ABC transporters are ATP binding cassette proteins which play an important role in pharmacokinetics of drugs. P-glycoprotein (P-gp, ABCB1) belongs to the ABCB subfamily. It recognizes a large number of structurally diverse xenotoxic compounds and exports them out of the cell, thereby protecting cells from their lethal effects. Many natural product drugs are used in the treatment of human disease and therefore expression of P-gp is able to elicit a multidrug resistant phenotype. It also plays an important role in drug disposition. The mechanism of substrate binding and transport remains unresolved. The present study was conducted to better understand how drugs are translocated by P-gp.
The membrane spanning portion of P-gp lacks charged residues. The fact that P-gp arose from a homodimeric and rotationally symmetric ancestor by gene duplication made us consider that pseudosymmetry would have been retained and solute might interact with it in a dual mode. In order to support this hypothesis, pseudosymmetric glutamine residues in helices 2 and 8 were mutated to positively charged arginine residues. The underlying idea was that positively charged ligands would be affected by this mutation, while uncharged compounds would not. In order to detect a potential (undesired) interaction of these residues with ligands, glutamines were also changed to neutral alanine residues. Transport and inhibition assays indicated that these residues are not direct interaction partners. In addition rhodamine123 prefers one path (translocation path 1, TP1), while positively charged propafenone analogues, verapamil and vinblastine prefer the other (translocation path 2, TP2). The major conclusion from these experiments is the existence of two (pseudo)symmetric drug translocation pathways, which reflect the transporter's evolutionary origin from a homodimeric ancestor. These paths are preferentially, but not exclusively used by solutes. Because of the fact that H-bonding interactions have been shown in the past to be important for P-gp solute interactions and tyrosines often represent major small molecule interaction partners, we further investigated the role of the two tyrosine residues Y307 and Y310 in translocation path 1. The reason for choosing TP1 as compared to TP2 was the lower number of tyrosine residues (2 vs. 6). Both tyrosines were mutated to phenylalanine either individually or together to probe into H-bonding rather than aromatic interactions. The mutations were also done in Q132R and Q773R background to direct positively charged ligands to use one of the translocation paths exclusively. A significant decrease in rhodamine123 efflux was observed in the double tyrosine mutant (Y307F.Y310F), as well as the triple mutant Y307F.Y310F.Q132R.
This indicates that hydrogen bonds are formed between tyrosines and rhodamine123. In contrast, the interaction with propafenone analogues is not affected. For the first time to our knowledge, the concept of dual translocation paths allowed studying and understanding the role of individual amino acid residues of P-gp for solute interaction. This thesis also describes the discovery of novel chalcone derivatives with strong P-gp inhibitory activity, which contains a core structure related to the phenylpropiophenone moiety of propafenones. Some of these planar compounds are more active than the most active propafenones and show a biological activity which is comparable to that of third generation P-gp inhibitors.