**1. Introduction**

The most affluent blood protein present in vertebrates is serum albumin (S.A.) and it is mainly responsible for transporting both endogenous and exogenous molecules to their target sites. It is the principal constituent of the plasma proteins (≈60%) [1] and plays a significant role in the pharmacokinetics, pharmacodynamics, and toxicity of drugs [2]. The two most common serum albumin used as model transport proteins are BSA and HSA (human serum albumin). There is almost 75.6% sequence homology and similar ligand binding characteristics between the two. Therefore, BSA is commonly used as an alternative in protein–ligand binding studies because of its ready availability and costeffectiveness [3,4]. The two most common ligand binding sites on BSA are Sudllow Site I and II positioned in subdomain IIA and IIIA [5–7].

Simultaneous administration of two or more drugs might lead to a competition between the two drugs to bind to a similar site present on the BSA. The conformation of the

protein binding pockets can be altered on interaction with ligands, thereby influencing the binding of other ligands to the same binding pockets [8,9]. The therapeutic or adverse effects of the drug depend on the free fraction of the drug available in the plasma, and this property of drugs is taken into account when prescribing two or more drugs in a multidrug therapy due to possible competition at the plasma protein binding level [10,11].

Erlotinib (ERL), a tyrosine kinase inhibitor, has anticancer properties and is approved to treat non-small cell lung cancer. There is a high epidermal growth factor receptor (EGRF) expression in certain cancers, and ERL inhibits EGFR [12]. One of the mechanisms of action suggested for the ERL acts by disabling the phosphorylation ability of EGFR, thus inhibiting the signal transduction cascade leading to malignant cell apoptosis. Even though several mechanisms of action for ERL are proposed, the exact mechanism is yet unknown [13,14]. The binding of ERL to serum proteins from different species such as humans, mice, and rats are 92%, 95%, and 92%, respectively [15].

Fresh fruits and vegetables contain phenolic compounds such as flavonoids and tannins, and one such flavonoid is quercetin [16]. Quercetin (QUR) is also present in various food supplements and has antioxidant properties [17]. Quercetin has chemoprotective and radioprotective properties. In addition, it protects normal cells from the adverse effects of chemotherapy and radiotherapy [18]. The pharmacokinetics of quercetin is variable in humans due to their diet history, genetic polymorphism, and metabolic variation because of gut microbiota [19,20]. Enhancement in the antitumor activity of a breast cancer drug on concurrent use of QUR has been reported [21].

Quercetin is highly metabolized, and the amount of QUR in systemic circulation varies based on inter-individual pharmacokinetics and the source of QUR. In addition, although different organs metabolize quercetin, its transport to these organs is dependent on systemic circulation [20,22]. Thus, quercetin may interfere with the binding of the other concomitantly used drugs, impacting their pharmacokinetics and displacing them from their albumin binding sites. Such interference may lead to a toxic or a subtherapeutic response of the drug in the presence of QUR [23–26]. In addition, the pharmacokinetics of some drugs is affected by concurrent usage of QUR [7,23,25]. For example, a study reported the sudden death of experimental animals on the simultaneous use of QUR and digoxin [26].

The flavonoid QUR binds to Site I, subdomain IIA of BSA. Further, the interaction of QUR with other concomitantly used drugs has also been investigated [27–31]. In addition, possible competition between ERL and QUR to bind to BSA may affect the binding of ERL to BSA and lead to undesired effects of ERL [27–34]. Therefore, the current study investigated the ERL and BSA interaction with multispectroscopic and computational methods. Additionally, the impact of QUR was analyzed on the binding of ERL to BSA.
