A new drug binding subsite on human serum albumin and drug–drug interaction studied by X-ray crystallography

https://doi.org/10.1016/j.jsb.2007.12.004Get rights and content

Abstract

3′-Azido-3′-deoxythymidine (AZT) is the first clinically effective drug for the treatment of human immunodeficiency virus infection. The drug interaction with human serum albumin (HSA) has been an important component in understanding its mechanism of action, especially in drug distribution and in drug–drug interaction on HSA in the case of multi-drug therapy. We present here crystal structures of a ternary HSA–Myr–AZT complex and a quaternary HSA–Myr–AZT–SAL complex (Myr, myristate; SAL, salicylic acid). From this study, a new drug binding subsite on HSA Sudlow site 1 was identified. The presence of fatty acid is needed for the creation of this subsite due to fatty acid induced conformational changes of HSA. Thus, the Sudlow site 1 of HSA can be divided into three non-overlapped subsites: a SAL subsite, an indomethacin subsite and an AZT subsite. Binding of a drug to HSA often influences simultaneous binding of other drugs. From the HSA–Myr–AZT–SAL complex structure, we observed the coexistence of two drugs (AZT and SAL) in Sudlow site 1 and the competition between these two drugs in subdomain IB. These results provide new structural information on HSA–drug interaction and drug–drug interaction on HSA.

Introduction

Human serum albumin (HSA) is the most abundant protein constituent of blood plasma and serves as a major protein storage component for endogenous and external compounds. HSA has three homologous domains (named I, II, and III); each domain is made up of two separate subdomains (named A and B) connected by a random coil (He and Carter, 1992). Extensive biochemical studies in the 1970s resulted in the widely accepted proposal of two main drug-binding sites in HSA (Sudlow et al., 1975), denoted as Sudlow site 1 and Sudlow site 2. Sudlow site 1 (located in subdomain IIA) was shown to prefer large heterocyclic and negatively charged compounds, while Sudlow site 2 (located in subdomain IIIA) was the preferred site for small aromatic carboxylic acids (Sudlow et al., 1975, Sudlow et al., 1976). In addition, there are a number of minor binding sites (Bhattacharya et al., 2000a, Ghuman et al., 2005, Sengupta and Hage, 1999, Zunszain et al., 2003) that allow multiple drug molecules to be bound simultaneously onto HSA and lead to higher drug binding capacity of HSA. HSA plays a central role in drug pharmacokinetics (Herve et al., 1994, Vorum, 1999), and thus affects therapeutic drug dosage. Out of the four aspects of pharmacokinetics (absorption, distribution, metabolism, excretion), distribution is the one that this protein controls, because most drugs travel in plasma and reach the target tissues bound to this protein (Colmenarejo, 2003, Herve et al., 1994).

3′-Azido-3′-deoxythymidine (AZT, structure 1) (Hoffman et al., 1985), is the first clinically effective drug used for the treatment of human immunodeficiency virus infection. It is a synthetic pyrimidinic analogue that differs from natural nucleoside thymidine (dThd) in having an azido substituent in place of the hydroxyl group at the 3′-position of the deoxyribose ring. As a variant of dThd, AZT typically has the same reactivity as dThd and follows the same metabolic pathways in the cell although with radically altered kinetic parameters (Samuels, 2006). AZT itself is a pro-drug and needs to be converted into the pharmaceutically active form 3′-azido-3′-deoxythymidine-5′-triphosphate (AZTTP). AZTTP can then interact with DNA polymerases and be incorporated into a replicating DNA strand in the place of thymidine triphosphate (dTTP). The azido group prevents further replication of the DNA molecule, leading to chain termination (Bradshaw et al., 2005, Kedar et al., 1990, Reardon and Miller, 1990). In today’s anti-HIV therapy regiment, AZT is administrated at 600 mg total per day (either 200 mg three times per day or 300 mg twice per day), resulting in a steady-state serum AZT concentration of ∼0.8 mM (Fletcher et al., 2002). Such high dosage is needed because of the short half-life time of AZT in plasma (≈1.1 h) (DiPiro, 1997). The AZT binding to plasma proteins, especially albumin, controls the concentration of the free drug, and hence affects the drug’s pharmacokinetics, storage, toxicity, transportability to the tissue and through cell membranes. It is known that AZT exhibits poor binding to human serum albumin (HSA) (Quevedo et al., 2001). Such characteristic was believed to be one of the main reasons for its short half-life time (Luzier and Morse, 1993, Quevedo et al., 2001). However, the structure basis of such characteristic has not been reported in details.

Interaction of AZT with HSA has been studied by capillary electrophoresis, FTIR and CD spectroscopic methods (Gaudreau et al., 2002). Capillary electrophoresis and spectroscopic results showed two major binding sites of AZT on HSA with binding constants K1 of 1.9 × 106 M−1 and K2 of 2.1 × 104 M−1. AZT exhibits a higher affinity to therapeutic HSA than to purified HSA (Quevedo et al., 2001). A crystallographic study in 1992 confirmed AZT binding with HSA (He and Carter, 1992), but did not give details on the binding interactions because of the limited resolution of the data. Therefore, a higher resolution crystal structure of the HSA–AZT complexes is useful to show the detailed drug binding modes and to identify residues of HSA that are key determinants of binding specificity.

Drug–drug interaction on HSA often occurred in multi-drug therapy (Aubry et al., 1995, Brenner et al., 2002, Mubashar et al., 2002, Schmit et al., 1996, Schwarzenbach, 2002). HSA is a flexible protein and has a number of drug binding sites (He and Carter, 1992, Sengupta and Hage, 1999, Sudlow et al., 1975, Sudlow et al., 1976). Binding of a drug can influence simultaneous binding of other drugs (Bertucci and Domenici, 2002). This can lead to (1) the displacement in the binding site of the drug by the one that has a higher affinity to the transporting albumin, and/or (2) the alteration of the albumin structure, resulting in the change (increase or decrease) of the protein affinity toward the drugs (Kragh-Hansen et al., 2002, Sulkowska et al., 2006). The competition of two drugs usually decreases the amount of drug bound to the albumin (Onks et al., 1991). Alterations caused by drug–drug interactions in the HSA binding of drugs may alter the volume of distribution, clearance, and elimination of a drug and may modulate its therapeutic effect. It is useful to classify drug-binding sites so that the risk of drug interactions can be evaluated. Therefore, identifying coexistence or competition of drugs on HSA is of great importance. The mechanism of cooperative binding of both cytarabine (araC) and fluorouracil, used in combination therapy, to BSA has been investigated using UV and NMR spectroscopy (Sulkowska et al., 2004). The study showed the competition of these two drugs and the removal of fluorouracil by araC from the common binding site. Study on the competition between araC and aspirin binding with BSA suggested that araC reduced the affinity of albumin toward aspirin. On the other hand, the interaction between araC and BSA was weaker in the presence of aspirin (Sulkowska et al., 2006). Over the last decade, several techniques have been developed and applied to study the drug–drug interaction on binding to serum albumin (HSA or bovine serum albumin, BSA) (Bai et al., 2005, Cui et al., 2004, Kuchimanchi et al., 2001, Sowell et al., 2001). However, there are not many detailed structural studies of such drug–drug interaction (Ghuman et al., 2005).

In this study, we use salicylic acid (SAL) as a competitive drug to determine the extent and nature of AZT binding to HSA by X-ray crystallography. The interactions of SAL with HSA have been extensively investigated for decades (He and Carter, 1992, Honma et al., 1991, Kragh-Hansen et al., 1990, Lee et al., 1995, Ozer and Tacal, 2001, Parsons and Sathe, 1991, Yang et al., 2007). This study provides new structural information on HSA–drug interaction and drug–drug interaction on HSA.

Section snippets

Protein purification, complex formation and crystallization

Fatty acid free HSA was purchased from Sigma Inc. (catalogue number A3782) and further purified to remove HSA dimer, according to the published protocols (Curry et al., 1998, Yang et al., 2007). The protein was dissolved in a 20 mM potassium phosphate buffer (pH 7.5) to about 100 mg/ml and stored in a −80 °C refrigerator before use. Sodium myristate, 3′-azido-3′-deoxythymidine, and salicylic acid were purchased from Sigma Inc.

Sodium myristate was dissolved into ethanol, and then was diluted to 2.5 

Structure of the HSA–Myr–AZT complex and model reliability

The crystal structure of HSA–AZT in the absence of myristate was previously reported (He and Carter, 1992). Without fatty acids, this HSA–AZT crystal diffracted only to a resolution of 4 Å, limiting the detailed analysis of molecular interaction between HSA and AZT. In this study, we crystallized HSA in the presence of sodium myristate, which very often facilitates the crystal formation (Yang et al., 2007). Under physiological conditions, 0.1–2 molecules of fatty acid (FA) are bound to each HSA

A new drug binding subsite on HSA

Comparison of our HSA–Myr–AZT structure with other HSA–Myr–drug complexes reveals a new drug binding subsite on HSA in the presence of myristate (Fig. 3). The existence of this new subsite was also observed, and thus further confirmed by another crystal structure (HSA–Myr–AZT–SAL) in this study. In general, most drugs studied are found to bind in the central portion of Sudlow site 1 that is within the core of subdomain IIA. For example, SAL occupies the predominantly apolar compartment of

Acknowledgments

This work was supported by Grants from the Natural Science Foundation of China (30430190, 30625011) and 973 (2007CB914304) to M.H., and NSF-EPSCoR of USA. Use of the Advanced Photon Source was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under contract No. W-31-109-Eng-38. We thank the staffs of the APS SER-CAT beamline 22ID for help with data collection.

References (52)

  • I. Ozer et al.

    Method dependence of apparent stoichiometry in the binding of salicylate ion to human serum albumin: a comparison between equilibrium dialysis and fluorescence titration

    Anal. Biochem.

    (2001)
  • I. Petitpas et al.

    Crystal structure analysis of warfarin binding to human serum albumin: anatomy of drug site I

    J. Biol. Chem.

    (2001)
  • M.A. Quevedo et al.

    Human serum albumin binding of novel antiretroviral nucleoside derivatives of AZT

    Biochem. Biophys. Res. Commun.

    (2001)
  • J.E. Reardon et al.

    Human immunodeficiency virus reverse transcriptase. Substrate and inhibitor kinetics with thymidine 5′-triphosphate and 3′-azido-3′-deoxythymidine 5′-triphosphate

    J. Biol. Chem.

    (1990)
  • H. Schwarzenbach

    A diagnostic tool for monitoring multidrug resistance expression in human tumor tissues

    Anal. Biochem.

    (2002)
  • J.R. Simard et al.

    Location of high and low affinity fatty acid binding sites on human serum albumin revealed by NMR drug-competition analysis

    J. Mol. Biol.

    (2006)
  • A.A. Spector

    Structure and lipid binding properties of serum albumin

    Methods Enzymol.

    (1986)
  • F. Yang et al.

    Effect of human serum albumin on drug metabolism: structural evidence of esterase activity of human serum albumin

    J. Struct. Biol.

    (2007)
  • A.F. Aubry et al.

    The effect of co-administered drugs on oxaprozin binding to human serum albumin

    J. Pharm. Pharmacol.

    (1995)
  • R. Bahr et al.

    Effect of exercise on recovery changes in plasma levels of FFA, glycerol, glucose and catecholamines

    Acta Physiol. Scand.

    (1991)
  • C. Bertucci et al.

    Reversible and covalent binding of drugs to human serum albumin: methodological approaches and physiological relevance

    Curr. Med. Chem.

    (2002)
  • P.C. Bradshaw et al.

    A computational model of mitochondrial AZT metabolism

    Biochem. J.

    (2005)
  • B.G. Brenner et al.

    Persistence and fitness of multidrug-resistant human immunodeficiency virus type 1 acquired in primary infection

    J. Virol.

    (2002)
  • R. Brodersen et al.

    Multiple fatty acid binding to albumin in human blood plasma

    Eur. J. Biochem.

    (1990)
  • A.T. Brunger et al.

    Crystallography & NMR system: A new software suite for macromolecular structure determination

    Acta Crystallogr. D Biol. Crystallogr.

    (1998)
  • G. Colmenarejo

    In silico prediction of drug-binding strengths to human serum albumin

    Med. Res. Rev.

    (2003)
  • Cited by (0)

    View full text