Betulinic acid binding to human serum albumin: A study of protein conformation and binding affinity

Abstract

Betulinic acid (BA) has anti cancer and anti-HIV activity and has been proved to be therapeutically effective against cancerous and HIV-infected cells. Human serum albumin (HSA) is the predominant protein in the blood. Most drugs that bind to HSA will be transported to other parts of the body. Using micro TOF-Q mass spectrometry, we have shown, for the first time that BA isolated from a plant (Tephrosia calophylla) binds to HSA. The binding constant of BA to HSA was calculated from fluorescence data and found to be K_BA = 1.685 ± 0.01 × 10⁶ M⁻¹, indicating a strong binding affinity. The secondary structure of the HSA–BA complex was determined by circular dichroism. The results indicate that the HSA in this complex is partially unfolded. Further, binding of BA at nanomolar concentrations of BA to free HSA was detected using micro TOF-Q mass spectrometry. The study revealed a mass increase from 65199 Da (free HSA) to 65643 Da (HSA + drug), where the additional mass of 444 Da was due to bound BA. Based on the results of this study, it is suggested that micro TOF-Q mass spectrometry is useful technique for drug binding studies.

Introduction

Human serum albumin (HSA) is an important plasma protein responsible for the binding and transport of many endogenous and exogenous substances such as hormones and fatty acids, as well as foreign molecules such as drugs [1], [2], [3], [4], [5], [6], [7], [8], [9], [10]. HSA is a widely studied protein because its primary structure is well known and its tertiary structure has been determined by X-ray crystallography [1]. HSA is synthesized in and secreted from liver cells, and is the most abundant protein among plasma, transport, and storage proteins. It is also important for maintaining normal osmolarity in plasma as well as in interstitial fluid. HSA is a 67 kDa single chain, non glycosylated polypeptide that folds into a heart-shaped structure containing approximately 67% α-helix [1], [7].

The exceptional ability of HSA to interact with many organic and inorganic molecules and function as an important regulator of intercellular fluxes, as well as the pharmacokinetic behavior of many drugs [1], [2], [3], [4], [5], [6], [7], [8], [9], [10], may be attributed to the presence of multiple binding sites on its surface. Albumin binding sites on HSA may be responsible for certain drug interactions observed in patients during therapy [11], [12]. For example, patients with various liver and kidney diseases may experience altered albumin binding of drugs; such that the distribution, metabolism, elimination, and pharmacological effects of the drugs are substantially changed [13], [14]. Most drugs are reversibly bound to plasma proteins, but the extent and nature of the binding varies with the specific drug. Most acidic, neutral, and basic drugs bind to HSA, although a few basic drugs bind almost exclusively to α₁-acid glycoprotein [7], [15]. The degree of binding between a drug and plasma proteins can govern its distribution into tissues, affect its elimination from the body, and, consequently, affect its therapeutic or toxic effects. It is generally believed that only the unbound form of a drug interacts with its receptor to produce a pharmacological effect.

Betulinic acid (BA) is a naturally occurring pentacyclic triterpenoid (Fig. 1) possessing anti-retroviral, anti-malarial, and anti-inflammatory properties. It is noteworthy that, through its inhibition of topoisomerase, BA shows also potential as an anticancer agent [16], [17], [18], [19], [20]. Studies of BA utilizing various microorganisms have predicted potential mammalian metabolites [21], [22], [23]. In addition, molecular modeling experiments have predicted that BA may be a substrate for cytochrome P450 [24]. In other reports, BA derivatives acylated on the C-3-hydroxyl group inhibited HIV-1 replication by interfering with HIV-1 maturation [25], [26].

Multiple studies on HSA structure and its interactions with different ligands exist in literature [8], [9], [10], [11], [27], [28], [29], [30]. Ghuman and co-workers have shown that the distribution, free concentration, and the metabolism of various drugs can be significantly altered as a result of binding to HSA [7]. Thus, interaction with plasma proteins, especially HSA, is an important factor to consider in drug development [1], [2], [3], [4], [5], [6], [7], [8], [9], [10]. However, to our knowledge, there are no existing reports on the BA binding affinity of HSA and its effect on protein conformation. Moreover, there have been no studies of HSA-ligand binding using quadrupole time-of-flight (micro TOF-Q) mass spectrometry.

We report here, for the first time, the binding at nanomolar concentration (5 nM) of BA to HSA, as determined by sensitive mass spectrometry (micro TOF-Q). We also present the conformational changes, protein-drug complex and binding studies of HSA with BA.