Prediction of drug-induced immune-mediated hepatotoxicity using hepatocyte-like cells derived from human embryonic stem cells

Kim, Dong Eon,Jang, Mi-Jin,Kim, Young Ran,Lee, Joo-Young,Cho, Eun Byul,Kim, Eunha,Kim, Yeji,Kim, Mi Young,Jeong, Won-il,Kim, Seyun,Han, Yong-Mahn,Lee, Seung-Hyo Elsevier 2017 Toxicology Vol.387 No.-

<P><B>Abstract</B></P> <P>Drug-induced liver injury (DILI) is a leading cause of liver disease and a key safety factor during drug development. In addition to the initiation events of drug-specific hepatotoxicity, dysregulated immune responses have been proposed as major pathological events of DILI. Thus, there is a need for a reliable cell culture model with which to assess drug-induced immune reactions to predict hepatotoxicity for drug development. To this end, stem cell-derived hepatocytes have shown great potentials. Here we report that hepatocyte-like cells derived from human embryonic stem cells (hES-HLCs) can be used to evaluate drug-induced hepatotoxic immunological events. Treatment with acetaminophen significantly elevated the levels of inflammatory cytokines by hES-HLCs. Moreover, three human immune cell lines, Jurkat, THP-1, and NK92MI, were activated when cultured in conditioned medium obtained from acetaminophen-treated hES-HLCs. To further validate, we tested thiazolidinedione (TZD) class, antidiabetic drugs, including troglitazone withdrawn from the market because of severe idiosyncratic drug hepatotoxicity. We found that TZD drug treatment to hES-HLCs resulted in the production of pro-inflammatory cytokines and eventually associated immune cell activation. In summary, our study demonstrates for the first time the potential of hES-HLCs as an <I>in vitro</I> model system for assessment of drug-induced as well as immune-mediated hepatotoxicity.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Generation and characterization of hES-HLCs for evaluation of drug-induced immune cell-mediated hepatotoxicity. </LI> <LI> The secretion of inflammatory cytokines is highly enhanced from APAP-treated hES-HLCs. </LI> <LI> Immune cells are activated and produce pro-inflammatory cytokines by conditioned medium from hES-HLCs cultured with APAP. </LI> <LI> Hepatotoxic results in hES-HLCs are consistent with those of primary human hepatocytes. </LI> <LI> Hepatotoxic drugs such as TZD, and non-hepatotoxic drugs such as aspirin and metformin, are also validated with our drug screening system. </LI> </UL> </P>

Analysis of therapeutic targets for COVID 19-nCoV/SARS-CoV-2 and discovery by virtual screening methods

Sharma, Sushil Kumar,Mishra, Ajay Kumar Cellmed Orthocellular Medicine and Pharmaceutical 2020 셀메드 (CellMed) Vol.10 No.2

Experimental drug development is very time consuming, expensive and limited to a relatively small number of targets. However, recent studies show that repositioning of existing drugs can function more efficiently then the novo experimental drug development to minimize cost and risks. Few previous studies have proven that network analysis is a versatile platform for this purpose, as the biological networks are used to model interactions between many different biological concepts. The present study is an attempt to review network based methods in predicting drug targets for drug repositioning. Human coronaviruses (HCoVs), including severe acute respiratory syndrome coronavirus (SARS-CoV) and 2019 novel coronavirus (2019-nCoV, also known as SARS-CoV-2), lead global epidemics with high morbidity and mortality. However, there are currently no effective drugs targeting 2019-nCoV/SARS-CoV-2. Drug repurposing, representing as an effective drug discovery strategy from existing drugs, could shorten the time and reduce the cost compared to de novo drug discovery. In this study, we present an integrative, antiviral drug repurposing methodology implementing a systems pharmacology-based network medicine platform, quantifying the interplay between the HCoV-host interactome and drug targets in the human protein-protein interaction network. Phylogenetic analyses of 15 HCoV whole genomes reveal that 2019-nCoV/SARS-CoV-2 shares the highest nucleotide sequence identity with SARS-CoV (79.7%). Specifically, the envelope and nucleocapsid proteins of 2019-nCoV/SARS-CoV-2 are two evolutionarily conserved regions, having the sequence identities of 96% and 89.6%, respectively, compared to SARS-CoV. Using network proximity analyses of drug targets and HCoV-host interactions in the human interactome, we prioritize 16 potential anti-HCoV repurposable drugs (e.g., melatonin, mercaptopurine, and sirolimus) that are further validated by enrichment analyses of drug-gene signatures and HCoV-induced transcriptomics data in human cell lines. We further identify three potential drug combinations (e.g., sirolimus plus dactinomycin, mercaptopurine plus melatonin, and toremifene plus emodin) captured by the "Complementary Exposure" pattern: the targets of the drugs both hit the HCoV-host subnetwork, but target separate neighborhoods in the human interactome network. In summary, this study offers powerful network-based methodologies for rapid identification of candidate repurposable drugs and potential drug combinations targeting 2019-nCoV/SARS-CoV-2.

Development of an Extracellular Matrix Plate for Drug Screening Using Patient-Derived Tumor Organoids

정용훈,박경원,김민섭,오현직,최동희,안진철,이샛별,나규환,Min Byung Soh,김진아,정석 한국바이오칩학회 2023 BioChip Journal Vol.17 No.2

With the advances in organoid culture, patient-derived organoids are utilized in diverse fields to broaden our understanding of conventional 2-dimensional (2D) culture methods and animal models. Patient-derived organoids have found new applications, such as screening for patient-matching drugs, immune checkpoint drugs, and mutation-target drugs, in the field of drug screening. However, conventional dome-shaped Matrigel drop-based screening methods using 24-, 48-, and 96-well plates are not effective for carrying out large-scale drug screening using organoids. Here, we present a newly developed 96-well plate-based method for the effective screening of patient-derived tumor organoids embedded in Matrigel. The new screening plate has a central hole with a diameter of 3 or 5 mm to provide a definite space for placing Matrigel in a cylindrical shape. Compared to the conventional dome-shaped Matrigel where the Matrigel drop is located arbitrarily, a cylinder-shaped Matrigel position in confined central wells allowed for faster and cost-effective tumor organoid screening. Importantly, the cylinder-shaped Matrigel ensures better consistency in high-throughput image-based analysis, which is used worldwide. Our results demonstrate the possibility of replacing the conventional 24-, 48-, and 96-well plates with the newly developed plates for effective tumor organoid screening.

Fully automated high-throughput cardiac toxicity screening platform using interlocking-structured 192 SU-8 cantilever arrays

Kim, Jong Yun,Oyunbaatar, Nomin-E.,Lee, Dong-Weon Elsevier 2019 Sensors and actuators. B Chemical Vol.285 No.-

<P><B>Abstract</B></P> <P>This paper proposes a high-throughput drug-screening platform integrated with 48 well plates containing 192 SU-8 cantilevers that assess cardiac toxicity levels by measuring changes in the contractile force of cardiomyocytes in vitro environment. To improve the reliability and reproducibility of the measured data, four interlocking-structured cantilevers were employed in each well and the displacement changes were measured at the nanometer scale using a laser-based sensor. The use of the mean value of the cantilever displacement in each well greatly improved the accuracy of the drug toxicity results. In addition, microgrooves patterned onto the SU-8 cantilevers greatly enhanced the alignment of cardiomyocytes, resulting in an increase in the contraction force by approximately 2.4 times. After the preliminary experiment, the contractile behaviors of cardiomyocytes on the surface-patterned SU-8 cantilevers were measured under various drugs, namely, Bay K8644, Verapamil, Isoproterenol, Quinidine, Lidocaine, E-4031, and Astemizole. In the case of Bay K8644, Verapamil, and Isoproterenol, the contractile force of cardiomyocytes was increased or decreased by 30%, 50%, and 20%, respectively, at the value of IC<SUB>50</SUB>. However, Quinidine only increased the half-value duration (HaVD) by approximately 22%, even though it did not affect the contractile force at IC<SUB>50</SUB>. Potassium-channel inhibitors E-4031 and Astemizole also showed no change in contractile force at low concentrations. However, the HaVD increased by approximately 3.4 times and abnormal peaks were observed at higher drug concentrations. The fully automated high-throughput screening capability of the proposed platform has immense potential as a preliminary drug-screening system in the next generation.</P> <P><B>Highlights</B></P> <P> <UL> <LI> 192 SU-8 cantilever arrays integrated with a 48-well plate is proposed for high-throughput screening in drug-induced cardiac toxicity. </LI> <LI> Changes in cantilever displacement and beating frequency of cardiomyocytes treated with various drugs were systematically analyzed. </LI> <LI> The fully automated high-throughput screening approach has great potential to further improve the accuracy of drug discovery studies. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>The manuscript introduced a novel method of fully automated high-throughput cardiac toxicity screening system platform. This system can precisely and continuously measure the displacement of 192 cantilever arrays using a laser vibrometer and a motorized XY-stage for the purposed of high-throughput drug screening.</P> <P>[DISPLAY OMISSION]</P>

Invited Review : Caenorhabditis elegans: A Model System for Anti-Cancer Drug Discovery and Therapeutic Target Identification

( Robert A Kobet ),( Xiao Ping Pan ),( Bao Hong Zhang ),( Stephen C Pak ),( Adam S Asch ),( Myon Hee Lee ) 한국응용약물학회 2014 Biomolecules & Therapeutics(구 응용약물학회지) Vol.22 No.5

The nematode Caenorhabditis elegans (C. elegans) offers a unique opportunity for biological and basic medical researches due to its genetic tractability and well-defined developmental lineage. It also provides an exceptional model for genetic, molecular, and cellular analysis of human disease-related genes. Recently, C. elegans has been used as an ideal model for the identification and functional analysis of drugs (or small-molecules) in vivo. In this review, we describe conserved oncogenic signaling pathways (Wnt, Notch, and Ras) and their potential roles in the development of cancer stem cells. During C. elegans germline development, these signaling pathways regulate multiple cellular processes such as germline stem cell niche specification, germline stem cell maintenance, and germ cell fate specification. Therefore, the aberrant regulations of these signaling pathways can cause either loss of germline stem cells or overproliferation of a specific cell type, resulting in sterility. This sterility phenotype allows us to identify drugs that can modulate the oncogenic signaling pathways directly or indirectly through a high-throughput screening. Current in vivo or in vitro screening methods are largely focused on the specific core signaling components. However, this phenotype based screening will identify drugs that possibly target upstream or downstream of core signaling pathways as well as exclude toxic effects. Although phenotype-based drug screening is ideal, the identification of drug targets is a major challenge. We here introduce a new technique, called Drug Affinity Responsive Target Stability (DARTS). This innovative method is able to identify the target of the identified drug. Importantly, signaling pathways and their regulators in C. elegans are highly conserved in most vertebrates, including humans. Therefore, C. elegans will provide a great opportunity to identify therapeutic drugs and their targets, as well as to understand mechanisms underlying the formation of cancer.

Application of liquid chromatography tandem mass spectrometry for the simultaneous quantification of multiple non-opioid drugs in human plasma

김광렬,강주희,김철우,남문석 대한독성 유전단백체 학회 2011 Molecular & cellular toxicology Vol.7 No.2

The simultaneous, quantitative and rapid analysis of plasma concentrations of multiple drugs is important to determine the clinical decision and to expect the prognosis in patients administered in emergency unit with intoxication. Here, we developed the liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based drug screening method for analyzing 12 drugs (acetaminophen, amitriptyline, chlorpromazine,cimetidine, diazepam, doxylamine, ephedrine, imipramine, metoclopramide, propranolol, tramadol, and zolpidem) with frequent events of intoxication throughout the country and evaluated its clinical applicability. The overall sensitivity (low limit of quantitation, 0.1-0.5 μg/mL), specificity, precision and accuracy for the quantification of 12 drugs were reliable and all drugs can be analyzed within 6 min. Among 12 drugs in samples for quality control, the REMEDi HS-based method detected only 6 drugs with low accuracy, while the LC-MS/MS system was able to precisely quantify all drugs. In addition, pilot analysis of patient samples with unknown drug intoxication was superior to the conventional LC-based drug profiling system, and was rapid and cost effective. In conclusion, LC-MS/MS-based drug screening is a good replacement for conventional LC-based REMEDi analyzer and has the better clinical applicability.

Method for the Rapid Screening of Drug Candidates Using Single-Protein Tracking in a Living Cell

김동균,김영숙,김찬식,이남기 대한화학회 2021 Bulletin of the Korean Chemical Society Vol.42 No.3

Screening drug candidates rapidly is the first step for developing new pharmaceutical drugs. One of the most promising ways to reduce the number of screening steps and cost is to directly use living cells for screening instead of using purified target proteins. Compounds screened using living cells will have increased biological activity compared to those screened with in vitro assays. Here, we report a robust method for screening drug candidates in living cells based on single-protein imaging. We employed single-protein tracking to observe the variation in the diffusion coefficient of membrane proteins treated with the candidate compounds. The diffusion coefficient shift was introduced as a criterion for selecting the potential candidate compounds. We tested three different membrane proteins, epidermal growth factor receptor, ErbB2, and ErbB3, and found effective natural compounds for each protein. The screening method we introduce will be widely used for screening potential drug candidates using living cells.

Role of computer-aided drug design in modern drug discovery

Stephani Joy Y. Macalino,홍선혜,GOSUVIJAYAKUMAR,최선 대한약학회 2015 Archives of Pharmacal Research Vol.38 No.9

Drug discovery utilizes chemical biology andcomputational drug design approaches for the efficientidentification and optimization of lead compounds. Chemical biology is mostly involved in the elucidation ofthe biological function of a target and the mechanism ofaction of a chemical modulator. On the other hand, computer-aided drug design makes use of the structuralknowledge of either the target (structure-based) or knownligands with bioactivity (ligand-based) to facilitate thedetermination of promising candidate drugs. Various virtualscreening techniques are now being used by bothpharmaceutical companies and academic research groupsto reduce the cost and time required for the discovery of apotent drug. Despite the rapid advances in these methods,continuous improvements are critical for future drug discoverytools. Advantages presented by structure-based andligand-based drug design suggest that their complementaryuse, as well as their integration with experimental routines,has a powerful impact on rational drug design. In thisarticle, we give an overview of the current computationaldrug design and their application in integrated rational drugdevelopment to aid in the progress of drug discoveryresearch.

Drug screening system development with high-throughput sequencing targeting skin-specific T cells in atopic dermatitis

( Chang Ook Park ) 대한피부과학회 2020 대한피부과학회 학술발표대회집 Vol.72 No.1

Atopic dermatitis (AD) is a common inflammatory skin disease, which has increased 2-3 times over the last century, especially in the developed countries. Specific treatments for AD are still limited and the most commonly used treatments are topical steroids and calcineurin inhibitors. Thus, we aimed to develop drug screening system targeting tissue-resident memory T (TRM) cells which have emerged as a major component of skin-specific T cells. CD69 + and CD103+ TRM cells have been observed to reside in the skin tissue of AD patients. In this study, we characterized immune and cell signaling signatures that exhibit significant expression in skin-specific T cells from AD patients using high-throughput sequencing techniques and selected candidate drugs, A, B, C. The MTT assay confirmed that CD69+ and CD103+ TRM cells were reduced by setting the appropriate drug concentration and confirming the effects of the drug on AD skin tissues. Then, we used the flaky tail mice, which essentially lack filaggrin, an important component of AD skin barrier. We developed house dust mite (HDM)- treated AD mouse model and evaluated drug effects on these AD mice. Epidermal thickening, hyperkeratosis, and inflammatory cell infiltration were observed in these mice. Finally, we validated the change of skin-specific T cells through flow cytometry and ELISA assays to evaluate skin-specific effects of the candidate drugs. This drug screening system targeting skin-specific T cells in AD may contribute to the development of new skin-targeted therapeutics in AD near future.

약물처리된 심장세포의 세포 수축력 측정을 위한 병렬 폴리머 캔틸레버 제작

김동수 ( Dong-su Kim ),이동원 ( Dong-weon Lee ) 한국센서학회 2020 센서학회지 Vol.29 No.2

Thus far, several in vivo biosensing platforms have been proposed to measure the mechanical contractility of cultured cardiomyocytes. However, the low sensitivity and screening rate of the developed sensors severely limit their practical applications. In addition, intensive research and development in cardiovascular disease demand a high-throughput drug-screening platform based on biomimetic engineering. To overcome the drawbacks of the current state-of-the-art methods, we propose a high-throughput drug-screening platform based on 16 functional high-sensitivity well plates. The proposed system simulates the physiological accuracy of the heart function in an in vitro environment. We fabricated 64 cantilevers using highly flexible and optically transparent silicone rubber and placed in 16 independent wells. Nanogrooves were imprinted on the surface of the cantilever to promote cell alignment and maturation. The adverse effects of the cardiovascular drugs on the cultured cardiomyocytes were systematically investigated. The 64 cantilevers demonstrated a highly reliable and reproducible mechanical contractility of the drug-treated cardiomyocytes. Real-time high-throughput screening and simultaneous evaluation of the cardiomyocyte mechanical contractility under multiple drugs verified that the proposed system could be used as an efficient drug-toxicity test platform.