Correction: A screen of drug-like molecules identifies chemically diverse electron transport chain inhibitors in apicomplexan parasites.

[This corrects the article DOI: 10.1371/journal.ppat.1011517.].

Analysis of Plasmodium falciparum Mitochondrial Electron Transport Chain Activity Using Seahorse XFe96 Extracellular Flux Assays.

The mitochondrial electron transport chain (ETC) is a multi-component pathway that mediates the transfer of electrons from metabolic reactions that occur in the mitochondrion to molecular oxygen (O2). The ETC contributes to numerous cellular processes, including the generation of cellular ATP through oxidative phosphorylation, serving as an electron sink for metabolic pathways such as de novo pyrimidine biosynthesis and for maintaining mitochondrial membrane potential. Proper functioning of the mitochondrial ETC is necessary for the growth and survival of apicomplexan parasites including Plasmodium falciparum, a causative agent of malaria. The mitochondrial ETC of P. falciparum is an attractive target for antimalarial drugs, due to its essentiality and its differences from the mammalian ETC. To identify novel P. falciparum ETC inhibitors, we have established a real-time assay to assess ETC function, which we describe here. This approach measures the O2 consumption rate (OCR) of permeabilized P. falciparum parasites using a Seahorse XFe96 flux analyzer and can be used to screen compound libraries for the identification of ETC inhibitors and, in part, to determine the targets of those inhibitors. Key features • With this protocol, the effects of candidate inhibitors on mitochondrial O2 consumption in permeabilized asexual P. falciparum parasites can be tested in real time. • Through the sequential injection of inhibitors and substrates into the assay, the molecular targets of candidate inhibitors in the ETC can, in part, be determined. • The assay is applicable for both drug discovery approaches and enquiries into a fundamental aspect of parasite mitochondrial biology.

A screen of drug-like molecules identifies chemically diverse electron transport chain inhibitors in apicomplexan parasites.

Apicomplexans are widespread parasites of humans and other animals, and include the causative agents of malaria (Plasmodium species) and toxoplasmosis (Toxoplasma gondii). Existing anti-apicomplexan therapies are beset with issues around drug resistance and toxicity, and new treatment options are needed. The mitochondrial electron transport chain (ETC) is one of the few processes that has been validated as a drug target in apicomplexans. To identify new inhibitors of the apicomplexan ETC, we developed a Seahorse XFe96 flux analyzer approach to screen the 400 compounds contained within the Medicines for Malaria Venture 'Pathogen Box' for ETC inhibition. We identified six chemically diverse, on-target inhibitors of the ETC in T. gondii, at least four of which also target the ETC of Plasmodium falciparum. Two of the identified compounds (MMV024937 and MMV688853) represent novel ETC inhibitor chemotypes. MMV688853 belongs to a compound class, the aminopyrazole carboxamides, that were shown previously to target a kinase with a key role in parasite invasion of host cells. Our data therefore reveal that MMV688853 has dual targets in apicomplexans. We further developed our approach to pinpoint the molecular targets of these inhibitors, demonstrating that all target Complex III of the ETC, with MMV688853 targeting the ubiquinone reduction (Qi) site of the complex. Most of the compounds we identified remain effective inhibitors of parasites that are resistant to Complex III inhibitors that are in clinical use or development, indicating that they could be used in treating drug resistant parasites. In sum, we have developed a versatile, scalable approach to screen for compounds that target the ETC in apicomplexan parasites, and used this to identify and characterize novel inhibitors.

Analysis of sex-specific lipid metabolism of Plasmodium falciparum points to the importance of sphingomyelin for gametocytogenesis.

Male and female Plasmodium falciparum gametocytes are the parasite lifecycle stage responsible for transmission of malaria from the human host to the mosquito vector. Not only are gametocytes able to survive in radically different host environments, but they are also precursors for male and female gametes that reproduce sexually soon after ingestion by the mosquito. Here, we investigate the sex-specific lipid metabolism of gametocytes within their host red blood cell. Comparison of the male and female lipidome identifies cholesteryl esters and dihydrosphingomyelin enrichment in female gametocytes. Chemical inhibition of each of these lipid types in mature gametocytes suggests dihydrosphingomyelin synthesis but not cholesteryl ester synthesis is important for gametocyte viability. Genetic disruption of each of the two sphingomyelin synthase genes points towards sphingomyelin synthesis contributing to gametocytogenesis. This study shows that gametocytes are distinct from asexual stages, and that the lipid composition is also vastly different between male and female gametocytes, reflecting the different cellular roles these stages play. Taken together, our results highlight the sex-specific nature of gametocyte lipid metabolism, which has the potential to be targeted to block malaria transmission. This article has an associated First Person interview with the first author of the paper.

Sex-specific Separation of Plasmodium falciparum Gametocyte Populations.

Plasmodium falciparum is a unicellular eukaryotic parasite that causes malaria in humans. The parasite is spread by Anopheles mosquitoes after ingestion of sexual stage parasites known as gametocytes. Malaria transmission depends on parasites switching from the disease-causing asexual blood forms to male and female gametocytes. The current protocol allows the simultaneous isolation of male and female parasites from the same population to study this critical lifecycle stage in a sex-specific manner. We have generated a transgenic P. falciparum cell line that expresses a GFP-tagged parasite protein in female, but not male, parasites. Gametocyte production is stress induced and, through a series of steps, sexual stage parasites are enriched relative to uninfected red blood cells or red blood cells infected with asexual stage parasites. Finally, male and female gametocytes are separated by fluorescence-activated cell sorting. This protocol allows for the separation of up to 12 million live male and female parasites from the same population, which are amenable to further analysis.

Novel Method for the Separation of Male and Female Gametocytes of the Malaria Parasite Plasmodium falciparum That Enables Biological and Drug Discovery.

We developed a flow-cytometry-based method to separate and collect cocultured male and female Plasmodium falciparum gametocytes responsible for malaria transmission. The purity of the collected cells was estimated at >97% using flow cytometry, and sorted cells were observed by Giemsa-stained thin-smear and live-cell fluorescence microscopy. The expression of validated sex-specific markers corroborated the sorting strategy. Collected male and female gametocytes were used to confirm three novel sex-specific markers by quantitative real-time PCR that were more enriched in sorted male and female gametocyte populations than existing sex-specific markers. We also applied the method as a proof-of-principle drug screen that allows the identification of drugs that kill gametocytes in a sex-specific manner. Since the developed method allowed for the separation of male and female parasites from the same culture, we observed for the first time a difference in development time between the sexes: females developed faster than males. Hence, the ability to separate male and female gametocytes opens the door to a new field of sex-specific P. falciparum gametocyte biology to further our understanding of malaria transmission.IMPORTANCE The protozoan Plasmodium falciparum causes the most severe form of human malaria. The development of sexual forms (so-called gametocytes) is crucial for disease transmission. However, knowledge of these forms is severely hampered by the paucity of sex-specific markers and the inability to extract single sex gametocytes in high purity. Moreover, the identification of compounds that specifically affect one sex is difficult due to the female bias of the gametocytes. We have developed a system that allows for the separation of male and female gametocytes from the same population. Applying our system, we show that male and female parasites mature at different rates, which might have implications for transmission. We also identified new sex-specific genes that can be used as sex markers or to unravel sex-specific functions. Our system will not only aid in the discovery of much needed gametocidal compounds, but it also represents a valuable tool for exploring malaria transmission biology.

Dinaciclib, a cyclin-dependent kinase inhibitor, is a substrate of human ABCB1 and ABCG2 and an inhibitor of human ABCC1 in vitro.

Dinaciclib is a novel cyclin-dependent kinase inhibitor (CDKI) with significant activity against various cancers in vitro and in vivo. ABC efflux transporters play an important role in drug disposition and are responsible for multidrug resistance in cancer cells. Inhibitors and substrates of these transporters may participate in pharmacokinetic drug-drug interactions (DDIs) that alter drug disposition during pharmacotherapy. To assess such risks associated with dinaciclib we evaluated its possible effects on efflux activities of ABCB1, ABCC1 and ABCG2 transporters in vitro. Monolayer transport, XTT cell proliferation, ATPase and intracellular accumulation assays were employed. Here, we show that the transport ratio of dinaciclib was far higher across monolayers of MDCKII-ABCB1 and MDCKII-ABCG2 cells than across MDCKII parental cell layers, demonstrating that dinaciclib is a substrate of ABCB1 and ABCG2. In addition, overexpression of ABCB1, ABCG2 and ABCC1 conferred resistance to dinaciclib in MDCKII cells. In ATPase assays, dinaciclib decreased stimulated ATPase activity of ABCB1, ABCG2 and ABCC1, confirming it has interactive potential toward all three transporters. Moreover, dinaciclib significantly inhibited ABCC1-mediated efflux of daunorubicin (EC50=18 µM). The inhibition of ABCC1 further led to a synergistic effect of dinaciclib in both MDCKII-ABCC1 and human cancer T47D cells, when applied in combination with anticancer drugs. Taken together, our results suggest that ABC transporters can substantially affect dinaciclib transport across cellular membranes, leading to DDIs. The DDIs of dinaciclib with ABCC1 substrate chemotherapeutics might be exploited in novel cancer therapies.

Interactions of cyclin-dependent kinase inhibitors AT-7519, flavopiridol and SNS-032 with ABCB1, ABCG2 and ABCC1 transporters and their potential to overcome multidrug resistance in vitro.

PURPOSE: ATP-binding cassette (ABC) transporters play an important role in multidrug resistance (MDR) toward anticancer drugs. Here, we evaluated interactions of cyclin-dependent kinase inhibitors (CDKi) AT-7519, flavopiridol and SNS-032 with the following ABC transporters in vitro: P-glycoprotein (ABCB1), breast cancer resistance protein (ABCG2) and multidrug resistance-associated protein 1 (ABCC1). METHODS: Inhibitory potency of studied CDKi to the transporters was evaluated by accumulation assays using fluorescent substrates and MDCKII cells overexpressing human ABCB1, ABCG2 or ABCC1. Resistance of transporter-expressing cells to the CDKi was evaluated by XTT proliferation assay. Observed interactions of CDKi were verified by ATPase assay in ABC transporter-expressing Sf9 membrane vesicles. Combination index analysis was additionally performed in ABC transporter-expressing cancer cell lines, HepG2 and T47D. RESULTS: Flavopiridol showed a significant inhibitory potency toward ABCG2 and ABCC1. SNS-032 also decreased ABCG2-mediated efflux, while AT-7519 failed to inhibit ABCB1, ABCG2 or ABCC1. Both flavopiridol and SNS-032 showed synergistic antiproliferative effects in combination with relevant ABC transporter substrates such as daunorubicin and topotecan in cancer cells. ABCB1 was found to confer significant resistance to AT-7519 and SNS-032, but not to flavopiridol. In contrast, ABCG2 and ABCC1 conferred resistance to flavopiridol, but not to AT-7519 and SNS-032. CONCLUSION: Our data provide detailed information on interactions of flavopiridol, SNS-032 and AT-7519 with ABC transporters, which may help elucidate the pharmacokinetic behavior and toxicity of these compounds. Moreover, we show the ability of flavopiridol and SNS-032, but not AT-7519, to overcome ABC transporter-mediated MDR.

Purvalanol A, olomoucine II and roscovitine inhibit ABCB1 transporter and synergistically potentiate cytotoxic effects of daunorubicin in vitro.

Cyclin-dependent kinase inhibitors (CDKi) have high potential applicability in anticancer therapy, but various aspects of their pharmacokinetics, especially their interactions with drug efflux transporters, have not yet been evaluated in detail. Thus, we investigated interactions of five CDKi (purvalanol A, olomoucine II, roscovitine, flavopiridol and SNS-032) with the ABCB1 transporter. Four of the compounds inhibited efflux of two ABCB1 substrates, Hoechst 33342 and daunorubicin, in MDCKII-ABCB1 cells: Olomoucine II most strongly, followed by roscovitine, purvalanol A, and flavopiridol. SNS-032 inhibited ABCB1-mediated efflux of Hoechst 33342 but not daunorubicin. In addition, purvalanol A, SNS-032 and flavopiridol lowered the stimulated ATPase activity in ABCB1 membrane preparations, while olomoucine II and roscovitine not only inhibited the stimulated ATPase but also significantly activated the basal ABCB1 ATPase, suggesting that these two CDKi are ABCB1 substrates. We further revealed that the strongest ABCB1 inhibitors (purvalanol A, olomoucine II and roscovitine) synergistically potentiate the antiproliferative effect of daunorubicin, a commonly used anticancer drug and ABCB1 substrate, in MDCKII-ABCB1 cells as well as in human carcinoma HCT-8 and HepG2 cells. We suggest that this pronounced synergism is at least partly caused by (i) CDKi-mediated inhibition of ABCB1 transporter leading to increased intracellular retention of daunorubicin and (ii) native cytotoxic activity of the CDKi. Our results indicate that co-administration of the tested CDKi with anticancer drugs that are ABCB1 substrates may allow significant dose reduction in the treatment of ABCB1-expressing tumors.

Olomoucine II, but not purvalanol A, is transported by breast cancer resistance protein (ABCG2) and P-glycoprotein (ABCB1).

Purine cyclin-dependent kinase inhibitors have been recognized as promising candidates for the treatment of various cancers; nevertheless, data regarding interaction of these substances with drug efflux transporters is still lacking. Recently, we have demonstrated inhibition of breast cancer resistance protein (ABCG2) by olomoucine II and purvalanol A and shown that these compounds are able to synergistically potentiate the antiproliferative effect of mitoxantrone, an ABCG2 substrate. In this follow up study, we investigated whether olomoucine II and purvalanol A are transported by ABCG2 and ABCB1 (P-glycoprotein). Using monolayers of MDCKII cells stably expressing human ABCB1 or ABCG2, we demonstrated that olomoucine II, but not purvalanol A, is a dual substrate of both ABCG2 and ABCB1. We, therefore, assume that pharmacokinetics of olomoucine II will be affected by both ABCB1 and ABCG2 transport proteins, which might potentially result in limited accumulation of the compound in tumor tissues or lead to drug-drug interactions. Pharmacokinetic behavior of purvalanol A, on the other hand, does not seem to be affected by either ABCG2 or ABCB1, theoretically favoring this drug in the potential treatment of efflux transporter-based multidrug resistant tumors. In addition, we observed intensive sulfatation of olomoucine II in MDCKII cell lines with subsequent active efflux of the metabolite out of the cells. Therefore, care should be taken when performing pharmacokinetic studies in MDCKII cells, especially if radiolabeled substrates are used; the generated sulfated conjugate may largely contaminate pharmacokinetic analysis and result in misleading interpretation. With regard to chemical structures of olomoucine II and purvalanol A, our data emphasize that even drugs with remarkable structure similarity may show different pharmacokinetic behavior such as interactions with ABC transporters or biotransformation enzymes.