Unravelling the interaction mechanism between clioquinol and bovine serum albumin by multi-spectroscopic and molecular docking approaches

Highlights

• Clioquinol interacts with BSA by a combined mechanism of static and dynamic processes.

• Clioquinol favors to bind in multiple pockets on BSA with an extremely high affinity constant at 10⁸ M⁻¹.

• The findings suggest a potential role of serum albumin as a clioquinol carrier in the vascular system.

• Clioquinol potentially impairs endogenous proteins and causes adverse effects by inducing protein aggregation.

• The redesigned or modified molecular structure of clioquinol may reduce its toxicity and improve its bioavailability

Abstract

Clioquinol has recently been proposed for the treatment of Alzheimer's disease. It is able to diminish β-amyloid protein aggregation and to restore cognition of Alzheimer's mice. However, its therapeutic benefits for Alzheimer's disease in human remain controversy and need further confirmation. Herein, we have explored the interaction mechanism of clioquinol toward bovine serum albumin (BSA) by means of multi-spectroscopic and docking simulation approaches. Clioquinol interacts with BSA by a combined mechanism of static and dynamic processes. Application of the Hill's equation to fluorescence quenching experiment revealed that the binding constant of the BSA-clioquinol complex is extremely high at 10⁸ M⁻¹ level. Competitive displacement and docking analysis consistently suggested that there are the multiple binding modes of clioquinol toward BSA. Competitive binding study showed that clioquinol shares the binding sites with ibuprofen and digitoxin on albumin, referring to be site II and site III binding compounds. Besides, partial binding in site I was also observed. Docking simulation confirmed that clioquinol favors to bind in site I, site II, site III, fatty acid binding site 5, and the protein cleft between subdomain IB and IIIB of the BSA. Due to its small size and electric dipole property, clioquinol may easily fit in multiple pockets of the BSA. Our finding suggests the potential role of BSA as a clioquinol carrier in the vascular system. Nonetheless, clioquinol-induced BSA aggregation has been observed by the three-dimensional fluorescence technique. This phenomenon may not only impair the BSA, but may also affect other endogenous proteins, which eventually causes adverse effects to human. Therefore, the redesigned or modified molecular structure of clioquinol may reduce its toxicity and improve its bioavailability.

Introduction

Clioquinol (5-chloro-7-iodo-8-hydroxyquinoline, Fig. 1) is a member of hydroxyquinoline family and commercially available in cream preparation for treatment of inflammatory skin disorders such as eczema, ringworm, and athlete's foot. The previous study demonstrated that clioquinol exerts high antimicrobial activity against the multidrug resistant Neisseria gonorrhoeae [1]. In addition, clioquinol was also reported to exhibit anticancer activity toward cholangiocarcinoma cells [2] and displayed neuroprotective effects against high glucose toxicity [3]. Currently, clioquinol and hydroxyquinoline derivatives have been proposed for treatment of neurodegenerative disorders including Alzheimer's, Huntington's, and Parkinson's diseases [4]. In this context, clioquinol plays many key roles in the brain including reduction of beta-amyloid burden, restoration of metal homeostasis, and improvement of cognition. It acts as a metal chelator toward Cu and Zn, which in turn depletes β-amyloid protein aggregation [[5], [6], [7], [8], [9], [10], [11], [12]] and restores cognition of transgenic Alzheimer's mice [8,[13], [14], [15]]. Unfortunately, adverse effects of clioquinol have been noted. Because of its association with subacute myelo-optic neuropathy (SMON); thus, oral clioquinol has been terminated since the 1970s [16,17]. The mechanism of such toxicity still remains unclear. It is believed that clioquinol reduces vitamin B12 bioavailability resulting in sensory and motor disorders of the lower limbs and the visual sign [[18], [19], [20]]. It also inhibits 20s proteasome and leads to misfolded protein aggregation and cell death [21]. Sampson and colleagues have mentioned that clinical trial of clioquinol for treatment of Alzheimer's participants showed no statistically significant difference among the active treatment and the placebo groups. However, one participant in the clioquinol-treated group developed neurological symptoms [22,23]. Therefore, the planned phase III trial of clioquinol had been terminated [23].

Owing to a controversy in balancing the clinical benefits and the side effects of clioquinol, further explorations in several aspects are still needed to be elucidated, for examples protein-drug interaction, pharmacokinetics, pharmacodynamics, pharmacological effects, and toxicity studies. Clioquinol undergoes first-pass metabolism to form glucuronide and sulfate conjugates. In human, clioquinol is less metabolized than that in rodents, and its half-life is about 10–14 h [24]. The serum level of clioquinol has been found in a range of 13–25 μM after orally administered to Alzheimer's disease patient [22]. Furthermore, several lines of evidence have revealed that the higher accumulation of clioquinol may exert more toxicity to human [17,25,26]. Interaction of drug with serum protein is one of the major factors that influence on half-life and free drug concentrations in the blood stream. Serum albumin is an abundant protein in circulatory system of mammal. It is responsible for many functions in our body, especially storing and transporting various endogenous and exogenous compounds, controlling physiological pH, and maintaining oncotic pressure. In some circumstances, binding of drug with serum albumin may enhance its transportation to drug target or vice versa. Bovine serum albumin (BSA) is typically exploited as a representative of serum proteins for in vitro study of protein-drug interaction because of its low cost, structural similarity to human serum albumin (HSA), and easy accessibility [27]. BSA is a globular non-glycoprotein comprising of 583 amino acids on a single polypeptide chain. Its structure can be categorized into three homologous domains including I (residue 1–195), II (residue 196–383), and III (residue 384–583) domains, where each domain contains sub-domains A and B. The hydrophobic pockets in subdomains IIA and IIIA, respectively named as site I and site II, have been identified as the binding sites for aromatic and heterocyclic compounds [28,29]. The third binding pocket within subdomain IB (site III) has recently been identified as the primary binding site for many compounds e.g. bilirubin, hemin, and sulfonamide derivatives. Seven binding sites for fatty acids have been recognized on subdomains IB, IIIA, IIIB, and subdomain interfaces [29,30]. BSA contains two tryptophan residues located on the surface of subdomain IA (Trp-134) and in the hydrophobic pocket of subdomain IIB (Trp-212), in which it can be employed as intrinsic fluorescence indicators for probing the BSA-ligand interaction.

Although there is extensive consideration of clioquinol repositioning for anti-Alzheimer's and anti-cancer drugs, the information about its interaction with serum proteins is rarely available and remains a challenge for elucidation. Therefore, this study aims to explore the interaction between clioquinol and serum albumin by using BSA as a representative. To postulate the interaction mechanism and the binding parameters, we exploited various mathematic models on the fluorescence quenching data, especially Stern-Volmer's, Hill's, and thermodynamic equations. Besides, molecular docking was also taken to explore structural information of the BSA-clioquinol interaction, including the favored binding site, the preferred orientation, and the binding energy.