A novel method for measurements of sleep/wake states, feeding and drinking behaviors using the tracking technique of 3D positions in freely moving mice.

We have previously developed a 3D video tracking system which enables us to analyze long-term quantitative analysis of gene expression in freely moving mice. In the present study, we improved 3D video tracking and developed a system that analyzes more detailed behavioral data. We succeeded in simultaneously analyzing sleep-wake, feeding, and drinking behavior rhythms in the same individual using our tracking system. This system will make it possible to measure gene expression in each tissue in vivo in real time in relation to the various behavioral rhythms mentioned above.

Synthesis and Bioluminescence of 'V'-Shaped Firefly Luciferin Analogues Based on A Novel Benzobisthiazole Core.

The design of π-extended conjugation 'V'-shaped red shifted bioluminescent D-luciferin analogues based on a novel benzobisthiazole core is described. The divergent synthetic route allowed access to a range of amine donor substituents through an SN Ar reaction. In spectroscopic studies, the 'V'-shaped luciferins exhibited narrower optical band gaps, more red-shifted absorption and emission spectra than D-luciferin. Their bioluminescence characteristics were recorded against four different luciferases (PpyLuc, FlucRed, CBR2 and PLR3). With native luciferase PpyLuc, the 'V'-shaped luciferins demonstrated more red-shifted emissions than D-luciferin (λbl =561 nm) by 60 to 80 nm. In addition, the benzobisthiazole luciferins showed a wide range of bioluminescence spectra from the visible light region (λbl =500 nm) to the nIR window (>650 nm). The computational results validate the design concept which can be used as a guide for further novel D-luciferin analogues based upon other 'V'-shaped heterocyclic cores.

Comparison of Bioluminescent Substrates in Natural Infection Models of Neglected Parasitic Diseases.

The application of in vivo bioluminescent imaging in infectious disease research has significantly increased over the past years. The detection of transgenic parasites expressing wildtype firefly luciferase is however hampered by a relatively low and heterogeneous tissue penetrating capacity of emitted light. Solutions are sought by using codon-optimized red-shifted luciferases that yield higher expression levels and produce relatively more red or near-infrared light, or by using modified bioluminescent substrates with enhanced cell permeability and improved luminogenic or pharmacokinetic properties. In this study, the in vitro and in vivo efficacy of two modified bioluminescent substrates, CycLuc1 and AkaLumine-HCl, were compared with that of D-luciferin as a gold standard. Comparisons were made in experimental and insect-transmitted animal models of leishmaniasis (caused by intracellular Leishmania species) and African trypanosomiasis (caused by extracellular Trypanosoma species), using parasite strains expressing the red-shifted firefly luciferase PpyRE9. Although the luminogenic properties of AkaLumine-HCl and D-luciferin for in vitro parasite detection were comparable at equal substrate concentrations, AkaLumine-HCl proved to be unsuitable for in vivo infection follow-up due to high background signals in the liver. CycLuc1 presented a higher in vitro luminescence compared to the other substrates and proved to be highly efficacious in vivo, even at a 20-fold lower dose than D-luciferin. This efficacy was consistent across infections with the herein included intracellular and extracellular parasitic organisms. It can be concluded that CycLuc1 is an excellent and broadly applicable alternative for D-luciferin, requiring significantly lower doses for in vivo bioluminescent imaging in rodent models of leishmaniasis and African trypanosomiasis.

Coix Seed Extract Enhances the Anti-Pancreatic Cancer Efficacy of Gemcitabine through Regulating ABCB1- and ABCG2-Mediated Drug Efflux: A Bioluminescent Pharmacokinetic and Pharmacodynamic Study.

A deep insight into the function and kinetics of ATP-binding cassette (ABC) transporters may aid in the development of pharmaceutics that can minimize the particular facet of chemo-resistance. We utilized bioluminescence imaging to monitor the ABC transporter mediated intracellular drug efflux function. We also investigated the potential association between the intracellular bioluminescent pharmacokinetic profiles and the anti-tumor efficacy of the coix seed extract and gemcitabine against pancreatic cancer cells in vitro and in vivo. The bioluminescent pharmacokinetic parameters and pharmacodynamic index (IC50 and TGI) were determined. The expression levels ABCB1 and ABCG2 were assessed. Results showed that coix seed extract could synergistically enhance the anti-cancer efficacy of gemcitabine (p < 0.05). Meanwhile coix seed extract alone or in combination with gemcitabine could significantly increase the AUCluc while decreasing the Kluc (p < 0.01). Western blot and immunohistochemistry assay demonstrated that coix seed extract could significantly mitigate gemcitabine-induced upregulation of ABCB1 and ABCG2 protein. The Pearson correlation analysis demonstrated that the bioluminescent pharmacokinetic parameters and pharmacodynamic index have strong association in vitro and in vivo. In conclusion coix seed extract could augment the efficacy of gemcitabine therapy in pancreatic cancer cells may at least partly due to the alteration of ABC transporter-mediated drug efflux function.

A Chemo-Enzymatic Cascade for the Smart Detection of Nitro- and Halogenated Phenols.

The flavin-dependent monooxygenase, HadA, catalyzes the dehalogenation and denitration of the toxicants, nitro- and halogenated phenols, to benzoquinone. The HadA reaction can be applied in one-pot reactions towards the de novo synthesis of d-luciferin by coupling with d-Cys condensation. d-luciferin, a valuable chemical widely used in biomedical applications, can be used as a substrate for the reaction of firefly luciferase to generate bioluminescence. As nitro- and halogenated phenols are key indicators of human overexposure to pesticides and pesticide contamination, the technology provides a sensitive and convenient tool for improved biomedical and environmental detection at ppb sensitivity in biological samples without the requirement for any pre-treatment. This dual-pronged method combines the advantages of waste biodetoxification to produce a valuable chemical as well as a smart detection tool for environmental and biomedical detection.

Organic anion transporter 1 (OAT1/SLC22A6) enhances bioluminescence based on d-luciferin-luciferase reaction in living cells by facilitating the intracellular accumulation of d-luciferin.

Bioluminescence (BL) imaging based on d-luciferin (d-luc)-luciferase reaction allows noninvasive and real-time monitoring of luciferase-expressing cells. Because BL intensity depends on photons generated through the d-luc-luciferase reaction, an approach to increase intracellular levels of d-luc could improve the detection sensitivity. In the present study, we showed that organic anion transporter 1 (OAT1) is useful, as a d-luc transporter, in boosting the BL intensity in luciferase-expressing cells. Functional screening of several transporters showed that the expression of OAT1 in HEK293 cells stably expressing Pyrearinus termitilluminans luciferase (HEK293/eLuc) markedly enhanced BL intensity in the presence of d-luc. When OAT1 was transiently expressed in HEK293 cells, intracellular accumulation of d-luc was higher than that in control cells, and the specific d-luc uptake mediated by OAT1 was saturable with a Michaelis constant (Km) of 0.23 µM. The interaction between OAT1 and d-luc was verified using 6-carboxyfluorescein, a typical substrate of OAT1, which showed that d-luc inhibited the uptake of 6-carboxyfluorescein mediated by OAT1. BL intensity was concentration-dependent at steady states in HEK293/eLuc cells stably expressing OAT1, and followed Michaelis-Menten kinetics with an apparent Km of 0.36 µM. In addition, the enhanced BL was significantly inhibited by OAT1-specific inhibitors. Thus, OAT1-mediated transport of d-luc could be a rate-limiting step in the d-luc-luciferase reaction. Furthermore, we found that expressing OAT1 in HEK293/eLuc cells implanted subcutaneously in mice also significantly increased the BL after intraperitoneal injection of d-luc. Our findings suggest that because OAT1 is capable of transporting d-luc, it can also be used to improve visualization and monitoring of luciferase-expressing cells.

A synthetic luciferin improves in vivo bioluminescence imaging of gene expression in cardiovascular brain regions.

Bioluminescence imaging is an effective tool for in vivo investigation of molecular processes. We have demonstrated the applicability of bioluminescence imaging to spatiotemporally monitor gene expression in cardioregulatory brain nuclei during the development of cardiovascular disease, via incorporation of firefly luciferase into living animals, combined with exogenous d-luciferin substrate administration. Nevertheless, d-luciferin uptake into the brain tissue is low, which decreases the sensitivity of bioluminescence detection, particularly when considering small changes in gene expression in tiny central areas. Here, we tested the hypothesis that a synthetic luciferin, cyclic alkylaminoluciferin (CycLuc1), would be superior to d-luciferin for in vivo bioluminescence imaging in cardiovascular brain regions. Male C57B1/6 mice underwent targeted delivery of an adenovirus encoding the luciferase gene downstream of the CMV promoter to the subfornical organ (SFO) or paraventricular nucleus of hypothalamus (PVN), two crucial cardioregulatory neural regions. While bioluminescent signals could be obtained following d-luciferin injection (150 mg/kg), CycLuc1 administration resulted in a three- to fourfold greater bioluminescent emission from the SFO and PVN, at 10- to 20-fold lower substrate concentrations (7.5-15 mg/kg). This CycLuc1-mediated enhancement in bioluminescent emission was evident early following substrate administration (i.e., 6-10 min) and persisted for up to 1 h. When the exposure time was reduced from 60 s to 1,500 ms, minimal signal in the PVN was detectable with d-luciferin, whereas bioluminescent images could be reliably captured with CycLuc1. These findings demonstrate that bioluminescent imaging with the synthetic luciferin CycLuc1 provides an improved physiological genomics tool to investigate molecular events in discrete cardioregulatory brain nuclei.

The Drug Excipient Cyclodextrin Interacts With d-Luciferin and Interferes With Bioluminescence Imaging.

Cyclodextrins are well-characterized, barrel-shaped molecules that can solubilize organic small molecules in aqueous solution via host-guest interactions. As such, cyclodextrins are used as excipients for experimental therapeutics in vivo. We observed unanticipated modifications to bioluminescence imaging (BLI) signal intensity when 2-hydroxy-propyl-ß-cyclodextrin (HPCD) was coinjected as an excipient. We hypothesized that HPCD bindsd-luciferin and interferes with the BLI signal. Using luciferase-expressing cell lines, we showed that HPCD lowers the BLI signal in a concentration-dependent manner. Flow cytometry revealed that HPCD resulted in reduced cellular accumulation ofd-luciferin, and mass spectrometry revealedd-luciferin HPCD species, confirming a direct interaction. In vivo imaging using a luciferase mouse model demonstrated that HPCD reduced luciferin-mediated BLI compared to luciferin alone. The implications of using HPCD as an excipient in BLI studies are discussed.

A Simple and Rapid Bioluminescence-Based Functional Assay of Organic Anion Transporter 1 as a D-Luciferin Transporter.

Organic anion transporter 1 (SLC22A6/OAT1) plays a key role in renal tubular excretion of endo- and exogenous anionic substances including drugs. Since the inhibition of OAT1 function by a concomitant drug may cause pharmacokinetic drug-drug interactions (DDIs) in clinical practice, an in vitro uptake study to evaluate the inhibition potency of OAT1 is useful for the prediction and avoidance of DDIs and recommended for drug candidates in drug development. In this chapter, we describe a rapid and highly sensitive functional assay of OAT1 based on bioluminescence (BL) detection using D-luciferin as a substrate in living cells. The principle of measurement simply relies on the biochemical feature of D-luciferin to be recognized as a substrate of OAT1, and the BL intensity depending on intracellular D-luciferin level and luciferase activity, thereby allowing the quantitative analysis of OAT1-mediated D-luciferin transport. The BL measurement can be completed within 1 min without experimental procedures for removing extracellular uptake solution and washing cells, both of which involve in the conventional uptake studies using isotope-labeled or fluorescent compounds. The present method is applicable to high-throughput screening to identify and avoid potential OAT1 inhibitors in drug development.

A Simple, Robust, and Affordable Bioluminescent Assay for Drug Screening Against Infective African Trypanosomes.

This chapter introduces a simple and robust in vitro viability assay to screen bioactive small molecules (e.g., natural, synthetic) against the monomorphic and infective (bloodstream) form of Trypanosoma brucei brucei. The assay relies on a bioluminescent transgenic parasite harboring a genetically encoded copy of a thermostable redshifted firefly luciferase from Photinus pyralis.The major advantages of the assay are simplicity and cost efficiency, along with excellent quality parameters. The bioassay allows estimating parasite numbers and viability (and metabolic state) as a function of bioluminescence (BL) signal. Parasites are grown in the presence of the molecules of interest in a 96-well microplate, and 24 h later, BL is determined with a simple protocol lacking washing steps, using cost-efficient reagents with a reasonable readout time for high-throughput applications.

Injectable peptide hydrogel as intraperitoneal triptolide depot for the treatment of orthotopic hepatocellular carcinoma.

Chemotherapy is among the limited choices approved for the treatment of hepatocellular carcinoma (HCC) at intermediate and advanced stages. Preferential and prolonged drug exposure in diseased sites is required to maximize the therapeutic index of the drug. Here, we report an injectable supramolecular peptide hydrogel as an intraperitoneal depot for localized and sustained release of triptolide for the treatment of orthotopic HCC. We chose peptide amphiphile C16-GNNQQNYKD-OH-based nanofibers as gelators and carriers for triptolide. Sustained triptolide release from the hydrogel was achieved over 14 days in vitro, with higher accumulation in and cytotoxicity against human HCC Bel-7402 in comparison with L-02 fetal hepatocytes. After intraperitoneal injection, the hydrogel showed prolonged retention over 13 days and preferential accumulation in the liver, realizing HCC growth inhibition by 99.7 ± 0.1% and animal median survival extension from 19 to 43 days, without causing noticeable pathological changes in the major organs. These results demonstrate that injectable peptide hydrogel can be a potential carrier for localized chemotherapy of HCC.

Synthesis and quantitative characterization of coumarin-caged D-luciferin.

Caged luciferin compounds of firefly luciferins have recently drawn much attention since firefly bioluminescence, in which D-luciferin acts as a substrate, is widely used in noninvasive gene-expression imaging, studies of in vivo cell trafficking, and the detection of enzyme activity. The objectives of this study are the development of new caged luciferins and the quantitative determination of the photophysical parameters of their photo-decomposition. We synthesized 7-(diethylaminocoumarin)-4-(yl)methyl caged D-luciferin (DEACM-caged D-luciferin) and quantitatively characterized its absorption spectrum, bioluminescence, and photoproducts using chiral HPLC chromatography, as a function of light-irradiation time. We observed that 4 min of UV irradiation generated maximum D-luciferin concentrations, which corresponds to 16.2% of the original DEACM-caged-D-luciferin concentration. Moreover, we evaluated not only the rate of photocleavage (0.20/min) from DEACM-caged D-luciferin to luciferin but also the rate of caged-luciferin degradation that did not produce luciferin (0.28/min) and the rate of luciferin decomposition (0.20/min) after exposure to irradiation with a 70 mW/cm2 high-pressure mercury lamp (254-600 nm). The formation rate of L-luciferin via DEACM-caged-D-luciferin photocleavage was smaller by a factor of 1/10 compared with that of D-luciferin. These quantitative measurements and simultaneous evaluations of photocleavage, degradation, and decomposition are the most important and original methodology presented in this study.

Effect of ß-elemene on the kinetics of intracellular transport of d-luciferin potassium salt (ABC substrate) in doxorubicin-resistant breast cancer cells and the associated molecular mechanism.

In order to explore the mechanism of the reversing multidrug resistance (MDR) phenotypes by ß-elemene (ß-ELE) in doxorubicin (DOX)-resistant breast cancer cells (MCF-7/DOX), both the functionality and quantity of the ABC transporters in MCF-7/DOX were studied. Bioluminescence imaging (BLI) was used to study the efflux of d-luciferin potassium salt, the substrate of ATP-binding cassette transporters (ABC transporters), in MCF-7/DOX cells treated by ß-ELE. At the same time three major ABC transport proteins and genes-related MDR, P-glycoprotein (P-gp, ABCB1) and multidrug resistance-associated protein 1 (MRP, ABCC1) as well as breast cancer resistance protein (BCRP, ABCG2) were analyzed by q-PCR and Western blot. To investigate the efflux functionality of ABC transporters, MCF-7/DOXFluc cell line with stably-overexpressed luciferase was established. BLI was then used to real-time monitor the efflux kinetics of d-luciferin potassium salt before and after MCF-7/DOXFluc cells being treated with ß-ELE or not. The results showed that the efflux of d-luciferin potassium salt from MCF-7/DOXFluc was lessened when pretreated with ß-ELE, which means that ß-ELE may dampen the functionality of ABC transporters, thus decrease the efflux of d-fluorescein potassium or other chemotherapies which also serve as the substrates of ABC transporters. As the effect of ß-ELE on the expression of ABC transporters, the results of q-PCR and Western blot showed that gene and protein expression of ABC transporters such as P-gp, MRP, and BCRP were down-regulated after the treatment of ß-ELE. To verify the efficacy of ß-ELE on reversing MDR, MCF-7/DOX cells were treated with the combination of DOX and ß-ELE. MTT assay showed that ß-ELE increased the inhibitory effect of DOX on the proliferation of MCF-7/DOX, and the IC50 of the combination group was much lower than that of the single DOX or ß-ELE treatment. In all, ß-ELE may reverse MDR through the substrates of ABC transporters by two ways, to lessen the ABC protein efflux by weakening their functionality, or to reduce the quantity of ABC gene and protein expression.

In Vivo Bioluminescent Imaging of ATP-Binding Cassette Transporter-Mediated Efflux at the Blood-Brain Barrier.

We provide a detailed protocol for imaging ATP-binding cassette subfamily G member 2 (ABCG2) function at the blood-brain barrier (BBB) of transgenic mice. D-Luciferin is specifically transported by ABCG2 found on the apical side of endothelial cells at the BBB. The luciferase-luciferin enzymatic reaction produces bioluminescence, which allows a direct measurement of ABCG2 function at the BBB. Therefore bioluminescence imaging (BLI) correlates with ABCG2 function at the BBB and this can be measured by administering luciferin in a mouse model that expresses luciferase in the brain parenchyma. BLI allows for a relatively low-cost alternative for studying transporter function in vivo compared to other strategies such as positron emission tomography. This method for imaging ABCG2 function at the BBB can be used to investigate pharmacokinetic inhibition of the transporter.

Hydrazide d-luciferin for in vitro selective detection and intratumoral imaging of Cu(2.).

Copper is an essential micronutrient involved in fundamental life processes but using a bioluminescence (BL) probe to selectively sense Cu(2+)in vitro or image Cu(2+)in vivo is still unavailable. Herein, a latent BL probe hydrazide d-luciferin (1) was rationally designed and successfully applied it for selective detection of Cu(2+)in vitro and imaging Cu(2+) in living cells and in tumors. Upon the catalysis of Cu(2+), 1 was converted to d-luciferin and turned on the BL in the presence of firefly luciferase (fLuc). In vitro tests indicated that 1 could be applied for highly selective sensing Cu(2+) within the range of 0-80µM with a limit of detection (LOD) of 39.0nM. Cell and animal experiments indicated that 1 could be applied for specific BL imaging of Cu(2+) in living cells and tumors and the BL signal of 1 was more stable and longer than that of d-luciferin. We envision that this unique probe 1 might serve as an elucidative tool for further exploration of the biological roles of Cu(2+) in physiological and pathological processes in the near future.

Intracellular Self-Assembly of Cyclic d-Luciferin Nanoparticles for Persistent Bioluminescence Imaging of Fatty Acid Amide Hydrolase.

Fatty acid amide hydrolase (FAAH) overexpression induces several disorder symptoms in nerve systems, and therefore long-term tracing of FAAH activity in vivo is of high importance but remains challenging. Current bioluminescence (BL) methods are limited in detecting FAAH activity within 5 h. Herein, by rational design of a latent BL probe (d-Cys-Lys-CBT)2 (1), we developed a "smart" method of intracellular reduction-controlled self-assembly and FAAH-directed disassembly of its cyclic d-luciferin-based nanoparticles (i.e., 1-NPs) for persistent BL imaging of FAAH activity in vitro, in cells, and in vivo. Using aminoluciferin methyl amide (AMA), Lys-amino-d-luciferin (Lys-Luc), and amino-d-luciferin (NH2-Luc) as control BL probes, we validated that the persistent BL of 1 from luciferase-expressing cells or tumors was controlled by the activity of intracellular FAAH. With the property of long-term tracing of FAAH activity in vivo of 1, we envision that our BL precursor 1 could probably be applied for in vivo screening of FAAH inhibitors and the diagnosis of their related diseases (or disorders) in the future.

Label-Free Cell Phenotypic Identification of D-Luciferin as an Agonist for GPR35.

D-Luciferin (also known as beetle or firefly luciferin) is one of the most widely used bioluminescent reporters for monitoring in vitro or in vivo luciferase activity. The identification of several natural phenols and thieno[3,2-b]thiophene-2-carboxylic acid derivatives as agonists for GPR35, an orphan G protein-coupled receptor, had motivated us to examine the pharmacological activity of D-Luciferin, given that it also contains phenol and carboxylic acid moieties. Here, we describe label-free cell phenotypic assays that ascertain D-Luciferin as a partial agonist for GPR35. The agonistic activity of D-Luciferin at the GPR35 shall evoke careful interpretation of biological data when D-Luciferin or its analogues are used as probes.

Identification of new MRP4 inhibitors from a library of FDA approved drugs using a high-throughput bioluminescence screen.

Multidrug resistance protein 4 (MRP4) effluxes a wide variety of drugs and endogenous signaling molecules from cells and has been proposed as an attractive therapeutic target in several solid tumors, including neuroblastoma and colorectal cancer. MRP4 also regulates the pharmacokinetics of its drug substrates and its absence can increase their tissue penetration. We observed that MRP4 can efflux the bioluminescence substrate d-luciferin, and exploited this phenomenon to develop a robust, high throughput, live cell-based bioluminescent screen to identify new MRP4 inhibitors. We applied this screen to a combined library of 3600 compounds, all of which were either FDA-approved drugs or bioactive compounds with defined mechanisms of action. From the primary screen, 36 compounds effectively inhibited MRP4 (>4-fold increase in bioluminescence), with inhibitors of receptor tyrosine kinases and phosphodiesterases highly over-represented. Selected compounds were tested for their ability to sensitize MRP4-overexpressing cell lines to the MRP4 substrate drugs 6-mercaptopurine and SN-38, with sensitization up to 6.5-fold with the ryanodine receptor antagonist dantrolene. These newly identified MRP4 inhibitors are readily available and are either established drugs or well-characterized bioactive compounds. As such, they should be immediately useful as investigative tools, and suitable for testing both in vitro and in vivo.

The use of optical imaging to assess the potential for embryo-fetal exposure to an exogenous material after intravaginal administration.

A ß-actin-luc transgenic mouse model was used to evaluate whether embryo-fetal exposure could occur after intravaginal administration of a compound. A bioluminescent substrate, d-luciferin, was delivered intravaginally to mimic compound exposure to the female reproductive track and the embryo-fetus. Bioluminescence was observed throughout the reproductive tract during diestrus, but not during estrus, 2-5min after intravaginal d-luciferin administration to female ß-actin-luc mice. Intravaginal administration of d-luciferin to wild-type females mated with male ß-actin-luc mice indicated that the substrate reached the developing embryo-fetus, with bioluminescence corresponding to transgene expression in the embryo-fetus. d-Luciferin substrate rapidly reached the embryo-fetus regardless of the administration route (intravaginal, intraperitoneal, subcutaneous, or intravenous). Vaginal ligation appeared to block at least some direct exposure to the embryo-fetus, but did not prevent d-luciferin from eventually reaching the embryo-fetus. Additional work will be necessary to form the basis for a reliable assessment of the human risk for male-mediated teratogenicity.

High-throughput screening identifies Ceefourin 1 and Ceefourin 2 as highly selective inhibitors of multidrug resistance protein 4 (MRP4).

Multidrug resistance protein 4 (MRP4/ABCC4), a member of the ATP-binding cassette (ABC) transporter superfamily, is an organic anion transporter capable of effluxing a wide range of physiologically important signalling molecules and drugs. MRP4 has been proposed to contribute to numerous functions in both health and disease; however, in most cases these links remain to be unequivocally established. A major limitation to understanding the physiological and pharmacological roles of MRP4 has been the absence of specific small molecule inhibitors, with the majority of established inhibitors also targeting other ABC transporter family members, or inhibiting the production, function or degradation of important MRP4 substrates. We therefore set out to identify more selective and well tolerated inhibitors of MRP4 that might be used to study the many proposed functions of this transporter. Using high-throughput screening, we identified two chemically distinct small molecules, Ceefourin 1 and Ceefourin 2, that inhibit transport of a broad range of MRP4 substrates, yet are highly selective for MRP4 over other ABC transporters, including P-glycoprotein (P-gp), ABCG2 (Breast Cancer Resistance Protein; BCRP) and MRP1 (multidrug resistance protein 1; ABCC1). Both compounds are more potent MRP4 inhibitors in cellular assays than the most widely used inhibitor, MK-571, requiring lower concentrations to effect a comparable level of inhibition. Furthermore, Ceefourin 1 and Ceefourin 2 have low cellular toxicity, and high microsomal and acid stability. These newly identified inhibitors should be of great value for efforts to better understand the biological roles of MRP4, and may represent classes of compounds with therapeutic application.