Research & Technology Service

Innovative Screening Service
Selected Assay

IPK renders access to our innovative drug screening expertise and technologies in biosafety level-2/-3 laboratories providing domestic and international partners the opportunity to advance their own drug development programs. The highly experienced professionals of IPK provide various services through all stages of the screening process including biochemical, cell-based, or custom assay design and adaptation, combinatorial screening, proof-of-concept screening, small to large scale screenings, dose-response studies, as well as chemical analysis and molecular modeling.

IPK’s cell-based screening allows new drug
candidates to be identified faster and more reliably.

Over the fifteen years since our founding, IPK has established itself an international reputation as a go-to partner for pre-clinical screening in infectious diseases. In this context, we are sought by many partners from academia, public/private research institutes, NGOs, and industry.

Specifically, this was exemplified in 2020 and 2021 as IPK expedited fee-for-service support for COVID-19 related screening requests for over 120 organizations that approached us for support and partnership. These activities included the evaluation of antiviral efficacy for candidate compounds and antibodies against SARS-CoV-2 using our already developed cell- based assays. Indeed, our own studies revealed potent candidates now undergoing clinical trials by partners.

Importantly, as a member of Korea’s pan-governmental COVID-19 treatment and vaccine R&D supporting group, IPK has been helping domestic companies with in vitro drug efficacy evaluation. We are also operating a flagship laboratory of the National Institute of Infectious Diseases for drug antiviral efficacy evaluation and participating in the Consortium of Infectious Disease Research initiated to respond to the national crisis of (re)emerging infectious diseases.

IPK has been active in collaborating with major domestic and foreign pharmaceutical and bio venture companies, covering various research fields. In particular, we have been carrying out screening to accelerate drug discovery for diseases such as Leishmaniasis, Chagas disease, and COVID-19 by means of our long-term cooperation with DNDi.

We welcome researchers and industry partners interested in exploring opportunities for screening services, scientific partnerships, and other commercialization initiatives.

For Inquiries: BDT-TEAM@ip-korea.org

Service Procedure

COVID-19 drug screen: Analysis of antiviral activity through image-based drug screening utilizing Vero or Calu-3 cells. Much of the analysis is performed on low molecular weight compounds or natural compounds. Mechanism of hit compounds are investigated using various methods.

Microneutralization assay: Quantitative analysis of the neutralizing capacity of vaccines and therapeutic antibodies during their development.

Flow cytometry analysis: Precise analysis of cell surface proteins, intracellular proteins, and nuclear proteins using the Beckman Coulter CytoFLEX LX, which can measure up to 20 parameters per single cell with 6 lasers. Analysis in tube and 96-well plate format.

Cytokine/chemokine bead-array: Analysis of up to 13 cytokines and chemokines from a single cell culture medium, serum, etc., using a commercially available cytokine bead-array kit. Analysis through flow cytometry after staining with fluorescent beads and capturing antibody.

Immune stimulation assay: Measures various substances’ capacity to stimulate immunity utilizing dendritic cells differentiated from bone marrow cells of mice. Analysis of dendritic cell activation through flow cytometry.

Vaccine immunogenicityassessment (mouse model): Evaluates vaccine immunogenicity by analyzing immune responses such as antibody and T-cell responses induced by the vaccine. Antibody titer can be measured by ELISA, and in the case of T cell response, IFN-γ secretion following antigen peptide re-stimulation is measured through flow cytometry or ELISA.

Vaccine immunogenicity assessment (human): Evaluates vaccine immunogenicity using collected human blood samples. First, whole blood is received and separated into PBMC and plasma, then, PBMC is tested for T cells secreting IFN-γ through flow cytometry or ELISA, and plasma is tested for antibody titer through ELISA.

Vaccine efficacy test: If the pathogen can be managed at IPK, vaccine efficacy can be evaluated in mice through vaccination models. For example, using the current mouse models for SARS-CoV-2 and influenza virus, vaccine efficacy can be evaluated by measuring the weight change, fatality rate, and the amount of virus in vivo.

QIM assay: This ex vivo assay in Mycobacterium tuberculosis- infected macrophages is used to monitor TB infection inside the cell by confocal fluorescent microscopy. This assay is the first cellular model to visualize the infection process of Mycobacterium tuberculosis inside human macrophages.

QUM assay: This is a bacteriostatic assay using the M. tuberculosis-gfp strain. This assay can be measured by Victor 3.

MDR/XDR assay: This is a QIM/QUM assay that uses MDR and XDR clinical isolates.

Drug combination assay (Checker board): This is an in vitro combination assay based on resazurin (compusyn analysis).

Drug combination assay in ex vivo: This is an in vitro combination assay based on QIM (compusyn analysis).

Pseudomonas aeruginosa assay in optimized Artificial Sputum Media (ASM): High-throughput, high-content drug efficacy screening for effective antibiotic development using artificial sputum media (ASM) containing pneumocytes and components mimicking the environment of P. aeruginosa lung infection in the human body.

Streptococcus pneumonia assay for HTS system: A high-throughput drug screening method optimized for efficient discovery of drugs against pneumococcus, a major cause of pneumonia which is difficult to cultivate in vitro.

Caco-2 Infection model of Staphylococcus aureus: This enterocyte infection model system of Staphylococcus aureus enables high-speed antibacterial efficacy search including basic research of Staphylococcus aureus infection, as well as drug permeation and initial toxicity analysis.

Co-culture pneumocytes with Pseudomonas aeruginosa: This assay is used to select non-cytotoxic small molecules that protect human pneumocytes from P. aeruginosa-induced cell death. In addition, the assay allows for the selection of unconventional compounds that inhibit virulence factors of the bacteria without killing it. This assay is performed in 384-well plate format with advantages of observing the cytotoxicity and efficacy of multiple compounds at a time.

HBV infection assay: This cell-based assay using 384-well plates is used to evaluate effect of a compound on early stages (entry, decapsidation, ccDNA synthesis, etc.) of the viral lifecycle.

HBV replication assay: This cell-based assay is used to evaluate the effect of a compound on the DNA replication stage of HBV lifecycle through real-time PCR in 384-well plates.

HBV virucidal assay: This test-tube assay measures the efficacy of antiviral drugs in killing HBV directly.

HCV infection assay: This cell-based assay using 384-well plates is used to evaluate the effect of a compound on all stages of the viral lifecycle.

HCV RNA replication assay: This cell-based assay using 384-well plates is used to evaluate the effect of a compound on the RNA replication stage of HCV lifecycle.

HCV pseudoparticle entry assay: This cell-based assay using 384-well plates is used to evaluate E1/E2-mediated entry of HCV.

HCV virucidal assay: This in vitro assay is used to evaluate virucidal activity of antivirals.

HDV infection assay: This cell-based assay using 384-well plates is used to evaluate the effect of a compound in each stage of HDV lifecycle.

HDV replication assay: This cell-based assay is used to analyze the effect of a compound on the RNA replication stage of HDV lifecycle through real-time PCR in 384-well plates.

HEV replication assay: This cell-based assay using 384-well plates is used to analyze the effect of a compound on the RNA replication stage of HEV lifecycle.

Visceral Leishmaniasis (VL) and cutaneous Leishmaniasis (CL) intracellular assay: This assay is used to monitor intracellular Leishmania growth inhibitory effects of testing inhibitors. The image-based assay system has the advantage of observing the cytotoxicity and efficacy of multiple compounds at a time. Leishmania donovani causing visceral Leishmaniasis, and L. major and L. amazonensis causing cutaneous Leishmaniasis are used for this assay.

Visceral Leishmaniasis (VL) and cutaneous Leishmaniasis (CL) extracellular assay: This assay is used to test the effect of inhibitors against extracellular leishmania. Leishmania donovani and Leishmania tropica are treated with compounds and the number of live parasites are detected using Resazurin.

T.cruzi (Chagas) intracellular assay: The T. cruzi intracellular assay is used to find inhibitors for Chagas for disease treatment. The image-based assay system has advantages of observing the cytotoxicity and efficacy of multiple compounds at a time. T. cruzi Y strain, a highly infective strain among T.cruzi, is used for this assay.

T. brucei cell-based assay: This cell-based assay is used to test the effect of inhibitors on T. brucei growth. T. brucei are treated with compounds, then the number of live parasites is detected using Resazurin.

Visceral leishmaniasis in vivo assay: The Leishman Donovan Unit assay is used to quantify the degree of infection within the liver after infecting Balb/c with Leishmania donovani and administering a drug.

Cutaneous leishmaniasis in vivo assay: This assay confirms the degree of infection by quantifying the thickness of the footpad after infecting Balb/c on the footpad with Leishmania tropica and administering a drug.

T. brucei in vivo assay: This assay confirms the concentration of protozoa by sampling the blood of Balb/c after infecting with T. brucei and administering a drug.

Hepatocellular carcinoma (HCC) stem cell assay: This assay is used to measure cytotoxicity through a mixed culture containing normal hepatocytes, cancer stem cells, and cancer cells.

3D culture system in HTS/HCS: In this assay, 3D spheroids are utilized for drug screening of HCC. Three dimensional cultures of hepatocytes are the proper culture models to sustain liver-specific functions, as well as liver-specific architecture, similar to those found in vivo. Features of spheroids are extracted at 3 different levels: the signal level, the cell level and the organoids level.

Angiogenesis assay: This assay measures the vascular regeneration capacity of venous endothelial cells following exposure to drugs.

Dose response curve assay in multicellular tumor spheroid (3D): This assay measures the cell proliferation capacity at various drug concentrations by considering the size of multicellular tumor spheroids (3D culture).

Anti-fibrosis 3D assay: Assay for selecting antifibrotic drugs using multicellular tumor spheroids, which is a liver fibrosis model.

Caspase 3 activity assay: This assay is used to evaluate drug toxicity by measuring the activity of caspase-3, one of the markers of apoptosis.

DNA damage assay & micro nuclei assay: This assay is used to detect DNA damage and genotoxicity following exposure to drugs.

EdU assay: This assay is used to detect proliferation activity after exposure to drugs.

ROS detection assay: This assay is used to measure reactive oxygen species (ROS), a major inducer of apoptosis.

Autophagy detection: Studies have shown that accumulation of autophagosome induces cell death. For detection of autophagy, active form-LC3B is measured by HCS or Western blot.

Cell-based assay development and optimization for HCS: This entails developing and optimizing cell-based, high-throughput screening and high-content assays; acquiring and analyzing screening data; and compiling and reporting the final results. All assay development is performed in 96- or 384-well plates.

Cell-based assay development and optimization for target based / PPI: This entails developing, optimizing, and validating biochemical assays for target-based or protein- protein interaction quantification. Data are collected and analyzed by high-throughput, high-content screening, then the final result is reported. All assay development is performed in 96- or 384-well plates.

High-throughput screening for chronic/infectious disease using adapted biosafety facility: We perform automated HTS/HCS using a multimode plate reader or automated confocal microscope in biosafety level 1-3 facilities. Experiments using pathogens, including bacteria, parasites and viruses, are conducted in the biosafety facilities.

HTS chemical screening: Institut Pasteur Korea’s chemical library consists of approximately 180,000 diverse molecules including synthetic compounds and natural products. The chemical library includes compounds synthesized directly at IPK, compounds purchased through verified suppliers such as Sigma-Aldrich, and those filtered according to the Lipinski rules.

HTS RNAi screening: The RNAi libraries at IPK consist of pooled and single siRNAs options, as well as a shRNA library available for screening.
■ Dharmacon ON-TARGETplus SMART pool siRNA: pooled format containing 4 siRNAs for every 18,000 genes
■ Life Technologies Silencer Select: arrayed format containing 3 siRNA duplexes for every 22,000 genes
■ Sigma-Aldrich Mission shRNA: arrayed format containing 5 shRNA hairpins for every 16,000 genes

Anti-EBOLA cell-based assay: This anti-EBOLA cell-based assay is a high- throughput, high-content screening method using 384-well plates, which confirms the antiviral activity of drugs against each stage of the Ebola virus lifecycle.

Anti-Dengue cell-based assay: This anti-dengue cell-based assay is an HTS/HCS screening method using 384-well plates, which can confirm the antiviral activity of drugs against dengue virus types 1-4.

Cell-based assay development and optimization for PPI: This entails developing and optimizing live cell- based assays for protein-protein interaction (PPI) quantification, acquiring and analyzing screening data, and compiling and reporting the final results. Assay development is performed in 96- or 384-well plates.

Tau protein phosphorylation assay: This cell-based, high-content screening assay using 384-well plates is used to evaluate tau protein expression and phosphorylation.

Gamma secretase activity assay: This cell-based, high-content screening assay using 384-well plates is used to evaluate gamma- secretase activity.

Beta secretase activity assay: This cell-based, high-content screening assay using 384-well plates is used to evaluate beta-secretase 1 or 2 activity.

5-HT2C receptor internalization assay: This cell-based, high-content screening assay using 384-well plates is used to evaluate 5-HT2C GPCR receptor internalization.

Compound synthesis: This includes chemical synthesis of compounds in small to medium scale; synthesis of nucleosides, peptides and natural products; synthesis of assay development tools and reagents; synthesis of cold- labeled (C13, H2, N15) compounds; purification of active materials from extracts; structure determination; and performing structure-activity relationship (SAR) studies.

Chembioinformatics & Molecular Modeling: Cheminformatics and molecular modeling are being used at Institut Pasteur Korea to accelerate the drug discovery process. At the early stage of the drug discovery program, cheminformatics is applied once hits are obtained from high-throughput screening. These hits are clustered by chemical structural information and therefore we obtain the structure activity relationship (SAR) information of hit compounds. We also gather the information about druggability, novelty and patentability of compounds providing chemists the criteria for decision making at hit confirmation and scaffold selection stage. At the lead optimization stage, molecular modeling is required in both ligand-based and structure-based approaches. We apply ligand-based modeling such as structure conformation analysis and pharmacophore modeling when targets are unknown. Also, physicochemical properties of chemicals are predicted providing information for improving compound properties. In the case of target-based drug discovery, structure-based techniques such as docking and putative 3D structure modeling provide good information for optimization step. In addition, virtual screening is performed to select possible binding compounds to the known target without the necessity to run high- throughput screening.

Compound solubility test: The solubility test determines solubility of compounds in various solvents. High-throughput solubility assays in drug discovery are often kinetic solubility assays that are based on the detection of precipitation of compounds in aqueous solutions.

400MHz FT-NMR analysis: Analysis of the structural and motional properties of molecules. It is possible to determine and analyze the structure of synthetic low-molecular or high- molecular organic substances, natural substance molecules, proteins, and synthetic high-molecular substances. We possess one type of multi probe, and can obtain chemical shift for nuclei 1H, 13C, 19F, and 31P.

High Performance Liquid Chromatography (HPLC) analysis: Separation and analysis of non-volatile or thermally unstable mixtures by dissolving the compound mixture in a solvent. Each component is separated into pure substance by chemical action with the stationary phase in a separation tube, and the separated components are analyzed qualitatively and quantitatively.

LC/MS analysis: Qualitative and quantitative analysis by separating each component in a mixture through LC then analyzing with a mass spectrometer. The chemical structure, chemical reaction, molecular weight, etc., of the substance can be identified through the mass spectrometer.

Combiflash column chromatography: Separation is induced through a speed difference in the movement at stationary phase due to difference in the polarity of organic molecules part of the organic reaction mixture. Compounds can be separated and analyzed with high efficiency as it carries out the function of automated flash column chromatography.