DOI: 10.1002/cmdc.201000216 Specific Inhibitors of the Breast Cancer Resistance Protein (BCRP) Anne Pick, Werner Klinkhammer, and Michael Wiese* [a] Introduction The current leading cause of failure in cancer chemotherapy is the development of multidrug resistance (MDR), in which cancer cells become simultaneously resistant to structurally un- related chemotherapeutic agents. The ATP binding cassette (ABC) transporters, especially P-glycoprotein (P-gp, ABCB1), the multidrug-resistance-associated protein 1 (MRP1, ABCC1), and the breast cancer resistance protein (BCRP, ABCG2) play key roles in the development of MDR. [1] By using the energy of ATP hydrolysis these members of the ABC family transport a wide variety of structurally unrelated substrates out of cells against a concentration gradient, leading to decreased intracellular concentrations and failure of chemotherapy. Furthermore, ABC transporters such as P-gp, BCRP, and MRP1 are widely ex- pressed in excreting organs and physiological barriers, where they play a physiological role in protection against toxic agents. [2, 3] To date 48 human ABC transporters have been identified and classified into seven subfamilies. [4] P-gp, which was discov- ered first, belongs to the ABCB subfamily. [5] It consists of two similar domains, each containing six transmembrane segments and a nucleotide binding domain. MRP1, a member of the ABCC subfamily, has an additional N-terminal extension with five transmembrane segments. [6] The most recently discovered ABC transporter, BCRP, [7] belongs to the ABCG subfamily and is considered to be a half-transporter. BCRP is composed of only a single N-terminal intracellular nucleotide binding domain, fol- lowed by six transmembrane segments. BCRP achieves its functionality by either homo- or heterodimerization. [8] P-gp transports a wide range of structurally unrelated drugs. They are usually amphipathic, [9] hydrophobic compounds with aromatic ring systems and molecular weights between 400 and 1900 Da. [10] In contrast to P-gp, MRP1 functions mainly as a transporter of amphipathic organic anions. BCRP is able to efflux miscellaneous molecules, both positively and negatively charged, with amphiphilic character. Transported substrates in- clude sulfated hormone metabolites and glucuronidated me- thotrexate. [11] Further substrates of BCRP include the anticancer drugs mitoxantrone, topoisomerase 1 inhibitors such as topo- tecan and irinotecan, the podophyllotoxins etoposid and teni- posid, and flavopiridol. [3, 12] An important approach to overcome MDR is the inhibition of these proteins by modulators with high potency. According to Ahmed-Belkacem et al., the known BCRP inhibitors can be divided into four categories: 1) ABCG2-specific inhibitors, 2) compounds that also inhibit P-gp and/or MRP1 (broad-spec- trum inhibitors), 3) naturally occurring flavonoids and deriva- tives, and 4) tyrosine kinase inhibitors (TKIs). [13] Specific inhibi- tors of ABCG2 include fumitremorgin C (FTC) [14] and novobio- cin. [15] More recently, a nontoxic, synthetic analogue of FTC, Ko143, was identified as a potent and specific modulator of BCRP, inhibiting BCRP with high affinity. [14] With the exception A new class of specific breast cancer resistance protein (BCRP) inhibitors was identified, showing no inhibition of the ATP binding cassette (ABC) transporters P-gp and MRP1. Some of these modulators inhibit BCRP with high potency; they are only slightly less potent than Ko143 and could serve as promis- ing lead structures for the design of novel effective BCRP in- hibitors. These inhibitors are structurally related to tariquidar (XR9576) and belong to a library of multidrug-resistance mod- ulators synthesized by our research group. The absence of the tetrahydroisoquinoline substructure appears to play a crucial role for specificity; we found that the presence of this sub- structure is not essential for interaction with BCRP. To deter- mine the type of interaction between pheophorbide A and compounds with and without the tetrahydroisoquinoline sub- structure, various substrate pheophorbide A concentrations were used in enzyme kinetics assays. The resulting data show that these compounds share a noncompetitive-type interaction with pheophorbide A. Experiments with imatinib and pheo- phorbide A revealed a mixed-type interaction. The combina- tion of imatinib and compounds with and without the tetrahy- droisoquinoline substructure resulted in a positive cooperative effect, indicating that imatinib engages a binding site distinct from that of the new compounds on one side and distinct from that of pheophorbide A on the other side as well. The re- sults of this study suggest that the category of BCRP-specific inhibitors, which includes only fumitremorgin C, Ko143 and an- alogues, and novobiocin needs to be extended by this new class of inhibitors, which possess three key characteristics: spe- cificity, potency, and low toxicity. [a] A. Pick, Dr. W. Klinkhammer, Prof. Dr. M. Wiese Institute of Pharmacy, University of Bonn An der Immenburg 4, 53121 Bonn (Germany) Fax: (+ 49) 228-737929 E-mail : mwiese@uni-bonn.de 1498  2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemMedChem 2010, 5, 1498 – 1505 MED