Mechanistic basis of breast cancer resistance protein inhibition by new indeno[1,2-b]indoles.

The ATP-binding cassette transporter ABCG2 mediates the efflux of several chemotherapeutic drugs, contributing to the development of multidrug resistance (MDR) in many cancers. The most promising strategy to overcome ABCG2-mediated MDR is the use of specific inhibitors. Despite many efforts, the identification of new potent and specific ABCG2 inhibitors remains urgent. In this study, a structural optimization of indeno[1,2-b]indole was performed and a new generation of 18 compounds was synthesized and tested as ABCG2 inhibitors. Most compounds showed ABCG2 inhibition with IC50 values below 0.5 µM. The ratio between cytotoxicity (IG50) and ABCG2 inhibition potency (IC50) was used to identify the best inhibitors. In addition, it was observed that some indeno[1,2-b]indole derivatives produced complete inhibition, while others only partially inhibited the transport function of ABCG2. All indeno[1,2-b]indole derivatives are not transported by ABCG2, and even the partial inhibitors are able to fully chemosensitize cancer cells overexpressing ABCG2. The high affinity of these indeno[1,2-b]indole derivatives was confirmed by the strong stimulatory effect on ABCG2 ATPase activity. These compounds did not affect the binding of conformation-sensitive antibody 5D3 binding, but stabilized the protein structure, as revealed by the thermostabilization assay. Finally, a docking study showed the indeno[1,2-b]indole derivatives share the same binding site as the substrate estrone-3-sulfate.

Uncompetitive nanomolar dimeric indenoindole inhibitors of the human breast cancer resistance pump ABCG2.

Multidrug resistance membrane pumps reduce the efficacy of chemotherapies by exporting a wide panel of structurally-divergent drugs. Here, to take advantage of the polyspecificity of the human Breast Cancer Resistance Protein (BCRP/ABCG2) and the dimeric nature of this pump, new dimeric indenoindole-based inhibitors from the monomeric α,ß-unsaturated ketone 4b and phenolic derivative 5a were designed. A library of 18 homo/hetero-dimers was synthesised. Homo-dimerization shifted the inhibition efficacy from sub-micromolar to nanomolar range, correlated with the presence of 5a, linked by a 2-6 methylene-long linker. Non-toxic, the best dimers displayed a therapeutic ratio as high as 70,000. It has been found that the high potency of the best compound 7b that displays a KI of 17 nM is due to an uncompetitive behavior toward mitoxantrone efflux and specific for that drug, compared to Hoechst 33342 efflux. Such property may be useful to target such anticancer drug efflux mediated by ABCG2. Finally, at a molecular level, an uncompetitive mechanism by which substrate promotes inhibitor binding implies that at least 2 ligands should bind simultaneously to the drug-binding pocket of ABCG2.

Structure-based design and profiling of novel 17ß-HSD14 inhibitors.

The human enzyme 17ß-hydroxysteroid dehydrogenase 14 (17ß-HSD14) oxidizes the hydroxyl group at position 17 of estradiol and 5-androstenediol using NAD+ as cofactor. However, the physiological role of the enzyme remains unclear. We recently described the first class of nonsteroidal inhibitors for this enzyme with compound 1 showing a high 17ß-HSD14 inhibitory activity. Its crystal structure was used as starting point for a structure-based optimization in this study. The goal was to develop a promising chemical probe to further investigate the enzyme. The newly designed compounds revealed mostly very high inhibition of the enzyme and for seven of them the crystal structures of the corresponding inhibitor-enzyme complexes were resolved. The crystal structures disclosed that a small change in the substitution pattern of the compounds resulted in an alternative binding mode for one inhibitor. The profiling of a set of the most potent inhibitors identified 13 (Ki = 9 nM) with a good selectivity profile toward three 17ß-HSDs and the estrogen receptor alpha. This inhibitor displayed no cytotoxicity, good solubility, and auspicious predicted bioavailability. Overall, 13 is a highly interesting 17ß-HSD14 inhibitor, which might be used as chemical probe for further investigation of the target enzyme.

New, highly potent and non-toxic, chromone inhibitors of the human breast cancer resistance protein ABCG2.

Breast cancer resistance protein (BCRP/ABCG2) is one of the major transporters involved in the efflux of anticancer compounds, contributing to multidrug resistance (MDR). Inhibition of ABCG2-mediated transport is then considered a promising strategy for overcoming MDR in tumors. We recently identified a chromone derivative, namely MBL-II-141 as a selective ABCG2 inhibitor, with relevant in vivo activity. Here, we report the pharmacomodulation of MBL-II-141, with the aim of identifying key pharmacophoric elements to design more potent selective and non-toxic inhibitors. Through rational structural modifications of MBL-II-141, using simple and affordable chemistry, we obtained highly active and easily-made inhibitors of ABCG2. Among the investigated compounds, derivative 4a, was found to be 3-fold more potent than MBL-II-141. It was similarly efficient as the reference inhibitor Ko143 but with the advantage of a lower intrinsic cytotoxicity, and therefore constitutes the best ABCG2 inhibitor ever reported displaying a very high therapeutic ratio.

Phenolic indeno[1,2-b]indoles as ABCG2-selective potent and non-toxic inhibitors stimulating basal ATPase activity.

Ketonic indeno[1,2-b]indole-9,10-dione derivatives, initially designed as human casein kinase II (CK2) inhibitors, were recently shown to be converted into efficient inhibitors of drug efflux by the breast cancer resistance protein ABCG2 upon suited substitutions including a N (5)-phenethyl on C-ring and hydrophobic groups on D-ring. A series of ten phenolic and seven p-quinonic derivatives were synthesized and screened for inhibition of both CK2 and ABCG2 activities. The best phenolic inhibitors were about threefold more potent against ABCG2 than the corresponding ketonic derivatives, and showed low cytotoxicity. They were selective for ABCG2 over both P-glycoprotein and MRP1 (multidrug resistance protein 1), whereas the ketonic derivatives also interacted with MRP1, and they additionally displayed a lower interaction with CK2. Quite interestingly, they strongly stimulated ABCG2 ATPase activity, in contrast to ketonic derivatives, suggesting distinct binding sites. In contrast, the p-quinonic indenoindoles were cytotoxic and poor ABCG2 inhibitors, whereas a partial inhibition recovery could be reached upon hydrophobic substitutions on D-ring, similarly to the ketonic derivatives.