Redefining the structure-activity relationships of 2,6-methano-3-benzazocines. Part 8. High affinity ligands for opioid receptors in the picomolar Ki range: oxygenated N-(2-[1,1'-biphenyl]-4-ylethyl) analogues of 8-CAC.

N-[2-(4'-methoxy[1,1'-biphenyl]-4-yl)ethyl]-8-CAC (1) is a high affinity (K(i)=0.084 nM) ligand for the µ opioid receptor and served as the lead compound for this study. Analogues of 1 were made in hopes of identifying an SAR within a series of oxygenated (distal) phenyl derivatives. A number of new analogues were made having single-digit pM affinity for the µ receptor. The most potent was the 3',4'-methylenedioxy analogue 18 (K(i)=1.6 pM).

Brain P450 epoxygenase activity is required for the antinociceptive effects of improgan, a nonopioid analgesic.

The search for the mechanism of action of improgan (a nonopioid analgesic) led to the recent discovery of CC12, a compound that blocks improgan antinociception. Because CC12 is a cytochrome P450 inhibitor, and brain P450 mechanisms were recently shown to be required in opioid analgesic signaling, pharmacological and transgenic studies were performed in rodents to test the hypothesis that improgan antinociception requires brain P450 epoxygenase activity. Intracerebroventricular (i.c.v.) administration of the P450 inhibitors miconazole and fluconazole, and the arachidonic acid (AA) epoxygenase inhibitor N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH) potently inhibited improgan antinociception in rats at doses that were inactive alone. MW06-25, a new P450 inhibitor that combines chemical features of CC12 and miconazole, also potently blocked improgan antinociception. Although miconazole and CC12 were weakly active at opioid and histamine H(3) receptors, MW06-25 showed no activity at these sites, yet retained potent P450-inhibiting properties. The P450 hypothesis was also tested in Cpr(low) mice, a viable knock-in model with dramatically reduced brain P450 activity. Improgan (145 nmol, i.c.v.) antinociception was reduced by 37% to 59% in Cpr(low) mice, as compared with control mice. Moreover, CC12 pretreatment (200 nmol, i.c.v.) abolished improgan action (70% to 91%) in control mice, but had no significant effect in Cpr(low) mice. Thus, improgan's activation of bulbospinal nonopioid analgesic circuits requires brain P450 epoxygenase activity. A model is proposed in which (1) improgan activates an unknown receptor to trigger downstream P450 activity, and (2) brainstem epoxygenase activity is a point of convergence for opioid and nonopioid analgesic signaling.

Aromatic thiol pKa effects on the folding rate of a disulfide containing protein.

The production of proteins via recombinant DNA technology often requires the in vitro folding of inclusion bodies, which are protein aggregates. To create a more efficient redox buffer for the in vitro folding of disulfide containing proteins, aromatic thiols were investigated for their ability to increase the folding rate of scrambled RNase A. Scrambled RNase A is fully oxidized RNase A with a relatively random distribution of disulfide bonds. The importance of the thiol pK(a) value was investigated by the analysis of five para-substituted aromatic thiols with pK(a) values ranging from 5.2 to 6.6. Folding was measured at pH 6.0 where the pK(a) value of the thiols would be higher, lower, or equal to the solution pH. Thus, relative concentrations of thiol and thiolate would vary across the series. At pH 6.0, the aromatic thiols increased the folding rate of RNase A by a factor of 10-23 over that observed for glutathione, the standard additive. Under optimal conditions, the apparent rate constant increased as the thiol pK(a) value decreased. Optimal conditions occurred when the concentration of protonated thiol in solution was approximately 2 mM, although the total thiol concentration varied considerably. The importance of the concentration of protonated thiol in solution can be understood based on equilibrium effects. Kinetic studies suggest that the redox buffer participates as the nucleophile and/or the center thiol in the key rate determining thiol disulfide interchange reactions that occur during protein folding. Aromatic thiols proved to be kinetically faster and more versatile than classical aliphatic thiol redox buffers.