Analysis of multi-site drug–protein interactions by high-performance affinity chromatography: Binding by glimepiride to normal or glycated human serum albumin

Abstract

High-performance affinity chromatography (HPAC) was used in a variety of formats to examine multi-site interactions between glimepiride, a third-generation sulfonylurea drug, and normal or in vitro glycated forms of the transport protein human serum albumin (HSA). Frontal analysis revealed that glimepiride interacts with normal HSA and glycated HSA at a group of high affinity sites (association equilibrium constant, or K a , 9.2-11.8 × 10 5 M −1 at pH 7.4 and 37°C) and a group of lower affinity regions (K a , 5.9-16.2 × 10 3 M −1 ). Zonal elution competition studies were designed and carried out in both normal-and reversed-role formats to investigate the binding by this drug at specific sites. These experiments indicated that glimepiride was interacting at both Sudlow sites I and II. Allosteric effects were also noted with R-warfarin at Sudlow site I and with tamoxifen at the tamoxifen site on HSA. The binding at Sudlow site I had a 2.1-to 2.3-fold increase in affinity in going from normal HSA to the glycated samples of HSA. There was no significant change in the affinity for glimepiride at Sudlow site II in going from normal HSA to a moderately glycated sample of HSA, but a slight decrease in affinity was seen in going to a more highly glycated HSA sample. These results demonstrated how various HPAC-based methods can be used to profile and characterize multi-site binding by a drug such as glimepiride to a protein and its modified forms. The information obtained from this study should be useful in providing a better understanding of how drug-protein binding may be affected by glycation and of how separation and analysis methods based on HPAC can be employed to study systems with complex interactions or that involve modified proteins.

Chromatographic Studies of Protein-Based Chiral Separations

“…Another use of zonal elution is to obtain information on the interactions between an injected solute and an immobilized protein. This approach can provide information on the strength of solute–protein binding, the changes in binding that occur as the mobile phase composition or temperature is changed, the location and types of binding sites for the solute on the protein, and the effect of altering the solute or protein structures on the interactions of these agents [32,36,37]. Zonal elution can be used to provide this information when a small amount of the solute of interest is injected onto a protein-based CSP in the presence of a competing agent or a mobile phase with a known composition and temperature.…”

Analysis of Biological Interactions by Affinity Chromatography: Clinical and Pharmaceutical Applications

“…One common approach is to use zonal elution (8,10). Zonal elution involves the injection of a small sample plug onto a chromatographic system, followed by separation of the peaks that result from this injection, as is illustrated in Figure 1A (11). This is the format that is most commonly used in other types of liquid chromatography for chemical measurement and identification.…”

Analysis of stereoselective drug interactions with serum proteins by high-performance affinity chromatography: A historical perspective

“…This led to the identification of other site-selective probes that could be used for either HSA or AGP [50–52,60,76,103–105]. Some applications that followed included the development of affinity maps for drugs on these proteins [7,22,106,107] and reports that examined how modifications of these proteins affected local drug interactions at given sites [22,108–110]. …”

“…This method has been utilized to see how the modification of HSA at specific amino acids (e.g., Tyr-411, Trp-214 or Cys-34) will affect its ability to bind to drugs or site-specific probes [124–128]. This same method has been recently used to see how non-enzymatic glycation, as occurs during diabetes, affects the ability of HSA to bind to sulfonylureas and other drugs or solutes [108–110]. …”

“…However, it is now also common to employ more complex models and equations that may involve non-linear fits [7,22]. Examples of such models include those that involve multi-site binding, non-saturable interactions, and combinations of saturable plus non-saturable interactions, such as have been observed for the interactions of some drugs with modified forms of HSA and with AGP or lipoproteins [6,22,76–78,80–84,108–110,132–134]. This approach has further been employed in the study of other systems, such as drug-receptor interactions [135] and the binding of various solutes with lectins or enzymes [136–139].…”