Hydrophobic and ionic factors in the binding of local anesthetics to the major variant of human alpha1-acid glycoprotein.

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 alpha1-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 (K(D) = 4.0 +/- 0.6 microM and >32 microM, 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.