Development of Novel Rifampicin-Derived P-Glycoprotein Activators/Inducers. Synthesis, In Silico Analysis and Application in the RBE4 Cell Model, Using Paraquat as Substrate

Abstract

P-glycoprotein (P-gp) is a 170 kDa transmembrane protein involved in the outward transport of many structurally unrelated substrates. P-gp activation/induction may function as an antidotal pathway to prevent the cytotoxicity of these substrates. In the present study we aimed at testing rifampicin (Rif) and three newly synthesized Rif derivatives (a mono-methoxylated derivative, MeORif, a peracetylated derivative, PerAcRif, and a reduced derivative, RedRif) to establish their ability to modulate P-gp expression and activity in a cellular model of the rat’s blood–brain barrier, the RBE4 cell line P-gp expression was assessed by western blot using C219 anti-P-gp antibody. P-gp function was evaluated by flow cytometry measuring the accumulation of rhodamine123. Whenever P-gp activation/induction ability was detected in a tested compound, its antidotal effect was further tested using paraquat as cytotoxicity model. Interactions between Rif or its derivatives and P-gp were also investigated by computational analysis. Rif led to a significant increase in P-gp expression at 72 h and RedRif significantly increased both P-gp expression and activity. No significant differences were observed for the other derivatives. Pre- or simultaneous treatment with RedRif protected cells against paraquat-induced cytotoxicity, an effect reverted by GF120918, a P-gp inhibitor, corroborating the observed P-gp activation ability. Interaction of RedRif with P-gp drug-binding pocket was consistent with an activation mechanism of action, which was confirmed with docking studies. Therefore, RedRif protection against paraquat-induced cytotoxicity in RBE4 cells, through P-gp activation/induction, suggests that it may be useful as an antidote for cytotoxic substrates of P-gp.

Reversing cancer multidrug resistance: insights into the efflux by ABC transports from in silico studies

“…Finally, similar conclusions on the location of the DBS and residues involved in drug binding were reported recently . In contrast, a recent study toward P‐gp activation with rifampicine‐derived molecules identified two molecules that may induce P‐gp expression against paraquat‐induced cytotoxicity, thus constituting a new approach for the treatment of toxicologic phenomena.…”

Cellular Models and In Vitro Assays for the Screening of modulators of P-gp, MRP1 and BCRP

“…Although the mechanism of action of P-gp inducers remains unclear, it is known that P-gp induction is regulated by nuclear factors, such as pregnane X receptor (PXR), constitutive androstane receptor (CAR), nuclear factor erythroid-derived 2-related factor (Nrf2), Y-box binding protein-1 (YB-1), nuclear factor Y (NF-Y), nuclear factor-κB (NF-κB), liver X receptor (LXR), farnesoid X receptor (FXR) and peroxisome proliferator-activated receptors α and γ (PPARα and PPARγ) [ 3 , 51 , 55 , 56 , 57 ]. The mechanism of action of P-gp activators suggests the involvement of the NBDs: (a) speed-up of the transport velocity, resulting in an increased conversion of ATP or (b) enhanced ATP affinity for the NBDs, leading to increased binding [ 58 ]; or conformational changes in the TMDs due to the binding of the compound in a specific ligand-binding site, which stimulates the substrate efflux on another ligand-binding site [ 20 ].…”

“…In humans, ABC transporters comprise 49 proteins distributed by seven subfamilies (ABCA–G) with about 20 carriers involved in xenobiotics transport [ 3 , 14 ]. Several in vitro and in vivo studies have shown that the expression and activity levels of efflux transporters are modulated by the presence of inhibitors, inducers and/or activators [ 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 ].…”

Quantification of 1‐(propan‐2‐ylamino)‐4‐propoxy‐9H‐thioxanthen‐9‐one (TX5), a newly synthetized P‐glycoprotein inducer/activator, in biological samples: method development and validation

“…Currently, it is known that P‐gp is present in both tumor and normal cells, exporting xenobiotics in the intestine, liver, kidney, pancreatic and bile ducts, renal proximal tubule, placenta, testis and blood–brain barrier, as well as in lymphocytes (Choudhuri & Klaassen, ; Vilas‐Boas et al ., ). P‐gp is inducible by many drugs, including dexamethasone, rifampicin, hypericin and hyperforin (St John's wort antidepressants), and chemotherapeutic agents such as doxorubicin, daunorubicin and vimblastine (Chaudhary & Roninson, ; Dinis‐Oliveira et al ., ; Hu et al ., ; Tian et al ., ; Vilas‐Boas et al ., ; Zhou, ). Considering all of the mechanisms in which P‐gp is involved, it can be concluded that P‐gp plays a very important role in defending cells against its toxic substrates, avoiding their intracellular accumulation.…”

“…Considering all of the mechanisms in which P‐gp is involved, it can be concluded that P‐gp plays a very important role in defending cells against its toxic substrates, avoiding their intracellular accumulation. Accordingly, strategies to increase P‐gp expression and/or activity may be viewed as potential antidotal pathways to prevent P‐gp substrate‐mediated toxicity and, therefore, an alternative therapeutic strategy to implement in the near future, in intoxication scenarios (Dinis‐Oliveira et al ., ; Silva et al ., ; Vilas‐Boas et al ., ).…”