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Identification and characterization of the canine multidrug resistance-associated protein
Liandong Ma1, Susan E Pratt, Jin Cao
1Division of Cancer Research, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285, USA.
Abstract:
Human multidrug resistance protein 1 (MRP1) confers resistance to the Vinca alkaloids, the anthracyclines, and the epipodophyllotoxins. It is also capable of binding to and transporting the glutathione S-conjugate leukotriene C4 (LTC4) in isolated membrane vesicles. To explore species differences that exist between MRP orthologs, we cloned and characterized the mRNA encoding a canine ortholog of human MRP1-designated canine MRP1 (canMRP1). The canMRP1 mRNA encodes a protein of identical length as MRP1. Sequence alignment revealed that canMRP1 was 92% identical to MRP1 and 88% identical to murine mrp1. Five polymorphisms were identified in the canMRP1 cDNA coding sequence, including one resulting in an amino acid change from alanine to serine at aa149 (canMRP1-A and B alleles, respectively). canMRP1 was expressed and functionally characterized in HeLa and A2780 cells. Both alleles conferred an increased resistance to vincristine and etoposide and transported LTC4. The compound LY402913, a modulating agent developed against human MRP1, was able to sensitize canMRP1-expressing cells to vincristine. The modulation of canMRP1 by LY402913 was additionally confirmed by the calcein-AM accumulation assay. LY402913 inhibited the efflux of calcein in canMRP1-expressing cells. Thus, canMRP1 is similar to MRP1 in conferring resistance to vincristine and etoposide, transporting calcein-a.m., and being inhibited by LY402913. However, despite the high degree of sequence identity and functional similarity to MRP1, canMRP1 transgene failed to confer resistance to doxorubicin either in HeLa or A2780 cells. Knowledge of species differences between canine and human proteins will aid in the design of appropriate pharmacokinetic and toxicokinetic studies for the preclinical evaluation of MRP1 modulators.
Insights
Canine multidrug resistance protein 1 (canMRP1) shares similarities with human MRP1 in drug resistance and transport functions. However, canMRP1 does not confer resistance to doxorubicin, highlighting species-specific differences for drug development.
Area of Science:
- Molecular Biology
- Pharmacology
- Biochemistry
Background:
- Human multidrug resistance protein 1 (MRP1) is crucial for resistance against various chemotherapy drugs, including Vinca alkaloids and anthracyclines.
- MRP1 also transports glutathione S-conjugates like leukotriene C4 (LTC4), indicating broader substrate specificity.
- Understanding species differences in MRP orthologs is vital for accurate preclinical drug evaluation.
Purpose of the Study:
- To clone and characterize the canine ortholog of human MRP1, named canine MRP1 (canMRP1).
- To investigate the functional similarities and differences between canMRP1 and human MRP1.
- To assess the potential of human MRP1 modulators in canine models.
Main Methods:
- Cloning and sequencing of canine MRP1 (canMRP1) mRNA.
- Expression of canMRP1 in HeLa and A2780 cells.
- Functional assays including drug resistance profiling (vincristine, etoposide, doxorubicin), LTC4 transport, and calcein-AM accumulation assays.
- Evaluation of the MRP1 modulator LY402913 on canMRP1 function.
Main Results:
- Canine MRP1 (canMRP1) shares high sequence identity (92%) with human MRP1 and encodes a protein of identical length.
- Both identified alleles of canMRP1 conferred resistance to vincristine and etoposide and transported LTC4.
- The MRP1 modulator LY402913 effectively sensitized canMRP1-expressing cells to vincristine and inhibited calcein-AM efflux.
- Crucially, canMRP1 failed to confer resistance to doxorubicin in tested cell lines, unlike human MRP1.
Conclusions:
- Canine MRP1 (canMRP1) exhibits significant functional and structural similarities to human MRP1, including drug transport and modulation by LY402913.
- The inability of canMRP1 to confer doxorubicin resistance represents a key species-specific difference compared to human MRP1.
- These findings underscore the importance of considering interspecies variations in MRP1 function for designing effective preclinical pharmacokinetic and toxicokinetic studies of MRP1 modulators.