Screening System For Compounds Research Articles

Development of an in vitro compound screening system that replicate the in vivo spine phenotype of idiopathic ASD model mice.

Autism Spectrum Disorder (ASD) is a developmental condition characterized by core symptoms including social difficulties, repetitive behaviors, and sensory abnormalities. Aberrant morphology of dendritic spines within the cortex has been documented in genetic disorders associated with ASD and ASD-like traits. We hypothesized that compounds that ameliorate abnormalities in spine dynamics might have the potential to ameliorate core symptoms of ASD. Because the morphology of the spine is influenced by signal inputs from other neurons and various molecular interactions, conventional single-molecule targeted drug discovery methods may not suffice in identifying compounds capable of ameliorating spine morphology abnormalities. In this study, we focused on spine phenotypes in the cortex using BTBR T + Itpr3 tf /J (BTBR) mice, which have been used as a model for idiopathic ASD in various studies. We established an in vitro compound screening system using primary cultured neurons from BTBR mice to faithfully represent the spine phenotype. The compound library mainly comprised substances with known target molecules and established safety profiles, including those approved or validated through human safety studies. Following screening of this specialized library containing 181 compounds, we identified 15 confirmed hit compounds. The molecular targets of these hit compounds were largely focused on the 5-hydroxytryptamine receptor (5-HTR). Furthermore, both 5-HT1AR agonist and 5-HT3R antagonist were common functional profiles in hit compounds. Vortioxetine, possessing dual attributes as a 5-HT1AR agonist and 5-HT3R antagonist, was administered to BTBR mice once daily for a period of 7days. This intervention not only ameliorated their spine phenotype but also alleviated their social behavior abnormality. These results of vortioxetine supports the usefulness of a spine phenotype-based assay system as a potent drug discovery platform targeting ASD core symptoms.

High-throughput "read-on-ski" automated imaging and label-free detection system for toxicity screening of compounds using personalised human kidney organoids.

Organoid models are used to study kidney physiology, such as the assessment of nephrotoxicity and underlying disease processes. Personalized human pluripotent stem cell-derived kidney organoids are ideal models for compound toxicity studies, but there is a need to accelerate basic and translational research in the field. Here, we developed an automated continuous imaging setup with the "read-on-ski" law of control to maximize temporal resolution with minimum culture plate vibration. High-accuracy performance was achieved: organoid screening and imaging were performed at a spatial resolution of 1.1 μm for the entire multi-well plate under 3 min. We used the in-house developed multi-well spinning device and cisplatin-induced nephrotoxicity model to evaluate the toxicity in kidney organoids using this system. The acquired images were processed via machine learning-based classification and segmentation algorithms, and the toxicity in kidney organoids was determined with 95% accuracy. The results obtained by the automated "read-on-ski" imaging device, combined with label-free and non-invasive algorithms for detection, were verified using conventional biological procedures. Taking advantage of the close-to-in vivo-kidney organoid model, this new development opens the door for further application of scaled-up screening using organoids in basic research and drug discovery.

Atractylon, a novel dopamine 2 receptor agonist, ameliorates Parkinsonian like motor dysfunctions in MPTP-induced mice

BackgroundMotor symptoms of Parkinson’s disease (PD) are characterized by bradykinesia, resting tremor, rigidity, slow movement, impaired gait and postural instability, resulting from progressive loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). Atractylon is a natural furan compound in Atractylodes rhizomes, exhibiting anticancer, anti-inflammation, antiviral and gastroprotective activities, and so on. However, it is still unknown whether atractylon is beneficial to motor dysfunctions of PD. MethodsGPCR-targeted piggyBac-TANGO compound screening system, cAMP assay, and immunostaining of p-CREB and BDNF were used to identify dopamine 2 receptor (DRD2) activation. The effects of atractylon on motor deficits and gait disturbances, as well as tyrosine hydroxylase (TH) in the SNpc were investigated in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice. ResultsAtractylon treatment increased the eGFP expression in dose-dependent manner in piggyBac-TANGO assay, decreased cAMP production, and enhanced the levels of p-CREB and BDNF in DRD2 highly expresseding SY-SY5Y cells. In MPTP-induced mice, atractylon improved the slow movement, diminished voluntary locomotion, and abnormal gait parameters, such as duration, cadence, average speed, step cycle, stride length, and so on. Moreover, atractylon rescued the TH positive cells in SNpc and TH positive nerve fibers in striatum. ConclusionsAtractylon could effectively activate DRD2, attenuate motor deficits and gait disorders, and protect dopaminergic neurons in MPTP-induced PD mice. Our findings stretch out the therapeutic potential of atractylon for motor symptoms of PD.

Design and Fabrication of Biological Wires for Cardiac Fibrosis Disease Modeling.

Extensive progress has been made in developing engineered models for elucidating human cardiac disease. Cardiac fibrosis is often associated with all forms of cardiac disease and has a direct deleterious effect on cardiac function. As currently there is no effective therapeutic strategy specifically designed to target fibrosis, in vitro diagnostic platforms for drug testing have generated significant interest. In this context, we have developed an innovative approach to generate human cardiac fibrotic tissues on Biowire II platform and established a compound screening system. The disease model is constructed to recapitulate contractile, biomechanical, and electrophysiological complexities of fibrotic myocardium. Additionally, an integrated model with fibrotic and healthy cardiac tissues coupled together can be created to mimic focal fibrosis. The methods for constructing the Biowire fibrotic model will be described here.

Abstract 20: HiBiT tagging system for high throughput chemical screening for chemotherapy

Abstract Chemical screening is an essential procedure for finding novel drugs of cancer treatments. Especially, cell-based screenings are mostly used for discovery of novel compounds or drug repositioning of already-used drugs. Cell viability or cell proliferation are usually used for evaluation of effects of candidate novel compounds for cancer treatments. However, high throughput system for rapid evaluation of endogenous target protein expression changes has not been established so far. Therefore, an easy and quick method for detection of endogenous target protein expression changes has been strongly needed for drug discovery. Herein, we established a novel chemical compound screening system using the HiBiT-tagging system for this aim using PD-L1 as an example. HiBiT tag is a small peptide tag composed of only 11 amino acids forming a dimer with LgBiT, and functions as a Nanoluciferase. The shortness of the peptide tag is useful for insertion of tag using genome editing technology. Thereby, we established a rapid chemical screening method using HiBiT-tagged cell lines created by CRISPR-Cas9 system. With this method, we made it possible to detect endogenous target protein expression changes within 10 minutes by just adding lytic buffer and LgBiT. For validation of this method, we performed chemical screening of PD-L1 using a HiBiT-tagged lung adenocarcinoma cell line with 1,280 compounds. As a result, we identified microtubule inhibitors as compounds upregulating PD-L1, and validated its upregulation of PD-L1 with qPCR and western blotting. In conclusion, we succeeded in establishing the method for chemical compounds detecting endogenous target protein expression changes with HiBiT-tagging system. Furthermore, we validated this method by performing chemical screening with PD-L1, and succeeded in identifying microtubules inhibitors as up-regulators of PD-L1. Citation Format: Yutaro Uchida, Takahide Matsushima, Ryota Kurimoto, Tomoki Chiba, Yuki Inutani, Hiroshi Asahara. HiBiT tagging system for high throughput chemical screening for chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 20.

Time-Resolved Luminescent High-Throughput Screening Platform for Lysosomotropic Compounds in Living Cells.

Lysosomes are membrane-bound organelles that regulate protein degradation and cellular organelle recycling. Homeostatic alteration by lysosomotropic compounds has been suggested as a potential approach for the treatment of cancer. However, because of the high false-negative rate resulting from strong fluorescent background noise, few luminescent high-throughput screening methods for lysosomotropic compounds have been developed for cancer therapy. Imidazole is a five-membered heterocycle that can act within the acidic interior of lysosomes. To develop an efficient lysosomotropic compound screening system, we introduced an imidazole group to iridium-based complexes and designed a long-lifetime lysosomal probe to monitor lysosomal activity in living cells. By integrating time-resolved emission spectroscopy (TRES) with the novel iridium-based lysosomal probe, a high-throughput screening platform capable of overcoming background fluorescent interference in living cells was developed for discovering lysosomotropic drugs. As a proof-of-concept, 400 FDA/EMA-approved drugs were screened using the TRES system, revealing five compounds as potential lysosomotropic agents. Significantly, the most promising potent lysosomotropic compound (mitoxantrone) identified in this work would have showed less activity if screened using a commercial lysosomal probe because of interference from the intrinsic fluorescence of mitoxantrone. We anticipate that this TRES-based high-throughput screening system could facilitate the development of more lysosomotropic drugs by avoiding false results arising from the intrinsic fluorescence of both bioactive compounds and/or the cell background.

Single Cell Analysis of Drug Susceptibility of Mycobacterium Abscessus During Macrophage Infection.

Mycobacterium abscessus is an emerging health risk to immunocompromised individuals and to people with pre-existing pulmonary conditions. As M. abscessus possesses multiple mechanisms of drug resistance, treatments of M. abscessus are of poor efficacy. Therefore, there is an urgent need for new therapeutic strategies targeting M. abscessus. We describe an experimental system for screening of compounds for their antimicrobial activity against intracellular M. abscessus using flow cytometry and imaging flow cytometry. The assay allows simultaneous analysis of multiple parameters, such as proportion of infected host cells, bacterial load per host cell from the infected population, and host cell viability. We verified the suitability of this method using two antibiotics with known activity against M. abscessus: clarithromycin and amikacin. Our analysis revealed a high degree of infection heterogeneity, which correlated with host cell size. A higher proportion of the larger host cells is infected with M. abscessus as compared to smaller host cells, and infected larger cells have higher intracellular bacterial burden than infected smaller cells. Clarithromycin treatment has a more pronounced effect on smaller host cells than on bigger host cells, suggesting that heterogeneity within the host cell population has an effect on antibiotic susceptibility of intracellular bacteria.

An innovative automated active compound screening system allows high-throughput optimization of somatic embryogenesis in Coffea arabica

Somatic embryogenesis (SE) faces many challenges in fulfilling the growing demand for elite materials. A high-throughput approach is required to accelerate the optimization of SE protocols by multiplying experimental conditions within a limited time period. For the first time in plant micropropagation, we have developed a miniaturized and automated screening system to meet high-throughput standards. Coffea arabica embryo regeneration, classically achieved in 250-ml Erlenmeyer flasks, was successfully miniaturized in 24-well plates, allowing a volume downscaling factor of 100 and a space saving of 53 cm2/well. Cell clusters were ground and filtered to fit the automated pipetting platform, leading to fast, reproducible and uniform cluster distribution (23.0 ± 5.5 cell clusters/well) and successful regeneration (6.5 ± 2.2 embryos/well). Pilot screening of active compounds on SE was carried out. Compounds belonging to the histone deacetylase inhibitor family were tested for embryo regeneration efficiency. Cells treated with 1 µM Trichostatin A showed a marked 3-fold increase in the number of regenerated embryos. When re-tested in 250-ml flasks, the same enhancement was obtained, thereby validating the miniaturized and automated screening method. These results showed that our screening system is reliable and well suited to screening hundreds of compounds, offering unprecedented perspectives in plant micropropagation.

Identifying Inhibitors of the HBx-DDB1 Interaction Using a Split Luciferase Assay System.

There is an urgent need for novel therapeutic agents for hepatitis B virus (HBV) infection. Although currently available nucleos(t)ide analogs potently inhibit viral replication, they have no direct effect on the expression of viral proteins transcribed from a viral covalently closed circular DNA (cccDNA). As high viral antigen load may play a role in this chronic and HBV-related carcinogenesis, the goal of HBV treatment is to eradicate viral proteins. HBV regulatory protein X (HBx) binds to the host DNA damage-binding protein 1 (DDB1) protein to degrade structural maintenance of chromosomes 5/6 (Smc5/6), resulting in activation of viral transcription from cccDNA. Here, using a split luciferase complementation assay system, we present a comprehensive compound screening system to identify inhibitors of the HBx-DDB1 interaction. Our protocol enables easy detection of interaction dynamics in real time within living cells. This technique may become a key assay to discover novel therapeutic agents for treatment of HBV infection.

A pull-down and slot blot-based screening system for inhibitor compounds of the podoplanin-CLEC-2 interaction.

Podoplanin, a transmembrane glycoprotein, is overexpressed in certain types of tumors and induces platelet aggregation by binding to C-type lectin-like receptor 2 (CLEC-2) on the platelet membrane. Activated platelets release granule components, which in turn, trigger epithelial-mesenchymal transition and confer invasive capacity to the tumor cells. Therefore, blocking the podoplanin-CLEC-2 interaction by a small-molecule compound is a potential therapeutic strategy to prevent cancer metastasis and invasion. To effectively identify such inhibitory compounds, we have developed a pull-down-based inhibitory compound screening system. An immunoglobulin Fc domain-CLEC-2 fusion protein was used as a bait to capture podoplanin derived from podoplanin-overexpressing HeLa cells in the presence and absence of the test compound. The protein complex was then pulled down using protein A beads. To shorten the turnaround time, increase throughput, and decrease the workload for the operators, centrifugal filter units were employed to separate free and bound podoplanin, instead of using customary aspiration-centrifugation washing cycles. Slot blotting was also utilized in lieu of gel electrophoresis and electrical transfer. Thus, the use of our pull down screening system could facilitate the effective selection of potential inhibitor compounds of the podoplanin-CLEC-2 interaction for cancer therapy. Importantly, our methodology is also applicable to targeting other protein-protein interactions.

Novel Notch signaling inhibitor NSI‑1 suppresses nuclear translocation of the Notch intracellular domain

The Notch receptor serves a fundamental role in the regulation of cell fate determination through intracellular signal transmission. Mutation of the Notch receptor results in abnormal active signaling, leading to the development of diseases involving abnormal cell growth, including malignant tumors. Therefore, the Notch signaling pathway is a useful pharmacological target for the treatment of cancer. In the present study, a compound screening system was designed to identify inhibitors of the Notch signaling targeting Notch intracellular domain(NICD). A total of 9,600compounds were analyzed using the Michigan Cancer Foundation‑7(MCF7) human breast adenocarcinoma cell line and the SH‑SY5Y human neuroblastoma cell line with the reporter assay system using an artificial protein encoding a partial Notch carboxyl‑terminal fragment fused to the Gal4 DNA‑binding domain. The molecular mechanism underlying the inhibition of Notch signaling by a hit compound was further validated using biochemical and cell biological approaches. Using the screening system, a potential candidate, Notch signaling inhibitor‑1 (NSI‑1), was isolated which showed 50%inhibition at 6.1µM in an exogenous Notch signaling system. In addition, NSI‑1 suppressed the nuclear translocation of NICD and endogenous gene expression of hairy and enhancer of split‑1, indicating that NSI‑1 specifically targets Notch. Notably, NSI‑1 suppressed the cell viability of MCF7 cells and another human breast adenocarcinoma cell line, MDA‑MB‑231 exhibiting constitutive and high Notch signaling activity, whereas no significant effect was observed in the SH‑SY5Y cells bearing a lower Notch signaling activity. NSI‑1 significantly suppressed the viability of SH‑SY5Y cells expressing exogenous human Notch1. These results indicate that NSI‑1 is a novel Notch signaling inhibitor and suggest its potential as a useful drug for the treatment of diseases induced by constitutively active Notch signaling.

Development of a Novel Cell-Based Assay System for High-Throughput Screening of Compounds Acting on Background Two-Pore Domain K+ Channels

Two-pore domain K+ (K2P) channels are thought to be druggable targets. However, only a few agents specific for K2P channels have been identified, presumably due to the lack of an efficient screening system. To develop a new high-throughput screening (HTS) system targeting these channels, we have established a HEK293-based “test cell” expressing a mutated Na+ channel (Nav1.5) with markedly slowed inactivation, as well as a K+ channel (Kir2.1) that sets the membrane potential quite negative, close to K+ equilibrium potential. We found in this system that Kir2.1 block by 100 μM Ba2+ application consistently elicited a large depolarization like a long-lasting action potential. This maneuver resulted in cell death, presumably due to the sustained Na+ influx. When either the TWIK-related acid-sensitive K+ (TASK)-1 or TASK-3 channel was expressed in the test cells, Ba2+-induced cell death was markedly weakened. Stronger activation of TASK-1 by extracellular acidification further decreased the cell death. In contrast, the presence of K2P channel blockers enhanced cell death. IC50 values for TASK-1 and/or TASK-3 blockers acquired by measurements of relative cell viability were comparable to those obtained using patch-clamp recordings. Both blockers and openers of K2P channels can be accurately assessed with high efficiency and throughput by this novel HTS system.

Establishment and Validation of Whole-Cell Based Fluorescence Assays to Identify Anti-Mycobacterial Compounds Using the Acanthamoeba castellanii - Mycobacterium marinum Host-Pathogen System

Tuberculosis is considered to be one of the world’s deadliest disease with 2 million deaths each year. The need for new antitubercular drugs is further exacerbated by the emergence of drug-resistance strains. Despite multiple recent efforts, the majority of the hits discovered by traditional target-based screening showed low efficiency in vivo. Therefore, there is heightened demand for whole-cell based approaches directly using host-pathogen systems. The phenotypic host-pathogen assay described here is based on the monitoring of GFP-expressing Mycobacterium marinum during infection of the amoeba Acanthamoeba castellanii. The assay showed straight-forward medium-throughput scalability, robustness and ease of manipulation, demonstrating its qualities as an efficient compound screening system. Validation with a series of known antitubercular compounds highlighted the advantages of the assay in comparison to previously published macrophage-Mycobacterium tuberculosis-based screening systems. Combination with secondary growth assays based on either GFP-expressing D. discoideum or M. marinum allowed us to further fine-tune compound characterization by distinguishing and quantifying growth inhibition, cytotoxic properties and antibiotic activities of the compounds. The simple and relatively low cost system described here is most suitable to detect anti-infective compounds, whether they present antibiotic activities or not, in which case they might exert anti-virulence or host defense boosting activities, both of which are largely overlooked by classical screening approaches.

Caenorhabditis elegans as a model for the screening of anthelminthic compounds: Ultrastructural study of the effects of albendazole

This study investigated the effects of albendazole on the viability, morphology and ultrastructure of different life stages of Caenorhabditis elegans. The albendazole EC50 value after seven days of treatment was 18.43μM. This concentration was very efficient against all the stages. Light and electron microscopy analysis showed damage to the body wall of the adults and larvae. An intense desquamation of the cuticle of larvae and of the surface of the eggs was observed, preventing their hatching and development. The main ultrastructural damage detected was the degeneration of the mitochondria in the noncontractile muscle of the body wall, which appeared as large vacuoles. This study reaffirmed the use of C. elegans as a screening system for compounds with potential anthelmintic activity and showed the effects of albendazole on the different life stages of these worms.

A Gene Expression–Based Screening System for Compounds Influencing Differentiation of Mouse Embryonic Stem Cells

Low molecular weight compounds have been shown to be useful for controlling stem cells, and various high-throughput screening systems have been developed for identifying compounds that regulate the differentiation of stem cells. However, the effects of such compounds on stem cell differentiation are usually evaluated by assessing a single parameter, which is insufficient for proper monitoring of the cellular status. In this study, to classify a number of compounds, the authors established a gene expression–based screening system using mouse embryonic stem (ES) cells that monitored multiple parameters. ES cells were differentiated into three germ layers by embryoid body formation and then treated with the test compounds. Next, cellular changes were assessed by analyzing the expression of multiple genes with the multiplex quantitative reverse transcriptase polymerase chain reaction. By screening a library of pharmacologically active compounds with this system, the authors were able to classify 52 compounds that influenced the gene expression profile of ES cells. They also found that some compounds identified by screening could enhance osteogenic or adipogenic differentiation of human mesenchymal stem cells. These results indicate that the screening system is effective for identifying compounds involved in regulating the differentiation of both ES cells and adult stem cells.

A Secreted Placental Alkaline Phosphatase-Based Reporter Assay System for Screening of Compounds Acting at an Octopamine Receptor Stably Expressed in a Mammalian Cell Line

Octopamine receptors are attractive insecticide targets. To screen compounds acting at octopamine receptors simply and rapidly, we constructed a chemiluminescent reporter gene assay system that detects secreted placental alkaline phosphatase transcriptionally regulated by the cAMP response element for a silkworm octopamine receptor. This system proved useful in high-throughput screening to develop octopamine receptor-specific insecticides.

Towards Coleoptera-specific high-throughput screening systems for compounds with ecdysone activity: development of EcR reporter assays using weevil ( Anthonomus grandis)-derived cell lines and in silico analysis of ligand binding to A. grandis EcR ligand-binding pocket

Molting in insects is regulated by ecdysteroids and juvenile hormones. Several synthetic non-steroidal ecdysone agonists are on the market as insecticides. These ecdysone agonists are dibenzoylhydrazine (DBH) analogue compounds that manifest their toxicity via interaction with the ecdysone receptor (EcR). Of the four commercial available ecdysone agonists, three (tebufenozide, methoxyfenozide and chromafenozide) are highly lepidopteran specific, one (halofenozide) is used to control coleopteran and lepidopteran insects in turf and ornamentals. However, compared to the very high binding affinity of these DBH analogues to lepidopteran EcRs, halofenozide has a low binding affinity for coleopteran EcRs. For the discovery of ecdysone agonists that target non-lepidopteran insect groups, efficient screening systems that are based on the activation of the EcR are needed. We report here the development and evaluation of two coleopteran-specific reporter-based screening systems to discover and evaluate ecdysone agonists. The screening systems are based on the cell lines BRL-AG-3A and BRL-AG-3C that are derived from the weevil Anthonomus grandis, which can be efficiently transduced with an EcR reporter cassette for evaluation of induction of reporter activity by ecdysone agonists. We also cloned the almost full length coding sequence of EcR expressed in the cell line BRL-AG-3C and used it to make an initial in silico 3D-model of its ligand-binding pocket docked with ponasterone A and tebufenozide.

백화옥수수 잎절편을 이용한 안토시아닌 생합성 조절제 탐색용 신규 검정법 확립

This study was carried out to establish a novel bioassay system for screening of compounds affecting aromatic amino acid or anthocyanin biosynthesis through investigating a degree of sucrose-induced anthocyanin formation such as size of plant material, buffer conditions, light intensity and irradiated duration, incubation temp., etc were determined and standard procedure (suitable experimental condition) was set up as follows. The second leaf blade of white corn seeding induced by fluridone treatment were segmented into a size of 5<TEX>${\times}$</TEX>5 min. The segments were floated on the solution of 1% sucrose in 1.0mM MES buffer (pH6.0∼6.5) and incubated at 26<TEX>$^{\circ}C$</TEX> for 2days under the continuous light condition(70∼100<TEX>$\mu$</TEX>mol m<TEX>$^{-2}$</TEX> s<TEX>$^{-1}$</TEX> ). Anthocyanin in the purpled tissues was extracted with methanol containing 1% HCl and the optical density of the clear supematants was determined at 528mm. Influences of some chemicals were tested using this system. Glyphosate, 5-enolpyruvylshikimate 3-phosphate synthase inhibitor, showed most sensitive response with I<TEX>$_{50}$</TEX> value at 3.3<TEX>$\mu$</TEX>M. Dicyclohexylcarbodiimide(DCCD) and parachloromercuribenzenesulfonic acid(PCMBS) had a relatively strong ingibition with I50 value at 7.1<TEX>$\mu$</TEX>M and 10.2<TEX>$\mu$</TEX>M, respectively. These results show that sucrose-induced anthocyanin formation in white com leaf segment provide a very simple and rapid system for searching new compounds affecting aromatic amino acid or anteocyanin biosynthesis by screening at less than 10<TEX>$\mu$</TEX>M.

Techniques for Studying Stimulation of Fetal Hemoglobin Production in Human Erythroid Cultures

This report describes in detail the procedures for growing human erythroid cells in liquid culture for evaluating the potential of pharmacological agents to affect hemoglobin production. The procedure consists of two phases: an erythropoietin-independent phase in which peripheral blood mononuclear cells are first cultured in the presence of a combination of hemopoietic growth factors, but in the absence of erythropoietin, where early erythroid committed progenitors proliferate and differentiate into more mature progenitors. In the second phase, the latter cells, cultured in an erythropoietin-supplemented medium, continue to proliferate and mature into orthochromatic normoblasts and enucleated erythrocytes. This procedure produces large cultures of relatively pure and synchronized erythroid cell populations derived from normal donors or patients with β hemoglobinopathies. The cultured cells recapitulate many aspects of erythropoiesis in vivo, including the donor's pattern of hemoglobin production (types and proportions). Tested compounds, at different concentrations, are added at different stages of the culture. The various types of hemoglobins and globin chains produced can be measured by high performance liquid chromatographic techniques and their cellular distribution analyzed by flow cytometry using fluorescently labeled antibodies against specific hemoglobins. This approach provides a screening system for compounds with potential therapeutic efficacy in patients with β hemoglobinopathies.

Microplate alamar blue assay versus BACTEC 460 system for high-throughput screening of compounds against Mycobacterium tuberculosis and Mycobacterium avium

In response to the need for rapid, inexpensive, high-throughput assays for antimycobacterial drug screening, a microplate-based assay which uses Alamar blue reagent for determination of growth was evaluated. MICs of 30 antimicrobial agents against Mycobacterium tuberculosis H37Rv, M. tuberculosis H37Ra, and Mycobacterium avium were determined in the microplate Alamar blue assay (MABA) with both visual and fluorometric readings and compared to MICs determined in the BACTEC 460 system. For all three mycobacterial strains, there was < or = 1 dilution difference between MABA and BACTEC median MICs in four replicate experiments for 25 to 27 of the 30 antimicrobics. Significant differences between MABA and BACTEC MICs were observed with 0, 2, and 5 of 30 antimicrobial agents against H37Rv, H37Ra, and M. avium, respectively. Overall, MICs determined either visually or fluorometrically in MABA were highly correlated with those determined in the BACTEC 460 system, and visual MABA and fluorometric MABA MICs were highly correlated. MICs of rifampin, rifabutin, minocycline, and clarithromycin were consistently lower for H37Ra compared to H37Rv in all assays but were similar for most other drugs. M. tuberculosis H37Ra may be a suitable surrogate for the more virulent H37Rv strain in primary screening of compounds for antituberculosis activity. MABA is sensitive, rapid, inexpensive, and nonradiometric and offers the potential for screening, with or without analytical instrumentation, large numbers of antimicrobial compounds against slow-growing mycobacteria.