Human Serum Albumin Binding in a Vial: A Novel UV-pH Titration Method To Assist Drug Design.

Gergő Dargó,Kristóf Simon,Judit Müller,György T. Balogh,Dávid Bajusz

doi:10.1021/acs.jmedchem.0c00046

Abstract

The knowledge on human serum albumin (HSA) binding is of utmost importance as it affects pharmacokinetic behavior and bioavailability of drugs. In this article, we report a novel method to screen for ionizable molecules with high HSA binding affinity based on pKa shifts using UV-pH titration. We investigated the HSA binding of 27 drugs and compared the results to experimental data from conventional methods. In most cases, significant shifts (ΔpKa > 0.1) were observed for drugs with high HSA binding, while no change could be detected for low-affinity binders. We showed the pivotal role of ionization centers in the formation of strong interactions between drug and HSA using molecular docking studies. We also verified our findings by testing five modified analogues designed by structural considerations. Significant decreases in their HSA binding proved that the UV-pH titration method combined with an in silico support can be used as a medicinal chemistry tool to assist rational molecular design.

Highlights

Active pharmaceutical ingredients (API) entering the systematic circulation may interact with various components of the blood, which directly affects their pharmacokinetic and pharmacodynamic behaviors.[1−5] Blood plasma consists of approximately 7−9% of plasma proteins, which contribute a major part to API-specific interactions
We investigated the possibility of measuring human serum albumin (HSA) binding of APIs based on changes of proton dissociation constants of their free and complexed molecular forms using UV-pH titration
We demonstrated the applicability of UV-pH titration as an orthogonal method for the identification of APIs with high-affinity binding

Summary

Introduction

Active pharmaceutical ingredients (API) entering the systematic circulation may interact with various components of the blood, which directly affects their pharmacokinetic and pharmacodynamic behaviors.[1−5] Blood plasma consists of approximately 7−9% of plasma proteins (albumins, glycoproteins, and fibrinogen), which contribute a major part to API-specific interactions. Human serum albumin (HSA) is the most abundant (about 54−60% of blood proteins),[6−8] which is present in the blood at high concentrations (35−50 g/L).[8−11] It plays an important role in maintaining the osmotic pressure of the blood, serves as a transport protein for endogenous substances (e.g., fatty acids and steroid hormones), and it is the main contributor to the binding of drug molecules.[5,7,8,10] The protein’s heartshaped structure is composed of three main domains (I−III), each containing two subdomains (A and B).[8−10] Besides the several recently identified low- to high-affinity binding sites of HSA,[12,13] the two specific drug binding sites (site I: warfarin site on the IIA subdomain and site II: indole-benzodiazepine site on the IIIA subdomain) are considered to be the most significant, regarding plasma protein binding.[8,11,14−18] Drug molecules usually form reversible complexes with HSA by electrostatic and hydrophobic interactions These HSA−API complexes cannot cross biological membranes; they become therapeutically inactive.[5,19] strong binding may have a significant effect on the pharmacokinetic (PK) behavior of APIs (e.g., altered clearance,[20−22] distribution,[19−21] drug−drug interactions,[19,23] and toxicity[24]). The HSA binding affinity shows wide diversity for drug-like compounds; it is essential to predict the unbound fraction of APIs from the early stages of drug discovery for the estimation of PK behavior

Objectives

Results

Conclusion