dibenzyl-h-cc-CGRP(8-37), all possess binding affinities for CGRP receptors, similar to the endogenous ligand CGRP, in the low nanomolar range. These studies show that the addition of functional groups to CGRP(8-37), rather than truncation, may be an improved strategy for the development of high affinity antagonists.|The goal of my research was to design, synthesize and pharmacologically characterize novel CGRP receptor antagonists. The antagonists were synthesized using rapid solid phase methods employing in situ neutralization, purified by reversed phase-high performance liquid chromatography, characterized by amino acid analysis and electrospray ionization mass spectrometry and pharmacologically characterized using the mouse thoracic aorta and the human SK-N-MC cell line.|During the synthesis of these antagonists using rapid solid phase methodology, I observed that coupling yields for the synthesis of CGRP(8-37) were highly dependent on the solvent used. I investigated the relationship between solvent properties and solvation of the (para- methylbenzhydrylamine)copoly(styrene-1 % DVB) (resin) and resin covalently bound to the fully protected amino acid sequence of CGRP(8-37) (peptide-resin)|in order to obtain satisfactory coupling yields for the rapid solid phase peptide synthesis of the 30-amino acid residue N-truncated fragment of human-a- calcitonin gene-related peptide, CGRP(8-37). CGRP(8-37) was synthesized manually using rapid solid phase peptide synthesis in various polar aprotic solvents. Also, I measured solvation of the resin and peptide-resin, in solvents with varying hydrogen-bonding (5h) and Hildebrand solubility (5) parameters and contour solvation plots of 5h versus 8 were constructed to correlate solvation of the resin or peptide-resin with 8h or 8. Maximum resin solvation occurred with NMP, NMP:DMSO (8:2) and DMSO, however, inefficient solvation of the peptide- resin occurred with these solvents and resulted in poor syntheses. Superior peptide-resin solvation was obtained using DMA and DMF, resulting in significantly higher average coupling yields. The region of maximum peptide- resin solvation shifted to solvents with higher 8h values. DMF provided the most effective peptide-resin solvation and was the only solvent from which CGRP(8- 37) was obtained as a single major product in the crude cleaved material.|Based on the known structure of CGRP(8-37) and the human CGRP receptor I designed an analogue which incorporated hydrophobic benzoyl and benzyl groups into the N-terminus and His10 side-chain, respectively, to promote high affinity and selectivity for human CGRP receptors. The affinity (KB) of the putative human selective competitive antagonist at CGRP receptors was determined using the mouse thoracic aorta and the human SK-N-MC cells. In aorta, CGRP(8-37), bzl-CGRP(8-37) and bzl-bn-CGRP(8-37) caused rightward shifts in the concentration-response relaxation curve for CGRP with KB values of 1000, 88 and 50 nM, respectively. In human SK-N-MC cells CGRP(8-37), bzl- CGRP(8-37) and bzl-bn-CGRP(8-37) caused rightward shifts in the concentration-response curve for CGRP stimulated cAMP production with KB values of 797, 15 and 0.63 nM, respectively. Thus, CGRP(8-37) had the same affinity for human and mouse CGRP receptors while bzl-CGRP(8-37) and bzl-bn- CGRP(8-37) displayed 6-fold and 80-fold higher affinity, respectively, for human CGRP receptors. In Addition, the selectivity of the antagonists for human CGRP receptors was highly correlated with the antagonist hydrophobicity index. These high affinity, species selective antagonists provide novel tools to differentiate structural and functional features that are unique to the human CGRP receptor. Thus, these analogues may be useful compounds for development of drugs to treat migraine headache and other cardiovascular diseases.|Also, I designed, synthesized and pharmacologically characterized the first irreversible CGRP receptor antagonists. Fluorosulfonyl or bis-(2- chloroethyl)amino moieties were incorporated into para-position of the N-terminal benzoyl group of a potent competitive antagonist, N-a-benzoyl-h-a-CGRP(8-37). In order to improve potency, a second pair of analogues was synthesized with histidine at position 10 benzylated, at C4 of the imidazole side-chain. All analogues blocked the actions of CGRP on mouse thoracic aorta and SK-N-MC cells and reduced maximal CGRP-mediated responses. The inability to obtain a maximal response to agonist after treatment and removal of either analogue is consistent with the analogues binding irreversibly to CGRP receptors. The bis-(2- chloroethyl)amino-modified analogues were more potent than the fluorosulfonyl- modified analogues. Benzylation of the His10 side-chain increased the potency of the bis-(2-chloroethyl)amino-modified irreversible antagonists but had no effect on the potency of the fluorosulfonyl-modified irreversible antagonist. The most potent analogue was N-a-4-[bis-(2-chloroethyl)amino]benzoyl-[His(4-benzyl)10]-h- a-CGRP(8-37). The addition of electrophillic alkylating moieties to potent CGRP receptor competitive antagonists yielded the first irreversible antagonists of CGRP receptors.