Abstract
Understanding the interaction of local anesthetics (LAs) with plasma proteins is essential to understanding their systemic pharmacology and toxicology. The molecular determinants of LA binding to the major variant (F1*S) of human α1-acid glycoprotein (AGP) were therefore investigated spectrofluorometrically using whole AGP and a novel, F1*S variant-selective probe previously developed in our laboratory. Equilibrium- competitive displacement of this probe by LAs, observed by the recovery of AGP's fluorescence as the quenching probe was displaced from its high-affinity site, was characterized by inhibitory dissociation constants for the various LAs. The importance of electrostatic factors was assessed by examining the pH dependent binding of an ionizable LA, lidocaine, using the quaternary lidocaine derivative QX-314 [N-(2,6-dimethylphenylcarbamoylmethyl) triethylammonium chloride] to control for pH dependent ionization of AGP. Uncharged lidocaine bound with at least 8 times the affinity of protonated lidocaine (KD = 4.0 ± 0.6 μM and >32 μM, respectively). This result is inconsistent with the current model of the AGP-binding site, which depicts a buried pocket having a negatively charged region that interacts with the amino termini of basic drugs. Consistent with the model, however, two sets of structurally homologous LAs (mepivacaine, ropivacaine, bupivacaine, and lidocaine, RAD-240, RAD-241, RAD-242, L-30, W-6603) demonstrated a strong positive correlation between hydrophobicity (measured as the octanol:buffer partition coefficient of the neutral species) and their free energies of dissociation. Given that the tertiary structure of AGP has proven refractory to resolution, these structure-activity studies should contribute to understanding the nature of the binding site on this important protein.
Footnotes
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↵1 These authors contributed equally to this work.
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Supported in part by National Institutes of Health Grant GM 35647 to G.R.S.
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DOI: 10.1124/jpet.102.042028
- Abbreviations:
- LA
- local anesthetic
- AGP
- α1-acid glycoprotein, human
- RAD-240
- N-(2,6-dimethylphenylcarbamoylmethyl) ethylmethylamine (HCl salt)
- RAD-241
- N-(2,6-dimethylphenylcarbamoylmethyl)ethylpropylamine (HCl salt)
- RAD-242
- N-(2,6-dimethylphenylcarbamoylmethyl)ethylpentylamine (HCl salt)
- L-30
- N-(2,6-dimethylphenylcarbamoylethyl)diethylamine
- W-6603
- N-(2,6-dimethylphenylcarbamoylpropyl)diethylamine
- QX-314
- N-(2,6-dimethylphenylcarbamoylmethyl)triethylammonium chloride
- DEDIC
- 2-hydroxy-3,5-diiodo-N-[2-(diethylamino)ethyl]benzamide
- F1*S
- refers collectively to the closely related F1 and S variants of AGP which together comprise roughly 70% of pooled source AGP
- SAM
- standard aqueous medium, containing 0.15 M NaCl and 5 mM MOPS buffer adjusted to pH 7.40
- F(λex/λem)
- corrected, normalized fluorescence with excitation and emission wavelengths of λex and λem
- Received July 23, 2002.
- Accepted September 11, 2002.
- The American Society for Pharmacology and Experimental Therapeutics
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