We thank Dr. Yves L. Janin for his fruitful comments and suggestions. We would like to thank H.H. Gad for CHIK/Ren and C. Combredet for her technical support. This work was supported by the Institut Pasteur, the Centre National de la Recherche Scientifique (CNRS), the Institut National de la Sant&#233; Et de la Recherche M&#233;dicale (INSERM), the Institut Carnot - Pasteur Maladies Infectieuses (Programme STING to P.O.V and H.M.-L), the Agence Nationale pour la Recherche (ANR-RPIB, Programme STING 2.0 to P.O.V), and the &#34;Conseil R&#233;gional d'Ile-de-France&#34; (Chemical Library Project, grants n&deg; I 06-222/R and I 09-1739/R to H.M.-L.). The work on CHIKV/Ren was supported by the project ArbOAS (ANR grant 2010-INTB-1601-02).

1. Preparation of 96-well Daughter Plates with Compounds
<ol>
	<li>Move 96-well mother plates containing 10 mM stock solutions of chemical compounds in DMSO from -20 &#176;C to room temperature. Dilute compounds 5x&#160;in DMSO to obtain intermediate 96-well dilution plates at 2 mM. Dilution is achieved by pipetting 5 &#956;l&#160;into 20 &#956;l of DMSO and mixing.</li>
	<li>Pipette 1 &#956;l from dilution plates into dry wells of white, bar-coded tissue culture 96-well plates. This first set of daughter plates (D1) will be used to evaluate the toxicity of compounds at a final concentration of 20 &#956;M. Store daughter plates at -20 &#176;C until use.</li>
	<li>Dilute compounds again 1:10 by pipetting 4 &#956;l from dilution plates into 36 &#956;l of DMSO and mixing. Pipette 1 &#956;l into dry wells of white, flat bottom, bar-coded tissue culture 96-wells plates. This second set of daughter plates (D2) will be used to evaluate the inhibition of MV replication by compounds at a final concentration of 2 &#956;M. Store daughter plates at -20 &#176;C until use.</li>
</ol>
2. Preparation of Cell Cultures to Determine Compound Toxicity
<ol>
	<li>Grow HEK-293T cells at 37 &#176;C and 5% CO2 in Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) with stabilized L-glutamine and supplemented with 10% fetal calf serum (FCS), streptomycin (100 &#956;g/ml) and penicillin (100 IU/ml). Cells are passaged by trypsinization every 4-5 days, and diluted 1:10 into a fresh flask. Cells should be in log phase for experiments.</li>
	<li>On the day of the experiment, recover cells by trypsinization and perform cell counting. Pellet cells by centrifugation, and resuspend them at 3 x 105 cells/ml in culture medium. Consider that 12.5 ml of cell suspension are required for each screening plate.</li>
	<li>Transfer cells to a trough, and dispense 100 &#956;l of cell suspension in D1 plates containing spiked chemical compounds. Cell suspension within the trough is regularly agitated to avoid sedimentation.</li>
	<li>Spike control wells A1, C1, E1, G1, A12, C12, E12 and G12 with 1 &#956;l of DMSO. Corresponding wells will be used as reference for living cells (no toxicity). Spike control wells B1, D1, F1, H1, B12, D12, F12 and H12 with 1 &#956;l of DMSO and 2.5 &#956;l of a 0.5% IGEPAL solution to kill cells. Corresponding wells will be used as a reference for dead cells (high toxicity).</li>
	<li>Incubate cells for 24 hr&#160;at 37 &#176;C and 5% CO2.</li>
</ol>
3. Preparation of Cell Cultures Infected by rMV2/Luc
<ol>
	<li>Grow and recover cells as described in 2.1 and 2.2. Again, resuspend cells at 3 x 105 cells/ml in culture medium. Consider that 12.5 ml of cell suspension are required for each screening plate.</li>
	<li>Optional: supplement culture medium with uridine at 20 &#956;g/ml. 
	Note: When screening chemical libraries for viral replication inhibitors, it seems that compounds targeting early steps of pyrimidine biosynthesis pathway are frequently isolated13,16,27,28. The addition of uridine to culture medium is used to filter out such antiviral molecules. Indeed, this efficiently restores viral replication when upstream enzymes of pyrimidine biosynthesis pathway are blocked.</li>
	<li>Save 1:10 of cell suspension for control wells with non-infected cells, and proceed to infection with remaining volume.</li>
	<li>Thaw the appropriate volume of rMV2/Luc stock solution. MV stock solutions are usually at 106-108 infectious particles per ml. Culture must be infected with 0.1 infectious particles of rMV2/Luc per target cells, which corresponds to 0.1 multiplicity of infection (MOI). Note: rMV2/Luc is derived from MV vaccine (Schwarz strain) and is manipulated in a BSL2 environment.</li>
	<li>Add the virus to cell suspension and mix gently. Transfer infected cells to a trough, and dispense 100 &#956;l of infected cell suspension in columns 2 to 11 of D2 plates containing spiked chemical compounds. Cell suspension within the trough is regularly mixed gently.</li>
	<li>In columns 1 and 12, dispense 100 &#956;l of non-infected cells a well on two. Infected cells are dispensed in the others wells.</li>
	<li>Incubate cells for 24 hr&#160;at 37 &#176;C and 5% CO2.</li>
</ol>
4. Luciferase Activity Measures and Data Analysis
<ol>
	<li>Remove D1 plates from the incubator, and determine cellular viability by adding 50 &#956;l of luciferase-based viability reagent directly to culture wells. Mix and incubate for 10 min at room temperature. Read plates with a luminometer. Integration time is set at 100 msec per well.</li>
	<li>Remove D2 plates from the incubator, and determine luciferase activity by adding 50 &#956;l of luciferase substrate directly to culture wells. Incubate for 6 min at room temperature, and read plates with a luminometer as described above.</li>
	<li>Calculate a Z&#39;-factor for each D1 plate of the toxicity assay to warrant the quality of the screen29. Z&#39; = 1-3*(&#963;+ + &#963;-)/(&#181;+ - &#181;-), where &#181;+ and &#963;+ correspond to means of luminescence and standard deviations for HEK-293T cells with DMSO alone (no toxicity), and &#181;- and &#963;- are means of luminescence and standard deviations for culture wells treated with IGEPAL to kill cells. Z&#39; is expected to be above 0.5, or the corresponding plate is discarded.</li>
	<li>Set toxicity threshold at &#181;+/2. Discard compounds with luminescence values below this cut-off (Figure 3A).</li>
	<li>Calculate a Z&#39;-factor for each D2 plate of the antiviral assay. Here, &#181;+ and &#963;+ correspond to means of luminescence and standard deviations for infected cells cultured with DMSO alone, and &#181;- and &#963;- are means of luminescence and standard deviations for non-infected cells. Again, plates with Z&#39; values below 0.5 are discarded.</li>
	<li>Calculate the inhibition of viral replication as follow: percentage of inhibition = (&#181;+ -&#160;luciferase activity for compound X)/(&#181;+ - &#181;-)*100. Set inhibition threshold at 75%, and select compounds that both reduce luminescence values below this cut-off (Figure 3B) and are not toxic according to criteria described in 4.4.</li>
</ol>
5. Secondary Screen for Toxicity and Broad-spectrum Antiviral Activity
<ol>
	<li>Make 1:2 serial dilutions for each hit compound in DMSO, starting from 500 &#181;M to 4 &#181;M (8 dilutions). Fill white, bar-coded tissue culture plates with 50 &#181;l of culture medium. Add 1 &#181;l of each compound dilution in culture wells. Repeat twice to have 3 culture plates with duplicate serial dilutions for each hit compound.</li>
	<li>Prepare 37.5 ml of a HEK-293T cell suspension at 6 x 105 cells/ml in culture medium as described above (in 2.1 and 2.2). Dispense 2x 12.5 ml in falcon tubes and infect cells with either rMV2/Luc at MOI = 0.1 or recombinant CHIKV expressing Renilla luciferase (CHIKV/Ren) at MOI = 0.2. Mix by inverting. 
	Note: CHIKV/Ren is derived from a wild-type strain of CHIKV and therefore should be manipulated in a BSL3 environment. Plates can be moved from BSL3 to BSL1 once Renilla substrate has been added to culture wells (see below).</li>
	<li>Dispense 50 &#181;l of uninfected, rMV2/Luc-infected, or CHIKV/Ren-infected cells in culture plates containing serial dilutions of hit compounds. Incubate 24 hr at 37 &#176;C.</li>
	<li>Add 50 &#181;l of firefly luciferase substrate to determine firefly luciferase activity in rMV2/Luc-infected wells. Add 50 &#181;l of Renilla luciferase substrate to determine Renilla luciferase activity in CHIKV/Ren-infected wells. Finally, add 50 &#181;l of luciferase-based viability assay reagent to uninfected cells to determine cellular viability.</li>
	<li>Plot data (Figure 4) and determine hit compound concentrations that inhibit MV and CHIKV replication by 50%. Disregard compound showing some toxicity in this assay.</li>
</ol>

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The authors declare that they have no competing financial interests.

The screening pipeline described here aims at selecting compounds with a suitable profile as broad-spectrum inhibitors of RNA viruses. When a library of 10,000 compounds was screened with this protocol and aforementioned filtering criteria were applied, i.e. inhibition of MV replication superior to 75%, 40 compounds (0.4%) scored positive. Besides, about half of them showed some toxicity in the luciferase-based viability assay, and were disregarded for this reason. Finally, a dozen of hits readily available in l...

RNA viruses are responsible for a large variety of human infections, and have a huge impact on populations worldwide both in terms of public health and economical cost. Efficient vaccines have been developed against several human RNA viruses, and are widely used as prophylactic treatments. However, there is still a critical lack of therapeutic drugs against RNA virus infections. Indeed, efficient vaccines are not available against major human pathogens such as dengue virus, Hepatitis C virus or human respiratory syncytial virus (hRSV). Besides, RNA viruses are responsible for a majority of emerging diseases, which have increased in frequency because of global exchanges and human impact on ecological systems. Against this threat that represent RNA viruses, our therapeutic arsenal is extremely limited and relatively inefficient1-3. Current therapies are essentially based on recombinant type I interferons (IFN-&#945;/&#946;) to stimulate innate immunity, or the administration of ribavirin. Although the mode of action of this ribonucleoside analog is controversial and probably relies on various mechanisms, the inhibition of cellular IMPDH (inosine monophosphate dehydrogenase), which depletes intracellular GTP pools, is clearly essential4. Ribavirin, in combination with pegylated IFN-&#945;, is the main treatment against Hepatitis C virus. However, IFN-&#945;/&#946;&#160;and ribavirin treatments are of relatively poor efficacy in vivo against most RNA viruses as they efficiently blunt IFN-&#945;/&#946;&#160;signaling through expression of virulence factors5 and often escape ribavirin3. This added to the fact that ribavirin treatment is raising important toxicity issues, although it was recently approved against severe hRSV disease with controversial benefits6. More recently, some virus-specific treatments have been marketed, in particular against influenza virus with the development of neuraminidase inhibitors3. However, the large diversity and permanent emergence of RNA viruses precludes the development of specific treatments against each one of them in a relatively close future. Altogether, this stresses the need for efficient strategies to identify and develop potent antiviral molecules in the near future.
It is trivial to say that a broad-spectrum inhibitor active against a large panel of RNA viruses would solve this problem. Although such a molecule is still a virologist&#39;s dream, our better understanding of cellular defense mechanisms and innate immune system suggest that some possibilities exist7,8. Several academic and industrial laboratories are now seeking molecules that stimulate specific facets of cellular defense mechanisms or metabolic pathways to blunt viral replication. Although such compounds will probably show significant side effects, treatments against acute viral infections will be administered for a relatively short time, making them acceptable despite some potential toxicity on the long term. Various strategies have been developed to identify such broad-spectrum antiviral molecules. Some research programs aim at finding molecules that target specific defense or metabolic pathways. This includes, for example, pathogen recognition receptors to elicit antiviral gene expression9 and activate antiviral factors such as RNaseL10, autophagy machinery to promote virus degradation11, nucleoside synthesis pathways12,13, or apoptotic cascades to precipitate death of virus-infected cells14. Other groups have developed phenotypic screens that are not target-based13,15-17. In that case, antiviral molecules are simply identified by their capacity to block viral replication in a given cellular system. The general assumption is that a compound inhibiting 2-3 unrelated RNA viruses would have a suitable profile for a broad-spectrum antiviral molecule. The mode of action of hit compounds selected with such an empirical approach is only determined in a second time and eventually, may lead to the identification of novel cellular targets for antivirals. Interestingly, a retrospective analysis of new drugs approved by the US Food and Drug Administration between 1999 and 2008 has shown that in general, such phenotypic screenings tend to perform better than target-based approaches to discover first-in-class small-molecule drugs18.
Viral replication in high-throughput cell-based assays is usually determined from virus cytopathic effects. Cells are infected and cultured in 96- or 384-well plates in the presence of tested compounds. After few days, cellular layers are fixed and stained with dyes such as crystal violet. Finally, absorbance is determined with a plate reader and compounds inhibiting viral replication are identified by their capacity to preserve cellular layers from virus-induced cytopathic effect. Alternatively, virally-mediated cytopathic effects are assessed using standard viability assays such as MTS reduction. Such assays are highly tractable and cost-effective, but suffer from three major limitations. First, they require a virus-cell combination where viral replication is cytopathic in only few days but this is not always possible, thus calling for alternative approaches19. Second, they are poorly quantitative since they are based on an indirect measure of viral replication. Finally, toxic compounds can be scored as positive hits, and therefore must be eliminated with a counter screen measuring cellular viability. To overcome some of these hurdles, recombinant viruses or replicons have been engineered by reverse genetics to express reporter proteins, such as EGFP or luciferase, from an additional transcription unit or in frame with viral protein genes (few examples are 20-23). When these viruses replicate, reporter proteins are produced together with viral proteins themselves. This provides a very quantitative assay to measure viral replication and evaluate the inhibitory activity of candidate molecules. This is particularly true for recombinant viruses expressing luciferase (or other enzymes capable of bioluminescence) since this reporter system exhibits a wide dynamic range with a high sensitivity and virtually no background. Furthermore, there is no excitation light source, thus preventing interference with compound fluorescence24.
Here, we detail a high-throughput protocol to screen chemical libraries for broad-spectrum inhibitors of RNA viruses. Compounds are tested first on human cells infected with a recombinant measles virus (MV) expressing firefly luciferase25 (rMV2/Luc,&#160;Figure 1, primary screen). MV belongs to Mononegavirales order, and is often considered as a prototypical member of negative-strand RNA viruses. As such, MV genome is used as a template by the viral polymerase to synthesize mRNA molecules encoding for viral proteins. In the recombinant MV strain called rMV2/Luc, luciferase expression is expressed from an additional transcription unit inserted between P and M genes (Figure 2A). In parallel, compounds are tested for their toxicity on human cells using a commercial luciferase-based reagent that evaluates, by ATP quantification, the number of metabolically active cells in culture (Figure 1, primary screen). Entire chemical libraries can be easily screened with these two assays in order to select compounds that are not toxic and efficiently block MV replication. Then, hits are retested for dose-response inhibition of MV replication, the lack of toxicity as well as for their capacity to impair chikungunya virus (CHIKV) replication (Figure 1, secondary screen). CHIKV is a member of Togaviridae family and its genome is a positive single-strand RNA molecule. As such, it is completely unrelated to measles and compounds inhibiting both MV and CHIKV stand a great chance to inhibit a large panel of RNA viruses. CHIKV nonstructural proteins are directly translated from the viral genome, whereas structural proteins are encoded by transcription and translation of a subgenomic mRNA molecule. Our in vitro replication assay for CHIKV is based on a recombinant strain called CHIKV/Ren, which expresses Renilla luciferase&#160;enzyme as a cleaved part of the nonstructural polyprotein through an insertion of the reporter gene between nsP3 and nsP4 sequences26 (Figure 2B). The measure of Renilla luciferase activity allows the monitoring of viral replication at the early stage of CHIKV life cycle.
This high-throughput protocol was used to quickly identify compounds with a suitable profile for broad-spectrum antivirals in a commercial library of 10,000 molecules enriched for chemical diversity. Compounds were essentially following Lipinski&#39;s rule of five, with molecular weights ranging from 250 to 600 daltons, and log D values below 5. Most of these molecules were new chemical entities not available in other commercial libraries.

<table border="0" cellpadding="0" cellspacing="0" width="756">
	<colgroup>
		<col />
		<col /> 
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Freedom EVO platform</td>
			<td style="width:105px;">TECAN</td>
			<td style="width:119px;"> </td>
			<td style="width:316px;">Robotic platform</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">96-Well Polystyrene Cell Culture Microplates, White</td>
			<td style="width:105px;">Greiner Bio One</td>
			<td style="width:119px;">655083</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">CellTiter-Glo Luminescent Cell Viability Assay</td>
			<td style="width:105px;">Promega</td>
			<td style="width:119px;">G7570</td>
			<td>Luciferase-based viability assay</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Bright-Glo Luciferase Assay System</td>
			<td style="width:105px;">Promega</td>
			<td style="width:119px;">E2610</td>
			<td>Reagent containing firefly luciferase substrate</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Renilla-Glo Luciferase Assay System</td>
			<td style="width:105px;">Promega</td>
			<td style="width:119px;">E2710</td>
			<td>Reagent containing Renilla luciferase substrate</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Britelite plus Reporter Gene Assay System</td>
			<td style="width:105px;">Perkin-Elmer</td>
			<td style="width:119px;">6016761</td>
			<td style="width:316px;">Reagent containing firefly luciferase substrate. Can be used as an alternative to Brigh-Glo reagent to determine luciferase activity</td>
		</tr>
	</tbody>
</table>

high-throughput screening for broad-spectrum chemical inhibitors of rna viruses

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. A good example of such an emerging situation is chikungunya virus epidemics of 2005-2006 in the Indian Ocean. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. Some broad-spectrum antiviral compounds have been identified with host target-oriented assays. However, measuring the inhibition of viral replication in cell cultures using reduction of cytopathic effects as a readout still represents a paramount screening strategy. Such functional screens have been greatly improved by the development of recombinant viruses expressing reporter enzymes capable of bioluminescence such as luciferase. In the present report, we detail a high-throughput screening pipeline, which combines recombinant measles and chikungunya viruses with cellular viability assays, to identify compounds with a broad-spectrum antiviral profile.

This screening pipeline relies first on the selection of compounds that inhibit MV replication and do not show any significant cellular toxicity (Figure 1, primary screen). It takes advantage of luciferase-based assays to determine cellular toxicity and the inhibition of viral replication. All pipetting steps and data acquisition can be performed in a high-throughput setting with a robotic platform.
Cellular toxicity of compounds is assessed using a luciferase-based viability ...

Watch this Scientific Journal Video about High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses at JoVE.com

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

In vitro assays to measure virus replication have been greatly improved by the development of recombinant RNA viruses expressing luciferase or other enzymes capable of bioluminescence. Here we detail a high-throughput screening pipeline that combines such recombinant strains of measles and chikungunya viruses to isolate broad-spectrum antivirals from chemical libraries.

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat ...

high-throughput-screening-for-broad-spectrum-chemical-inhibitors-rna

Research

JoVE Journal

Immunology and Infection

13.7K 조회수.  Institut Pasteur, CNRS UMR3569.  바이러스 복제를 측정하는 시험 관내 분석에서 크게 루시페라아제 또는 생물 발광 가능한 다른 효소를 발현하는 재조합 RNA 바이러스의 개발에 의해 개선되었다. 여기 세부 홍역 및 화학 도서관에서 폭 넓은 스펙트럼 항 바이러스를 분리하기 Chikungunya의 바이러스와 같은 재조합 균주를 결합하여 높은 처리량 검사 파이프 라인을. 

비디오: High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5.
Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8.
Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.

We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since several decades, the sudden burst of multi- and extensively-drug resistant strains is a serious threat for the control of tuberculosis. Therefore, it is essential to identify new targets and pathways critical for the causative agent of the tuberculosis, Mycobacterium tuberculosis (Mtb) and to search for novel chemicals that could become TB drugs. One approach is to set&#160;up methods suitable for the genetic and chemical screens of large scale libraries enabling the search of a needle in a haystack. To this end, we developed a phenotypic assay relying on the detection of fluorescently labeled Mtb within fluorescently labeled host&#160;cells using automated confocal microscopy. This in vitro assay allows an image based quantification of the colonization process of Mtb&#160;into the host and was optimized for the 384-well microplate format, which is proper for screens of siRNA-, chemical compound- or Mtb mutant-libraries. The images are then processed for multiparametric analysis, which provides read&#160;out inferring on the pathogenesis of Mtb&#160;within host cells.

Here, we describe a phenotypic assay applicable to the High-throughput/High-content screens of small-interfering synthetic RNA (siRNA), chemical compound, and Mycobacterium tuberculosis mutant libraries. This method relies on the detection of fluorescently labeled Mycobacterium tuberculosis within fluorescently labeled host&#160;cell using automated confocal microscopy.

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since ...

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify new compounds that can inhibit infection by the common drug resistant HIV-1 strains with minimal toxicity. Here we describe an efficient assay that can be used to rapidly determine the cellular cytotoxicity and efficacy of a compound against WT and mutant viral strains.
The desired target cell line is seeded in a 96-well plate and, after a 24 hr incubation, serially dilutions of the compounds to be tested are added. No further manipulations are necessary for cellular cytotoxicity assays; for anti HIV assays a predetermined amount of either a WT or drug resistant HIV-1 vector that expresses luciferase is added to the cells. Cytotoxicity is measured by using an ATP dependent luminescence assay and the impact of the compounds on infectivity is measured by determining the amount of luciferase in the presence or the absence of the putative inhibitors.
This screening assay takes 4 days to complete and multiple compounds can be screened in parallel. Compounds are screened in triplicate and the data are normalized to the infectivity/ATP levels in absence of target compounds. This technique provides a quick and accurate measurement of the efficacy and toxicity of potential anti HIV compounds.

Here we describe cellular cytotoxicity and single round infectivity assays that allow for the rapid and accurate screening of compounds to determine their cellular cytotoxicity (CC50) and IC50 values against WT and drug resistant HIV-1.

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify ...

Nucleocapsid Annealing-Mediated Electrophoresis (NAME) Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral proteins involved in the regulation of the virus activity recognize several nucleic acids. The nucleocapsid protein NCp7 (NC) is a key protein regulating several processes during virus replication. NC is in fact a chaperone destabilizing the secondary structures of RNA and DNA and facilitating their annealing. The inactivation of NC is a new approach and an interesting target for anti-HIV therapy. The Nucleocapsid Annealing-Mediated Electrophoresis (NAME) assay was developed to identify molecules able to inhibit the melting and annealing of RNA and DNA folded in thermodynamically stable tridimensional conformations, such as hairpin structures of TAR and cTAR elements of HIV, by the nucleocapsid protein of HIV-1. The new assay employs either the recombinant or the synthetic protein, and oligonucleotides without the need of their previous labeling. The analysis of the results is achieved by standard polyacrylamide gel electrophoresis (PAGE) followed by conventional nucleic acid staining. The protocol reported in this work describes how to perform the NAME assay with the full-length protein or its truncated version lacking the basic N-terminal domain, both competent as nucleic acids chaperones, and how to assess the inhibition of NC chaperone activity by a threading intercalator. Moreover, NAME can be performed in two different modes, useful to obtain indications on the putative mechanism of action of the identified NC inhibitors.

Nucleocapsid Annealing-Mediated Electrophoresis NAME Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

This protocol describes NAME, an assay that allows the rapid identification of molecules able to inhibit in vitro the chaperone activities of HIV-1 nucleocapsid protein.

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by direct contact with infected animals or by ingestion of contaminated food and can lead to severe chronic infections.
Brucella can invade professional and non-professional phagocytic cells and replicates within endoplasmic reticulum (ER)-derived vacuoles. The host factors required for Brucella entry into host cells, avoidance of lysosomal degradation, and replication in the ER-like compartment remain largely unknown. Here we describe two assays to identify host factors involved in Brucella entry and replication in HeLa cells. The protocols describe the use of RNA interference, while alternative screening methods could be applied. The assays are based on the detection of fluorescently labeled bacteria in fluorescently labeled host cells using automated wide-field microscopy. The fluorescent images are analyzed using a standardized image analysis pipeline in CellProfiler which allows single cell-based infection scoring.
In the endpoint assay, intracellular replication is measured two days after infection. This allows bacteria to traffic to their replicative niche where proliferation is initiated around 12 hr after bacterial entry. Brucella which have successfully established an intracellular niche will thus have strongly proliferated inside host cells. Since intracellular bacteria will greatly outnumber individual extracellular or intracellular non-replicative bacteria, a strain constitutively expressing GFP can be used. The strong GFP signal is then used to identify infected cells.
In contrast, for the entry assay it is essential to differentiate between intracellular and extracellular bacteria. Here, a strain encoding for a tetracycline-inducible GFP is used. Induction of GFP with simultaneous inactivation of extracellular bacteria by gentamicin enables the differentiation between intracellular and extracellular bacteria based on the GFP signal, with only intracellular bacteria being able to express GFP. This allows the robust detection of single intracellular bacteria before intracellular proliferation is initiated.

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

Two assays for microscopy-based high-throughput screening of host factors involved in Brucella infection are described. The entry assay detects host factors required for Brucella entry and the endpoint assay those required for intracellular replication. While applicable for alternative approaches, siRNA screening in HeLa cells is used to illustrate the protocols.

Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by ...

A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or cellular processes. With the advancement of biotechnologies and the high translational potential of small molecules, a number of innovative approaches in drug discovery have evolved, which explains the resurgent interest in the use of HTS. The oncology field is currently the most active research area for drug screening, with no major breakthrough made for the identification of new immunomodulatory compounds targeting transplantation-related complications or autoimmune ailments. Here, we present a novel in vitro murine fluorescent-based lymphocyte assay easily adapted for the identification of new immunomodulatory compounds. This assay uses T or B cells derived from a transgenic mouse, in which the Nur77 promoter drives GFP expression upon T- or B-cell receptor stimulation. As the GFP intensity reflects the activation/transcriptional activity of the target cell, our assay defines a novel tool to study the effect of given compound(s) on cellular/biological responses. For instance, a primary screening was performed using 4,398 compounds in the absence of a &#34;target hypothesis&#34;, which led to the identification of 160 potential hits displaying immunomodulatory activities. Thus, the use of this assay is suitable for drug discovery programs exploring large chemical libraries prior to further in vitro/in vivo validation studies.

We present in the current study a novel fluorescence-based assay using lymphocytes derived from a transgenic mouse. This assay is suitable for high-throughput screening (HTS) of small molecules endowed with the capacity of either inhibiting or promoting lymphocyte activation.

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

A High-throughput Cre-Lox Activated Viral Membrane Fusion Assay to Identify Inhibitors of HIV-1 Viral Membrane Fusion

This assay is designed to specifically report on HIV-1 fusion via the expression of green fluorescent protein (GFP) detectable by flow cytometry or fluorescence microscopy. An HIV-1 reporter virus (HIV-1 Gag-iCre) is generated by inserting Cre recombinase into the HIV-1 genome between the matrix and the capsid proteins of the Gag polyprotein. This results in a packaging of Cre recombinase into virus particles, which is then released into a target cell line stably expressing a Cre recombinase-activated red fluorescent protein (RFP) to GFP switch cassette. In the basal state, this cassette expresses RFP only. Following the delivery of Cre recombinase into the target cell, the RFP, flanked by loxP sites, excises, resulting in GFP expression. This assay can be used to test any inhibitors of viral entry (specifically at the fusion step) in cell-free and cell-to-cell infection systems and has been used to identify a class of purinergic receptor antagonists as novel inhibitors of HIV-1 viral membrane fusion.

We describe a cell-based assay to report on HIV-1 fusion via the expression of green fluorescent protein detectable by flow cytometry or fluorescence microscopy. It can be used to test inhibitors of viral entry (specifically at the fusion step) in cell-free and cell-to-cell infection systems.

This assay is designed to specifically report on HIV-1 fusion via the expression of green fluorescent protein (GFP) detectable by flow cytometry or ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...