Modulation of multidrug resistance via inhibition of toxin efflux pumps

Research project P 13851 Modulation of Multidrug Resistance Peter CHIBA 11.10.1999 Multidrug transporters are plasma membrane proteins that are able to expel a broad range of toxic molecules from cells. Antimicrobial, antiparasitic as well as anticancer therapy relies heavily on the administration of natural toxin drugs. The expression or overexpression of active efflux systems prevents natural toxins from reaching their intracellular targets and thereby renders cells drug resistant. One of the most well characterized members of the multidrug transporter family is P-glycoprotein, which has been correlated with the resistance of human tumors to cytotoxins. A variety of efflux pumps which share structural and functional homology and which have therefore been subsumed in the Pgp-cluster of ATP-binding-casette transporters have been described in such diverse organisms as plasmodium falciparum. , the causative agent of malaria, fungi such as candida albicans, Leishmania donovani and Entamoeba histolytica, which causes amebiasis. Over the last decade, resensitization of resistant tumor cells to cytotoxic drugs has been attempted by the simultaneous administration of toxins with low molecular weight inhibitors of Pgp. A large number of clinical studies have been initiated to prove a potential benefit of these combination .regimens in cancer patients. Despite intensive research, the mode of action of modulators on a molecular basis remains to be elucidated. This is not surprising,. since the interaction of substrates/modulators with Pgp takes place within the plasma membrane which up to now precludes crystallographic and protein modelling methods from being applied successfully. The present application proposes ligand-based design as well as an advanced photoaffinity labeling strategy as the most promising experimental approaches to characterize the Pgp - modulator interaction on a molecular basis. The concept of ligand based design relies on structural variation of propafenone type modulators using a combinatorial approach. The rule of altered lipophilicity distribution within the molecules is investigated using conventional and 3D-QSAR method as well as membrane interaction measurements based on NMR-spectroscopy. The photoaffinity approach takes advantage of the property of the carbonyl group in propafenones to be intrinsically photoactivatable. In addition this group represents a pharmacophoric substructure. Purification of ligand labeled fragments will be accomplished by chemical derivatisation of the 1,2 aminoalcohol substructure. This new ligand directed derivatisation and subsequent affinity purification via a chemical capture group may also proof applicable for other ligand/protein pairs. (370 words)

Multidrug resistance (MDR) is a significant challenge in the treatment of cancer and infectious disease. The World Health Organization has reported that MDR can account for up to 60% of all hospital acquired infections globally (WHO Press Release 41, WHO, Geneva, 2000). Drug resistant bacterial strains that cause pneumonia, cholera and tuberculosis are widespread and difficult to treat. In human cancers, a similar mechanism of MDR is a major reason for the failure of cytostatic drugs. For patients with resistant disease treatment alternatives are limited. A major proportion of bacterial and cancer drug resistance is attributed to the expression of MDR-transporters which extrude toxic compounds at the expense of ATP-hydrolysis or by exploiting proton gradients. By the action of these pumps drugs are prevented from accessing their intracellular sites of action and thus are rendered ineffective. Most compromising to a successful therapy is the promiscuity of these transport pumps. A large number of structurally and functionally diverse cytostatics and antimicrobial agents, including antibiotics, antifungals and antiparasitics are substrates of these transporters. One of the most intensely studied members of this group of proteins is P- glycoprotein (P-gp, ABCB1), an ATP-dependent multidrug efflux transporter. P-gp represents a paradigm for a number of structurally and functionally related microbial drug transporters. Though the biochemistry of P-gp is well understood, little is known about the atomic structure of the protein and the mechanism by which it extrudes drugs. These issues have been addressed by our group using photoaffinity labeling, high resolution mass spectrometry and protein homology modeling techniques. Results aid in understanding the molecular basis of the transport process and thus have a significant impact on the development of new therapeutics used to treat cancer and infectious disease.