Cyclohexyl-linked tricyclic isoxazoles are potent and selective modulators of the multidrug resistance protein (MRP1).

Structure-activity relationship (SAR) studies on the tricyclic isoxazole series of MRP1 modulators have resulted in the identification of potent and selective inhibitors containing cyclohexyl-based linkers. These studies ultimately identified compound 21b, which reverses drug resistance to MRP1 substrates, such as doxorubicin, in HeLa-T5 cells (EC(50)=0.093microM), while showing no inherent cytotoxicity. Additionally, 21b inhibits ATP-dependent, MRP1-mediated LTC(4) uptake into membrane vesicles prepared from the MRP1-overexpressing HeLa-T5 cells (EC(50)=0.064microM) and shows selectivity (1115-fold) against the related transporter, P-glycoprotein, in HL60/Adr and HL60/Vinc cells. Finally, when dosed in combination with the oncolytic MRP1 substrate vincristine, 21b showed tumor regression and growth delay in MRP1-overexpressing tumors in vivo.

Akt activation, but not extracellular signal-regulated kinase activation, is required for SDF-1alpha/CXCR4-mediated migration of epitheloid carcinoma cells.

Emerging evidence shows that the stromal cell-derived factor 1 (SDF-1)/CXCR4 interaction regulates multiple cell signaling pathways and a variety of cellular functions such as cell migration, proliferation, and survival. There is little information linking the cellular functions and individual signaling pathways mediated by SDF-1 and CXCR4 in human cancer cells. In this study, we have shown that human epitheloid carcinoma HeLa cells express functional CXCR4 by reverse transcription-PCR, immunofluorescent staining, and 125I-SDF-1alpha ligand binding analyses. The treatment of HeLa cells with recombinant SDF-1alpha results in time-dependent Akt and extracellular signal-regulated kinase 1/2 (ERK1/2) activations. The SDF-1alpha-induced Akt and ERK1/2 activations are CXCR4 dependent as confirmed by their total inhibition by T134, a CXCR4-specific peptide antagonist. Cell signaling analysis with pathway-specific inhibitors reveals that SDF-1alpha-induced Akt activation is not required for ERK1/2 activation and vice versa, indicating that activations of Akt and ERK1/2 occur independently. Functional analysis shows that SDF-1alpha induces a CXCR4-dependent migration of HeLa cells. The migration can be totally blocked by phosphoinositide 3-kinase inhibitors, wortmannin or LY294002, whereas mitogen-activated protein/ERK kinase inhibitors, PD98059 and U0126, have no significant effect on SDF-1alpha-induced migration, suggesting that Akt activation, but not ERK1/2 activation, is required for SDF-1alpha-induced migration of epitheloid carcinoma cells.

Evaluation of the binding of the tricyclic isoxazole photoaffinity label LY475776 to multidrug resistance associated protein 1 (MRP1) orthologs and several ATP- binding cassette (ABC) drug transporters.

Several of the ATP-binding cassette (ABC) transporters confer resistance to anticancer agents and/or antiviral agents when overexpressed in drug-sensitive cells. Recently a MRP1 (ABCC1) tricyclic isoxazole inhibitor, LY475776 was shown to be a glutathione-dependent photoaffinity label of human MRP1 and showed poor labeling of murine mrp1, an ortholog that does not confer anthracycline resistance. In the present study, the specificity of LY475776 was examined for its ability to modulate or photolabel orthologs of MRP1 and several other drug efflux transporters of the ABC transporter family. LY475776 modulated MRP1 and Pgp-mediated resistance (MDR, ABCB1) in, respectively, HeLa-T5 and CEM/VLB(100) cells to both vincristine and doxorubicin. LY475776 photolabeled 170kDa Pgp and was inhibited by the potent Pgp inhibitor LY335979 (Zosuquidar.3HCl). The labeling of the 190kDa MRP1 protein in membranes of HeLa-T5 cells was inhibited by substrates of MRP1 such as leukotriene C(4), vincrisine, and doxorubicin and by the inhibitor, MK571. LY475776 did not photolabel human MRP2 (ABCC2), MRP3 (ABCC3), MRP5 (ABCC5) or breast cancer resistance protein (ABCG2). Because LY475776 photolabels murine mrp1 less well than human MRP1 and binds to a region believed important for anthracycline binding, studies were conducted with monkey and canine MRP1 which also show a reduced ability to confer resistance to anthracyclines. Unlike murine mrp1, both orthologs were photolabeled well by LY475776. These studies indicate that the specificity of LY475776 is fairly limited to Pgp and MRP1 and further studies will help to define the binding regions.

Tricyclic isoxazoles are novel inhibitors of the multidrug resistance protein (MRP1).

Tricyclic isoxazoles were identified from a screen as a novel class of selective multidrug resistance protein (MRP1) inhibitors. From a screen lead, SAR efforts resulted in the preparation of LY 402913 (9h), which inhibits MRP1 and reverses drug resistance to MRP1 substrates, such as doxorubicin, in HeLa-T5 cells (EC(50)=0.90 microM), while showing no inherent cytotoxicity. Additionally, LY 402913 inhibits ATP-dependent, MRP1-mediated LTC(4) uptake into membrane vesicles prepared from the MRP1-overexpressing HeLa-T5 cells (EC(50)=1.8 microM). LY 402913 also shows selectivity ( approximately 22-fold) against the related transporter, P-glycoprotein, in HL60/Adr and HL60/Vinc cells. Finally, when dosed in combination with the oncolytic MRP1 substrate vincristine, LY 402913 delays the growth of MRP1-overexpressing tumors in vivo.