Hydrophobic and Ionic Factors in the Binding of Local Anesthetics to the Major Variant of Human α₁-Acid Glycoprotein

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 ␣ 1 -acid glycoprotein (AGP) were therefore investigated spectrofluorometrically using whole AGP and a novel, F1*S variant-selective probe previously developed in our laboratory. Equilibriumcompetitive 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 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 ϭ 4.0 Ϯ 0.6 M and Ͼ32 M, 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.Understanding the interaction of local anesthetics with plasma proteins is essential to predicting their systemic pharmacology and toxicology. Many basic drugs, including local anesthetics (LAs) and class I antiarrythmics, are bound in blood principally or in part by AGP (Routledge, 1986;Stanski and Watkins, 1986;Wood, 1986;Kremer et al., 1988). The binding of a drug to a plasma protein reduces its free fraction in serum, thus reducing its availability for active uptake or diffusion into surrounding tissue and significantly influencing its pharmacokinetics, toxicity, etc. (Wood, 1986).For example, the local anesthetic bupivacaine binds tightly to AGP (with K D ϭ 1 M) (Essassi et al., 1989), and this binding may mitigate the systemic toxicity of bupivacaine (Wulf et al., 1991;Mazoit et al., 1996).Indeed, altered pharmacokinetics have been observed following the binding of AGP to the LAs lidocaine and cocaine, and the same holds true with other clinically relevant amphipathic amines: amitriptyline, chlorpromazine, nicardipine, and verapamil (Benet and Hoener, 2002). For example, AGP concentration has been shown to correlate inversely with the free fraction of the LA ropivacaine 60 min after epidural injection (Porter et al., 2001). Also, one of the AGP variants...

Drug‐binding energetics of human α‐1‐acid glycoprotein assessed by isothermal titration calorimetry and molecular docking simulations

“…Moreover, the thermodynamic parameters did not show any correlation with the drug octanol/water partition coefficients or polar surface area (PSA) of the test compounds (Figure B,C and Table S1). This observation contrasts with the direct correlation between ∆ H º, ∆ S º, and ∆Gº and hydrophobicity (log P ) reported for the binding of a series of local anesthetics and phenothiazine neuroleptics and AGP (Miyoshi et al ., ; Urien et al ., ; Taheri et al ., ). However, it should be noted that these studies employed equilibrium dialysis or fluorometric binding assays to generate affinity constants from which the thermodynamic parameters were indirectly derived.…”

Enantioselective drug–protein interaction between mexiletine and plasma protein

“…From the above results, the number of binding sites ( n ) which were less than 1 for both R ‐ and S ‐mexiletine indicated that mexiletine may have selectively bound with hAGP variants [29] . Mifepristone and imipramine were the specific ligands of the F1‐S and A variants of hAGP, respectively [30,31] . In this competition binding study we investigated the binding of mexiletine enantiomers with different genetic variants of hAGP and determined the possible enantioselectivity (as shown in Table 1).…”

High‐throughput analysis of drug dissociation from serum proteins using affinity silica monoliths

“…Several techniques have been used to study the interactions and affinities of drugs with either HSA or AGP 5–15. These methods include ultrafiltration 5, 9, equilibrium dialysis 6, 8, fluorescence assays 7, 12, capillary electrophoresis 10, UV–Vis spectroscopy 11, and solid‐phase microextraction 13–15. Another technique used to study drug–protein interactions is high‐performance affinity chromatography (HPAC) 1, 16.…”