ASSAY METHOD, PEPTIDES AND REAGENT KITS FOR TESTING MRP-SPECIFIC INHIBITORS

Abstract: The invention relates to a method for testing L0-specific inhibitors of trasporter proteins of the MRP-family and for testing inhibitors specific to the predicted amphipatic helical region of the L0 region, the tested inhibitors L0 peptide, mutants thereof, polynucleotides coding the peptides, vectors and hosts cells comprising the polynucleotides and reagent kits for testing and developing the inhibitors. The invention is especially useful for combatting multidrog resistance in cancer patients and in the field of related research.

Description: Assay method, peptides and reagent kits for testing MRP-specific inhibitors The invention relates to an assay method for testing L 0 -specific inhibitors of transporter proteins of the MRP-family, L 0 peptide and reagent kits for testing and developing the inhibitors and L 0 -specific inhibitors of the MRP-family provided by using the method or reagent kit of the invention. The invention is especially useful in the field of multidrog resistance research and cancer treatment. MDRl (P-glycoprotein) and MRP1 (multidrug resistance protein 1) are members of the ABC (ATP-Binding Cassette) transporter family that are able to extrude chemotherapeutic drugs from tumor cells in an ATP-dependent manner. Whereas MDRl preferentially transports hydrophobic, slightly positive molecules, MRP1 transports glutathione conjugates (e.g. leukotriene C ) and other large hydrophobic molecules conjugated to negatively charged ligands like sulfate and glucuronide [Gottesman, M.M. and Pastan, I. (1993) Annu. Rev. Biochem. 62, 385-427; McGrath, T., Latoud, C, Arnold, S.T., Safe, A.R., Felsted, E.R., and Center, M.S. (1989) Biochem. Pharmacol. 38, 3611-3619; Muller, M., Meijer, C, Zaman, G. J. R., Borst, P., Scheper, R. J., Mulder, N. H., de Vries, E. G. E., and Jansen, P. L. M. (1994) Proc. Natl. Acad. Sci. USA 91, 13033-13037; Jedlitschky, G., Leier, I, Buchholz, U., Center, M.S., and Keppler, D. (1994) Cancer Res. 54, 4833-4836; Jedlitschky, G., Leier, I., Buchholz, U., Barnouin, K., Kurz, G., and Keppler, D. (1996) Cancer Res. 56, 988-994; Loe, D. W., Almquist, K. C, Cole, S. P. C, and Deeley, R. G. (1996) J. Biol. Chem. 271, 9683-9689]. MRP1 also transports unmodified neutral and basic organic compounds, probably by a co-transport mechanism together with glutathione [Loe, D. W., Almquist, K. C, Cole, S. P. C, and Deeley, R. G. (1996) J. Biol. Chem. 271, 9675-9682; Loe, D. W., Deeley, R. G., and Cole, S. P. C. (1998) Cancer Res. 58, 5130-5136; Evers, r., de Haas, M., Sparidans, R., Bijnen, J., Wielenga, P. R, Lankelma, J., and Borst, P. Br. J. Cancer, in press]. MDRl Pgp and MRP1 seem to be major players in chemotherapy resistance in numerous forms of cancer, although homologs of these two transporters, as well as the recently identified breast cancer resistance protein (BCRP or, in other name, ABCG2) may also be involved in this clinical phenomenon [Doyle, L. A., Yang, W., Abruzzo, L. V., Krogmann, T., Gao, Y., Rishi, A. K., and Ross, D. D. (1998) Proc. Natl. Acad. Sci. USA 95, 15665-15670]. MRP1 has been shown to be a high affinity primary active transporter for the glutathione- conjugated eicosanoid, leukotriene C 4 (LTC ) [Leier, I., Jeditschky, G., Buchholz, U, Cole, S.P.C., Deeley, R. C, and Keppler, D. (1994) J. Biol. Chem., 969, 27807-27810 and Muller, M., (1994), see above] and to transport various other compounds that are conjugated to glutathione, sulfate or glucuronide [Leier, I, (1994), Jedlitschky, G. 5 (1996), Loe, D. W., (1996), Zaman, G.J. R., Cnubben. N. H. P., van Bladeren, P.J., Evers, R., and Borst, P., (1996) KBS Lett., 391, 125-130], The physiological functions of MRP1 range from the mediation of an inflammatory response to the elimination of certain xenobiotics [Hollo, Zs., Homolya L., Hegedus, T., and Sarkadi B. (1996) FEBS Lett., 383, 99-104; Grant, C.E, Valdimarsson, G., Hipfeer, D.R., Almquist, K. C, Cole, S.P.C, and Deeley, R.G., (1994) Cancer Res., 51, 357-361; Wijnholds, J., Evers, R. van Leusden, M.R., Mol. C.A., Zaman, G.J. Mayer, U., Reijnen, J. M., van der Valk, M., KJrimpenfort, P., and Borst, P. (1997), Nat. Med. 3, 1275-1279; Lorico, A., Rappa G., Finch, R.A., Yand. D., Flavell, R.A. and Sarcorelli, A.C., (1997) Cancer Res. 57, 5238-5242; Rappa G., Lorico, A., Flavell. R.A. and Sartorella. A.C., 5 (1997), Cancer Res., 57, 5232-5237] and this protein may play a role in the chemotherapy resistance of several types of cancer. Vanadate inhibits ATP-dependent drug transport both by MDRl and MRPl [Scarborough G. A. (1995) J Bioenerg Biomembr. 27, 37-41; Bakos, E., Evers, R., Sinkό, E., Varadi, A., Borst, P., and Sarkadi, B. (2000) Mol. Pharmacol. 57, 760-768; Mao, Q., Leslie, E. M., Deeley, R.G., and Cole, S.P. 10 (1999) Biochim Biophys Acta 1461. 69-82; Chang, X. B., Hou, Y. X., and Riordan, J. R. (1998) J. Biol. Chem. 273, 23844-23848] and in the presence of vanadate, the trapping of an adenine nucleotide in these proteins has been demonstrated [Urbatsch, I. L., Sankaran, R, Weber, J. and Senior, A.E (1995), J. Biol. Chem., 270, 19383-19390; Taguchi, Y., Yoshida, A., Takada, Y., Komano, T., and Ueda, K. (1997) FEBS Lett. 401. 11-14]. Transported compounds specifically increase the rate of vanadate- 15 dependent nucleotide trapping in MRPl [Taguchy Y et al., FEBS Lett. 401, 11-14 (1997)], similarly to what has been shown for hydrophogic drugs in the case of MDRl Pgp [Szabό, K. et al, J. Biol. Chem 273. 10132-10138 (1998)]. Vanadate dependent, drug stimulated nucleotide trapping reflects a partial reaction of the multidrug transporters and thus can be used to examine their functional characteristics. Other inhibitors of MRPl are also known in the art. 20 One of them is PAK-105P which efficiently increased CPT-11- and SN-38-accumulation in C- A120- and KB/MRP cells in a 10 nM concentration [Chen et al., Mol. Pharmacol. 55(5), 921-8 (1999)]. Draper et al [Br. J. Cancer 75(6), 810-15 (1997)] studied the effect of indomethacine, a known inhibitor of cyclooxigenase and glutathione-S-transferase and a modulator of anion transport, on murine and human cells expressing MRP and found that the indomethacine effectively inhibited the 25 efflux of a fluorescent dye, BCECF and increased its accumulation. Further measurements and a comparision with the MDRl protein suggested that the inhibition is MRPl -specific; however, the question that whether indomethacine acts via the inhibition of glutathione-S-transferase or competes directly with the transported drug at the transport site, could not be decided. The problem that a decrease in drug resistance is not the result of the potential inhibitor's effect 50 on MRPl, is mentioned also in the work of Curtin and Turner [Eur. J. Cancer 35(6), 1020-5 (1999)]. The authors studied the sensitizing effect of an inhibitor of nucleoside transport, dipiridamol (DP) on cells overexpressing MRPl and found that sensitization is rather due to a decrease in the cellular GSH level than a modification in MRPl mediated drug efflux. Versantvoort et al. [Br. J. Cancer 72(1) 82-9 (1995)] came to a similar conclusion upon studying buthyonine-sulfoxide, an inhibitor of glutathione »5 synthesis. Said problem may emerge in case of any test which is aimed to detect MRPl activity indirectly, in living cells, via drug efflux or accumulation. In the state of art, however, most often such tests are applied. One of the most well-known MRPl inhibitor is MK-571, a leukotriene-D4-receptor antagonist. Van der Kolk et al [Clin. Cancer Res. 4(7) 1727-36 (1998)] worked out a functional assay for measuring MRPl activity, which is based on carboxyfluorescein (CF) transport by MRPl and its inhibition by MK-571. This method might turn out to be suitable for testing the effect of other MRPl inhibitors as well, though this possibility in not mentioned by the authors. The inhibitors of the art were not developed by purposeful design, thus, their mechanism of action is not known. Moreover, they probably influence other biochemical pathways of the organism, as well. Therefore, there is an existing need for an assay method or test system by which a potential inhibitor can be tested for definite MRP functions. For that purpose in proteins of the MRP family the key structural region, for which the inhibitor is specific, should be distinguished functionally, or its ructions should be detectable so that an inhibitor specific for this particular, important structural region may be tested. However, no test, suitable for detecting functions associated with various regions of the molecule has been known so far. To provide such a test, information is required on functions and interactions of individual regions or domains of this large molecule characterized by complicated domain-domain interactions. Relevant data of the art in respect of functions and "co-operation" of some significant structural units of MRPl are briefly summarized below. MDRl and MRPl share a similar core structure, containing a tandem repeat of transmembrane domains (TMDs) and cytoplasmic ATP-binding cassette (ABC) regions. However, MRPl and several homologues form a subfamily of ABC-transporters, characterized by a triple membrane-bound domain struchire. These include an MDRl -like core region, containing two ABC units and two membrane- bound domains (TMDi and TMD 2 ), and an N-terminal polypeptide region of about 280 amino acids, forming a cytoplasmic loop and a membrane-bound domain (L 0 and TMD 0 , respectively, see Fig. 1A; [see the Figure a, and Tusnady, G.E, Bakos, E., Varadi, A., and Sarkadi, B. (1997) FEBS Lett. 402, 1-3 and Bakos, E., Evers, R., Szakacs, G., Tusnady, G.E., Welker, E., Szabό, K., de Haas, M., van Deemter, L., Borst, P., Varadi, A., and Sarkadi, B. (1998) J. Biol. Chem. 273, 32161-32175]). Intact, whole MRPl was expressed in baculovirus infected insect cells [Bakos, E., Hegedύs, T., Hollo, Zs., Welker, E., Tusnady, G.E., Zaman, G.J.R., Flens, M.J., Varadi, A., and Sarkadi, B. (1996) J. Biol. Chem. 271, 12322-12326; Gao, M., Loe, D.W., Grant, C.E., Cole, S.P.C. and Deeley, R.G. (1996), J. Biol. Chem., 271, 27782-27787; and Gao, M, Yamazaki, M., Loe, D.W, Westlake, C.J., Grant, C.E., Cole, S.P.C, and Deeley, R.G. (1998) J. Biol. Chem. 273, 10733-10740] and, though the protein proved to be underglycosylated in this expression system, its basic structural and transport properties were the identical with those found in mammalian cells [Gao, M et al, (1996), see above]. Further, MRPl mutants were expressed in insect cells, which mutants did not contain the N-terminal TMD 0 Lo region of the protein and which were not active, while if the N-terminal TMD 0 L 0 region was coexpressed with the C-terminal core region an functional LTC 4 transporter was obtained [Gao, M et al, (1996), see above]. In polarised cells MDRl is routed to the apical membrane, while MRPl to the basolateral membrane [Bakos, E., Evers, R, Szakacs, G., Tusnady, G.E., Welker, E., Szabό, K., de Haas, M., van Deemter, L., Borst, P., Varadi, A., and Sarkadi, B. (1998) J. Biol. Chem. 273, 32161-32175; Evers, R., Zaman, G.J., van Deemter, L., Jansen, H., Calafat, J., Oomen, L.C., Oude Elferink, R.P., Borst, P., and Schinkel, A.H. (1996) J. Clin. Invest. 97, 1211-1218]. In these publications, just prior to the elaboration of the invention, the role of N-terminal regions of MRPl and delivery of human MRPl to plasmamembrane were studied. It was shown that the MDR like core region has practically no transport activity and nucleotid occlusion activity [Bakos et al, (1 98), see above]. However, transport function could be restored by co-expressing the whole N-terminal TMD 0 L 0 region and the MDRl -like core region [see also Gao et al (1998), like above]. Further, carrying out experiments with mutants Bakos, E. and co-workers found that mutant MRPl comprising the core region and L 0 region but not comprising the TMD 0 domain proved to be an active transporter molecule with a correct, i.e. basolateral membrane routing [Bakos et al, (1998), see above]. This earlier study suggested that the presence of L 0 region in the molecule is required to MRPl mediated transport activity and basolateral membrane routing of the protein, in polarised cells. However, certain confusion or ambiquity existed in the prior art regarding the role of TMD 0 region and the possible importance of the L 0 region itself to tlie molecular activity. Namely, Gao et al [Gao et al (1998), see above] found that removal of the whole TMD 0 domain or only its first N-terminal transmembrane helix abolished MRPl function (when removing the whole TMDo region Gao et al removed a very short part of tlie L 0 region as defined in tlie description). This led these authors to conclude that the entire TMD 0 L 0 region is essential for transport activity. This is clearly contrary to the above results leading to the assumption that the L 0 ΔMRP protein is fully functional. The above results strongly suggest that tlie intact character of functions of some of the molecule's structural regions (i.e. whether die said function is retained or not) highly depends on the molecular environment and on interactions with ether parts or regions of the molecule. It could not be expected therefore that L 0 forms a distinguishable structural unit and, as such, can be studied in itself and also that other structural units of the molecule can be studied in themselfes. In the state of art there is no definite suggestion regarding the role of L 0 . Earlier it was considered as a cytoplasmatic (or sometimes even extracellular) loop, which only links (hence its name: linker region) die two large structural unit, the core region and the N-terminal transmembrane domain. Furthermore, no definite structural feature have been assigned to the Lo region. At first, aiming at elaborating the invention, tlie present inventors also supposed that in order to develop a method or test system for testing an L 0 region specific inhibitor the L 0 region has to be expressed in the form of a chimeric protein, covalently linked to a protein (e.g. MDR) or a domain homologous but functionally unrelated to natural structural units of the MRP. The inventors hoped that thereby the significance of die L 0 region can be clarified. These experiments are described below. Unexpectedly, it was found that if L 0 region is used, preferably expressed, as a separate molecule (Lo peptide), optionally co-expressed with other MRP regions, its functions can be detected or assayed specifically and studied by tiiemselfes; furthermore, novel functions can be found. Thus, the L 0 peptide can be a suitable tool for testing inhibitors. Furthermore, surprisingly it was found that a shorter segment of the L 0 region has especially significant in respect of the functional character of die L 0 peptide and the whole transporter protein, and if said segment is neutralized or disabled, most of the L 0 -functions (or MRP-functions) are damaged. Thus, an object of the invention is to provide a method suitable for testing L 0 region specific inhibitors of transporter proteins of the MRP-family. An other object of the invention is to provide a smaller segment within the L 0 region, which segment is itself important to the activity (operability) of the molecule. A further object of die invention is to provide tools, e.g. peptides, recombinant constructs, expression vectors, suitable host cells and reagent kits useful in testing the inhibitors and in study of transporter proteins of the MRP family, e.g. in research of multidrug resistance. Thus, the invention relates to an assay method for testing L 0 -specific inhibitors of transporter proteins of the MRP family, comprising assaying an L 0 peptide exposed to the effect of a potential inhibitor, to determine whether at least one L 0 -function was retained or at least partially lost. Preferably, the L 0 peptide is expressed and optionally the L 0 peptide is exposed to the effect of the potential inhibitor before the final formation of its native structure (processing). According to a further preferred embodiment assaying the L 0 peptide is carried out by using a deletion mutant protein comprising an MRP-family transporter protein core region or its fragment capable of functioning and assaying at least one L 0 -function manifesting itself only upon contact with the core region. Preferably, botii the L 0 peptide and the core region are prepared by expression, or optionally by coexpression. As an Lo-function e.g. the following functions can be assayed or determined: membrane binding, rescuing the core region of the protein to the physiological membrane compartment and/or restoring transporter protein function. Preferably, when die question is whether the transporter protein function is restored or not by the L 0 peptide, it is also determined whether at least one of the following functions of die whole transporter protein was retained or at least partially lost: nucleotide trapping in membrane preparations; membrane routing, preferably to a specific membrane area of polarised cells; ATP-ase activity; membrane transport of substrates, preferably substrate uptake in inside-out vesicles; vectorial membrane transport into or from polarised cells. The invention further relates to an assay method for screening potential inhibitors of MRP family transporter protein specific for a segment of the Lo region predicted to be an amphipatic helix comprising determining whether die potential inhibitor exhibits affinity for the segment predicted to be an amphipatic helix. In a further preferred embodiment the invention relates to an assay method for testing inhibitors of MRP family transporter proteins specific for an L 0 region segment predicted to be an amphipatic helix, comprising assaying a transporter protein of the MRP family having exposed to the effect of a potential inhibitor; a functional transporter protein fragment comprising L 0 region; or an L 0 peptide and a protein consisting of a transporter protein core region (or a functional fragment thereof) said peptide and said protein being not linked covalently; a) in respect of specific membrane routing and/or glycosilation of the protein or its fragment, and b) in respect of any other function, preferably transporting capability of the protein, membrane binding of the L 0 peptide and/or MRPl function restoring capability of the L 0 peptide, and if the assayed functions of a) are retained and if the assayed functions of b) are damaged considering the result as the selective inhibiting effect of the inhibitor specific for the segment predicted to be an amphipatic helix. In a further aspect the invention relates to an L 0 peptide or a mutant or homologue thereof at least partly at least one Lo-fuction of which is retained for use in testing, designing or developing L 0 - specific inhibitors of transporter proteins of the MRP family, in multidrag resistance research or as a validated drug target. The peptide or its mutant comprises preferably amino acids of the amphipatic helix of the L 0 peptide of MRPl or a part thereof, e.g. the segment comprising amino acids 216 to 227 or a part diereof, e.g amino acids 221 to 227, 220 to 228, 219 to 230 or 223 to 232. Furthermore, the invention relates to a mutant L 0 peptide, carrying a mutation in the segment of the peptide predicted to be an amphipatic helix, preferably in segment 221 to 227, said mutation being preferably a deletion or a substitution. The invention relates to a polynucleotide comprising a sequence coding for L 0 region of a transporter protein of the MRP family for use in expressing L 0 peptide. The polynucleotide can be an expression vector, preferably an expression vector capable of funtioning in a bacterial, an insect and/or a mammalian cell. The invention further relates to host cells carrying a vector of the invention. The invention further relates to reagent kits for developing or testing specific inhibitors of transporter proteins of die MRP family or for multidrug resistance research, said kit comprising a peptide, a polynucleotide or a host cell of the invention, and optionally further comprising an expression vector suitable to express die transporter protein or a fimctional mutant thereof, preferably a deletion mutant substantially consisting of the core region of the transporter protein, and/or host cells, preferebly bacterial, insect and/or mammalian cells, in which the expression vector is operable, and/or reagents and buffers for the expression and/or assaying functions of the expressed L 0 peptide. Preferably, in reagent kits according to die invention the vector is a vector capable of functioning in E. coli, Sf9 and/or MDCKII cells and the host cells are E. coli, Sf9 and/or MDCKII cells. Using the assay methods of die invention inhibitors can be obtained. Thus, die invention relates to drugs behaving as an L 0 -specific inhibitor of a protein of the MRP family if assayed by a method of the invention. Preferably, said drug is capable to effect as an inhibitor due to being contacted with an Lo region segment predicted to be an amphipatic helix or a part diereof. DEFINITIONS A "transporter protein of the MRP family" is a transporter protein having a domain structure of any of MRPl to 6, preferably MRPl; in particular, according our present knowledge about domain structure, a transporter protein having a core region, an L 0 -like, typical cytoplasmatic loop region and, in many cases but not necessarily, an N-terminal TMD 0 region. Such a protein is e.g. the SUR protein. Most recently, according to the HGNC gene family nomenclature, the MRP family is also called ABCC family. Thus, in an other aspect, the MRP family comprises also those transporter proteins which are classed as members of the ABCC family, e.g. upon genomic sequencing. "Core region" of a protein of the MRP family is meant as the MDR-like core region of the protein, in particular a region comprising two ATP-binding casette (ABC) and two membrane bound domain (TMDi and TMD 2 domains). "Corresponding regions" of proteins of the same family are meant as sequence regions proved to be homologous upon sequence alignment based on homology. Herein, a "function" of any protein or its fragment is meant as any capability, suitability or non- structural feature or property which is also characteristic of a variant having a sequence identical with the wild type. A "function" can appear either in vivo or in vitro. Preferably the function is an (at least in principle) detectable feature or property manifesting itself upon functioning. In this sense functioning is considered as any response of the protein or its fragment to an environmental effect during its whole life period from its synthesis to degradation. Thus, without any limitation, "functions" are e.g. the following: capability of the protein or its fragment to be glycosilized or folded properly, its targeting, assembly of the protein or participation of a fragment in such an assembly, activity or partial activity. Thus the terms "Lo-fuction", "MRPl -function" are used in this sense. Functions of proteins of the MRP family are e.g. the following: The term "activity" of MRP comprises e.g. transport of a drug through the membrane carrying the MRP protein, ATP-ase activity etc. In a broad sense "activity" is meant as any partial reaction (e.g. substrate binding) of the whole reaction cycle of the enzyme as well as a partially damaged activity, e.g. nucleotide occlusion (trapping). A further MRP-function is "routing" to a given membrane area. A function can be a form of a general function manifested in a particular environment of assay method, e.g. uptake of transported drugs into membrane vesicules comprising MRP protein or their transport through a membrane of polarized cells. A fragment of a protein, e.g. of the MRP family, may show typical functions, e.g. membrane binding; rescue of the core region by the L 0 peptide or by the N-terminal region to the plasmamembrane (or a specific membrane compartment); or restoration of the activity of the MRP protein. Herein, the meaning of a "fragment capable of functioning" or a "functional fragment" is a fragment of a region with definite funtions in a protein, a homologue or a mutant of said fragment at least one function of which is detectable. "Lo peptide" is a peptide molecule the amino acid sequence of which is identical with or a fragment, a homologue or a mutant of the amino acid sequence of the L 0 region of any MRP family proteins. Preferably, L 0 peptide is an L 0 region fragment capable of functioning. Preferably, the L 0 peptide is homologous to the sequence of the MRPl 204 to 281 amino acids, however, it should be understood that it may contain either more or less amino acids. The "segment predicted to be an amphipatic helix" of the L 0 peptide is a segment predicted to be an amphipatic helix by the helical wheel prediction method or a corresponding region. According to our results this region plays a role in the membrane binding of the L 0 region of MRP family transporter proteins. More closely, based on sequence homology, a "segment predicted to be an amphipatic helix" is a conservative region corresponding to the about 220 to 232 amino acids of MRPl or the helical part of this segment or a region comprising a part of this segment having features typical to said region. SHORT DESCRIPTION OF THE DRAWINGS Fig. 1. Schematic representation (Panel A) and immunoblot detection of the MRPl -MDRl chimeric proteins expressed in Sf9 cells (Panel B) and in MDCKII cells (Panels C and D) Panel A. Schematic representation of the MRPl -MDRl chimera proteins. Panel B. Western blot analysis of Sf9 cell membranes containing MDRl, L 0 MDR1, TMDoMDRl, TMDoLoMDRl, or MRPl. Isolated membranes of Sf9 cells (5 μg) were subjected to electrophoresis in a 6% Laemmli-type gel, and immunoblotted as described in the Materials and Methods section. Lanes 1-5 were stained with the polyclonal antibody 4077, specific for human MDRl, while lanes 6-10 were developed with mAb Rl, specific for human MRPl. Protein-antibody interactions were visualized using the enhanced chemiluminescence technique. Panel C. Western blot with lysates from MDCKII wild-type cells and cells producing L 0 MDR1, MDRl or MRPl. 20 μg of protein was size fractionated in a denaturing 7.5 % polyacrylamide gel. After electroblotting blots were incubated with either C219, recognising human MDRl (lanes 1-4) or mAb Rl (lanes 5-8). The identity of the cell lines is indicated over the lanes. Panel D. Western blots showing MDCKH-MDRl cells (lanes 1-4) and MDCKII-L 0 MDR1 cells (lanes 5-8) treated with tunicamycin (3 μg/ml) for 0, 12, 25 or 40 h. MDRl was visualized with mAb C219, and L 0 MDR1 with Fig. 2. LTC4 transport (Panel A) and nucleotide trapping (Panel B) in Sf9 cell membranes containing MRPl -MDRl chimeric proteins Panel A. Time-course of ATP-dependent LTC 4 uptake in Sf9 membrane vesicles, expressing MRPl, MDRl, TMDoMDRl, TMD 0 L 0 MDR1, L 0 MDR1, or β-galactosidase. Membrane preparations were incubated with 50 nM LTC at 23°C and ATP-dependent uptake was calculated by subtracting the values obtained in the presence of 4 mJVI AMP from tiiose in the presence of 4 mM ATP. Samples were taken at the time points indicated. Panel B. Vanadate-dependent nucleotide trapping by MRPl -MDRl chimeric proteins. Isolated membranes from Sf9 cells expressing MRPl (lanes 1-3), MDRl (lanes 4-6), L 0 MDR1 (lanes 7-9), TMDoMDRl (lanes 10-12), and TMD 0 L 0 MDR1 (lanes 13-15) were labeled with Mg-8-azido-[α- 32 P]- ATP in the presence of vanadate, as described in the Materials and Methods section [see also Szabό, K., et al, (1998), above]. The reaction buffer contained 30 μM verapamil in experiments documented in lanes 2, 5, 8, 11 and 14, and 600 nM LTC 4 in lanes 3, 6, 9, 12, and 15. Fig. 3. Localization and transport properties of the chimera LoMDRl in MDCKII cells Panel A. Immunolocalization of L 0 MDR1 in MDCKII monolayers by confocal laser scanning microscopy. L 0 MDR1 was detected by indirect immunofluorescence with mAb MRK16 (green signal, light signal on die figures). The upper part shows a top view of the monolayer, the lower part a vertical • X/Z section. The arrow head indicates the position where the section was made. Nucleic acids were counterstained with propidium iodide (red signal). Panel B. Transport of [ 3 H]vinblastine by MDCKII-derived monolayers. Transport of vinblastine by MDCKII, MDCKII-MDR1 and MDCKII-LoMDRl cells is shown. Transport is presented as die fraction of total radioactivity added at the beginning of the experiment (t = 0), appearing in the opposite compartment. PSC833 (0.1 μM) was present in both compartments to inhibit the endogenously present MDRl Pgp present in these cells [Bakos E. et al, (1998), see above]. Squares: translocation from apical to basolateral. Circles: translocation from basolateral to apical. The experiments was performed in duplicate and repeated twice. Fig. 4. Detection and function of MRPl variants in Sf9 cell membranes Panel A. Schematic representation of the MRPl protein and mutant proteins obtained from MRPl by deletion. Panel B: Western blot analysis of Sf9 cell membranes prepared from cells expressing MRPl (lanes 1 and 5), ΔMRP1 (lanes 2 and 6), ΔMRP1 + L 0 (lanes 3 and 7) or L 0 (lanes 4 and 8). Isolated membranes of Sf9 cells (5 μg) were subjected to electrophoresis in a 4-20% Laemmli-type gel, and immunoblotted. Blots were incubated with mAb Rl and M6, respectively. Panels C and D: Time-course of ATP-dependent LTC 4 (Panel C) and NEM-GS (Panel D) uptake in Sf9 cell membrane vesicles containing MRPl, ΔMRP1, ΔMRP1+L 0 , or β-galactosidase. Panel B: Membranes were incubated with 50 nM LTC at 23°C; Panel C: Membranes were incubated with 50 μM NEM-GS at 37°C. ATP-dependent uptake was calculated by subtracting the values obtained in the presence of 4 mM AMP from those in the presence of 4 mM ATP. Samples were taken at the time points indicated. Fig. 5. Vanadate-dependent nucleotide trapping by MR l variants Isolated membranes from Sf9 cells expressing MRPl (lanes 1-3), ΔMRP1 + L 0 (lanes 4-6), or ΔMRP1 (lanes 7-9), were labeled with Mg-8-azido-[ - 32 P]-ATP in the presence of vanadate as described in die Materials and Metiiods section [see also Szabό K. et al (1998), above]. The reaction buffer contained 600 nM LTC 4 (lanes 2, 5, and 8), or 500 μMNEM (lanes 3, 6, and 9). Fig. 6. Immunolocalization of Lo co-expressed with ΔMRP1 i MDCKII monolayers by confocal laser scanning microscopy Lo was detected by indirect immunofluorescence with mAb Rl (green color, light signal in the lateral membrane). The upper part shows a top view of the monolayer, the lower part a vertical X/Z section. The arrow head indicates the position where the section was made. Nucleic acids were counterstained with propidium iodide (red signal). Fig. 7. Membrane attachment of the L a region of MRPl expressed in Sf9 cells Panel A: Detection of the L 0 region of human MRPl, expressed in Sf9 cells by Western blotting. Samples were fractionated in a 20% Laemmli-type gel, immunoblotted and incubated with mAb Rl. C: cell extract (5μg); M: membrane fraction (5μg); CP: cytoplasmic fraction, representing equivalent volumes of die cytoplasm as compared to membrane fractions. Panel B: Effects of KC1 (1 M, lanes 2,3), Urea (5M, lanes 4,5), or Triton X-100 (1%, lanes 6,7) on the membrane attachment of the L 0 region of MRPl. Membrane samples were washed with tlie respective agents and then size fractionated as in Panel A. Lane 1: isolated Sf9 cell membranes, Lanes 2-7: membranes washed with the respective agents. P: membrane pellet fraction; S: supernatant fraction. Samples represent equivalent initial volumes of each fraction. Panel C: Computer-predicted schematic representation of an amphipafhic helix within the L 0 region of various MRP proteins. Helical wheel plot of residues 221-233 in MRPl, 216-228 in MRP2, 220-232 in MRP3, 24-36 in MRP4, 112-124 in MRP5, and 216-228 in MRP6. Hydrophobic residues are circled. Figure 8. Characterization of MRPlΔ(223-232) and L n Δ(223-232. in Sf9 cells Panel A: Western blot analysis of Sf9 cell membranes containing MRPl or MRPlΔ(223-232). Isolated membranes of Sf9 cells (5 μg) were subjected to electrophoresis in a 6% Laemmli-type gel, and immunoblotted as described in the Materials and Methods section, with monoclonal antibody Rl, specific for human MRP 1. Panel B: Time-course of ATP-dependent LTC 4 uptake in Sf9 cell membrane vesicles, expressing MRPl, MRPlΔ(223-232), or β-galactosidase. Membrane preparations were incubated with 50 nM LTC at 23°C and ATP-dependent uptake was calculated by subtracting the values obtained in the presence of 4 mM AMP from those in the presence of 4 mM ATP. Samples were taken at the times indicated. Panel C: Detection of the L 0 Δ(223-232) mutant region of human MRPl, expressed in Sf9 cells. Samples were fractionated on a 20% Laemmli-type gel, and immunoblotted by Rl antibody as described in the Materials and Methods section. C: cell extract (5μg); M: membrane fraction (5μg); CP: cytoplasmic fraction, representing an equivalent initial volume compared to the total membrane fraction. Figure 9. Characterization of MRPlΔ(223-232) in MDCKII cells Panel A. Western blot with lysates from MDCKII wild-type, MDCKII-MRPl and MDCKII- MRPlΔ(223-232) cells. Four or 20 μg of protein were size fractionated. The blot was incubated with mAb Rl. The identity of the cell lines is indicated over the lanes. Panel B. Western blot with lysates from MDCKII-MRPl and MDCKII-MRPlΔ(223-232) cells, treated with tunicamycin (3 μg/ml) for 0, 12, 25 or 40 h. The blots were incubated with mAb Rl. Panel C. Immunolocalization of MRPlΔ(223-232) in MDCKII monolayers. MRPlΔ(223-232) was detected as in Figure 6 with mAb Rl. Panel D. Transport of DNP-GS by MDCKII derived monolayers. Cells were incubated with [ 14 C]CDNB (2 μM) in both the apical and basal compartment. This hydrophobic compound difiuses over the plasma membrane and intracellularly reacts to glutathione by the mediation of glutathione S- transferases. Samples were taken at t = 0, 1, 3, 6, and 20 min from both compartments and extracted with ethyl acetate to separate free CDNB from intracellularly formed DNP-GS. Squares: transport to the basolateral compartment. Circles: transport to the apical compartment. The experiments were done in duplicate and repeated twice. Figure 10. Expression of Ln peptide in E. coli cells Figure 10 illustrates the expression of the fusion protein consisting of MRPl L 0 peptide and thioredoxine (TRX) by BL21 cells, in cell lysates and varioius cell fractions. It can be seen that the TRX-MRP-Lo was produced by inducing with IPTG and appeared in the soluble fraction of the cells. Figure 11 Amino acid sequence of MR l L Q peptide Figure 11 shows the amino acid sequence of MRPl L 0 peptide plus some further amino acids. The amino acid numbering corresponds to the numbering of the MRPl sequence. DETAILED DESCRIPTION OF THE INVENTION Below the invention is disclosed in more detail with reference to some examples. In order to develop die assay method of the invention the functional properties of the approximately 280 amino acid long N-terminal region of human MRPl were analyzed. The so called TMD 0 and L 0 regions of MRPl (see Fig. 4A) are present in several members of the MRP family, including the SUR proteins, and partially also in CFTR, but absent in the proteins of the MDR family (MDRl, MDR3 and sPGP). By fusing the N-terminal regions of MRPl to the human MDRl protein, and by performing various mutation and co-expression studies, we examined the role of these regions in processing, plasma membrane targeting and transport of the multidrug resistance proteins. Proteins were expressed both in Sf9 insect cells and MDCKII mammalian cells. In the first system, due to the high levels of expression in a heterologous cell type, functional studies and convenient biochemical assays could be carried out in isolated membranes with less interference from any unrelated proteins. These assays included measurements of labeled glutathione-conjugate and leukotriene C 4 transport, as well as vanadate dependent nucleotide occlusion in isolated membrane vesicles. The polarized MDCKII cell monolayers offered the possibility to efficiently study membrane routing and transcellular transport properties of the overproduced proteins. In order to study the role of the N-terminal regions of MRPl, inventors constructed chimeric MRPl -MDRl proteins, in which MDRl was extended either with the full-length N-terminal region of MRPl (TMDo+Lo), or with the two separate sub-components (TMD 0 or L 0 ) of this region. The object of these experiments was to create an assay system by which L 0 -function can be studied separately from the function of other parts of the MRPl -molecule, and thereby L 0 -specific inhibitors can be tested. It was found, however, that though all of these proteins were properly expressed in both the Sf9 and MDCKII cells, essentially preserved the transport, nucleotide occlusion and apical membrane routing features of the wild-type MDRl. A possible explanation for this unexpected result i.e that MDRl protein (though its core region is very similar to that of MRP 1) could not be converted to MRP 1 in respect of any feature, can be that specific interdomain interactions are required for the function of this region within MRP 1. Whereas tlie L 0 region undoubtedly has a significant role, it could not be expected from the above results that expressing it separately is useful and that said L 0 region can be studied as a separate molecule having its own functions. In spite of that, we have prepared a construct for expression of a separate L 0 -molecule in Sf9 cells. It was unexpectedly found that the MRPl core region which is unfunctional in itself, if coexpressed with a separate L 0 peptide, resulted in a functional MRPl protein having substantially the same transport and nucleotide trapping features as die wild type protein. Thus, there is no need for a covalent linkage between the L 0 peptide and the core region of MRPl. The complex assembled from the coexpressed L 0 and ΔMRP1 had similar kinetic properties to L 0 MRP1 [Bakos et al. (1998), see above]. When ΔMR 1 was co-expressed with the L 0 region in polarized MDCKII cells, we found that L 0 was routed to die lateral membrane (Fig. 6), most likely together with ΔMRP1. Similar results were obtained in co-expression experiments with die TMD 0 L 0 region in which we could show a partial relocalization of ΔMRP1 and TMD 0 L 0 to die plasma membrane (data not shown). Although in MDCKII cells, for technical reasons, transport by these co-produced proteins could not be measured, these results suggest that the isolated TMD 0 L 0 and L 0 peptides can properly fold, attach to the cell membrane and associate with ΔMRP1. All co-expression studies in Sf9 and MDCKII cells indicate that the Lo region is a dinstict protein domain within the MRPl protein that works in specific association with the core region of MRP 1. Independently from the inventive solution it is supposed that the L 0 region contains a routing signal sufficient to induce lateral routing of the ΔMRP1 fragment. However, we can not exclude that ΔMRP1 alone is not properly folded in MDCKII cells and therefore gets stuck in the endoplasmic reticulum. Association with L 0 may result in proper folding and transport to the plasma membrane. As described in the Examples, the L 0 peptide of the MRPl was prepared also in bacterial expression system (in E. coli BL21 cells), separated from the core region, in soluble form as a fusion peptide linked to tiiioredoxine (TRX). When the lysate was added to inactive ΔMRP1 prepared in a different expression system (Sf9 cells) the transport activity of the latter could be restored in inside-out Sf9-vesicles. This result shows that L 0 peptide produced in a bacterial system has a typical structure ensuring capability of functioning, and further suggests tiiat L 0 region is a distinct domain within MRPl. Using the bacterial expression system it is possible to prepare a native functional peptide easy to purify. The obtained L 0 peptide provides an opportunity to monitor interactions with this region of MRP family transporter proteins, and to develop or test specific inhibitors more effectively. Thus, witii the set of experiments disclosed above and by developing meanwhile peptide molecules, polynucleotides, vectors and host cells, actually an assay method was created for testing L 0 - specific inhibitors of transporter proteins of the MRP family, comprising assaying an L 0 peptide exposed to the effect of a potential inhibitor, to determine whether at least one L 0 -function was retained or at least partially lost. Preferably, in the presence and in the absence of the potential inhibitor, it is determined whether at least one Lo-function was retained or not and the results obtained in each case are compared. In a preferred embodiment L 0 peptide is expressed. If the fimction studied is not independent from translation, folding or processing (resulting in the final native structure), the inhibitor should be added to the system during these periods. In a further preferred embodiment, when assaying d e Lo-function, the core region of an MRP-family transporter protein or its fragment capable of functioning, is also examined in respect of at least one L 0 -function manifesting itself only upon contact with the core region. In a further preferred embodiment the core region or a protein comprising a functional fragment thereof is also expressed. L 0 -functions, which can be preferably assayed are e.g. membrane binding, rescuing the core region of die protein to the physiological membrane compartment and/or restoring transporter protein fimction. Preferably, it can be examined whether at least one L 0 -function manifesting itself only upon contact with the core region can be restored or not. Such functions are e.g.: nucleotide trapping (occlusion) in membrane preparations; membrane routing, preferably to a specific membrane area of polarized cells; ATP-ase activity; membrane transport, preferably substrate uptake in inside-out vesicles; vectorial membrane transport into or from polarized cells. The assay method can be carried out also by preparing the L 0 peptide and the core region in different expression systems (e.g. in bacterial cell and insect cell, respectively), and contacting them afterwards. Then the potential inhibitor may block (if added previously) or abolish (if added later) the MRP-function which could have been or was restored. Besides functional studies, it is expedient to study the potential inhibitor in respect of its binding to or affinity to L 0 peptide or its amphipatic helix, by using the L 0 peptide or its mutants or fragments of the invention. Technologies useful for that purpose, such as spectroscopic methods (e.g. infrared, ultraviolet or CD spectroscopic methods), sedimental measurements, measurements based on light scattering, mobility etc. or nuclear magnetic resonance experiments are well known by the skilled person. Peptides, polynucleotides, vectors and host cells disclosed according to the invention are also claimed. Some illustrative examples of these are described in the Examples section. Molecules and assay methods of the invention are useful as developing and testing drugs behaving as inhibitors and for facilitating inhibitor design. Due to the above embodiments study of the disclosed key regions of MRP-family proteins, obtaining a detailed structural information thereon and thereby preparation of molecules capable of binding with strong affinity becomes possible. Based on the above, the claimed peptides, polynucleotides, vectors and host cells can be combined into reagent kits for developing and/or testing inhibitors specific for transporter proteins, as disclosed herein. hi the experiments with Sf9 membranes we found that the isolated L 0 peptide, which was predicted to be localized in the cytoplasm, was attached to membranes and could be solubilized only by treatment with urea or detergents. This membrane interaction of L 0 did not require the presence of the ΔMRP1 protein. Computer-based secondary structure predictions indicated that the L 0 region has two alpha-helical regions, one of which (amino acids 221-233) is a characteristic amphipathic helix, conserved in all members of the MRP family (Fig. 7C). Such an amphipathic helix may be bound by hydrophobic interactions to die lipid phase of die cell membrane or with hydrophobic regions of other membrane proteins. In order to analyze the role of die predicted amphipathic alpha-helical region within L 0 , a mutant was generated in which this region was deleted. When MRPlΔ(223-232) was expressed in both Sf9 and MDCKII cells, we found that tiiis deletion eliminated MR l transport function, but did not affect glycosylation or membrane routing. Moreover, when the isolated L 0 Δ(223-232) was produced in Sf9 cells, the mutation abolished the hydrophobic membrane attachment of the L 0 region and eliminated any functional re-activation of the ΔMRP 1 construct. A similar result was obtained when the amphipatic helix of the MRPl was exchanged for the L 0 region amphipatiiic helix of another MRP-family protein (MRP2). A chimeric L 0 obtained this way and expressed in die above-described E. coli expression system was added to ΔMRP1, but could not restore its activity. However, if only die amino acids of the supposed hydrophilic site were replaced by the corresponding amino acids of the hydrophilic site of the MRP2 L 0 amphipatic helix, transport activity was restored. Thus, the experiments showed that the predicted and experimentally supported amphipatic helix found in die L 0 region participates in membrane binding and is absolutely necessary for maintaining the functional character of MRPl. Therefore, this region is particularly useful for testing inhibitors. It can be expected tiiat an inliibitor blocking specifically a function of this region will be a particularly specific and effective inhibitor of the whole MRPl molecule. Preferably, the design of the inhibitor structure can be based on die structure of this segment. This peptide segment is particularly useful in the study of MRP type proteins and thus in multidrug resistance research. The specificity of an assayed inliibitor for the segment predicted to be an amphipatic helix can be assessed, by using the methods described above, as follows: A transporter protein or a functional transporter protein fragment comprising L 0 region (or a transporter protein the function of which was restored, e.g. by coexpression) is assayed a) in respect of specific membrane routing and/or glycosilation of the protein or its fragment, and b) in respect of any other function, preferably transporting capability of the protein, membrane binding of the L 0 peptide and/or capability of the L 0 peptide to restore MRPl function, and if the assayed functions of a) are retained and if the assayed functions of b) are at least partially considering the inhibitor to have a selective inhibiting effect specific for the segment predicted to be an amphipatic helix. As will be understood the affinity to the helix can be preferably assayed also in tliis embodiment. Further, a reagent kit can be readily provided, said kit comprising an expression vector useful for expressing a mutant L 0 region, wherein the segment of said L 0 region predicted to be an amphipatic helix or a part thereof, preferably a segment corresponding to MRPl amino acids 221 to 232 or any part thereof is deleted or substituted. The invention is further illustrated below by detailed experimental examples. EXAMPLES 1. Materials and Methods 1.1 Generation of MRPl variants and recombinant baculoviruses In order to generate tlie MRP1-MDR1 chimeric constructs, the frill length MDRl cDNA was cloned into the BamHI site of Bluescript SK. The BamHI-EcoRI (nt -8-1177) fragment was removed from Bluescript-MDRl and subcloned into M13mpl8 and an Xbal site was introduced by site-directed mutagenesis at nt 4 of MDRl. The Xbal-EcoRI fragment from this construct and the EcoRI-Notl (Notl of the polycloning site) fragment from Bluescript-MDRl were ligated together into the pVL1393 baculovirus transfer vector, yielding pVL1393MDRl. Previously, an Xbal site was introduced at nt 606 of the MRPl cDNA in M13SBMRP1 [Bakos E. et al, (1998), see above]. To obtain TMD 0 MDR1 chimeric cDNA, a Draffl-BamHI fragment (nt 87-840) was removed from the pAcSG2MRP [Bakos E. 5 et al, (1998), see above] and replaced by the Dralll-BamHI insert of M13SBMRP1. An Xbal-Xbal fragment (nt 5-606) fragment was subcloned into tlie Xbal site of pVL1393MDRl. To generate the TMDoLoMDRl the pAcSG2MRPlinkerII [Bakos E. et al, (1998), see above] was digested with Nhel within the linker I preceeding the stop codon and the Xbal site at position -5. The fragment was subcloned into the Xbal site of pVL1393MDRl. To obtain L 0 MDR1, linker I [Bakos E. et al, (1998), 10 see above] was inserted into the unique BamHI site of the pAcSG2MRPlinkerII. The pAcSG2MRPlinkerII/linkerI was digested with Nhel within linker I and II. The fragment was subcloned into the Xbal site of pVL1393MDRl. The MR lΔ(223-232) mutant was generated by overlap PCR using pJ3Ω-MRPl (23) as the template. The following primers were used: 5'-gatgtcgacaccggcatggcgctccggggcttc-3' (primer 1; Sail 15 site underlined), 5'-cactgccctccaggggctggcgccagaaggtgatcctcgacagg-3' (primer 2), 5'-cctgtcgaggatcac cttctggcgccagcccctggagggcagtg-3' (primer 3, encoding amino acids 216-222 and 233-239, and complementary to primer 2), 5'ctcctcattcgcatccaccttggaactctc3' (primer 4, amino acids 287-297). The upstream fragment was generated using primers 1 and 2, the downstream fragment using primers 3 and 4. Both fragments were gel purified, combined and a PCR reaction with primers 1 and 4 was 20 performed. The amplified overlap product was digested with Sall-BamHI and die fragment was substituted for the original Sall-BamHI fragment in pJ3Ω-MRPl, resulting in pJ3Ω-MRPl(Δ223-232). To generate the isolated L 0 the pAcSG2MRPlinkerII/linkerI construct was partially digested with Nhel in linker II and Spel in linker I. The fragment (encoding amino acids MAL+204-281+LLA+Stop) was subcloned into the Xbal site of pVL1392. To obtain the L 0 Δ(223-232) fragment, the fragment >5 encoding amino acids 204-281 was amplified by PCR using pJ3ΩMRPl(?223-232) as the template. The forward primer used was 5'-taggctagccggcatggaccctaatccctgcccaga-3', the reverse primer used was 5'-cggctagatctaatccttggaggagtacac-3'. The primers included a Nhel site and Bglll site, respectively. Tlie PCR fragment was digested with Nhel and Bglll and the fragment was cloned into pVL1393. All constructs were verified by sequence analysis. :0 Sf9 (Spodoptera frugiperda) cells were cultured and infected with a baculovirus as described earlier (11). Recombinant baculoviruses were generated by using the BaculoGold Transfection Kit (PharMingene). 1.2 Generation of MDCKII cells expressing MRPl variants For expression of MRPl variants in MDCKII cells, the L 0 MDR1 chimera, ΔMRP1, and 5 MRP2Δ(223-232) were inserted as blunt-end fragments into the retroviral vector pCMV-neo as described [Bakos E et al, (1998), see above]. For co-expression experiments the L 0 fragment was cloned into the pBabe-Puro-CMV vector (kindly provided by Dr. J. Wijnholds, The Netherlands Cancer Institute, Amsterdam, The Netherlands). This vector was generated by cloning the blunt-ended Hindlll fragment from pCMV-neo (containing the CMV early promoter) into the blunt-ended BamHI site of the pBabe-puro vector [Morgenstern, J.P., and Land, H. (1990) Nucleic Acids Res. 18, 3587- 3596]. Cells were retrovirally-transduced as described before [Evers, R., Kool, M., van Deemter, L., Janssen, H., Calafat, J., Oomen, L.C., Paulusma, C.C., Oude Elferink, R.P., Baas, F., Schinkel, A.H., and Borst, P. (1998) J. Clin. Invest. 101, 1310-1319]. MDCKII cells stably transduced with the pBabe- Puro or pCMV-neo vector were selected for 4-6 days in medium containing puromycin (3 μg/ml) or for 7 days in medium containing G-418 (800 μg/ml), respectively. 1.3 Expression of MRPl L Q peptide inE. coli cells In order to produce a fusion protein of L 0 peptide with thioredoxin a fragment coding amino acids 204 to 281 of MRPl was PCR-amplified by using pVL1393-L0 as a template. The sequence of the "forward" primer 'a' was 5 '-attggtacccgaccctaatccctgg-3 ', and the sequence of the "reverse" primer 'd' was 5'-aagaagcttaatccttggaggagtacac-3'. The primers contained a Kpnl and a Hindiπ endonuclease restriction site, respectively. The PCR fragment was digested by enzymes Kpnl and Hindlll and subcloned to die Kpnl-Hindlll site of the pCLTRX vector (Crosslink), resulting in pCLTRX-LO. The MRP1-MRP2 chimeric L 0 , comprising the amphipatic helix of MRP2, was prepared by the overlap PCR method. As a template pVL1393-L0 was used. The 5' fragment was prepared by using forward primer 'a' and reverse primer 'b' (5'-tcaga atgat gctgt catac cagct gtagg tgatc gtcga cagg-3'). The complementer of primer 'b' codes for amino acids 216 to 220 of MRPl and amino acid 206 to 214 of MRP2. The 3' fragment was prepared by using forward primer 'c' (5'-tgaca gcatc attct gaaag gctac aagca gcccc tggag ggg-3') and reverse primer 'd'. Primer V codes for amino acids 210 to 218 of MRP2 and amino acids 234 to 238 of MRPl. Fragments were isolated and pooled then a further PCR was carried out by primers 'a' and 'd'. The obtained product was digested by Kpnl and Hindlll and cloned to pCLTRX vector. The obtained pCLTRX-LO vector was cloned to E. coli BL21 (DE3) cells, which in turn were grown in TYBG antibiotic broth (10 g tryptone, 10 g yeast extract, 4 g glucose, pH 7.2, 100 μg/ml ampicillin, 25 μg/ml kanamycine) at 37°C. When OD(600) exceeded 1.1 cells were pelleted by centrifugation, suspended in M9 medium, the incubated at 28°C for 30 min. Afterwards, expression was induced by IPTG for 2 hours. When the induction was finished, cells were pelleted by centrifugation and die pellet was washed with 20% sacharose, then suspended in a solution comprising 20% agarose, 10 mM Tris and 0.1 μg/ml lysosyme (pH 7.2) and incubated for 30 min at 4°C then sonicated. The lysed cells were pelleted by centrifugation (20000 g, 20 min). The so obtained lysate was used in the further experiments, for studying the restoration of the transport activity. 1.4 Membrane preparation and immunoblotting The virus-infected Sf9 cells were harvested, their membranes isolated and stored, and the membrane protein concentrations determined as described [Sarkadi B., (1992) see above]. Immunoblotting, detection of human MRPl with the Rl and M6 monoclonal antibodies, and detection of protein-antibody interactions using the enhanced chemiluminescence technique were as described previously [Bakos E, et al., (1996), see above]. 1.5 Tracer uptake in membrane vesicles and vanadate-dependent nucleotide trapping ( 3 H)LTC 4 (135 Ci/mmol) was obtained from DuPont NEN; ( 3 H)NEM-GS was prepared from ( 3 H)NEM (60 Ci/mmol, NEN Life Science Products) by mixing die isotope in 10 mM Tris-HCl (pH 7.0) with freshly dissolved GSH in a 1:1.1 molar ratio. For ( 3 H)NEM-GS and ( 3 H)LTC 4 transport measurement, isolated membranes (200 μg of protein) were incubated in the presence of 4 mM ATP or AMP in 500 μl of transport buffer (10 mM MgCl 2 , -40 mM MOPS-Tris, pH 7.0, 50 mM KC1) at 23°C (LTC 4 ) or at 37°C (NEM-GS). 100 μl aliquots of this suspension were added to 1 ml of ice-cold transport buffer at the time points indicated and subsequently filtered rapidly through nitrocellulose membranes (0.25 μm pore size). The filters were washed twice with .5 ml of cold transport buffer, and filter-bound radioactivity was measured in scintillation fluid (Optifluor, Packard). ATP-dependent transport was calculated by subtracting the activity values obtained with AMP from those in the presence of ATP. Vanadate-dependent nucleotide trapping was measured basically as described elsewhere [Szabό K. et al, (1998) see above]. 1.6 Confocal laser scanning microscopy For immunofluorescence experiments cells were grown on microporous polycarbonate filters (3 μm pore size, 24.5 mm diameter, Transwell™ 3414; Costar Corp., Cambridge, MA) at a density of 4 x 10 5 cells/well for 3 days. Antibody incubations with mAbs M6 and Rl were as described before [Bakos E et al., (1998) see above]. Incubations with the anti-MDRl mAb MRK16 was performed as above with the modification that cells were not permeabilized with Triton X-100. FITC-labeled secondary antibodies were from Zymed Laboratories, Inc. and from Roche diagnostics. 1.7 Transport assays with MDCKII Cells Transport assays were carried out as described [Evers et al, (1996) and (1998), see above with the modification that in [ 14 C]DNP-GS transport experiments, Hanks' buffered saline solution [Evers et al., (1998) see above] was used instead of Dulbeccos's modified Eagle's medium. l-chloro-2,4-dinitro[ 14 C]benzene (lOCi/mol), and [ 3 H]vinblastine (9.4 Ci/mmol) were obtained from Amersham Pharmacia Biotech. 2. Chimeric MRP1-MDR1 variants, their expression and functions We have inserted cDNAs into baculovirus and retroviral vectors to obtain the following molecules: (i) L 0 of MRPl (amino acids 204-281), covalently coupled to the N-terminus of MDRl (LoMDRl); (ii) TMD 0 of MRPl (amino acids 1-203), covalently coupled to the N-terminus of MDRl (TMDoMDRl); and (iii) die frill N-terminal region of MRPl (TMD 0 L 0 , amino acids 1-281), covalently coupled to the N-terminus of MDRl (TMD 0 L 0 MDR1; see Fig. 1A). The MRP1-MDR1 chimeras were expressed in baculovirus-infected Sf9 cells and in retrovirus-infected MDCKII cells. For this latter system we only present the data for the L 0 MDR1 chimera, since we focused on the development of an assay method for detecting L 0 -specific inhibitors. The protein levels and the transport properties of the various mutants were studied in intact cells and in isolated membrane preparations. Fig. 1 (panels B, C, and D) presents the immunoblots of the proteins detectable in membrane preparations of Sf9 cells and in retrovirally-transduced MDCKII cells, respectively (see example 1.4). The epitope for mAb Rl is within the L 0 region [amino acids 238-247; Ppfher, D. R, Gao, m., Scheffer, G, Scheper, R. J., Deeley, R. G., and Cole, S. C. P. (1998) Br. Journal of Cancer 78, 1134-1140], while those for the polyclonal Ab 4077 and mAb C219 are within MDRl Pgp. We obtained significant expression of the chimeric MRP1-MDR1 proteins, commensurable to those of the wild-type molecules in the same expression systems. A small amount of partial proteolytic cleavage was apparent in proteins produced in the baculovirus expression system (Fig. IB), at sites close to the artificially constructed joining regions. However, the majority of the produced proteins was found at molecular mass values corresponding to the full length chimeras. In MDCKII cells the L 0 MDR1 protein, like MDRl, was glycosylated and treatment of the cells with tunicamycin abolished this glycosylation (Fig. ID). In order to examine the functional character of the chimeric proteins, we first measured ATP- dependent LTC4 and NEM-GS transport in isolated Sf9 cell membrane vesicles (see example 1.5). Despite the appreciable amounts of protein detectable in the vesicles, we did not find an MRPl -like GS-conjugate transport activity exceeding that observed in the control, β-galactosidase containing membrane vesicles (Fig. 2A). hi d e absence of any MRPl -like transport activity it was important to determine if the MRP1- MDRl chimeras retained MDRl -like hydrophobic drug transport capacity. Smce direct vesicular transport measurements with hydrophobic MDRl substrates are difficult to perform, we examined the substrate-stimulated ATPase and nucleotide trapping activity of the MRPl -MDRl chimeric proteins in isolated Sf9 membranes. In all membranes containing die chimera MRPl -MDRl proteins we found a significant verapamil-stimulated ATPase activity (not shown). However, this activity may be the consequence of a partial proteolysis of die chimeras (see Fig. IB), yielding full-length MDRl, thus nucleotide trapping was considered more informative in this regard. Covalent photoaffinity labeling of the proteins was performed with Mg-8-azido-[α- 32 P]-ATP in the presence of vanadate as a trapping agent (see Materials and Methods). It has been known that the MDRl protein is capable of nucleotide trapping when stimulated by a hydrophobic compound (e.g. verapamil) and that it is not affected by LTC4. This activity in MRPl is significantly stimulated by LTC4, but not by verapamil [Szabό, K et al and Bakos, E. et al, (1998), see above]. As shown in Fig. 2B, the nucleotide trapping of the MRP1-MDR1 chimeras was easily measurable and was vanadate-dependent. Stimulation by verapamil was observed both in the protein bands representing the full-length MRPl -MDRl chimeras, as well as in the proteolytic fragments representing the MDRl core, hi all cases a full inhibition of nucleotide trapping was obtained by pretreatment with 1 mM N-ethylmaleimide (data not shown). These functional assays demonstrate that the MRPl -MDRl chimeras retained their MDRl -like nucleotide trapping function. In the following experiments, we examined the membrane localization and transport function of the LoMDRl chimeric protein in retrovirally transduced MDCKII cell monolayers by Confocal Laser Scanning Microscopy (CLSM). L 0 MDR1 was detected using mAb MRK16, an antibody recognizing a conformational epitope on the extracelular loops of MDRl Pgp [Ha ada, H, and Tsuruo, T. (1986) Proc. Natl. Acad. Sci. USA 83, Cancer Res. 7785-7789]. As shown in Fig. 3A, L 0 MDR1 was predominantly localized in the apical membrane of MDCKII cell monolayers. In accordance with an apical localization, the MDCKII-L 0 MDR1 and MDCKII-MDR1 cells showed an increased apical transport of vinblastine (Fig. 3B). The vectorial transport activity of L 0 MDR1 correlated with its protein levels as compared to that of MDRl (Fig. 1C). We found no increased transport of organic anions, more closely DNP-GS, by the MDCKII-L 0 MDR1 cells (data not shown). Tlie combination of these results clearly indicates that the MRP1-MDR1 chimeras were produced in both insect and mammalian cells and that the chimeric proteins retained the features of the MDRl protein. Since L 0 , when coupled to MDRl protein, does not result in any change in functions, its functional behaviour can not be studied in the system described above. Therefore, no assay system, suitable for testing of L 0 -specific inhibitors, could be created using chimeric constructions disclosed in this example. 3. Mutant MRPl variants, their expression and functions hi order to analyse the specific role of the N-terminal linker region of MRPl in more detail, we have expressed the following mutant MRPl constructs in both Sf9 and MDCKII cells: (i) An N- terminally truncated MRPl (amino acids 281-1531), containing the two ABC units and the 2 x 6 TM (transmembrane) helices [ΔMRP1; Bakos E et al., (1998), see above]; (ii) amino acids 204-281, representing the N-teπninal linker region of MRPl (L 0 ), (Fig. 4.A). In the experiments below the existence of MRPl functions was examined for each function, in case of ΔMRP1 and co-expressing ΔMRP1 and L 0 peptide, and analysed the behaviour of L 0 in itself and what functions can be restored by it. 3.1 Expression in Sf9 cells Fig. 4B demonstrates the immunoblot detection of the ΔMRP1 and L 0 variants in Sf9 cells by the anti-MRP 1 monoclonal antibodies Rl and M6. The epitope for mAb M6 is at the C-terminus of MRPl [ammo acids 1511-1520; Hipfiier et al., (1998) see above]. For all these molecules we obtained appreciable expression, similar to that of wild-type MRPl (Fig. 4B). It is important to note (see also below) that, like wild-type MRPl and ΔMRP1, die L 0 polypeptide was found to be almost entirely present in the membrane fraction of the cell lysates. The amount of this membrane-bound L 0 was independent of the presence or absence of ΔMRP1 in the Sf9 cell membranes. Slight variations in the expression levels of these mutant proteins between various membrane preparations were still observed and therefore the functional data were corrected for the specific protein expression levels (see below). 3.2 Transport of MRPl substrates ILTCA and NEM-GS. Muller. M. et al. (1994). Bakos E. et al. (1998). see abovel In order to correctly determine the transport function of the different MRPl proteins, we estimated the relative amount of transport-competent inside-out vesicles in the Sf9 membrane preparations [Bakos E. et al, (1998), see above], and corrected the transport rates according to the relative MRPl expression levels, as determined by immunoblotting. Fig. 4, panels C and D, shows the time-dependent uptake of [ 3 H]LTC4 (50 nM) and [ 3 H]NEM- GS (50 μM) in Sf9 membrane vesicles containing MRPl, ΔMRP1, or the co-expressed L 0 and ΔMRP1. Full-length MRPl produced an ATP-dependent increase in the vesicular radioactivity which for LTC 4 was linear for about 30 seconds at 23°C and was linear for NEM-GS for about 3 minutes at 37°C. The initial rate of ATP-dependent LTC4 transport by the MRPl containing membranes was about 60 pmol mg-1 min-1 while that of NEM-GS transport was about 250 pmol mg~l min" under these conditions. As shown previously (20) rapid washing of the vesicles with cold substrate-containing solutions did not alter the vesicular LTC4 and NEM-GS levels, while the addition of concentrated sucrose (1 M) or low concentrations of Triton X-100 (0.02%) to the media eliminated ATP-dependent tracer accumulation. As demonstrated in Fig. 4C and D, we found that the expression of ΔMRP1 did not result in any transport activity (as compared to transport measured in the control, β-galactosidase expressing vesicles). However, the co-expression of ΔMRP1 and the L 0 region resulted in significant uptake of LTC4 (at 50 nM about 90% of MRPl) and NEM-GS (at 50 μM NEM-GS about 40 % of MRPl). Again, in all these cases intravesicular tracer uptake was eliminated by the addition of Triton X-100 or 1 M sucrose. Examination of tlie kinetic parameters for the transport of LTC by the co-expressed ΔMRP1 and Lo showed that the Km values were similar as for wild-type MRPl (about 140 nM). However, for transport of NEM-GS the Km of wild-type MRPl was 150 μM, while that of the co-expressed ΔMRPl+Lo was about 400 μM. The maximum transport rate in this latter case was approaching the level observed for MRPl (data not shown). Thus, co-expression of L 0 region and ΔMRP1 provides a possibility to determine an L 0 -function separately, namely that L 0 is capable of restoring ΔMRP1 activity in respect of MRPl substrates. Thus, the system unexpectedly proved to be promising in testing L 0 -specific inhibitors. 3.3 vanadate sensitive nucleotide trapping in Sf9 cell membranes Next, we examined substrate-stimulated, vanadate sensitive nucleotide trapping in Sf9 cell membranes containing the various MRPl variants (Fig. 5). As reported before [Bakos E. et al, (1998), see above], ΔMRP1 did not show any nucleotide trapping. However, as shown in Fig. 5, when the L 0 peptide was co-expressed with the ΔMRP1 region, the full activity was restored and a stimulation of the nucleotide trapping by LTC was observed, similar to that found for wild-type MRPl. Again, in all cases a full inhibition of nucleotide trapping was obtained by pretreatment with 1 mM N- ethylmaleimide. Thus, it was found again that the Lo-function restoring MRPl activity can be tested and thereby a further method, suitable for assessing effectiveness of an L 0 -specific inhibitor, was provided. 3.4 Rescue of ΔMRP1 to the plasma membrane It was shown previously tiiat ΔMRP1 in MDCKII cells is localized in an intracellular compartment, probably the endoplasmic reticulum [Bakos E. et al, (1998), see above]. To investigate whether the L 0 peptide could rescue ΔMRP1 to the plasma membrane, we transduced MDCKII- ΔMRP1 cells with the pBabe-Puro-CMV-L o construct (Example 1.2). Upon selection with puromycin the localization of L 0 was determined using mAb Rl. In a pool of puromycin resistant cells clear lateral plasma membrane staining was observed in small groups of cells, although some intracellular staining was also present in these cells (Fig. 6). As only a low percentage of cells showed staining with mAb Rl, it was impossible to isolate single clones. Therefore, immunoblotting and transepithelial transport measurements could not be performed. No signal was obtained after infection of wild-type cells with the Lo containing construct, probably due to rapid degradation of this small protein in the absence of ΔMRPl. The studies documented above strongly suggest that tlie isolated L 0 polypeptide is able to associate with ΔMRP1 and that as a consequence the protein regains its function and routes to the lateral membrane. An L 0 -specific inhibitor will most probably inhibit this function, too. 3.5 Membrane association of L n peptide As mentioned above (see Fig. 4B), a significant amount of the L 0 protein produced in Sf9 cells was found to be asssociated with cellular membranes. Fig. 7A documents that in tlie Sf9 cell lysates, most of die L 0 protein was found in the membrane fraction and that only a small fraction was in the cytoplasm. Washing the Sf9 cell membranes with 1 M KCl did not change this membrane-bound localization of the L 0 peptide. However, washing the membranes with 5 M urea or 1% Triton X-100 completely solubilized the L 0 peptide (Fig. 7B). Co-expression of ΔMRP1 in the same cell did not affect the binding of L 0 to die membrane or sensitivity towards treatment with urea or Triton X-100. Treatment of membranes with urea had no effect on the membrane localization of MRPl or ΔMR 1, and even treatment with 1% Triton X-100 resulted only in a 20-40% solubilization of these membrane- spanning proteins (data not shown). Thus, membrane association of L 0 peptide can be studied separately and can be considered as an Lo-function which can be inhibited. It follows from the above that functions of L 0 peptide can be assayed in a system comprising one or more methods disclosed above. Expectedly it is true for each transporter protein of the MRP-family having an L 0 region homologous to or functionally corresponding to the L 0 region of MRP 1. 3.6 Expression of Lo peptide in a bacterial expression system Lo peptide was expressed in a soluble form, as a fusion protein, using an expression vector operable in BL21 bacterial cells (Promega). The MRPl L 0 region was cloned into pCLTRX vector (Crosslink) comprising a coding sequence for thioredoxin (TRX) fusion protein and obtained pCLTRX-Lo. Using this vector protein was produced in BL21 cells (see MATERIALS AND METHODS). Cell were lysed with lysosyme and the soluble fraction of them was separated by centrifugation. The photo of die gel stained by Coomassie shows that TRX-L 0 was produced in a soluble form (Fig. 10). The effect of TRX-L 0 to the transport activity of ΔMRP1 was assayed, too. Lysate of cells containing TRX-L 0 was added to membrane vesicles containing ΔMRP1, then ATP dependent vesicular LTC 4 uptake was measured. The transport activity of the inactive ΔMRP1 was restored. 3.7 The study of the conservative, hydrophobic. membrane binding segment of the Lo peptide The salt-insensitive, but denaturing agent- or detergent-sensitive membrane attachment of the L 0 region raised the possibility of the presence of a membrane-attaching amphipathic region (e.g. helix) within Lo. Indeed, as shown in Fig. 7C, secondary structure predictions indicated the presence of such a conserved amphipathic helix in all currently known MRP homologs (such a helix is also present in the MRP-related CFTR and SUR proteins; data not shown). In order to examine the role of this amphipathic helix within the L 0 region, we constructed a 10 amino acid deletion (Δ223-232) within this predicted helix, both in the context of the isolated L 0 fragment and in the full-length MRPl molecule. Fig 8A demonstrates that MRPlΔ(223-232) was produced in Sf9 cells in similar quantities as the wild-type MRPl protein. Fig. 8B shows LTC4 transport experiments in isolated Sf9 cells expressing MRPlΔ(223-232), and Fig. 8C shows the membrane interaction of L 0 Δ(223-232). In contrast to wild-type MRPl, MRPlΔ(223-232) had no appreciable transport activity and the L 0 Δ(223-232) did not attach to the cell membrane. When the L 0 Δ(223-232) was co-expressed with ΔMRP1 or when a concentrated supernatant containing L 0 Δ(223- 232) was added to isolated ΔMRP1 containing-membranes, no transport activity for LTC4 was observed (data not shown). Fig. 9 shows the expression, localization and function of the MRPlΔ(223-232) protein produced in MDCKII monolayers. As shown hi Fig. 9A, the MRPlΔ(223-232) protein was efficiently expressed and similar to MRPl, was almost completely glycosylated in these cells. Tunicamycin treatment resulted in the loss of glycosylation both in the MRPl and the MRPlΔ(223-232) expressing cells (Fig. 9B). Similar to wild-type MRPl [Bakos E. et al, (1998), see above], MRPlΔ(223-232) was localized in the lateral membrane in MDCKII cells (Fig. 9C). However, when the transport activities of MRPl and MRPlΔ(223-232) were compared in polarised cell monolayers, tiiere was no detectable increase in the export of DNP-GS by the MRPlΔ(223-232) protein (Fig. 9D) relative to the baseline export activity of the cells. In a further set of experiments the amphipatic helix of MRPl was exchanged with the L 0 region amphipatic helix of another member of the MRP-family (MRP2). When the so obtained chimeric L 0 was added to ΔMRP1, it could not restore ΔMRP1 activity. Expression systems and constructs desclosed in the description are illustrative and non-limiting. The segment of L 0 peptide predicted to be an amphipatic helix and having key importance, preferably the segment corresponding to amino acids 221 to 232, is particularly suitable for designing inhibitors specific to said segment. Using the constructs disclosed above and by assaying appropriate Lo-fuctions any potential inhibitor can be tested also for its specificity for this conservative region within the L 0 being particularly important for membrane binding. If such a preferred inhibitor is to be identified, a mutant construct can be created from which this region so important in membrane binding is deleted or in which is inactivated. Such a mutant may have certain functions which are different from those taught herein. It is to be understood that by deleting or substituting a shorter or a longer amino acid segment a molecule mutated on substantially the same principle can be prepared, which can be used for testing or developing inhibitors, for finding narrower key segments or key amino acids of proteins of the MRP- family, for understanding structural correlations and tiiereby disclosing functions. Informations obtained this way can be used in inhibitor or drug design. Since it is known that MRPl plays an important role in resistance of human tumors against clinical chemotherapy [Hipfher, D. R, Deeley, R. D, and Cole, S. P. C. (1999) Biochim Biophys Acta 1461. 359-376], the specific and effective assay method, molecules and host cells of the invention may have a significant part in research of transporter proteins of the MRP-family, testing and designing inhibitors of them and thereby combatting multidrug resistance.

Claims: CLAIMS 1. An assay method for testing L 0 -specific inhibitors of transporter proteins of the MRP family, comprising assaying an L 0 peptide exposed to the effect of a potential inliibitor, to determine whether at least one L 0 -function was retained or at least partially lost. 2. A method of claim 1 comprising expressing the L 0 peptide and optionally exposing the L 0 peptide to the effect of the potential inhibitor before the final formation of its native structure. 3. A method of any of claims 1 or 2 wherein assaying the L 0 peptide comprises the steps of using a deletion mutant protein prepared by expression or its fimctional fragment, said protein or fragment comprising an MRP -family transporter protein core region; and assaying at least one L 0 -function manifesting itself only upon contact with the core region. 4. A method of claim 3 comprising expressing, or optionally coexpressing, both the L 0 peptide and the core region or its fuctional fragment. 5. A method of any of claims 3 or 4 comprising assaying L 0 peptide to determine whether at least one of the following functions was retained: membrane binding, rescuing the core region of the protein to the physiological membrane compartment and/or restoring transporter protein function. 6. A method of any of claims 3 to 5 wherein the at least one L o -function manifesting itself only upon contact with the core region is selected from the following: nucleotide trapping in membrane preparations; membrane routing, preferably to a specific membrane area of polarised cells; ATP-ase activity; membrane transport, preferably substrate uptake in inside-out vesicles; vectorial membrane transport into or from polarised cells. 7. An assay method for screening potential inhibitors a transporter protein of the MRP family said inhibitor being specific for a segment of the L 0 region predicted to be an amphipatic helix comprising determining whether the potential inhibitor exhibits affinity for the segment predicted to be an amphipatic helix. 8. An assay method for testing inhibitors of MRP family transporter proteins specific for an L 0 region segment predicted to be an amphipatic helix, comprising assaying a transporter protein of the MRP family having exposed to die effect of a potential inhibitor; a functional transporter protein fragment comprising L 0 region; or an L 0 peptide and a protein consisting of a transporter protein core region (or a functional fragment thereof) said peptide and said protein being not linked covalently; a) in respect of specific membrane routing and/or glycosilation of the protein or its fragment, and b) in respect of any other fimction, preferably transporting capability of the protein, membrane binding of die L 0 peptide and/or MRPl function restoring capability of the L 0 peptide, and if die assayed functions of a) are retained and if the assayed functions of b) are at least partially lost considering the result as the selective inhibiting effect of the inliibitor specific for the segment predicted to be an amphipatic helix. 9. Lo peptide or a mutant or homologue thereof retaining at least partly at least one L 0 -fuction, for use in testing, designing or developing L 0 -specific inhibitors of transporter proteins of the MRP family, in multidrug resistance research or as a validated drug target. 10. Lo peptide or its mutant of claim 9 comprising amino acids 221 to 227 of MRPl L 0 peptide. 11. Mutant L o peptide carrying a mutation in the peptide segment predicted to be an amphipatic helix, preferably in tlie segment consisting of amino acids 221 to 227, said mutation being preferably a deletiojn-or a substitution. M ^ A polynucleotide comprising a sequence coding L 0 region of a transporter protein of the MRP family for use in expressing L 0 peptide. 13. A polynucleotide of claim 12 wherein said polynucleotide is an expression vector, preferably an expression vector capable of functioning in a bacterial, an insect and/or a mammalian cell. 14. A host cell carrying a vector of claim 13. 15. A reagent kit for developing or testing specific inhibitors of transporter proteins of the MRP family or for multidrug resistance research, said kit comprising a peptide of any of claims 9 to 11, a polynucleotide of any of claims 12 to 13 or a host cell of claim 14, and optionally further comprising an expression vector suitable to express the transporter protein or a functional mutant diereof, preferably a deletion mutant substantially consisting of the core region of the transporter protein, host cells, preferebly bacterial, insect and/or mammalian cells, in which the expression vector is operable, and reagents and buffers for the expression and/or assaying functions of the expressed L 0 peptide. 16. A reagent kit according to claim 15, wherein the vector is a vector operable in E. coli, Sf9 and/or MDCKII cells and the host cells are E. coli, Sf9 and/or MDCKII cells. 17. A drug behaving as an L 0 -specific inhibitor of a protein of the MRP family, if assayed by a method of any of claims 1 to 8. 18. A drug according to claim 17 behaving as an inhibitor, wherein said drug is capable to effect due to a contact with an L 0 region segment predicted to be an amphipatic helix or a part thereof.

Inventors: BAKOS EVA, EVERS RAYMOND, SARKADI BALAZS, TUSNADI GABOR, VARADI ANDRAS, BAKOS, EVA, EVERS, RAYMOND, SARKADI, BALAZS, TUSNADI, GABOR, VARADI, ANDRAS
Applicants: BAKOS EVA, EVERS RAYMOND, SARKADI BALAZS, SOLVO BIOTECHNOLOGY LTD, TUSNADI GABOR, VARADI ANDRAS, BAKOS, EVA, EVERS, RAYMOND, SARKADI, BALAZS, SOLVO BIOTECHNOLOGY LTD., TUSNADI, GABOR, VARADI, ANDRAS
Application year: 2001
Main classification: G01N33
IPC codes: A61K45, A61P35, A61P43, C07K14, C12N1, C12N5, C12N15, C12Q1, G01N33