A Photo-Activatable Peptide Mimicking Functions of Apolipoprotein A-I.

Apolipoprotein A-I (apoA-I) plays a critical role in high-density lipoprotein (HDL) biogenesis, function and structural dynamics. Peptides that mimic apoA-I have a short amphipathic α-helical structure that can functionally recapitulate many of the same biologic properties of full-length apoA-I in HDL. Hence, they might be expected to have clinical applications in the reduction of atherosclerosis. However, nonspecific cellular efflux of cholesterol induced by apoA-I mimetic peptides might cause side effects that are, as yet, unidentified. In this study, we developed a photo-activatable peptide, 2F*, which is an 18 amino acid peptide mimicking apoA-I bearing an internal photocleavable caging group that is designed to assume an α-helical structure in response to a light stimulus and trigger efflux of cholesterol from cells. Without light irradiation, 2F* peptide showed a low tendency for the formation of α-helices, and therefore did not associate with lipids and failed to induce efflux of cholesterol. In addition, 2F* did not cause hemolysis under our experimental condition. Mass spectrometry indicated that, after light exposure, the caging group detached from 2F* and it assumed the α-helical structure in the presence of lipids, and enhanced cholesterol efflux from cells. Photo-activatable peptides such as 2F* that control cholesterol efflux following light stimulus may be useful for future atherosclerosis-reducing therapies.

Targeted Degradation of Proteins Localized in Subcellular Compartments by Hybrid Small Molecules.

Development of novel small molecules that selectively degrade pathogenic proteins would provide an important advance in targeted therapy. Recently, we have devised a series of hybrid small molecules named SNIPER (specific and nongenetic IAP-dependent protein ERaser) that induces the degradation of target proteins via the ubiquitin-proteasome system. To understand the localization of proteins that can be targeted by this protein knockdown technology, we examined whether SNIPER molecules are able to induce degradation of cellular retinoic acid binding protein II (CRABP-II) proteins localized in subcellular compartments of cells. CRABP-II is genetically fused with subcellular localization signals, and they are expressed in the cells. SNIPER(CRABP) with different IAP-ligands, SNIPER(CRABP)-4 with bestatin and SNIPER(CRABP)-11 with MV1 compound, induce the proteasomal degradation of wild-type (WT), cytosolic, nuclear, and membrane-localized CRABP-II proteins, whereas only SNIPER(CRABP)-11 displayed degradation activity toward the mitochondrial CRABP-II protein. The small interfering RNA-mediated silencing of cIAP1 expression attenuated the knockdown activity of SNIPER(CRABP) against WT and cytosolic CRABP-II proteins, indicating that cIAP1 is the E3 ligase responsible for degradation of these proteins. Against membrane-localized CRABP-II protein, cIAP1 is also a primary E3 ligase in the cells, but another E3 ligase distinct from cIAP2 and X-linked inhibitor of apoptosis protein (XIAP) could also be involved in the SNIPER(CRABP)-11-induced degradation. However, for the degradation of nuclear and mitochondrial CRABP-II proteins, E3 ligases other than cIAP1, cIAP2, and XIAP play a role in the SNIPER-mediated protein knockdown. These results indicate that SNIPER can target cytosolic, nuclear, membrane-localized, and mitochondrial proteins for degradation, but the responsible E3 ligase is different, depending on the localization of the target protein.

Pharmacological interaction with sunitinib is abolished by a germ-line mutation (1291T>C) of BCRP/ABCG2 gene.

Sunitinib malate (Sutent, SU11248) is a small-molecule multitargeted tyrosine kinase inhibitor (TKI) used for the treatment of renal cell carcinoma and imatinib-resistant gastrointestinal stromal tumors. Some TKIs can overcome multidrug resistance conferred by ATP-binding cassette transporter, P-glycoprotein (P-gp)/ABCB1, multidrug resistance-associated protein 1 (MRP1)/ABCC1, and breast cancer resistance protein (BCRP)/ABCG2. Here, we analyzed the effects of sunitinib on P-gp and on wild-type and germ-line mutant BCRPs. Sunitinib remarkably reversed BCRP-mediated and partially reversed P-gp-mediated drug resistance in the respective transfectants. The in vitro vesicle transport assay indicated that sunitinib competitively inhibited BCRP-mediated estrone 3-sulfate transport and P-gp-mediated vincristine transport. These inhibitory effects of sunitinib were further analyzed in Q141K-, R482G-, R482S-, and F431L-variant BCRPs. Intriguingly, the F431L-variant BCRP, which is expressed by a germ-line mutant allele 1291T>C, was almost insensitive to both sunitinib- and fumitremorgin C (FTC)-mediated inhibition in a cell proliferation assay. Sunitinib and FTC did not inhibit (125)I-iodoarylazidoprazosin-binding to F431L-BCRP. Thus, residue Phe-431 of BCRP is important for the pharmacological interaction with sunitinib and FTC. Collectively, this is the first report showing a differential effect of a germ-line variation of the BCRP/ABCG2 gene on the pharmacological interaction between small-molecule TKIs and BCRP. These findings would be useful for improving our understanding of the pharmaceutical effects of sunitinib in personalized chemotherapy.

Substrate-dependent bidirectional modulation of P-glycoprotein-mediated drug resistance by erlotinib.

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) inhibit the function of certain adenosine triphosphate (ATP)-binding cassette transporters, including P-glycoprotein/ABCB1 and breast cancer resistance protein (BCRP)/ABCG2. We previously reported an antagonistic activity of gefitinib towards BCRP. We have now analyzed the effects of erlotinib, another EGFR-TKI, on P-glycoprotein and BCRP. As with gefitinib, erlotinib effectively reversed BCRP-mediated resistance to SN-38 (7-ethyl-10-hydroxycamptothecin) and mitoxantrone. In contrast, we found that erlotinib effectively suppressed P-glycoprotein-mediated resistance to vincristine and paclitaxel, but did not suppress resistance to mitoxantrone and doxorubicin. Conversely, erlotinib appeared to enhance P-glycoprotein-mediated resistance to mitoxantrone in K562/MDR cells. This bidirectional activity of erlotinib was not observed with verapamil, a typical P-glycoprotein inhibitor. Flow cytometric analysis showed that erlotinib co-treatment restored intracellular accumulation of mitoxantrone in K562 cells expressing BCRP, but not in cells expressing P-glycoprotein. Consistently, erlotinib did not inhibit mitoxantrone efflux in K562/MDR cells although it did vincristine efflux in K562/MDR cells and mitoxantrone efflux in K562/BCRP cells. Intravesicular transport assay showed that erlotinib inhibited both P-glycoprotein-mediated vincristine transport and BCRP-mediated estrone 3-sulfate transport. Intriguingly, Lineweaver-Burk plot suggested that the inhibitory mode of erlotinib was a mixed type for P-glycoprotein-mediated vincristine transport whereas it was a competitive type for BCRP-mediated estrone 3-sulfate transport. Collectively, these observations indicate that the pharmacological activity of erlotinib on P-glycoprotein-mediated drug resistance is dependent upon the transporter substrate. These findings will be useful in understanding the pharmacological interactions of erlotinib used in combinational chemotherapy.