In Silico Identification of Six Mushroom-Derived Sterol and Triterpenoid Compounds as Potential P-Glycoprotein Modulators in Multidrug Resistance  
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1 UCIBIO--Applied Molecular Biosciences Unit, Toxicologic Pathology Research Laboratory, University Institute of Health Sciences (UCIBIO-IUCS-CESPU), 4585-116 Gandra, Portugal; [EMAIL_ADDRESS]  
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P-glycoprotein (P-gp) or ABCB1, which is a member of the B superfamily of ATP-binding cassette transporter (ABCs), is one of the main causes of this phenomenon [1; 2]. This energy-dependent efflux pump, located in the plasma membrane, is responsible for transporting substrates across the cell membrane [3; 4]. Numerous tumor cell types have already been found to have overexpressed P-gp, which acts by increasing the efflux of chemotherapeutic drugs and reduces intracellular accumulation, thus decreasing the drug's efficacy [5]. Consequently, the failure of some chemotherapy treatments, including doxorubicin, paclitaxel, and topotecan, which are mediated by this efflux mechanism, is directly linked to the increased expression of P-gp [6]. P-gp is a polypeptide with 1280 residues and a mass of 170 kDa. P-gp's structure consists of two nucleotide-binding domains (NBDs) and two transmembrane domains (TMDs) (Figure 1) [3]. While the TMDs are composed of six hydrophobic \(\alpha\)-helices that allow the drug to cross the membrane through internal and external conformations, the NBDs are necessary for the binding and hydrolysis of ATP, which provides energy for the conformational change needed to transport the drug out of the cells [7]. The operational process of P-gp includes switching between inward- and outward-facing forms, in which ATP binds to the NBDs to initiate a conformational change that allows the bound substrate's release [3]. Given the importance of P-gp in MDR, different methods have been investigated to increase the effectiveness of drug administration. There are three ways to prevent P-gp activity: compounds blocking the binding sites, interrupting ATP degradation, and altering the lipid structure of the cell membrane [6]. There are currently three generations of P-gp inhibitors, but all of them have some level of toxicity. The first generation (e.g., verapamil and cyclosporine A) showed low MDR reversal activity and high toxicity; the second generation (e.g., biricodar) showed greater efficacy but unpredictable drug interactions, limiting its use; and the third generation (tariquidar) showed high specificity but also failed in clinical trials due to toxicity [7; 8]. Despite the efforts and advances made in developing P-gp inhibitors over the last few decades, the first three groups of inhibitors failed in the clinical testing phase, mainly due to the toxicity associated with the doses needed to block the action of P-gp [7]. Figure 2 shows the groups of inhibitors developed and their respective mechanisms of action and generations. Acknowledging these compounds' limitations, searching for natural P-gp inhibitors has emerged as a promising strategy. Approximately 70% of current anticancer drugs are derived from natural products [9]. Natural compounds, such as those from plants and fungi, are considered less harmful in reversing tumor resistance due to their relatively low toxicity to normal cells [2]. Thanks to this, and to the bioactive potential of their secondary metabolites, mushrooms have been used for many years in traditional Chinese medicine and have, therefore, often been considered a natural choice for cancer treatment [10]. Numerous investigations have demonstrated that the bioactive substances found in mushrooms can inhibit MDR [9; 11; 12; 13; 14]. Other studies have reported that these compounds could act as effective modulators of P-gp, improving drug retention in resistant cancer cells [15; 16; 17; 18]. A recent systematic review published by our research group discussed the potential of mushroom extracts in regulating P-gp expression [19].