Search

Abstract: Columns containing immobilized lipoproteins were prepared for the analysis of drug interactions with these particles by high-performance affinity chromatography (HPAC). This approach was evaluated by using it to examine the binding of high-density lipoprotein (HDL) to the drugs propranolol and verapamil. HDL was immobilized by the Schiff base method onto silica and gave HPAC columns with reproducible binding to propranolol over 4–5days of continuous operation at pH 7.4. Frontal analysis experiments indicated that two types of interaction were occurring between R- or S-propranolol and HDL at 37°C: saturable binding with an association equilibrium constant (K a) of 1.1–1.9×105 M−1 and nonsaturable binding with an overall affinity constant (n K a) of 3.7–4.1×104 M−1. Similar results were found at 4 and 27°C. Verapamil also gave similar behavior, with a K a of 6.0×104 M−1 at 37°C for the saturable sites and an n K a for the nonsaturable sites of 2.5×104 M−1. These measured affinities gave good agreement with solution phase values. The results indicated that HPAC can be used to study drug interactions with HDL, providing information that should be valuable in obtaining a better description of how drugs are transported within the body. [Copyright &y& Elsevier]

Abstract: Quantifying the interaction of drugs with carrier proteins in plasma is of importance for understanding effective drug delivery to disease-affected tissues. In this study, we employed analytical ultracentrifugation and steady-state fluorescence spectroscopy to characterize the interaction of a potential new anticancer drug, AG1478-mesylate, with plasma proteins in a suspension of normal serum albumin (NSA). We found that mesylate salt of AG1478, an epidermal growth factor receptor kinase inhibitor, sediments in 0.1%(w/v) NSA as a complex with a sedimentation coefficient of 3.8 S. This is consistent with the size of human serum albumin. This interaction was quantitated by meniscus depletion sedimentation and fluorescence titration analyses. AG1478-mesylate binds to albumin with an apparent single-site affinity (K d) of 120μM. In this article, we show that the cyclodextrin carrier molecule, Captisol, increases the apparent affinity of the hydrophobic AG1478-mesylate for albumin (K d =4–6μM), and we propose that the AG1478-mesylate–Captisol (1:1) complex binds to albumin with at least 10-fold higher affinity than does AG1478-mesylate ligand alone. A fluorenylmethoxycarbonyl–sulfonic acid (FMS) derivative of the 6-aminoquinazoline analog of AG1478, which was designed to have improved serum-binding properties, was shown by fluorescence analysis to bind with approximately 100-fold greater affinity than the parent compound. This has significant implications in the effective delivery of therapeutic agents in vivo. [Copyright &y& Elsevier]

A new method for localizing binding sites of noncovalent drugs on proteins is presented. We have developed an accurate and high-throughput method based on the mass spectrometric measurement of differential reaction yield of cysteine alkylation (MS–DRC). This method, essentially a semiquantitative footprinting approach, is applicable to any type of ligand targeting cysteine-rich proteins because the method measures the reactivity change of each cysteine toward an alkylating agent instead of monitoring the drug itself. Thus, no modification of the drug is needed. In this study, the method is evaluated using tubulin as a model system. Tubulin and drug-treated tubulin were alkylated separately with several alkylating reagents, followed by proteolysis and high-performance liquid chromatography (HPLC)–tandem mass spectrometry (MS/MS) and HPLC–MS. Relative alkylation yields of each cysteine toward the reagents were measured by mass spectrometric quantitation. The reaction yields of each cysteine of two samples were compared to detect a particular cysteine (or cysteines) for which reaction yield was markedly decreased following drug binding. Monobromobimane (mBrB) showed the highest differential.Thus, the MS–DRC method with mBrB was evaluated with various tubulin agents, including the covalent agent T138067 and the noncovalent agents colchicine, podophyllotoxin, and 2-methoxyestradiol. Conformational changes induced by drug binding, as well as sites of direct binding, may be identified. [Copyright &y& Elsevier]

This paper describes an expanded application of our recently reported method (Eskew et al., Analytical Biochemistry 621,1 2021) utilizing thermogram signals for thermal denaturation measured by differential scanning calorimetry. Characteristic signals were used to quantitatively evaluate ligand binding constants for human serum albumin. In our approach the ensemble of temperature dependent calorimetric responses for various protein-ligand mixtures and native HSA were compared, in a ratiometric manner, to extract binding constants and stoichiometries. Protein/ligand mixtures were prepared at various ligand concentrations and subjected to thermal denaturation analysis by calorimetry. Measurements provided the melting temperature, T m , and free-energy Δ G c a l (37 ° C) for melting ligand-bound Albumin as a function of ligand concentration. Concentration dependent behaviors of these parameters derived from protein/ligand mixtures were used to construct dose-response curves. Fitting of dose-response curves yielded quantitative evaluation of the ligand binding constant and semi-quantitative estimates of the binding stoichiometry. Many of the ligands had known binding affinity for Albumin with binding constants reported in the literature. Evaluated binding parameters for the ligands impressively agreed with reported literature values determined using other standard experimental methods. Results are reported for 29 drug ligands binding to Albumin. These validate our calorimetry-based process for applications in pre-clinical drug screening. [Display omitted] • Ratiometric analysis of DSC thermograms enabled quantitative evaluation of ligand binding constants to HSA. • Novel means for evaluating thermodynamic stability of HSA/ligand complexes. • Excellent agreement of measured binding constants with reported literature values. • Semi-quantitative estimates on ligand stoichiometry. • Binding analysis of insoluble or poorly soluble ligands without organic solvents. [ABSTRACT FROM AUTHOR]