The taste of ribonucleosides: Novel macronutrients essential for larval growth are sensed by Drosophila gustatory receptor proteins.

Animals employ various types of taste receptors to identify and discriminate between different nutritious food chemicals. These macronutrients are thought to fall into 3 major groups: carbohydrates/sugars, proteins/amino acids, and fats. Here, we report that Drosophila larvae exhibit a novel appetitive feeding behavior towards ribose, ribonucleosides, and RNA. We identified members of the gustatory receptor (Gr) subfamily 28 (Gr28), expressed in both external and internal chemosensory neurons as molecular receptors necessary for cellular and appetitive behavioral responses to ribonucleosides and RNA. Specifically, behavioral preference assays show that larvae are strongly attracted to ribose- or RNA-containing agarose in a Gr28-dependent manner. Moreover, Ca2+ imaging experiments reveal that Gr28a-expressing taste neurons are activated by ribose, RNA and some ribonucleosides and that these responses can be conveyed to Gr43aGAL4 fructose-sensing neurons by expressing single members of the Gr28 gene family. Lastly, we establish a critical role in behavioral fitness for the Gr28 genes by showing that Gr28 mutant larvae exhibit low survival rates when challenged to find ribonucleosides in food. Together, our work identifies a novel taste modality dedicated to the detection of RNA and ribonucleosides, nutrients that are essential for survival during the accelerated growth phase of Drosophila larvae.

Biochemical, cellular, and biophysical characterization of a potent inhibitor of mutant isocitrate dehydrogenase IDH1.

Two mutant forms (R132H and R132C) of isocitrate dehydrogenase 1 (IDH1) have been associated with a number of cancers including glioblastoma and acute myeloid leukemia. These mutations confer a neomorphic activity of 2-hydroxyglutarate (2-HG) production, and 2-HG has previously been implicated as an oncometabolite. Inhibitors of mutant IDH1 can potentially be used to treat these diseases. In this study, we investigated the mechanism of action of a newly discovered inhibitor, ML309, using biochemical, cellular, and biophysical approaches. Substrate binding and product inhibition studies helped to further elucidate the IDH1 R132H catalytic cycle. This rapidly equilibrating inhibitor is active in both biochemical and cellular assays. The (+) isomer is active (IC50 = 68 nm), whereas the (-) isomer is over 400-fold less active (IC50 = 29 µm) for IDH1 R132H inhibition. IDH1 R132C was similarly inhibited by (+)-ML309. WT IDH1 was largely unaffected by (+)-ML309 (IC50 >36 µm). Kinetic analyses combined with microscale thermophoresis and surface plasmon resonance indicate that this reversible inhibitor binds to IDH1 R132H competitively with respect to α-ketoglutarate and uncompetitively with respect to NADPH. A reaction scheme for IDH1 R132H inhibition by ML309 is proposed in which ML309 binds to IDH1 R132H after formation of the IDH1 R132H NADPH complex. ML309 was also able to inhibit 2-HG production in a glioblastoma cell line (IC50 = 250 nm) and had minimal cytotoxicity. In the presence of racemic ML309, 2-HG levels drop rapidly. This drop was sustained until 48 h, at which point the compound was washed out and 2-HG levels recovered.

Molecular basis for the high-affinity binding and stabilization of firefly luciferase by PTC124.

Firefly luciferase (FLuc), an ATP-dependent bioluminescent reporter enzyme, is broadly used in chemical biology and drug discovery assays. PTC124 (Ataluren; (3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid) discovered in an FLuc-based assay targeting nonsense codon suppression, is an unusually potent FLuc-inhibitor. Paradoxically, PTC124 and related analogs increase cellular FLuc activity levels by posttranslational stabilization. In this study, we show that FLuc inhibition and stabilization is the result of an inhibitory product formed during the FLuc-catalyzed reaction between its natural substrate, ATP, and PTC124. A 2.0 A cocrystal structure revealed the inhibitor to be the acyl-AMP mixed-anhydride adduct PTC124-AMP, which was subsequently synthesized and shown to be a high-affinity multisubstrate adduct inhibitor (MAI; K(D) = 120 pM) of FLuc. Biochemical assays, liquid chromatography/mass spectrometry, and near-attack conformer modeling demonstrate that formation of this novel MAI is absolutely dependent upon the precise positioning and reactivity of a key meta-carboxylate of PTC124 within the FLuc active site. We also demonstrate that the inhibitory activity of PTC124-AMP is relieved by free coenzyme A, a component present at high concentrations in luciferase detection reagents used for cell-based assays. This explains why PTC124 can appear to increase, instead of inhibit, FLuc activity in cell-based reporter gene assays. To our knowledge, this is an unusual example in which the "off-target" effect of a small molecule is mediated by an MAI mechanism.

Mechanism of PTC124 activity in cell-based luciferase assays of nonsense codon suppression.

High-throughput screening (HTS) assays used in drug discovery frequently use reporter enzymes such as firefly luciferase (FLuc) as indicators of target activity. An important caveat to consider, however, is that compounds can directly affect the reporter, leading to nonspecific but highly reproducible assay signal modulation. In rare cases, this activity appears counterintuitive; for example, some FLuc inhibitors, acting through posttranslational Fluc reporter stabilization, appear to activate gene expression. Previous efforts to characterize molecules that influence luciferase activity identified a subset of 3,5-diaryl-oxadiazole-containing compounds as FLuc inhibitors. Here, we evaluate a number of compounds with this structural motif for activity against FLuc. One such compound is PTC124 {3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid}, a molecule originally identified in a cell-based FLuc assay as having nonsense codon suppression activity [Welch EM, et al., Nature (2007) 447:87-91]. We find that the potency of FLuc inhibition for the tested compounds strictly correlates with their activity in a FLuc reporter cell-based nonsense codon assay, with PTC124 emerging as the most potent FLuc inhibitor (IC(50) = 7 +/- 1 nM). However, these compounds, including PTC124, fail to show nonsense codon suppression activity when Renilla reniformis luciferase (RLuc) is used as a reporter and are inactive against the RLuc enzyme. This suggests that the initial discovery of PTC124 may have been biased by its direct effect on the FLuc reporter, implicating firefly luciferase as a molecular target of PTC124. Our results demonstrate the value of understanding potential interactions between reporter enzymes and chemical compounds and emphasize the importance of implementing the appropriate control assays before interpreting HTS results.

Phosphocyclocreatine is the dominant form of cyclocreatine in control and creatine transporter deficiency patient fibroblasts.

Creatine transporter deficiency (CTD) is a metabolic disorder resulting in cognitive, motor, and behavioral deficits. Cyclocreatine (cCr), a creatine analog, has been explored as a therapeutic strategy for the treatment of CTD. We developed a rapid, selective, and accurate HILIC ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method to simultaneously quantify the intracellular concentrations of cCr, creatine (Cr), creatine-d3 (Cr-d3), phosphocyclocreatine (pcCr), and phosphocreatine (pCr). Using HILIC-UPLC-MS/MS, we measured cCr and Cr-d3 uptake and their conversion to the phosphorylated forms in primary human control and CTD fibroblasts. Altogether, the data demonstrate that cCr enters cells and its dominant intracellular form is pcCr in both control and CTD patient cells. Therefore, cCr may replace creatine as a therapeutic strategy for the treatment of CTD.

Gustatory perception and behavior in Drosophila melanogaster.

The sense of taste is essential for the survival of virtually all animals. Considered a 'primitive sense' and present in the form of chemotaxis in many bacteria, taste is also a sense of sophistication in humans. Regardless, taste behavior is a crucial activity for the world's most abundant (insects) and most successful (mammals) inhabitants, providing a means of discrimination between nutrient-rich substrates, such as sugars and amino acids, from harmful, mostly bitter-tasting chemicals present in many plants. In this review, we present an update on progress in understanding taste perception in the model fruit fly Drosophila melanogaster. An introduction to the fly's taste system will be presented first, followed by a description of relevant behavioral assays developed to quantify taste perception at the organismal level and a short overview of electrophysiological studies performed on taste cells. The focal point will be the recent molecular-genetic investigations of the gustatory receptor (Gr) genes, which is complemented by a comparison between Drosophila and mammalian taste perception and transduction. Finally, we provide a perspective on the future of Drosophila taste research, including three specific proposals that seem uniquely applicable to this exquisite model system and cannot, at least currently, be pursued elsewhere.

Atypical expression of Drosophila gustatory receptor genes in sensory and central neurons.

Members of the Drosophila gustatory receptor (Gr) gene family are generally expressed in chemosensory neurons and are known to mediate the perception of sugars, bitter substrates, CO(2), and pheromones. The Gr gene family consists of 68 members, many of which are organized in gene clusters of up to six genes, yet only expression of about 15 Gr genes has been characterized in detail prior to this study. Here we describe the first comprehensive expression analysis of six highly conserved Gr genes, Gr28a and Gr28b.a to Gr28b.e. Four of these Gr genes are not only expressed in the characteristic pattern associated with previously analyzed Gr genes-chemosensory neurons of the gustatory and olfactory system-but several other types of sensory neurons and neurons in the brain. Specifically, we show that several of the Gr28 genes are expressed in abdominal multidendritic neurons, putative hygroreceptive neurons of the arista, neurons associated with the Johnston's organ, peripheral proprioceptive neurons in the legs, neurons in the larval and adult brain, and oenocytes. Thus, our findings suggest that some Gr genes are utilized in nongustatory roles in the nervous system and tissues involved in proprioception, hygroreception, and other sensory modalities. It is also possible that the Gr28 genes have chemosensory roles in the detection of internal ligands.

A robotic platform for quantitative high-throughput screening.

High-throughput screening (HTS) is increasingly being adopted in academic institutions, where the decoupling of screening and drug development has led to unique challenges, as well as novel uses of instrumentation, assay formulations, and software tools. Advances in technology have made automated unattended screening in the 1,536-well plate format broadly accessible and have further facilitated the exploration of new technologies and approaches to screening. A case in point is our recently developed quantitative HTS (qHTS) paradigm, which tests each library compound at multiple concentrations to construct concentration-response curves (CRCs) generating a comprehensive data set for each assay. The practical implementation of qHTS for cell-based and biochemical assays across libraries of > 100,000 compounds (e.g., between 700,000 and 2,000,000 sample wells tested) requires maximal efficiency and miniaturization and the ability to easily accommodate many different assay formats and screening protocols. Here, we describe the design and utilization of a fully integrated and automated screening system for qHTS at the National Institutes of Health's Chemical Genomics Center. We report system productivity, reliability, and flexibility, as well as modifications made to increase throughput, add additional capabilities, and address limitations. The combination of this system and qHTS has led to the generation of over 6 million CRCs from > 120 assays in the last 3 years and is a technology that can be widely implemented to increase efficiency of screening and lead generation.

Astrocytes as targets for drug discovery.

Recent studies have illuminated the crucial role of astrocytes in maintaining proper neuronal health and function. Abnormalities in astrocytic functions have now been implicated in the pathogenesis of neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and amyotrophic lateral sclerosis (ALS). Historically, drug development programs for neurodegenerative diseases generally target only neurons, overlooking the contributions of astrocytes. Therefore, targeting both disease neurons and astrocytes offers a new approach for drug development for the treatment of neurological diseases. Looking forward, the co-culturing of human neurons with astrocytes could be the next evolutionary step in drug discovery for neurodegenerative diseases.

Repurposing a novel parathyroid hormone analogue to treat hypoparathyroidism.

BACKGROUND AND PURPOSE: Human parathyroid hormone (PTH) is critical for maintaining physiological calcium homeostasis and plays an important role in the formation and maintenance of the bone. Full-length PTH and a truncated peptide form are approved for treatment of hypoparathyroidism and osteoporosis respectively. Our initial goal was to develop an improved PTH therapy for osteoporosis, but clinical development was halted. The novel compound was then repurposed as an improved therapy for hypoparathyroidism. EXPERIMENTAL APPROACH: A longer-acting form of PTH was synthesised by altering the peptide to increase cell surface residence time of the bound ligand to its receptor. In vitro screening identified a compound, which was tested in an animal model of osteoporosis before entering human trials. This compound was subsequently tested in two independent animal models of hypoparathyroidism. KEY RESULTS: The peptide identified, LY627-2K, exhibited delayed internalization kinetics. In an ovariectomy-induced bone loss rat model, LY627-2K demonstrated improved vertebral bone mineral density and biomechanical properties at skeletal sites and a modest increase in serum calcium. In a Phase I clinical study, dose-dependent increases in serum calcium were reproduced. These observations prompted us to explore a second indication, hypoparathyroidism. In animal models of this disease, LY627-2K restored serum calcium, comparing favourably to treatment with wild-type PTH. CONCLUSIONS AND IMPLICATIONS: We summarize the repositioning of a therapeutic candidate with substantial preclinical and clinical data. Our results support its repurposing and continued development, from a common indication (osteoporosis) to a rare disease (hypoparathyroidism) by exploiting a shared molecular target. LINKED ARTICLES: This article is part of a themed section on Inventing New Therapies Without Reinventing the Wheel: The Power of Drug Repurposing. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.2/issuetoc.

Vomeronasal organ: pheromone recognition with a twist.

Pheromones are detected by the vomeronasal organ using members of two receptor superfamilies: the V1Rs and V2Rs. New studies show that MHC class I molecules are co-expressed in particular combinations with specific V2Rs in the vomeronasal organ. The role of these MHC molecules is unknown, but they may be of considerable biological significance.

Taste perception and coding in Drosophila.

BACKGROUND: Discrimination between edible and contaminated foods is crucial for the survival of animals. In Drosophila, a family of gustatory receptors (GRs) expressed in taste neurons is thought to mediate the recognition of sugars and bitter compounds, thereby controlling feeding behavior. RESULTS: We have characterized in detail the expression of eight Gr genes in the labial palps, the fly's main taste organ. These genes fall into two distinct groups: seven of them, including Gr66a, are expressed in 22 or fewer taste neurons in each labial palp. Additional experiments show that many of these genes are coexpressed in partially overlapping sets of neurons. In contrast, Gr5a, which encodes a receptor for trehalose, is expressed in a distinct and larger set of taste neurons associated with most chemosensory sensilla, including taste pegs. Mapping the axonal targets of cells expressing Gr66a and Gr5a reveals distinct projection patterns for these two groups of neurons in the brain. Moreover, tetanus toxin-mediated inactivation of Gr66a- or Gr5a-expressing cells shows that these two sets of neurons mediate distinct taste modalities-the perception of bitter (caffeine) and sweet (trehalose) taste, respectively. CONCLUSION: Discrimination between two taste modalities-sweet and bitter-requires specific sets of gustatory receptor neurons that express different Gr genes. Unlike the Drosophila olfactory system, where each neuron expresses a single olfactory receptor gene, taste neurons can express multiple receptors and do so in a complex Gr gene code that is unique for small sets of neurons.

Targeting Wolman Disease and Cholesteryl Ester Storage Disease: Disease Pathogenesis and Therapeutic Development.

Wolman disease (WD) and cholesteryl ester storage disease (CESD) are lysosomal storage diseases (LSDs) caused by a deficiency in lysosomal acid lipase (LAL) due to mutations in the LIPA gene. This enzyme is critical to the proper degradation of cholesterol in the lysosome. LAL function is completely lost in WD while some residual activity remains in CESD. Both are rare diseases with an incidence rate of less than 1/100,000 births for WD and approximate 2.5/100,000 births for CESD. Clinical manifestation of WD includes hepatosplenomegaly, calcified adrenal glands, severe malabsorption and a failure to thrive. As in CESD, histological analysis of WD tissues reveals the accumulation of triglycerides (TGs) and esterified cholesterol (EC) in cellular lysosomes. However, the clinical presentation of CESD is less severe and more variable than WD. This review is to provide an overview of the disease pathophysiology and the current state of therapeutic development for both of WD and CESD. The review will also discuss the application of patient derived iPSCs for further drug discovery.

Neural stem cells for disease modeling of Wolman disease and evaluation of therapeutics.

BACKGROUND: Wolman disease (WD) is a rare lysosomal storage disorder that is caused by mutations in the LIPA gene encoding lysosomal acid lipase (LAL). Deficiency in LAL function causes accumulation of cholesteryl esters and triglycerides in lysosomes. Fatality usually occurs within the first year of life. While an enzyme replacement therapy has recently become available, there is currently no small-molecule drug treatment for WD. RESULTS: We have generated induced pluripotent stem cells (iPSCs) from two WD patient dermal fibroblast lines and subsequently differentiated them into neural stem cells (NSCs). The WD NSCs exhibited the hallmark disease phenotypes of neutral lipid accumulation, severely deficient LAL activity, and increased LysoTracker dye staining. Enzyme replacement treatment dramatically reduced the WD phenotype in these cells. In addition, δ-tocopherol (DT) and hydroxypropyl-beta-cyclodextrin (HPBCD) significantly reduced lysosomal size in WD NSCs, and an enhanced effect was observed in DT/HPBCD combination therapy. CONCLUSION: The results demonstrate that these WD NSCs are valid cell-based disease models with characteristic disease phenotypes that can be used to evaluate drug efficacy and screen compounds. DT and HPBCD both reduce LysoTracker dye staining in WD cells. The cells may be used to further dissect the pathology of WD, evaluate compound efficacy, and serve as a platform for high-throughput drug screening to identify new compounds for therapeutic development.

High-Throughput Phenotypic Screening of Human Astrocytes to Identify Compounds That Protect Against Oxidative Stress.

UNLABELLED: Astrocytes are the predominant cell type in the nervous system and play a significant role in maintaining neuronal health and homeostasis. Recently, astrocyte dysfunction has been implicated in the pathogenesis of many neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis. Astrocytes are thus an attractive new target for drug discovery for neurological disorders. Using astrocytes differentiated from human embryonic stem cells, we have developed an assay to identify compounds that protect against oxidative stress, a condition associated with many neurodegenerative diseases. This phenotypic oxidative stress assay has been optimized for high-throughput screening in a 1,536-well plate format. From a screen of approximately 4,100 bioactive tool compounds and approved drugs, we identified a set of 22 that acutely protect human astrocytes from the consequences of hydrogen peroxide-induced oxidative stress. Nine of these compounds were also found to be protective of induced pluripotent stem cell-differentiated astrocytes in a related assay. These compounds are thought to confer protection through hormesis, activating stress-response pathways and preconditioning astrocytes to handle subsequent exposure to hydrogen peroxide. In fact, four of these compounds were found to activate the antioxidant response element/nuclear factor-E2-related factor 2 pathway, a protective pathway induced by toxic insults. Our results demonstrate the relevancy and utility of using astrocytes differentiated from human stem cells as a disease model for drug discovery and development. SIGNIFICANCE: Astrocytes play a key role in neurological diseases. Drug discovery efforts that target astrocytes can identify novel therapeutics. Human astrocytes are difficult to obtain and thus are challenging to use for high-throughput screening, which requires large numbers of cells. Using human embryonic stem cell-derived astrocytes and an optimized astrocyte differentiation protocol, it was possible to screen approximately 4,100 compounds in titration to identify 22 that are cytoprotective of astrocytes. This study is the largest-scale high-throughput screen conducted using human astrocytes, with a total of 17,536 data points collected in the primary screen. The results demonstrate the relevancy and utility of using astrocytes differentiated from human stem cells as a disease model for drug discovery and development.

High-throughput viability assay using an autonomously bioluminescent cell line with a bacterial Lux reporter.

Cell viability assays are extensively used to determine cell health, evaluate growth conditions, and assess compound cytotoxicity. Most existing assays are endpoint assays, in which data are collected at one time point after termination of the experiment. The time point at which toxicity of a compound is evident, however, depends on the mechanism of that compound. An ideal cell viability assay allows the determination of compound toxicity kinetically without having to terminate the assay prematurely. We optimized and validated a reagent-addition-free cell viability assay using an autoluminescent HEK293 cell line that stably expresses bacterial luciferase and all substrates necessary for bioluminescence. This cell viability assay can be used for real-time, long-term measurement of compound cytotoxicity in live cells with a signal-to-basal ratio of 20- to 200-fold and Z-factors of ~0.6 after 24-, 48- 72-, or 96-h incubation with compound. We also found that the potencies of nine cytotoxic compounds correlated well with those measured by four other commonly used cell viability assays. The results demonstrated that this kinetic cell viability assay using the HEK293(lux) autoluminescent cell line is useful for high-throughput evaluation of compound cytotoxicity.

Phenotypic screens as a renewed approach for drug discovery.

The significant reduction in the number of newly approved drugs in the past decade has been partially attributed to failures in discovery and validation of new targets. Evaluation of recently approved new drugs has revealed that the number of approved drugs discovered through phenotypic screens, an original drug screening paradigm, has exceeded those discovered through the molecular target-based approach. Phenotypic screening is thus gaining new momentum in drug discovery with the hope that this approach may revitalize drug discovery and improve the success rate of drug approval through the discovery of viable lead compounds and identification of novel drug targets.

Physicochemical characterization of a thermostable alcohol dehydrogenase from Pyrobaculum aerophilum.

In this work we characterize an alcohol dehydrogenase (ADH) from the hyperthermophilic archaeon Pyrobaculum aerophilum (PyAeADHII). We have previously found that PyAeADHII has no activity when standard ADH substrates are used but is active when α-tetralone is used as substrate. Here, to gain insights into enzyme function, we screened several chemical libraries for enzymatic modulators using an assay employing α-tetralone. The results indicate that PyAeADHII activity in the presence of α-tetralone was inhibited by compounds such as flunarizine. We also examined metal coordination of the enzyme in solution by performing metal substitution of the enzyme-bound zinc (Zn²âº) with cobalt. The solution-based absorption spectra for cobalt substituted PyAeADHII supports substitution at the structural Zn²âº site. To gain structural insight, we obtained the crystal structure of both wild-type and cobalt-substituted PyAeADHII at 1.75 Å and 2.20 Å resolution, respectively. The X-ray data confirmed one metal ion per monomer present only at the structural site with otherwise close conservation to other ADH enzymes. We next determined the co-crystal structure of the NADPH-bound form of the enzyme at 2.35 Å resolution to help define the active site region of the enzyme and this data shows close structural conservation with horse ADH, despite the lack of a catalytic Zn²âº ion in PyAeADHII. Modeling of α-tetralone into the NADPH bound structure suggests an arginine as a possible catalytic residue. The data presented here can yield a better understanding of alcohol dehydrogenases lacking the catalytic zinc as well as the structural features inherent to thermostable enzymes.

A homogeneous, high-throughput assay for phosphatidylinositol 5-phosphate 4-kinase with a novel, rapid substrate preparation.

Phosphoinositide kinases regulate diverse cellular functions and are important targets for therapeutic development for diseases, such as diabetes and cancer. Preparation of the lipid substrate is crucial for the development of a robust and miniaturizable lipid kinase assay. Enzymatic assays for phosphoinositide kinases often use lipid substrates prepared from lyophilized lipid preparations by sonication, which result in variability in the liposome size from preparation to preparation. Herein, we report a homogeneous 1536-well luciferase-coupled bioluminescence assay for PI5P4Kα. The substrate preparation is novel and allows the rapid production of a DMSO-containing substrate solution without the need for lengthy liposome preparation protocols, thus enabling the scale-up of this traditionally difficult type of assay. The Z'-factor value was greater than 0.7 for the PI5P4Kα assay, indicating its suitability for high-throughput screening applications. Tyrphostin AG-82 had been identified as an inhibitor of PI5P4Kα by assessing the degree of phospho transfer of γ-(32)P-ATP to PI5P; its inhibitory activity against PI5P4Kα was confirmed in the present miniaturized assay. From a pilot screen of a library of bioactive compounds, another tyrphostin, I-OMe tyrphostin AG-538 (I-OMe-AG-538), was identified as an ATP-competitive inhibitor of PI5P4Kα with an IC(50) of 1 µM, affirming the suitability of the assay for inhibitor discovery campaigns. This homogeneous assay may apply to other lipid kinases and should help in the identification of leads for this class of enzymes by enabling high-throughput screening efforts.

Evaluation of cholesterol reduction activity of methyl-ß-cyclodextrin using differentiated human neurons and astrocytes.

Recent advances in stem cell technology have enabled large-scale production of human cells such as cardiomyocytes, hepatocytes, and neurons for evaluation of pharmacologic effect and toxicity of drug candidates. The assessment of compound efficacy and toxicity using human cells should lower the high clinical attrition rates of drug candidates by reducing the impact of species differences on drug efficacy and toxicity from animal studies. Methyl-ß-cyclodextrin (MBCD) has been shown to reduce lysosomal cholesterol accumulation in skin fibroblasts derived from patients with Niemann Pick type C disease and in the NPC1-/- mouse model. However, the compound has never been tested in human differentiated neurons. We have determined the cholesterol reduction effect of MBCD in neurons differentiated from human neural stem cells (NSCs) and commercially available astrocytes. The use of NSCs for producing differentiated neurons in large quantities can significantly reduce the production time and enhance the reproducibility of screening results. The EC(50) values of MBCD on cholesterol reduction in human neurons and astrocytes were 66.9 and 110.7 µM, respectively. The results indicate that human neurons differentiated from the NSCs and human astrocytes are useful tools for evaluating pharmacologic activity and toxicity of drug candidates to predict their clinical efficacy.