Cell-based Screening System Research Articles

CBP-93872 inhibits NBS1-mediated ATR activation, abrogating maintenance of the DNA double-strand break-specific G2 checkpoint.

CBP-93872 was previously identified as a G2 checkpoint inhibitor using a cell-based high-throughput screening system. However, its molecular actions as well as cellular targets are largely unknown. Here, we uncovered the molecular mechanisms underlying abrogation of the G2 checkpoint by CBP-93872. CBP-93872 specifically abrogates the DNA double-stranded break (DSB)-induced G2 checkpoint through inhibiting maintenance but not initiation of G2 arrest because of specific inhibition of DSB-dependent ATR activation. Hence, ATR-dependent phosphorylation of Nbs1 and replication protein A 2 upon DSB was strongly suppressed in the presence of CBP-93872. CBP-93872 did not seem to inhibit DNA-end resection, but did inhibit Nbs1-dependent and ssDNA-induced ATR activation in vitro in a dose-dependent manner. Taken together, our results suggest that CBP-93872 is an inhibitor of maintenance of the DSB-specific G2 checkpoint and thus might be a strong candidate as the basis for a drug that specifically sensitizes p53-mutated cancer cells to DSB-inducing DNA damage therapy.

ALZHEIMER'S DISEASE MODEL CELLS DERIVED FROM HUMAN IPS CELLS

Alzheimer's disease (AD) is the most common cause of dementia characterized by impaired memory and cognitive dysfunction due to neurodegeneration. One of the many pathological features of the disease is loss of neuronal and synaptic neurons in the nucleus basalis of Meynert, cerebral cortex and hippocampus as well as the deposition of amyloid-beta-peptide (Aβ). This gives rise to the amyloid hypothesis that deposition of Aβ causes neurodegeneration. Furthermore, in familial AD, the likely cause of the disease is the mutation of the presenilin 1 (PS1) gene coding of component of γ-secretase which is involved in processing Aβ from amyloid precursor protein.The predicted growth of AD morbidity coupled with the socioeconomic impact of the disease places enormous pressure on the need to find an effect therapy. It is necessary to provide a physiologically relevant AD model cell and a scalable assay system for understanding the disease and for the development of novel therapies. To make the cell-based drug screening system for AD, we can provide AD model cells derived from human iPS cells incorporated with a mutated PS1 gene. We have two types of Alzheimer's disease model cells, ReproNeuro Ach-AD kit and ReproNeuro Glu-AD kit. ReproAch-AD kit contains cholinergic neurons and ReproNeuro Glu-AD kit contains glutamatergic neurons. The effect on production of Aβ40 and Aβ42 was measured using an AlphaLISA ® Human Amyloid β1-40/1-42 immunoassay kit (Perkin Elmer) for high throughput screening (HTS). These cells show an increase in the Aβ ratio similar to the in-vivo of AD patient. The investigations of other AD phenotype (increasing p-Tau, cell death, ect.) are proceeding. We expect these cells to be an a valuable addition to cell-based drug screening systems for AD.

Development of a cell-based assay system considering drug metabolism and immune- and inflammatory-related factors for the risk assessment of drug-induced liver injury

Drug-induced liver injury (DILI) is a major safety concern in drug development and clinical pharmacotherapy. However, prediction of DILI is difficult because the underlying mechanisms are not fully understood. To establish a novel cell-based screening system to suggest drugs with hepatotoxic potential in preclinical drug development, comprehensive gene expression analyses during in vivo DILI are necessary. Using in vivo mouse DILI models and 4 sets of hepatotoxic positive and non-hepatotoxic drugs, we found that the hepatic mRNA levels of S100A8; S100A9; “NATCH, LRR, and pyrin domain-containing protein 3” (NALP3); interleukin (IL)-1β; and the receptor for advanced glycation endproducts (RAGE) were commonly increased in hepatotoxic drug-administered mice compared to non-hepatotoxic drug-administered mice. To clarify whether these 5 in vivo biomarkers can be applied to a cell-based screening system, we adapted human liver microsomes (HLM) in the presence of NADPH to assess the metabolic activation reaction, and we also adapted human monocytic leukemia cells HL-60, K562, KG-1 and THP-1 to assess the effects on mRNA expression of immune- and inflammatory-related factors. We investigated 30 clinical drugs with different safety profiles with regard to DILI and found that the total sum score of gene expression levels of S100A8, S100A9, RAGE, NALP3 and IL-1β mRNA in HL-60 or K562 cells incubated with HLM, could identify drugs at high risk for hepatotoxicity. We proposed the use of the total sum score of gene expression level for assessing metabolic activation by drug-metabolizing enzymes and immune- and inflammatory-related factors for the risk assessment of DILI in preclinical drug development.

SIRT1 upregulators from high‐throughput screening as anti‐proliferation and anti‐migration agents in vascular smooth muscle cells (654.2)

Objective: To establish a cell‐based drug screening system for discovering up‐regulators of SIRT1 and confirmed their biological activities in rat aortic VSMCs. Methods and Results: A stable transfected with a human SIRT1‐promoter‐luciferase reporter gene construct cell line was obtained in 293A cells. Using Resveratrol as a positive control, a cell‐based SIRT1 up‐regulator high‐throughput screening (HTS) model was established in a 384‐well microplate format. Of 27,274 compounds, 86 compounds were identified to up‐regulate SIRT1 transcriptional activity in the cell‐based reporter assay. Furthermore, 10 compounds including a series of Resveratrol analogues showing low toxic in both VSMCs and HUVECs were demonstrated to not only stimulate the expression of SIRT1 on the transcriptional level in VSMCs, but also have anti‐proliferation and anti‐migration properties in PDGF‐BB (40 ng/ml)‐stimulated VSMCs. In addition, several natural compounds (Baicalin, Genistein, Chrysin, etc.) were identified to elevate SIRT1 transcriptional level, with the reported property of attenuating neointima formation following injury via suppression of VSMC proliferation, migration or both. Conclusion: A cell‐based drug screening model was established to screen SIRT1 up‐regulators. The active compounds originated from this HTS assay may be developed to drug candidates or lead compounds for new anti‐restenosis drugs.Grant Funding Source: This work was supported by the National Natural Science Foundation of China (No. 81102444).

Discovery of novel adenylyl cyclase inhibitor by cell-based screening.

We screened 2400 compounds to find novel inhibitors of the adenylyl cyclase (AC)-protein kinase A (PKA)-cAMP response-element-binding protein (CREB) signaling pathway (AC/PKA/CREB pathway). Using a multistep cell-based screening system employing split luciferase technique, we narrowed down the candidates effectively from 2400 chemical compounds and identified a novel AC inhibitor (compound 1). Since dysregulation of the AC/PKA/CREB pathway is known to cause diseases not only in the nervous system but also in other organs, compound 1 is expected to be developed as a medicine for these diseases.

Xylogranin B: A Potent Wnt Signal Inhibitory Limonoid from Xylocarpus granatum

Xylogranin B (2) was isolated from Xylocarpus granatum (Meliaceae) leaves, by use of a cell-based luciferase screening system targeting a Wnt signaling pathway. Compound 2 inhibited TCF/β-catenin transcriptional activity (IC50 48.9 nM) and exhibited strong cytotoxicity against colon cancer cell lines. Compound 2 significantly decreased β-catenin protein levels in nuclei but not in the cytosol. These results indicated that a decrease in β-catenin levels in nuclei by 2 resulted in the Wnt signal inhibitory effects of 2.

Spectomycin B1 as a Novel SUMOylation Inhibitor That Directly Binds to SUMO E2

Conjugation of small ubiquitin-like modifier (SUMO) to protein (SUMOylation) regulates multiple biological systems by changing the functions and fates of a large number of proteins. Consequently, abnormalities in SUMOylation have been linked to multiple diseases, including breast cancer. Using an in situ cell-based screening system, we have identified spectomycin B1 and related natural products as novel SUMOylation inhibitors. Unlike known SUMOylation inhibitors such as ginkgolic acid, spectomycin B1 directly binds to E2 (Ubc9) and selectively blocks the formation of the E2-SUMO intermediate; that is, Ubc9 is the direct target of spectomycin B1. Importantly, either spectomycin B1 treatment or Ubc9 knockdown inhibited estrogen-dependent proliferation of MCF7 human breast-cancer cells. Our findings suggest that Ubc9 inhibitors such as spectomycin B1 have potential as therapeutic agents against hormone-dependent breast cancers.

Cell-based high-throughput odorant screening system through visualization on a microwell array

The development of a cell-based high-throughput screening system has attracted much attention from researchers who study drug screening mechanisms and characterization of G-protein coupled receptors (GPCRs). Although olfactory receptors (ORs) constitute the largest group of GPCRs that play a critical role recognizing and discriminating odorants, only a few ORs have been characterized, and most remain orphan. The conventional cell-based assay system for characterizing GPCRs, including ORs, is very laborious, time consuming, and requires an expensive assay system. In this study, we developed a simple, low-cost miniaturized odorant screening method by combining Micro-Electro-Mechanical system (MEMs) technique and visualization technique for detecting an odorant response. We fabricated PEG microwell from a photocrosslinkable polyethylene glycol diacrylate (PEGDA) solution and applied it to cell culture and a reverse transfection platform for cell-based high-throughput screening. For the first time, the olfactory receptors were expressed on the microwell platform using reverse transfection technique. The various olfactory receptors can be expressed simultaneously using this technique and the microwell spotted with olfactory receptor genes can be used as a high-throughput screening platform. The odorant response was detected via fluorescence analysis on the microwell using a cAMP response element (CRE) reporter assay. We tested this platform using four de-orphaned ORs. This new cell-based screening method not only reduced numerous time-consuming steps but also allowed for simple, efficient, and quantitative screening and patterning of large numbers of GPCRs including ORs, which can help to visualize the OR response to odorants on a microwell.

Highlights of This Issue

c-Myc, as the central hub for cellular signals, is deregulated at multiple levels in almost all human cancers. Although this makes c-Myc an attractive candidate target for cancer therapy, drug development against c-Myc has been challenging. Fan-Minogue and colleagues have identified the new antineoplastic function of an antiparasitic agent, nitazoxanide, through their robust cell-based high throughput screening system by utilizing a c-Myc activation sensor. This new system allows large-scale and fast screening for compounds inhibiting c-Myc, thus potentially accelerating drug development against c-Myc–associated cancer.Aberrant activity of receptor tyrosine kinases has been associated with tumor progression in a wide variety of human malignancies. In the present study, Burbridge and colleagues demonstrate that S49076, a novel, potent inhibitor of MET, AXL/MER, and FGFR1/2/3, has specific targeting and strong antitumor effect in vitro and in vivo. S49076 not only showed synergy with bevacizumab in colon cancer xenograft models but also blocked the growth of bevacizumab-resistant tumors. A phase I clinical trial of S49076 is currently underway in patients with advanced solid tumors.Hypoxia is a critical microenvironmental factor in multiple myeloma. It has been shown previously that the hypoxia-activated prodrug TH-302 selectively targets hypoxic multiple myeloma cells. To further explore the application of TH-302 in multiple myeloma, Hu and colleagues evaluated in this study the antitumor effect of TH-302 in combination with the proteasome inhibitor bortezomib, which is already approved for multiple myeloma. The combination of TH-302 and bortezomib was found to synergistically induce apoptosis in human multiple myeloma cell lines in vitro and prolong the survival of 5T33MMvv multiple myeloma mice in vivo. This study provides a rationale for clinical evaluation of this combination in multiple myeloma patients.Patients with glioblastoma have an exceptionally grim prognosis. Although HER1/EGFR is the most frequently amplified oncogene in this disease, monotargeted agents against this receptor have fallen short of improving the clinical course. In order to increase the efficacy of HER1/EGFR inhibition, Karpel-Massler and colleagues screened rationally identified candidate molecular targets and found that combining NSC23766, an inhibitor of RAC1, with erlotinib results in a synergistic antiproliferative effect on primary and established glioblastoma cells. This novel therapeutic approach might be promising in developing a more efficient treatment for glioblastoma patients.

A cell-based screening system for influenza A viral RNA transcription/replication inhibitors

Although two classes of antivirals, NA inhibitors and M2 ion channel blockers, are licensed for influenza treatment, dual resistant mutants, including highly pathogenic H5N1 viruses, have appeared. Alternative treatment options are, therefore, needed. Influenza A viral RNA (vRNA) transcription/replication is a promising target for antiviral development, since it is essential for virus replication. Accordingly, an efficient and reliable method to identify vRNA transcription/replication inhibitors is desirable. Here, we developed a cell-based screening system by establishing a cell line that stably expresses influenza viral ribonucleoprotein complex (vRNP). Compound library screening using this cell line allowed us to identify a compound that inhibits vRNA transcription/replication by using reporter protein expression from virus-like RNA as a readout and virus replication in vitro. vRNP-expressing cells have potential as a simple and convenient high-throughput screening (HTS) system, and, thus, are promising to identify vRNA transcription/replication inhibitors for various RNA viruses, especially for primary screens.

Mitochondrial fragmentation caused by phenanthroline promotes mitophagy

Mitochondrial dynamics and mitophagy are thought to be important events for the quality control of mitochondria and mitochondria-associated diseases. To identify novel mitophagy modulators, we developed a cell-based screening system and selected 1,10-phenanthroline (Phen) as a target molecule. Phen treatment highly induced mitochondrial fragmentation and mitochondrial dysfunctions in a Drp1 dependent manner. Phen treatment also increased autophagy. Moreover, prolonged exposure of Phen increased mitochondria clearance through mitophagy. Phen-mediated loss of mitochondrial mass was more reduced in ATG5 deficient cells than in wild type cells. In addition, down-regulation of Drp1 decreased autophagy activation, suggesting that mitochondrial fission is involved in Phen-mediated mitophagy. Thus, our results demonstrate that the disruption of mitochondrial dynamics and mitochondrial dysfunctions provokes mitophagy in Phen-treated cells.

Micro-patterned porous substrates for cell-based assays

In the search for new therapeutic chemicals, lab-on-a-chip systems have recently emerged as innovative and efficient tools for cell-based assays and high throughput screening. Here, we describe a novel, versatile and simple device for cell-based assays at the bench-top. We created spatial variations of porosity on the surface of a membrane filter by microcontact printing with a biocompatible polymer (PDMS). We called such systems Micro-Printed Membranes (μPM). Active compounds dispensed on the porous areas, where the membrane pores are not clogged by the polymer, can cross the membrane and reach cells growing on the opposite side. Only cells immediately below those porous areas could be stimulated by chemicals. We performed proof-of-principle experiments using Hoechst nuclear staining, calcein-AM cell viability assay and destabilization of the cytoskeleton organisation by cytochalasin B. Resulting fluorescent staining properly matched the drops positioning and no cross-contaminations were observed between adjacent tests. This well-less cell-based screening system is highly flexible by design and it enables multiple compounds to be tested on the same cell tissue. Only low sample volumes in the microlitre range are required. Moreover, chemicals can be delivered sequentially and removed at any time while cells can be monitored in real time. This allows the design of complex, sequential and combinatorial drug assays. μPMs appear as ideal systems for cell-based assays. We anticipate that this lab-on-chip device will be adapted for both manual and automated high content screening experiments.

Inhibitors of SARS-CoV entry – Identification using an internally-controlled dual envelope pseudovirion assay

Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) emerged as the causal agent of an endemic atypical pneumonia, infecting thousands of people worldwide. Although a number of promising potential vaccines and therapeutic agents for SARS-CoV have been described, no effective antiviral drug against SARS-CoV is currently available. The intricate, sequential nature of the viral entry process provides multiple valid targets for drug development. Here, we describe a rapid and safe cell-based high-throughput screening system, dual envelope pseudovirion (DEP) assay, for specifically screening inhibitors of viral entry. The assay system employs a novel dual envelope strategy, using lentiviral pseudovirions as targets whose entry is driven by the SARS-CoV Spike glycoprotein. A second, unrelated viral envelope is used as an internal control to reduce the number of false positives. As an example of the power of this assay a class of inhibitors is reported with the potential to inhibit SARS-CoV at two steps of the replication cycle, viral entry and particle assembly. This assay system can be easily adapted to screen entry inhibitors against other viruses with the careful selection of matching partner virus envelopes.

A novel FK-506-binding-like protein that lacks peptidyl-prolyl isomerase activity is involved in intracellular infection and in vivo virulence of Burkholderia pseudomallei

Burkholderia pseudomallei is a facultative intracellular bacterial pathogen causing melioidosis, an often fatal infectious disease that is endemic in several tropical and subtropical areas around the world. We previously described a Ptk2 cell-based plaque assay screening system of B. pseudomallei transposon mutants that led to the identification of several novel virulence determinants. Using this approach we identified a mutant with reduced plaque formation in which the BPSL0918 gene was disrupted. BPSL0918 encodes a putative FK-506-binding protein (FKBP) representing a family of proteins that typically possess peptidyl-prolyl isomerase (PPIase) activity. A B. pseudomallei ΔBPSL0918 mutant showed a severely impaired ability to resist intracellular killing and to replicate within primary macrophages. Complementation of the mutant fully restored its ability to grow intracellularly. Moreover, B. pseudomallei ΔBPSL0918 was significantly attenuated in a murine model of infection. Structural modelling confirmed a modified FKBP fold of the BPSL0918-encoded protein but unlike virulence-associated FKBPs from other pathogenic bacteria, recombinant BPSL0918 protein did not possess PPIase activity in vitro. In accordance with this observation BPSL0918 exhibits several mutations in residues that have been proposed to mediate PPIase activity in other FKBPs. To our knowledge this B. pseudomallei FKBP represents the first example of this protein family which lacks PPIase activity but is important in intracellular infection of a bacterial pathogen.

Identification of low-molecular weight inhibitors of HIV-1 reverse transcriptase using a cell-based high-throughput screening system

A cell-based drug screening system that utilizes a green fluorescent protein (GFP)-tagged recombinant lentiviral vector has been used to screen a chemical library of 34,000 small molecules for antiretroviral compounds. Thirty-three initial hits were analyzed and four compounds were selected based on their anti-human immunodeficiency virus type 1 (HIV-1) activity (EC 50 values ranging from 0.17 to 1.9 μM) and low cellular toxicity (CC 50 values >50 μM). The four compounds blocked reverse transcription and were able to inhibit the replication of a panel of different HIV-1 strains, including non-B subtype and viruses resistant to different drug classes. Serial in vitro passages of HIV-1 B-HXB2 in the presence of increasing drug concentrations selected for viruses with reduced susceptibility. Mutations previously associated with resistance to non-nucleoside reverse transcriptase (RT) inhibitors (L100I and Y181C for CBL-17 and CBL-21, respectively) or linked to nucleoside analogue resistance (A62V for CBL-4.0 and CBL-4.1) were identified. Viruses with reduced susceptibility to CBL-17 and CBL-21 but not the ones resistant to CBL-4.0 or CBL-4.1 showed a decrease in replicative fitness. Interestingly, two of the small molecules (CBL-4.0 and CBL-4.1) are indolopyridinones that were previously described as nucleotide-competing RT inhibitors.

Discovery of a Small Peptide from Combinatorial Libraries That Can Activate the Plant Immune System by a Jasmonic Acid Signaling Pathway

Plants defend themselves by using an innate immune system that is activated in response to a variety of molecules derived from pathogens. These molecules have provided profound insights into the mechanisms of pathogen recognition and subsequent signaling pathways in plants. In the present study, we screened a combinatorial random hexapeptide library for peptides that activate the plant immune system, by using a cell-based high-throughput screening system in which H(2)O(2) generation was monitored. We discovered a novel small peptide (YGIHTH-amide, PIP-1) that triggered H(2)O(2) production in tobacco and tomato cells, but not in Arabidopsis cells. PIP-1 induced significant levels of phytoalexin biosynthesis and defense-related gene expression in tobacco cells; this is likely to be activated by a jasmonic acid pathway.

The small molecule harmine regulates NFATc1 and Id2 expression in osteoclast progenitor cells

Small molecule compounds that potently affect osteoclastogenesis could be useful as chemical probes for elucidating the mechanisms of various biological phenomena and as effective therapeutic strategies against bone resorption. An osteoclast progenitor cell-based high-throughput screening system was designed to target activation of NFAT, which is a key event for osteoclastogenesis. Orphan ligand library screening using this system identified the β-carboline derivative harmine, which is a highly potent inhibitor of dual-specificity tyrosine-phosphorylation regulated kinase 1A (DYRK1A), to be an NFAT regulator in osteoclasts. RAW264.7 cells highly expressed DYRK1A protein, and in vitro phosphorylation assay demonstrated that harmine directly inhibited the DYRK1A-mediated phosphorylation (in-activation) of NFATc1. Harmine promoted the dephosphorylation (activation) of NFATc1 in RAW264.7 cells within 24h, and it significantly increased the expression of NFATc1 in RAW264.7 cells and mouse primary bone marrow macrophages (BMMs) both in the presence and absence of RANKL stimulation. Although harmine promoted NFATc1 expression and stimulated target genes for osteoclastogenesis, cell-cell fusion and the formation of TRAP-positive multinucleated osteoclasts from RAW264.7 cells and BMMs was significantly inhibited by harmine treatment. Meanwhile, harmine remarkably promoted the expression of inhibitor of DNA binding/differentiation-2 (Id2), which is a negative regulator for osteoclastogenesis, in RAW264.7 cells and BMMs. An Id2-null-mutant showed slightly increased osteoclast formation from BMMs, and the harmine-mediated inhibition of osteoclast formation was abolished in the BMMs of Id2-null-mutant mice. These results suggest that harmine is a potent activator of NFATc1 that interferes with the function of DYRK1A in osteoclast precursors and also up-regulates Id2 protein, which may dominantly inhibit expression pathways associated with cell-cell fusion, thereby leading to the disruption of the fusion events mediating osteoclastogenesis. The small molecule harmine is therefore expected to provide an experimental tool for investigating signaling cascades in osteoclastogenesis, especially those centered on DYRK1A-mediated NFATc1 and Id2 regulation.

Discovery of polyoxometalate-based HDAC inhibitors with profound anticancer activity in vitro and in vivo

We obtained 5 positive novel histone deacetylase inhibitors (HDACIs) from a polyoxometalate (POM) library by using a cell-based screening system targeting the p21 gene promoter. Among them, PAC-320, a new tri-organic-tin-substitute germanotungstate, displayed remarkable extracellular inhibitory activity. Meanwhile, the crystal structure of PAC-320 was characterized by X-ray crystallography. PAC-320 could stably exist under physiological conditions as revealed by UV spectrum, CV and TG. PAC-320 possessed a strong inhibitory effect to intracellular HDAC activity. More significantly, PAC-320 inhibited the growth of a variety of cancer cells, and exhibited remarkable anticancer effect in a hepatocarcinoma H22 cell mice model. This study revealed, for the first time, that the HDAC inhibitory activity is a mechanism by which POMs exert their anticancer effect.

A New, Convenient Cell-Based Screening Method for Small-Molecule Glycolytic Inhibitors

To counteract active glycolysis in tumors, we developed a new, convenient cell-based screening system to identify an inhibitor of glycolysis. Using this system, we searched for an inhibitor in the synthetic Carbasugar library and found two candidates. It was found that both inhibited glycolysis by suppressing the glucose uptake step in tumor cells.

CYP2C9-Mediated Metabolic Activation of Losartan Detected by a Highly Sensitive Cell-Based Screening Assay

Drug-induced hepatotoxicity is a major problem in drug development, and reactive metabolites generated by cytochrome P450s are suggested to be one of the causes. CYP2C9 is one of the major enzymes in hepatic drug metabolism. In the present study, we developed a highly sensitive cell-based screening system for CYP2C9-mediated metabolic activation using an adenovirus vector expressing CYP2C9 (AdCYP2C9). Human hepatocarcinoma HepG2 cells infected with our constructed AdCYP2C9 for 2 days at multiplicity of infection 10 showed significantly higher diclofenac 4'-hydroxylase activity than human hepatocytes. AdCYP2C9-infected cells were treated with several hepatotoxic drugs, resulting in a significant increase in cytotoxicity by treatment with losartan, benzbromarone, and tienilic acid. Metabolic activation of losartan by CYP2C9 has never been reported, although the metabolic activations of benzbromarone and tienilic acid have been reported. To identify the reactive metabolites of losartan, the semicarbazide adducts of losartan were investigated by liquid chromatography-tandem mass spectrometry. Two CYP2C9-specific semicarbazide adducts of losartan (S1 and S2) were detected. S2 adduct formation suggested that a reactive metabolite was produced from the aldehyde metabolite E3179, but a possible metabolite from S1 adduct formation was not produced via E3179. In conclusion, a highly sensitive cell-based assay to evaluate CYP2C9-mediated metabolic activation was established, and we found for the first time that CYP2C9 is involved in the metabolic activation of losartan. This cell-based assay system would be useful for evaluating drug-induced cytotoxicity caused by human CYP2C9.