Role of Multidrug Resistance Protein 1 (MRP1) and Glutathione S-Transferase A1-1 in Alkylating Agent Resistance

Abstract

We investigated the role of phase II (conjugation) and phase III (efflux) detoxification of the anticancer drugs melphalan (MLP) and chlorambucil (CHB). Although both drugs are substrates of Alpha-class glutathione Stransferases (GST) and the monoglutathionyl conjugates formed in these enzymatic reactions are transported by MRP1, we found that GSTA1-1 and MRP1 acted in synergy to confer resistance to CHB but not to MLP (Morrow, C. S., Smitherman, P. K., Diah, S. K., Schneider, E., and Townsend, A. J. (1998) J. Biol. Chem. 273, 20114 -20120). To explain this selectivity of MRP1/GST-mediated resistance, we report results of side-byside experiments comparing the kinetics of MLP-versus CHB-glutathione conjugate: formation, product inhibition of GSTA1-1 catalysis, and transport by MRP1. The monoglutathionyl conjugate of CHB, CHB-SG, is a very strong competitive inhibitor of GSTA1-1 (K i 0.14 M) that is >30-fold more potent than that of the corresponding conjugate of MLP, MLP-SG (K i 4.7 M). The efficiency of GSTA1-1-mediated monoglutathionyl conjugate formation is more than 4-fold higher for CHB than MLP. Lastly, both CHB-SG and MLP-SG are efficiently transported by MRP1 with similar V max although the K m for CHB-SG (0.37 M) is significantly lower than for MLP-SG (1.1 M). These results indicate that MRP1 is required for GSTA1-1-mediated resistance to CHB in order to relieve potent product inhibition of the enzyme by intracellular CHB-SG formed. The kinetic properties of MRP1 are well suited to eliminate CHB-SG at pharmacologically relevant concentrations. For MLP detoxification, where product inhibition of GSTA1-1 is less important, GSTA1-1 does not confer resistance because of the relatively poorer catalytic efficiency of MLP-SG formation. Similar analyses can be useful for predicting the pharmacological and toxicological consequences of MRP and GST expression on cellular sensitivity to various other electrophilic xenobiotics.

A Fluorometric Screening Assay for Drug Efflux Transporter Activity in the Blood-Brain Barrier

“…While the present study is the first to quantitatively determine IC 50 and Imax values for P-gp and MRP-related transporters in the BBMEC, comparisons to other cells or other drug screening assays is quite favorable. The IC 50 value of 14.6 M obtained for risperidone in the R123 assay in the present study is comparable to the Km value of 18 M reported by Boulton et al in a P-gp ATPase assay (37).…”

Synthesis and Characterization of Some New Phase Ii Metabolites of the Alkylator Bendamustine and Their Identification in Human Bile, Urine, and Plasma From Patients With Cholangiocarcinoma

“…Numbering of the carbon skeleton was done according to Table 1 Fig. 1, was synthesized following the method reported by Paumi et al (2001). Accordingly, 0.4 mg/ml L-cysteine and 0.6 mg/ml 1 hydrochloride was dissolved in 100 ml of a solution of 0.1 M sodium bicarbonate and 0.2 M NaCl.…”

“…In vitro spontaneous and glutathione S-transferasemediated reaction of melphalan and chlorambucil, both structurally related to 1 (Fig. 1), with GSH has been investigated as reported in numerous articles (Dulik et al, 1986(Dulik et al, , 1990Ciaccio et al, 1990Ciaccio et al, , 1991Meyer et al, 1992;Awasthi et al, 1996;Horton et al, 1999;Paumi et al, 2001;Zhang and Lou, 2003;. However, no studies have been published describing the qualitative or quantitative determination of these conjugates in humans.…”

The MRP family of drug efflux pumps

“…It is presumed that MRP1 confers resistance to oxyanions such as arsenic trioxide by transporting these compounds as glutathione complexes, by analogy with the Leishmania MRP1 homologue PGPA and the yeast MRP1 homologue YCF1, which transport arsenite and cadmium, respectively, as glutathione complexes (Li et al, 1997;Legare et al, 2001). The ability of MRP1 to transport glutathione conjugates is also germane to chlorambucil, in that the pump is able to mediate the transport of monoglutathionyl-chlorambucil (Paumi et al, 2001). Interestingly, while most studies have found that MRP1 is unable to confer resistance to alkylating agents such as chlorambucil, the activity of the pump toward chlorambucil can be made manifest by coexpression with GSTa1-1, which catalyses the formation of monoglutathionyl-chlorambucil from the parent drug (Morrow et al, 1998).…”