Drug binding to human alpha 1-acid glycoprotein

Many basic drugs are bound principally or in part by the lipocalin alpha 1-acid glycoprotein (AGP), and this influences their systemic pharmacology and toxicology. As AGP's peptide and glycan heterogeneity have confounded years of efforts to obtain an experimental structure, here the molecular determinants of drug-AGP interactions were investigated using two approaches. First, a homology model of the major (F1*S) variant was constructed. The model revealed two topographically plausible hydrophobic sites: inside the barrel fold and at an external cleft. Notable is a hydrophilic portal region, gated by two oppositely charged residues, restricting access to the barrel site. Second, a novel fluorescence quenching, F1*S variant-selective probe (DEDIC) was developed to measure AGP association with amphipathic amines (e.g. local anesthetics, antiarrythmics, antidepressants). Using DEDIC as a probe, structure-activity studies on local anesthetics demonstrated a positive correlation between drug hydrophobicity and affinity for F1*S. The importance of electrostatic factors was assessed by examining the pH-dependent binding of an ionizable local anesthetic, lidocaine. Uncharged lidocaine was found to bind with at least eight times the affinity of its protonated counterpart. While this result is consistent with our model (if it is assumed that drugs' amino termini are electrostatically repelled principally by the positively charged residue in the portal), it is inconsistent with a current, published model, positing only a negatively charged region at the base of the binding site that interacts favorably with cationic ligands. DEDIC-F1*S association kinetics were studied using stopped-flow spectrofluorometry, revealing a fast, unresolvable process (tau < 1 msec) having an EC50 identical to that observed in equilibrium experiments for low affinity binding and several slower processes (tau = ca. 0.1 to 10 sec). Since binding to a sterically unencumbered cleft, as depicted by the model, should be near diffusion limited, the low affinity site was thereby assigned to the cleft region. The next fastest component exhibited an apparent rate that saturated at higher [DEDIC], consistent with the two-step mechanism for barrel binding predicted by the model: once the pre-association (portal) site saturates, barrel entry becomes rate limiting. The apparent high affinity site was therefore identified as the barrel site.