Ligands Devoid of Sulfur

Over 25 years ago, investigation of polyamines in vivo established that the tetramine disulfide, benextramine, was an a-adrenergic receptor (a-AR) antagonist. Further work established that inhibition by benextramine was irreversible, time-dependent, and essentially specific for a-ARs (muscarinic and nicotinic acetylcholine receptors were also affected, although these were considered ionic interactions). A hypothesis was proposed that a buried thiol group in the receptor was unmasked by benextramine binding; thiol-di thiol interchange resulted in covalent attachment to the receptor that could be alleviated using a cationic thiol such as cysteamine.48

More recently, an alkylating derivative of the a2-adrenergic agonist clonidine was found to bind irreversibly to a2-ARs. A covalent bond is formed with the sulfhydryl side chain of a cysteine residue in TM5 exposed in the binding cavity, leading to inactivation of the receptor. Subsequently, the recognition of inactive and active receptor conformations was claimed after differential labeling of receptor cysteines.49 Thus, the cysteine residues of adrenergic receptors have both structural and functional roles in ligand activation, with the receptor demonstrating a range of flexibility.

Benextramine (but not other thiol-reactive agents such as cysteamine or DTNB) also blockades the 7TMR for neuropeptide Y.50 Thiol-reducing agents reversed the effect of benextramine, freeing the receptor for subsequent NPY binding. Benextramine is an antagonist of a-adrenergic, and presumably NPY, hormone activity. Although covalent attachment of benextramine to these receptors was perceived, there was no evidence to suggest that other antagonists, or even agonist ligands, are capable of covalent linkage with the receptor.

FIGURE 10.9 Evidence for redox control of 7TMR binding and activation. A, the relative adenylate cyclase activity of 19 b-adrenergic partial agonists is correlated with their reductive potential measured by cyclic voltammetry, Ep, reproduced with permission from Wong A, et al, in Molecular Pharmacology, © 1987 American Society for Pharmacology and Experimental Therapeutics.51 B, exposure to either agonist or reducing agent produces a fully unfolded b-adrenergic receptor.52 C, the retention of RNAse by its horseshoe-shaped inhibitor is through disulfide exchange under redox control (1dfj) and a theoretical model for the LH/hCG receptor (1xul) places a cystine bridge adjacent to the receptor-determinant loop of the hormone which has intrinsic thioredoxin activity.53

FIGURE 10.9 Evidence for redox control of 7TMR binding and activation. A, the relative adenylate cyclase activity of 19 b-adrenergic partial agonists is correlated with their reductive potential measured by cyclic voltammetry, Ep, reproduced with permission from Wong A, et al, in Molecular Pharmacology, © 1987 American Society for Pharmacology and Experimental Therapeutics.51 B, exposure to either agonist or reducing agent produces a fully unfolded b-adrenergic receptor.52 C, the retention of RNAse by its horseshoe-shaped inhibitor is through disulfide exchange under redox control (1dfj) and a theoretical model for the LH/hCG receptor (1xul) places a cystine bridge adjacent to the receptor-determinant loop of the hormone which has intrinsic thioredoxin activity.53

Based on the redox sensitivities of p-ARs and involvement of the attendant molecules of the four-state ternary complex of ligand, receptor, and G protein, a redox mechanism for the action of agonists at b-adrenergic receptors was proposed nearly 20 years ago.51 The hypothesis was presented that agonists activate p-ARs by reducing them and this was examined by analyzing 41 agonists and antagonists. The structural features that determined binding affinity were shown to be distinct from those that determined agonist activity. Agonist activity was shown to be related to the oxidation-reduction properties of the ligands, which were determined primarily by the nature of the substituents on the phenyl ring. Of the 19 antagonists, all exhibited EP values greater than 0.75 V (electrochemical peak potential for the first oxidative wave in cyclic voltammetry), suggesting that they were difficult to oxidize. Partial agonists, however, exhibited a wide range of EP (0.25 to 0.7 V) with values lower than those of the antagonists (Figure 10.9A).

Also in the mid-1980s, Western blots with DTT-treated p-AR demonstrated the importance of receptor cysteines and their availability during ligand activation. Indeed, the behavior of the receptor in the presence and absence of thiol reduction led to a hypothesis of ligand binding controlled by disulfide bridge breakdown.52 Pure p-AR migrated with a size of 54 kDa on SDS-PAGE, which increased to 65

kDa after chemical reduction with reagents that cleave disulfide bridges (Figure 10.9B). A possible explanation for this behavior is that the smaller molecule was compact because of intramolecular disulfide bridges. Cleavage of these bonds would allow true unfolding of an elongated molecule that would migrate nearer to its true molecular size. Agonist exposure before purification of the receptor increased the proportion of the larger, unfolded molecular species, implying that this was concomitant with receptor activation. Because the cytosol of a cell is a strongly reducing environment and most intracellular cysteines exist as free sulfhydryl groups, the authors concluded that all the cysteines in the extracellular loops and also in the TM helices 2, 3, 6 and 7 were disulfide-bonded in the vacant receptor configuration. Agonist binding would promote disulfide exchange and transform the receptor into its transducing configuration. The authors52 consider "whether receptor disulfides are avenues to receptor activation or merely an unavoidable but provocative dead end." In the intervening years, this issue has still not been resolved — a problem mainly related to the advent of accessible molecular biology and the cloning boom of the late 1980s. Although many novel 7TMR sequences and ligands have since been discovered, biochemical pharmacology was seriously neglected in the 1990s and only recently underwent a rejuvenation, with SCAM and other mutation replacement and removal strategies for chemically reactive residues.

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