calcein-am and pheophorbide-a

A virtual screening protocol with combination of similarity search and pharmacophore modeling was applied to virtually screen a large compound library to gain new scaffolds regarding ABCC1 inhibition. Biological investigation of promising candidates revealed four compounds as ABCC1 inhibitors, three of them with scaffolds not associated with ABCC1 inhibition until now. The best hit molecule-a thienopyrimidine-was a moderately potent, competitive inhibitor of the ABCC1-mediated transport of calcein AM which also sensitized ABCC1-overexpressing cells toward daunorubicin. Further evaluation showed that it was a moderately potent, competitive inhibitor of the ABCB1-mediated transport of calcein AM, and noncompetitive inhibitor of the ABCG2-mediated pheophorbide A transport. In addition, the thienopyrimidine could also sensitize ABCB1- as well as ABCG2-overexpressing cells toward daunorubicin and SN-38, respectively, in concentration ranges that qualified it as one of the ten best triple ABCC1/ABCB1/ABCG2 inhibitors in the literature. Besides, three more new multitarget inhibitors were identified by this virtual screening approach.

Topics: ATP Binding Cassette Transporter, Subfamily B; ATP Binding Cassette Transporter, Subfamily G, Member 2; Cell Line, Tumor; Chlorophyll; Daunorubicin; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Fluoresceins; Humans; Irinotecan; Multidrug Resistance-Associated Proteins; Neoplasm Proteins; Pyrimidines; Small Molecule Libraries; Thiophenes

Five series of pyrrolo[3,2-d]pyrimidines were synthesized and evaluated with respect to potency and selectivity toward multidrug resistance-associated protein 1 (MRP1, ABCC1). This transport protein is a major target to overcome multidrug resistance in cancer patients. We investigated differently substituted pyrrolopyrimidines using the doxorubicin selected and MRP1 overexpressing small cell lung cancer cell line H69 AR in a calcein AM and daunorubicin cell accumulation assay. New compounds with high potency and selectivity were identified. Piperazine residues at position 4 bearing large phenylalkyl side chains proved to be beneficial for MRP1 inhibition. Its replacement by an amino group led to decreased activity. Aliphatic and aliphatic-aromatic variations at position 5 and 6 revealed compounds with IC50 values in high nanomolar range. All investigated compounds had low affinity toward P-glycoprotein (P-gp, ABCB1). Pyrrolopyrimidines with small substituents showed moderate inhibition against breast cancer resistance protein (BCRP, ABCG2).

Topics: Animals; Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Cell Line, Tumor; Chlorophyll; Daunorubicin; Dogs; Doxorubicin; Drug Evaluation, Preclinical; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Fluoresceins; Humans; Inhibitory Concentration 50; Madin Darby Canine Kidney Cells; Multidrug Resistance-Associated Proteins; Neoplasm Proteins; Pyridines; Pyrroles; Structure-Activity Relationship

Many studies have been performed with the aim of developing effective resistance modulators to overcome the multidrug resistance (MDR) of human cancers. Potent MDR modulators are being investigated in clinical trials. Many current studies are focused on dietary herbs due to the fact that these have been used for centuries without producing any harmful side effects. In this study, the effect of tetrahydrocurcumin (THC) on three ABC drug transporter proteins, P-glycoprotein (P-gp or ABCB1), mitoxantrone resistance protein (MXR or ABCG2) and multidrug resistance protein 1 (MRP1 or ABCC1) was investigated, to assess whether an ultimate metabolite form of curcuminoids (THC) is able to modulate MDR in cancer cells. Two different types of cell lines were used for P-gp study, human cervical carcinoma KB-3-1 (wild type) and KB-V-1 and human breast cancer MCF-7 (wild type) and MCF-7 MDR, whereas, pcDNA3.1 and pcDNA3.1-MRP1 transfected HEK 293 and MXR overexpressing MCF7AdrVp3000 or MCF7FL1000 and its parental MCF-7 were used for MRP1 and MXR study, respectively. We report here for the first time that THC is able to inhibit the function of P-gp, MXR and MRP1. The results of flow cytometry assay indicated that THC is able to inhibit the function of P-gp and thereby significantly increase the accumulation of rhodamine and calcein AM in KB-V-1 cells. The result was confirmed by the effect of THC on [(3)H]-vinblastine accumulation and efflux in MCF-7 and MCF-7MDR. THC significantly increased the accumulation and inhibited the efflux of [(3)H]-vinblastine in MCF-7 MDR in a concentration-dependent manner. This effect was not found in wild type MCF-7 cell line. The interaction of THC with the P-gp molecule was clearly indicated by ATPase assay and photoaffinity labeling of P-gp with transport substrate. THC stimulated P-gp ATPase activity and inhibited the incorporation of [(125)I]-iodoarylazidoprazosin (IAAP) into P-gp in a concentration-dependent manner. The binding of [(125)I]-IAAP to MXR was also inhibited by THC suggesting that THC interacted with drug binding site of the transporter. THC dose dependently inhibited the efflux of mitoxantrone and pheophorbide A from MXR expressing cells (MCF7AdrVp3000 and MCF7FL1000). Similarly with MRP1, the efflux of a fluorescent substrate calcein AM was inhibited effectively by THC thereby the accumulation of calcein was increased in MRP1-HEK 293 and not its parental pcDNA3.1-HEK 293 cells. The MDR reversing properties of THC on P-

Topics: Adenosine Triphosphate; Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily B; ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Beryllium; Cell Line, Tumor; Chlorophyll; Curcumin; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Etoposide; Fluoresceins; Fluorescent Dyes; Fluorides; Humans; Mitoxantrone; Molecular Structure; Multidrug Resistance-Associated Proteins; Neoplasm Proteins; Organic Anion Transporters; Rhodamine 123; Vinblastine