Luciferase-Based,High-Throughput Assay for Screening and Profiling Transmission-Blocking Compounds against Plasmodium falciparum Gametocytes

The discovery of new antimalarial drugs able to target both the asexual and gametocyte stages of Plasmodium falciparum is critical to the success of the malaria eradication campaign. We have developed and validated a robust, rapid, and cost-effective high-throughput reporter gene assay to identify compounds active against late-stage (stage IV and V) gametocytes. The assay, which is suitable for testing compound activity at incubation times up to 72 h, demonstrates excellent quality and reproducibility, with average Z′ values of 0.85 ± 0.01. We used the assay to screen more than 10,000 compounds from three chemically diverse libraries. The screening outcomes highlighted the opportunity to use collections of compounds with known activity against the asexual stages of the parasites as a starting point for gametocytocidal activity detection in order to maximize the chances of identifying gametocytocidal compounds. This assay extends the capabilities of our previously reported luciferase assay, which tested compounds against early-stage gametocytes, and opens possibilities to profile the activities of gametocytocidal compounds over the entire course of gametocytogenesis.     

Simple Colorimetric Trypanothione Reductase-Based Assay for High-Throughput Screening of Drugs against Leishmania Intracellular Amastigotes

Critical to the search for new anti-leishmanial drugs is the availability of high-throughput screening (HTS) methods to test chemical compounds against the relevant stage for pathogenesis, the intracellular amastigotes. Recent progress in automated microscopy and genetic recombination has produced powerful tools for drug discovery. Nevertheless, a simple and efficient test for measuring drug activity against Leishmania clinical isolates is lacking. Here we describe a quantitative colorimetric assay in which the activity of a Leishmania native enzyme is used to assess parasite viability. Enzymatic reduction of disulfide trypanothione, monitored by a microtiter plate reader, was used to quantify the growth of Leishmania parasites. An excellent correlation was found between the optical density at 412 nm and the number of parasites inoculated. Pharmacological validation of the assay was performed against the conventional alamarBlue method for promastigotes and standard microscopy for intracellular amastigotes. The activity of a selected-compound panel, including several anti-leishmanial reference drugs, demonstrated high consistency between the newly developed assay and the reference method and corroborated previously published data. Quality assessment with standard measures confirmed the robustness and reproducibility of the assay, which performed in compliance with HTS requirements. This simple and rapid assay provides a reliable, accurate method for screening anti-leishmanial agents, with high throughput. The basic equipment and manipulation required to perform the assay make it easy to implement, simplifying the method for scoring inhibitor assays.     

Novel Cell-Based Hepatitis C Virus Infection Assay for Quantitative High-Throughput Screening of Anti-Hepatitis C Virus Compounds

Therapy for hepatitis C virus (HCV) infection has advanced with the recent approval of direct-acting antivirals in combination with peginterferon and ribavirin. New antivirals with novel targets are still needed to further improve the treatment of hepatitis C. Previously reported screening methods for HCV inhibitors either are limited to a virus-specific function or apply a screening method at a single dose, which usually leads to high false-positive or -negative rates. We developed a quantitative high-throughput screening (qHTS) assay platform with a cell-based HCV infection system. This highly sensitive assay can be miniaturized to a 1,536-well format for screening of large chemical libraries. All candidates are screened over a 7-concentration dose range to give EC₅₀s (compound concentrations at 50% efficacy) and dose-response curves. Using this assay format, we screened a library of pharmacologically active compounds (LOPAC). Based on the profile of dose-dependent curves of HCV inhibition and cytotoxicity, 22 compounds with adequate curves and EC₅₀s of <10 μM were selected for validation. In two additional independent assays, 17 of them demonstrated specific inhibition of HCV infection. Ten potential candidates with efficacies of >70% and CC₅₀s (compound concentrations at 50% cytotoxicity) of <30 μM from these validated hits were characterized for their target stages in the HCV replication cycle. In this screen, we identified both known and novel hits with diverse structural and functional features targeting various stages of the HCV replication cycle. The pilot screen demonstrates that this assay system is highly robust and effective in identifying novel HCV inhibitors and that it can be readily applied to large-scale screening of small-molecule libraries.     

Rapid,Semiquantitative Assay To Discriminate among Compounds with Activity against Replicating or Nonreplicating Mycobacterium tuberculosis

The search for drugs that can kill replicating and nonreplicating Mycobacterium tuberculosis faces practical bottlenecks. Measurement of CFU and discrimination of bacteriostatic from bactericidal activity are costly in compounds, supplies, labor, and time. Testing compounds against M. tuberculosis under conditions that prevent the replication of M. tuberculosis often involves a second phase of the test in which conditions are altered to permit the replication of bacteria that survived the first phase. False-positive determinations of activity against nonreplicating M. tuberculosis may arise from carryover of compounds from the nonreplicating stage of the assay that act in the replicating stage. We mitigate these problems by carrying out a 96-well microplate liquid MIC assay and then transferring an aliquot of each well to a second set of plates in which each well contains agar supplemented with activated charcoal. After 7 to 10 days—about 2 weeks sooner than required to count CFU—fluorometry reveals whether M. tuberculosis bacilli in each well have replicated extensively enough to reduce a resazurin dye added for the final hour. This charcoal agar resazurin assay (CARA) distinguishes between bacterial biomasses in any two wells that differ by 2 to 3 log₁₀ CFU. The CARA thus serves as a pretest and semiquantitative surrogate for longer, more laborious, and expensive CFU-based assays, helps distinguish bactericidal from bacteriostatic activity, and identifies compounds that are active under replicating conditions, nonreplicating conditions, or both. Results for 14 antimycobacterial compounds, including tuberculosis (TB) drugs, revealed that PA-824 (pretomanid) and TMC207 (bedaquiline) are largely bacteriostatic.     

Development of a Cell-Based Hepatitis C Virus Infection Fluorescent Resonance Energy Transfer Assay for High-Throughput Antiviral Compound Screening

A major obstacle in the treatment of chronic hepatitis C virus (HCV) infection has been the lack of effective, well-tolerated therapeutics. Notably, the recent development of the HCV cell culture infection system now allows not only for the study of the entire viral life cycle, but also for the screening of inhibitors against all aspects of HCV infection. However, in order to screen libraries of potential antiviral compounds, it is necessary to develop a highly reproducible, accurate assay for HCV infection adaptable for high-throughput screening (HTS) automation. Using an internally quenched 5-FAM/QXL 520 fluorescence resonance energy transfer (FRET) substrate containing the HCV NS3 peptide cleavage sequence, we report the development of a simple, mix-and-measure, homogenous, cell-based HCV infection assay amendable for HTS. This assay makes use of synchronized, nondividing human hepatoma-derived Huh7 cells, which support more-reproducible long-term HCV infection and can be readily scaled down to a 96-well-plate format. We demonstrate that this stable cell culture method eliminates common problems associated with standard cell-based HTS, such as cell culture variability, poor reproducibility, and low signal intensity. Importantly, this HCV FRET assay not only can identify inhibitors that act throughout the viral life cycle as effectively as more-standard HCV assays, such as real-time quantitative PCR and Western blot analysis, but also exhibits a high degree of accuracy with limited signal variation (i.e., Z′ ≥ 0.6), providing the basis for a robust HTS campaign for screening compound libraries and identifying novel HCV antivirals.Hepatitis C virus (HCV) is an enveloped positive-strand RNA virus that infects and replicates in the liver of ∼170 million individuals worldwide. Although acute infection is typically asymptomatic, ∼80% of patients fail to clear the virus, resulting in a chronic infection associated with significant liver disease, including cirrhosis and hepatocellular carcinoma (HCC) (2). As such, in the United States, HCV-related HCC accounts for over 50% of HCC cases and over 30% of the liver transplants that are performed. With no vaccine available to protect against HCV infection and only a subset of chronically infected patients responding to current treatment options (1), there is an obvious and immediate need for new effective HCV antivirals.HCV is classified in the family Flaviviridae based on conservation of the viral RNA-dependent RNA polymerase and genome organization (32). The ∼9.6-kb RNA genome encodes a single open reading frame flanked by highly structured 5′ and 3′ untranslated regions. The 5′ untranslated region contains an internal ribosome entry site that is required for translation of an ∼3,010-amino acid viral polyprotein, which is proteolytically cleaved into structural and nonstructural proteins. The nonstructural viral proteins assemble on cytoplasmic cellular membranes to form the viral RNA replication complex in which negative-strand RNA synthesis is believed to occur (15). The negative strand then provides the template for ∼10-fold amplification of positive-strand genomic RNA, which can subsequently be used for additional translation, negative-strand synthesis, or packaging into progeny virus (32).Since its discovery in 1989 as the causative agent of non-A non-B hepatitis (10), the viral life cycle and host-virus interactions that determine infection outcome have been difficult to study because experimental HCV cell culture infection systems and small animal models have not been available. Nonetheless, significant advancements in the study of HCV have been made over the past 19 years using surrogate systems (4), subgenomic and full-length HCV replicons (6, 7, 20, 33), and pseudotyped particles (HCVpp) (3). While the HCV replicon and HCVpp systems were breakthroughs that overcame key experimental limitations, these systems afforded the study of only viral replication and entry, respectively, and did not recapitulate the entire viral life cycle. It was not until 2005 that the genotype 2a HCV consensus clone (JFH-1) was shown to replicate in the Huh7 human hepatoma-derived cell line and produce infectious HCV in cell culture (HCVcc) (31, 50, 56), recapitulating all aspects of the viral life cycle and providing a promising multifaceted opportunity for drug discovery and high-throughput screening (HTS).Although numerous types of HCV replicon-based HTS have been developed (8, 11, 16, 19, 24, 29, 30, 34, 38, 43, 58), the need to screen compounds that target all steps of the HCV life cycle is warranted. As such, in this report we describe the development of a cell-based HCVcc HTS assay for identification of HCV antivirals that target any aspect of the viral life cycle, including steps yet to be defined. Rather than using exogenous or foreign enzymatic reporters to measure HCV infection, the assay described herein uses NS3 protease activity as a virally encoded “enzymatic reporter” of HCV infection. This strategy is based on (i) the observation that NS3 protease activity parallels HCV infection kinetics and (ii) the ability of the viral NS3 protein to cleave internally quenched peptide substrates (5, 21), allowing for quantitative measurement of NS3 protease activity by fluorescence resonance energy transfer (FRET), which can be easily adapted to an HTS format. Moreover, to optimize reproducibility and signal intensity while reducing nonspecific effects that screened compounds might have on host cell growth, this assay uniquely incorporates synchronized, nondividing Huh7 cells. Here we show that the resulting mix-and-measure, homogenous, cell-based HCV infection assay can identify inhibitors targeting all aspects of the viral life cycle and exhibits an average assay window with a Z′ factor of greater than 0.6, making it suitable for compound library screening.     

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.     

Expression of Green Fluorescent Protein as a Marker for Effects of Antileishmanial Compounds In Vitro

Transgenic Leishmania infantum promastigotes, which constitutively express green fluorescent protein (GFP) in their cytoplasm, were used to monitor the effects of antileishmanial compounds in real time. The GFP-based assay provided a reliable measure of drug-induced inhibitory effects on protein expression, resulting in a dynamic picture of the responses of leishmanial promastigotes to the compounds tested.     

绿色荧光蛋白及其应用

源于水母（Aepuorea vietoria）等海洋无脊椎动物的绿色荧光蛋白（green fluorescent protein,GFP）是一种极具潜力的标记物。其内源荧光其团在受紫外光或蓝光激发时可高效发射清淅可见的绿光,且荧光性质稳定,与现在的标记物相比,GFP具有无可比拟的优势。因而自GFP被发现以来,一直作为一个监测完整细胞和组织内基因表达及蛋白定位的理想标记。广泛应用于转基因动物的研究、融合标记、基因治疗、蛋白在活细胞内功能定位及迁移变化,病原菌侵入活细胞的分子过程等。表明GFP是一类能在现代细胞生物学和分子生物学研究与临床检测中发现重要作用的较理想的基因表达标记物。     

Imaging-Based High-Throughput Screening Assay To Identify New Molecules with Transmission-Blocking Potential against Plasmodium falciparum Female Gamete Formation

In response to a call for the global eradication of malaria, drug discovery has recently been extended to identify compounds that prevent the onward transmission of the parasite, which is mediated by Plasmodium falciparum stage V gametocytes. Lately, metabolic activity has been used in vitro as a surrogate for gametocyte viability; however, as gametocytes remain relatively quiescent at this stage, their ability to undergo onward development (gamete formation) may be a better measure of their functional viability. During gamete formation, female gametocytes undergo profound morphological changes and express translationally repressed mRNA. By assessing female gamete cell surface expression of one such repressed protein, Pfs25, as the readout for female gametocyte functional viability, we developed an imaging-based high-throughput screening (HTS) assay to identify transmission-blocking compounds. This assay, designated the P. falciparum female gametocyte activation assay (FGAA), was scaled up to a high-throughput format (Z′ factor, 0.7 ± 0.1) and subsequently validated using a selection of 50 known antimalarials from diverse chemical families. Only a few of these agents showed submicromolar 50% inhibitory concentrations in the assay: thiostrepton, methylene blue, and some endoperoxides. To determine the best conditions for HTS, a robustness test was performed with a selection of the GlaxoSmithKline Tres Cantos Antimalarial Set (TCAMS) and the final screening conditions for this library were determined to be a 2 μM concentration and 48 h of incubation with gametocytes. The P. falciparum FGAA has been proven to be a robust HTS assay faithful to Plasmodium transmission-stage cell biology, and it is an innovative useful tool for antimalarial drug discovery which aims to identify new molecules with transmission-blocking potential.     

Simple Model for Testing Drugs against Nonreplicating Mycobacterium tuberculosis

Nonreplicating or dormant cells of Mycobacterium tuberculosis constitute a challenge to tuberculosis (TB) therapy because of their tolerance or phenotypic resistance to most drugs. Here, we propose a simple model for testing drugs against nongrowing cells that exploits the 18b strain of M. tuberculosis, a streptomycin (STR)-dependent mutant. Optimal conditions were established that allowed 18b cells to replicate in the presence of STR and to survive, but not multiply, following withdrawal of STR. In the presence of the antibiotic, M. tuberculosis 18b was susceptible to the currently approved TB drugs, isoniazid (INH) and rifampin (RIF), and to the experimental drugs TMC207, PA-824, meropenem (MER), benzothiazinone (BTZ), and moxifloxacin (MOXI). After STR depletion, the strain displayed greatly reduced susceptibility to the cell wall inhibitors INH and BTZ but showed increased susceptibility to RIF and PA-824, while MOXI and MER appeared equipotent under both conditions. The same potency ranking was found against nonreplicating M. tuberculosis 18b after in vivo treatment of chronically infected mice with five of these drugs. Despite the growth arrest, strain 18b retains significant metabolic activity in vitro, remaining positive in the resazurin reduction assay. Upon adaption to a 96-well format, this assay was shown to be suitable for high-throughput screening with strain 18b to find new inhibitors of dormant M. tuberculosis.Tuberculosis (TB) is one of the most important infectious diseases caused by a single pathogen and afflicts both healthy and immunocompromised individuals. One-third of the human population is reported to be latently infected with Mycobacterium tuberculosis, and millions of lives are lost every year (8). The emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains (10), together with a deadly synergy with HIV, has worsened the global problem and forced the research community to look for new strategies to fight M. tuberculosis. As a consequence, several drug discovery programs have been established worldwide with the goal of finding new therapeutic agents that may complement or even replace the existing directly observed therapy short course (DOTS) for TB. These programs have to cope with the ability of the pathogen to enter the so-called dormant or latent phase (5, 6), characterized by phenotypic drug resistance (11). Complete control of TB will therefore require finding drugs that are effective against the reservoir of nongrowing tubercle bacilli and, hence, the necessity of modeling this state in vitro and in vivo in order to screen for active compounds.Latent TB is the result of complex host-pathogen interactions (26, 35), and mimicking this state may thus be partial and challenging. Different models have been proposed so far, each one based on a particular stress that the pathogen may encounter in the host. In particular, the oxygen depletion model mimics the hypoxic environment of the granuloma (30, 31) and the nutrient starvation model recapitulates the lack of nutrients (4), whereas treatment with nitric oxide (15) and growth in acidic medium (9) represent two additional stresses M. tuberculosis has to deal with during infection. Most recently, these treatments were combined (7). While these models contributed to unraveling M. tuberculosis phenotypes in dormancy, they are too cumbersome for application to high-throughput screening (HTS) assays. A reproducible and easy-to-use model of nongrowing cells is therefore required.In this context, we felt that the streptomycin (STR)-dependent M. tuberculosis strain 18b might represent a good candidate for modeling dormant bacteria in a simple yet conditional way. M. tuberculosis 18b was isolated in Japan in 1955 from a patient with streptomycin-resistant tuberculosis (12). The bacterial phenotype was then recognized as streptomycin dependent, with the microorganism being unable to grow unless streptomycin was present. Additionally, the strain did not lose viability when maintained in streptomycin-free cultures for several weeks and grew again upon adding streptomycin back (12). The molecular reason for this unique phenotype was investigated, and insertion of a cytosine in the 530 loop of the 16S rRNA, known to be involved in STR resistance, was found to be associated with the dependency (13). Recent vaccine work exploited M. tuberculosis 18b for establishing dormant infection in mouse and guinea pig models (16, 17). Notably, those authors demonstrated that the strain replicates in the lungs and spleens of animals injected with STR, whereas it no longer grows and it enters the nonreplicating state upon STR withdrawal, thus leading to the conclusion that it mimics dormancy.In this work, we performed an in-depth characterization of M. tuberculosis 18b growth in vitro and in vivo, with an emphasis on drug susceptibility testing, in order to develop a new model for HTS approaches.     

Bioluminescence for Assessing Drug Potency against Nonreplicating Mycobacterium tuberculosis

Targeting dormant Mycobacterium tuberculosis represents a challenge to antituberculosis drug discovery programs. We previously reported and validated the use of the streptomycin (STR)-dependent M. tuberculosis 18b strain as a tool for assessing drug potency against nonreplicating bacteria both in vitro and in vivo. In this study, we generated a luminescent 18b strain, named 18b-Lux, by transforming the bacteria with a vector expressing the luxCDABE operon from Photorhabdus luminescens. Luciferase expression was demonstrated under replicating conditions, and, more importantly, luminescence levels significantly above background were detected following STR removal. The sensitivity of STR-starved 18b-Lux to approved and candidate antituberculosis therapeutic agents was evaluated by means of a luciferase assay in a 96-well format. Results mirrored the data obtained with the standard resazurin reduction microplate assay, and the luminescence readout allowed time course assessments of drug efficacy in vitro. Specifically, we proved that bedaquiline, the rifamycins, and sutezolid displayed time-dependent activity against dormant bacteria, while pyrazinamide and SQ109 showed bactericidal effects at the highest concentrations tested. Overall, we established the optimal conditions for an inexpensive, simple, and very sensitive assay with great potential for future applications.     

Efficacy of antimicrobial peptoids against Mycobacterium tuberculosis

Tuberculosis is a leading cause of death worldwide. Resistance of Mycobacterium to antibiotics can make treatments less effective in some cases. We tested selected oligopeptoids--previously reported as mimics of natural host defense peptides--for activity against Mycobacterium tuberculosis and assessed their cytotoxicity. A tetrameric, alkylated, cationic peptoid (1-C13(4mer)) was most potent against M. tuberculosis and least cytotoxic, whereas an unalkylated analogue, peptoid 1(4mer), was inactive. Peptoid 1-C13(4mer) thus merits further study as a potential antituberculosis drug.