Michael Wiese

Based on literature reports of the last two decades, a computer-aided pattern analysis ([email protected]) was implemented for the discovery of novel multitarget ABCB1 (P-gp), ABCC1 (MRP1), and ABCG2 (BCRP) inhibitors. [email protected] included basic scaffold identification, substructure search and statistical distribution, as well as novel scaffold extraction to screen a large virtual compound library. Over 45,000 putative and novel broad-spectrum ABC transporter inhibitors were identified, from which 23 were purchased for biological evaluation. Our investigations revealed five novel lead molecules as triple ABCB1, ABCC1, and ABCG2 inhibitors. [email protected] is the very first successful computational approach for the discovery of promiscuous ABC transporter inhibitors.

In search for highly effective modulators addressing ABCG2-mediated MDR, 23 pyrimidines were synthesized and biologically assessed. Seven derivatives with (a) nitrogen- and/or halogen-containing residue(s) had extraordinary potencies against ABCG2 (IC50 < 150 nM). The compounds competitively inhibited ABCG2-mediated Hoechst 33342 transport, but were not substrates of ABCG2. The most potent MDR reverser compound 19 concentration-dependently increased SN-38-mediated cancer cell death at 11 nM (EC50), time-dependently doubled SN-38 toxicity in a period of seven days at 10 nM, and half-maximally accelerated cell death combined with SN-38 at 17 nM. No induction of ABCG2 was observed. Furthermore, eleven pyrimidines were revealed as triple ABCB1/ABCC1/ABCG2 inhibitors. Five possessed IC50 values below 10 µM against each transporter, classifying them as some of the 50 most potent multi-target ABC transporter inhibitors. The most promising representative compound 37 reversed ABCB1-, ABCC1-, and ABCG2-mediated MDR, making it one of the 3 most potent ABC transporter inhibitors and reversers of ABC-transporters-mediated MDR.

ATP-binding cassette (ABC) transporters, such as breast cancer resistance protein (BCRP), are key players in resistance to multiple anti-cancer drugs, leading to cancer treatment failure and cancer-related death. Currently, there are no clinically approved drugs for reversal of cancer drug resistance caused by ABC transporters. This study investigated if a novel drug candidate, SCO-201, could inhibit BCRP and reverse BCRP-mediated drug resistance. We applied in vitro cell viability assays in SN-38 (7-Ethyl-10-hydroxycamptothecin)-resistant colon cancer cells and in non-cancer cells with ectopic expression of BCRP. SCO-201 reversed resistance to SN-38 (active metabolite of irinotecan) in both model systems. Dye efflux assays, bidirectional transport assays, and ATPase assays demonstrated that SCO-201 inhibits BCRP. In silico interaction analyses supported the ATPase assay data and suggest that SCO-201 competes with SN-38 for the BCRP drug-binding site. To analyze for inhibition of other transporters or cytochrome P450 (CYP) enzymes, we performed enzyme and transporter assays by in vitro drug metabolism and pharmacokinetics studies, which demonstrated that SCO-201 selectively inhibited BCRP and neither inhibited nor induced CYPs. We conclude that SCO-201 is a specific, potent, and potentially non-toxic drug candidate for the reversal of BCRP-mediated resistance in cancer cells.

Several mechanisms of pharmacokinetic, metabolic, and regulatory nature have been elucidated to take part or act in concert in the phenomenon of multidrug resistance (MDR). MDR is characterized by cross‐resistance of cells against chemotherapeutic agents, which are used for treatment of e.g., cancer, bacterial infections, or human immunodeficiency virus (HIV) infections. One group of proteins that combines all three stated aspects—the metabolism and distribution of drugs as well as their own regulation—is adenosine triphosphate‐binding cassette (ABC) transporters. These efflux pumps use the energy of adenosine triphosphate hydrolysis for drug translocation from the membrane and the cytosol to the extracellular space, often with cotransport of a cosubstrate. Multidrug resistance‐associated protein 1 (MRP1, ABCC1) had been discovered as one major key player in cancer‐related MDR. The xenobiotic substrates include anthracyclines, vinca alkaloids, podophyllotoxins, as well as glutathione (GSH)‐adducts of certain cytostatics. Contrary to other transport proteins involved in cancer‐related MDR the activity of MRP1 is related to the GSH content of cells. A modern strategy to overcome MRP1‐associated MDR is besides its inhibition the activation of GSH efflux, enforcing cell death due to cellular stress. In addition, it has recently been found that MRP1 contributes to the β‐amyloid protein clearance in Alzheimer's disease (AD). Collectively, transport activation of MRP1 is of therapeutic value, and furthermore helps to elucidate the transport protein function and the mechanisms behind it. This review is meant to summarize the known concepts of MRP1 activation, which might contribute to a further understanding of MRP1 in particular and ABC transporters in general.

A virtual screening protocol with combination of similarity search and pharmacophore-modelling was applied to virtually screen a large compound library to gain new scaffolds regarding ABCC1 inhibition. Biological investigation of promising candidates revealed nine compounds as ABCC1 inhibitors, five 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 pheophorbide A transport by ABCG2. 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 literature. Besides, seven more new multi-target inhibitors were identified by this virtual screening approach.

Several members of the ABC transporter superfamily play a decisive role in the development of multidrug resistance (MDR) in cancer. One of these MDR associated efflux transporters is ABCG2. One way to overcome this MDR is the co-administration of potent inhibitors of ABCG2. In this study we identified novel inhibitors containing a 2,4,6-substituted quinazoline scaffold. Introduction of a 6-nitro function led to extraordinarily potent compounds, that were highly selective for ABCG2 and also able to reverse the MDR toward the chemotherapeutic drugs SN-38 and mitoxantrone. The binding of substrate Hoechst 33342 and the two potent inhibitors 31 and 41 which differ in their mechanism of inhibition was rationalized using the recently published cryo-EM structures of ABCG2. For a better understanding of the interaction between the inhibitors and ABCG2, additional investigations regarding the ATPase activity, the interaction with Hoechst 33342 and with the conformational sensitive 5D3 antibody were carried out.