Cell-based High-throughput Screening Research Articles

A high-throughput cell-based screening method for Zika virus protease inhibitors discovery

Zika virus (ZIKV) continues to pose a significant global public health threat, with recurring regional outbreaks and potential for pandemic spread. Despite often being asymptomatic, ZIKV infections can have severe consequences, including neurological disorders and congenital abnormalities. Unfortunately, there are currently no approved vaccines or antiviral drugs for the prevention or treatment of ZIKV. One promising target for drug development is the ZIKV NS2B-NS3 protease due to its crucial role in the virus life cycle. In this study, we established a cell-based ZIKV protease inhibition assay designed for high-throughput screening (HTS). Our assay relies on the ZIKV protease's ability to cleave a cyclised firefly luciferase fused to a natural cleavage sequence between NS2B and NS3 protease within living cells. We evaluated the performance of our assay in HTS setting using the pharmacologic controls (JNJ-40418677 and MK-591) and by screening a Library of Pharmacologically Active Compounds (LOPAC). The results confirmed the feasibility of our assay for compound library screening to identify potential ZIKV protease inhibitors.

Structural and thermodynamic framework for PIEZO1 modulation by small molecules.

Mechanosensitive PIEZO channels constitute potential pharmacological targets for multiple clinical conditions, spurring the search for potent chemical PIEZO modulators. Among them is Yoda1, a widely used synthetic small molecule PIEZO1 activator discovered through cell-based high-throughput screening. Yoda1 is thought to bind to PIEZO1's mechanosensory arm domain, sandwiched between two transmembrane regions near the channel pore. However, how the binding of Yoda1 to this region promotes channel activation remains elusive. Here, we first demonstrate that cross-linking PIEZO1 repeats A and B with disulfide bridges reduces the effects of Yoda1 in a redox-dependent manner, suggesting that Yoda1 acts by perturbing the contact between these repeats. Using molecular dynamics-based absolute binding free energy simulations, we next show that Yoda1 preferentially occupies a deeper, amphipathic binding site with higher affinity in PIEZO1 open state. Using Yoda1's binding poses in open and closed states, relative binding free energy simulations were conducted in the membrane environment, recapitulating structure-activity relationships of known Yoda1 analogs. Through virtual screening of an 8 million-compound library using computed fragment maps of the Yoda1 binding site, we subsequently identified two chemical scaffolds with agonist activity toward PIEZO1. This study supports a pharmacological model in which Yoda1 activates PIEZO1 by wedging repeats A and B, providing a structural and thermodynamic framework for the rational design of PIEZO1 modulators. Beyond PIEZO channels, the three orthogonal computational approaches employed here represent a promising path toward drug discovery in highly heterogeneous membrane protein systems.

High-throughput screening of novel TFEB agonists in protecting against acetaminophen-induced liver injury in mice

Macroautophagy (referred to as autophagy hereafter) is a major intracellular lysosomal degradation pathway that is responsible for the degradation of misfolded/damaged proteins and organelles. Previous studies showed that autophagy protects against acetaminophen (APAP)-induced injury (AILI) via selective removal of damaged mitochondria and APAP protein adducts. The lysosome is a critical organelle sitting at the end stage of autophagy for autophagic degradation via fusion with autophagosomes. In the present study, we showed that transcription factor EB (TFEB), a master transcription factor for lysosomal biogenesis, was impaired by APAP resulting in decreased lysosomal biogenesis in mouse livers. Genetic loss-of and gain-of function of hepatic TFEB exacerbated or protected against AILI, respectively. Mechanistically, overexpression of TFEB increased clearance of APAP protein adducts and mitochondria biogenesis as well as SQSTM1/p62-dependent non-canonical nuclear factor erythroid 2-related factor 2 (NRF2) activation to protect against AILI. We also performed an unbiased cell-based imaging high-throughput chemical screening on TFEB and identified a group of TFEB agonists. Among these agonists, salinomycin, an anticoccidial and antibacterial agent, activated TFEB and protected against AILI in mice. In conclusion, genetic and pharmacological activating TFEB may be a promising approach for protecting against AILI.

Acoustic droplet ejection facilitates cell-based high-throughput screenings using natural products

Natural Products (NPs) are one of the main sources for drug discovery. Many clinical drugs are NPs or NP-inspired compounds, and recently discovered New Chemical Entities (NCEs) of NPs are emerging as promising new drugs. High-Throughput Screening (HTS) of large sample sets or libraries has grown to be vital for the drug discovery field. Industrial-scale HTS of NP libraries can be limited due to the difficulties entailed in working with tiny extract volumes and the variability in viscosity of NP extracts. For these reasons, the implementation of new technologies to miniaturize different reagent volumes grows to be fundamental. Since Acoustic Droplet Ejection (ADE) emerged as a helpful tool in HTS campaigns for the transference of compound libraries. The aim of this work was to test the effectiveness of ADE for the dispensation of NP extract libraries in cell-based HTS assays.

Development of novel cytoprotective small compounds inhibiting mitochondria-dependent cell death

We identified cytoprotective small molecules (CSMs) by a cell-based high-throughput screening of Bax inhibitors. Through a medicinal chemistry program, M109S was developed, which is orally bioactive and penetrates the blood-brain/retina barriers. M109S protected retinal cells in ocular disease mouse models. M109S directly interacted with Bax and inhibited the conformational change and mitochondrial translocation of Bax. M109S inhibited ABT-737-induced apoptosis both in Bax-only and Bak-only mouse embryonic fibroblasts. M109S also inhibited apoptosis induced by staurosporine, etoposide, and obatoclax. M109S decreased maximal mitochondrial oxygen consumption rate and reactive oxygen species production, whereas it increased glycolysis. These effects on cellular metabolism may contribute to the cytoprotective activity of M109S. M109S is a novel small molecule protecting cells from mitochondria-dependent apoptosis both invitro and invivo. M109S has the potential to become a research tool for studying cell death mechanisms and to develop therapeutics targeting mitochondria-dependent cell death pathway.

Quizartinib inhibits necroptosis by targeting receptor-interacting serine/threonine protein kinase 1.

Systemic inflammatory response syndrome (SIRS), at least in part driven by necroptosis, is characterized by life-threatening multiple organ failure. Blocking the progression of SIRS and consequent multiple organ dysfunction is challenging. Receptor-interacting serine/threonine protein kinase 1 (RIPK1) is an important cell death and inflammatory mediator, making it a potential treatment target in several diseases. Here, using a drug repurposing approach, we show that inhibiting RIPK1 is also an effective treatment for SIRS. We performed cell-based high-throughput drug screening of an US Food and Drug Administration (FDA)-approved drug library that contains 1953 drugs to identify effective inhibitors of necroptotic cell death by SYTOX green staining. Dose-response validation of the top candidate, quizartinib, was conducted in two cell lines of HT-22 and MEFs. The effect of quizartinib on necroptosis-related proteins was evaluated using western blotting, immunoprecipitation, and an in vitro RIPK1 kinase assay. The in vivo effects of quizartinib were assessed in a murine tumor necrosis factor α (TNFα)-induced SIRS model. High-throughput screening identified quizartinib as the top "hit" in the compound library that rescued cells from necroptosis in vitro. Quizartinib inhibited necroptosis by directly inhibiting RIPK1 kinase activity and blocking downstream complex IIb formation. Furthermore, quizartinib protected mice against TNFα-induced SIRS. Quizartinib, as an FDA-approved drug with proven safety and efficacy, was repurposed for targeted inhibition of RIPK1. This work provides essential preclinical data for transferring quizartinib to the treatment of RIPK1-dependent necroptosis-induced inflammatory diseases, including SIRS.

A phase 2 study of dasatinib in recurrent clear cell carcinoma of the ovary, fallopian tube, peritoneum or endometrium: NRG oncology/gynecologic oncology group study 0283

ObjectiveGynecologic cancers are traditionally managed according to their presumed site of origin, without regard to the underlying histologic subtype. Clear cell histology is associated with chemotherapy refractoriness and poor survival. Mutations in SWI/SNF chromatin remodeling complex member ARID1A, which encodes for BAF250a protein, are common in clear cell and endometriosis-associated endometrioid carcinomas. High-throughput cell-based drug screening predicted activity of dasatinib, a tyrosine kinase inhibitor, in ARID1A-mutant clear cell carcinoma. MethodsWe conducted a phase 2 clinical trial of dasatinib 140 mg once daily by mouth in patients with recurrent or persistent ovarian and endometrial clear cell carcinoma. Patients with measurable disease were enrolled and then assigned to biomarker-defined populations based on BAF250a immunohistochemistry. The translational endpoints included broad next-generation sequencing to assess concordance of protein expression and treatment outcomes. ResultsTwenty-eight patients, 15 of whom had tumors with retained BAF250a and 13 with loss of BAF250a were evaluable for treatment response and safety. The most common grade 3 adverse events were anemia, fatigue, dyspnea, hyponatremia, pleural effusion, and vomiting. One patient had a partial response, eight (28%) had stable disease, and 15 (53.6%) had disease progression. Twenty-three patients had next-generation sequencing results; 13 had a pathogenic ARID1A alteration. PIK3CA mutations were more prevalent in ARID1A-mutant tumors, while TP53 mutations were more prevalent in ARID1A wild-type tumors. ConclusionsDasatinib was not an effective single-agent treatment for recurrent or persistent ovarian and endometrial clear cell carcinoma. Studies are urgently needed for this rare gynecologic subtype.

Identification of signaling pathways modifying human dopaminergic neuron development using a pluripotent stem cell-based high-throughput screening automated system: purinergic pathways as a proof-of-principle.

Introduction: Alteration in the development, maturation, and projection of dopaminergic neurons has been proposed to be associated with several neurological and psychiatric disorders. Therefore, understanding the signals modulating the genesis of human dopaminergic neurons is crucial to elucidate disease etiology and develop effective countermeasures. Methods: In this study, we developed a screening model using human pluripotent stem cells to identify the modulators of dopaminergic neuron genesis. We set up a differentiation protocol to obtained floorplate midbrain progenitors competent to produce dopaminergic neurons and seeded them in a 384-well screening plate in a fully automated manner. Results and Discussion: These progenitors were treated with a collection of small molecules to identify the compounds increasing dopaminergic neuron production. As a proof-of-principle, we screened a library of compounds targeting purine- and adenosine-dependent pathways and identified an adenosine receptor 3 agonist as a candidate molecule to increase dopaminergic neuron production under physiological conditions and in cells invalidated for the HPRT1 gene. This screening model can provide important insights into the etiology of various diseases affecting the dopaminergic circuit development and plasticity and be used to identify therapeutic molecules for these diseases.

Development of a next-generation endogenous OCT4 inducer and its anti-aging effect in vivo

The identification of small molecules capable of replacing transcription factors has been a longstanding challenge in the generation of human chemically induced pluripotent stem cells (iPSCs). Recent studies have shown that ectopic expression of OCT4, one of the master pluripotency regulators, compromised the developmental potential of resulting iPSCs, This highlights the importance of finding endogenous OCT4 inducers for the generation of clinical-grade human iPSCs. Through a cell-based high throughput screen, we have discovered several new OCT4-inducing compounds (O4Is). In this work, we prepared metabolically stable analogues, including O4I4, which activate endogenous OCT4 and associated signaling pathways in various cell lines. By combining these with a transcription factor cocktail consisting of SOX2, KLF4, MYC, and LIN28 (referred to as "CSKML") we achieved to reprogram human fibroblasts into a stable and authentic pluripotent state without the need for exogenous OCT4. In Caenorhabditis elegans and Drosophila, O4I4 extends lifespan, suggesting the potential application of OCT4-inducing compounds in regenerative medicine and rejuvenation therapy.

A novel cell-based screen identifies chemical entities that reverse the immune-escape phenotype of metastatic tumours.

Genetic and epigenetic events have been implicated in the downregulation of the cellular antigen processing and presentation machinery (APM), which in turn, has been associated with cancer evasion of the immune system. When these essential components are lacking, cancers develop the ability to subvert host immune surveillance allowing cancer cells to become invisible to the immune system and, in turn, promote cancer metastasis. Here we describe and validate the first high-throughput cell-based screening assay to identify chemical extracts and unique chemical entities that reverse the downregulation of APM components in cell lines derived from metastatic tumours. Through the screening of a library of 480 marine invertebrate extracts followed by bioassay-guided fractionation, curcuphenol, a common sesquiterpene phenol derived from turmeric, was identified as the active compound of one of the extracts. We demonstrate that curcuphenol induces the expression of the APM components, TAP-1 and MHC-I molecules, in cell lines derived from both metastatic prostate and lung carcinomas. Turmeric and curcumins that contain curcuphenol have long been utilized not only as a spice in the preparation of food, but also in traditional medicines for treating cancers. The remarkable discovery that a common component of spices can increase the expression of APM components in metastatic tumour cells and, therefore reverse immune-escape mechanisms, provides a rationale for the development of foods and advanced nutraceuticals as therapeutic candidates for harnessing the power of the immune system to recognize and destroy metastatic cancers.

Development of pathway-oriented screening to identify compounds to control 2-methylglyoxal metabolism in tumor cells

Controlling tumor-specific alterations in metabolic pathways is a useful strategy for treating tumors. The glyoxalase pathway, which metabolizes the toxic electrophile 2-methylglyoxal (MG), is thought to contribute to tumor pathology. We developed a live cell-based high-throughput screening system that monitors the metabolism of MG to generate d-lactate by glyoxalase I and II (GLO1 and GLO2). It utilizes an extracellular coupled assay that uses d-lactate to generate NAD(P)H, which is detected by a selective fluorogenic probe designed to respond exclusively to extracellular NAD(P)H. This metabolic pathway-oriented screening is able to identify compounds that control MG metabolism in live cells, and we have discovered compounds that can directly or indirectly inhibit glyoxalase activities in small cell lung carcinoma cells.

HSF1 Pathway Inhibitor Clinical Candidate (CCT361814/NXP800) Developed from a Phenotypic Screen as a Potential Treatment for Refractory Ovarian Cancer and Other Malignancies.

CCT251236 1, a potent chemical probe, was previously developed from a cell-based phenotypic high-throughput screen (HTS) to discover inhibitors of transcription mediated by HSF1, a transcription factor that supports malignancy. Owing to its activity against models of refractory human ovarian cancer, 1 was progressed into lead optimization. The reduction of P-glycoprotein efflux became a focus of early compound optimization; central ring halogen substitution was demonstrated by matched molecular pair analysis to be an effective strategy to mitigate this liability. Further multiparameter optimization led to the design of the clinical candidate, CCT361814/NXP800 22, a potent and orally bioavailable fluorobisamide, which caused tumor regression in a human ovarian adenocarcinoma xenograft model with on-pathway biomarker modulation and a clean in vitro safety profile. Following its favorable dose prediction to human, 22 has now progressed to phase 1 clinical trial as a potential future treatment for refractory ovarian cancer and other malignancies.

Abstract 3473: High throughput screening of combination therapies to improve response to checkpoint inhibitors in solid tumors using a myelomonocytic and T cell co-culture assay

Abstract Background: Immune checkpoint inhibitors (ICI) targeting the PD-1/PD-L1 pathway are part of the new “standard of care” treatment for a number of advanced cancers. However, for many types of cancer, targeting either PD-1 or PD-L1 is associated with clinical benefit in a minority of patients. Combination strategies are needed to improve clinical benefit rates in ‘immune responsive’ cancers, and to expand treatment options for non-responsive cancers. Preclinical models have revealed that tumor-associated macrophages (TAMs) possess T cell-suppressive activities associated with resistance to PD-1/PD-L1 targeted therapies. To reveal therapeutic targets to relieve this suppression, we developed a robust cell-based high-throughput screening (HTS) assay to identify small molecules that can relieve myelomonocytic cell-dependent T cell-suppression, and that enhance T cell activities when combined with PD-1/PD-L1 ICI. Methods: Bone marrow-derived myelomonocytic cells and splenic T cells were used to develop the HTS assay. After exposure to libraries of small molecules, co-cultures were evaluated for enhanced T cell activity, followed by in vivo investigations of emerging small molecules in syngeneic murine models of breast cancer to reveal anti-tumor activity. Results: Over 1,400 FDA-approved drugs, used for both cancer or non-cancer indications, were screened in the HTS assay, 66 of which demonstrated activity in the HTS assay. Approximately 50% of these were known anti-inflammatory agents, with the highest density reflecting various COX-2 inhibitors (COX-2i). In vivo, combination therapy of the COX-2i with PD-1/PD-L1 ICI resulted in a significant reduction in tumor growth kinetics in both 4T1 and EMT6 breast cancer models, both of which are resistant to PD-1/PD-L1 inhibitors as monotherapy. In the 4T1 model, slowed tumor kinetics was associated with ICI increased T cell activation as compared to controls or monotherapy anti-PD-1/PD-L1 therapy. Conclusions: A myelomonocytic cell-dependent T cell-suppression assay can be used to develop a HTS platform for discovering combination therapies showing antitumor responses in preclinical models. COX-2 inhibitors can be repurposed to enhance therapeutic benefits of PD-1/PD-L1 targeting agents in breast cancer patients. Keywords: Myelomonocytic cells, T-cell, immunotherapy, PD-1, PD-L1, breast cancer Citation Format: Sushil Kumar, Dhanir Tailor, Arpit Dheeraj, Wenqi Li, Jee Min Lee, Kirsten Stefan, Dylan Nelson, Shivaani Kummar, Lisa M. Coussens, Sanjay V. Malhotra. High throughput screening of combination therapies to improve response to checkpoint inhibitors in solid tumors using a myelomonocytic and T cell co-culture assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3473.

High throughput screening for drugs that inhibit 3C-like protease in SARS-CoV-2.

The SARS coronavirus 2 (SARS-CoV-2) pandemic remains a major problem in many parts of the world and infection rates remain at extremely high levels. This high prevalence drives the continued emergence of new variants, and possibly ones that are more vaccine-resistant and that can drive infections even in highly vaccinated populations. The high rate of variant evolution makes clear the need for new therapeutics that can be clinically applied to minimize or eliminate the effects of COVID-19. With a hurdle of 10 years, on average, for first in class small molecule therapeutics to achieve FDA approval, the fastest way to identify therapeutics is by drug repurposing. To this end, we developed a high throughput cell-based screen that incorporates the essential viral 3C-like protease and its peptide cleavage site into a luciferase complementation assay to evaluate the efficacy of known drugs encompassing approximately 15,000 clinical-stage or FDA-approved small molecules. Confirmed inhibitors were also tested to determine their cytotoxic properties. Medicinal chemistry efforts to optimize the hits identified Tranilast as a potential lead. Here, we report the rapid screening and identification of potentially relevant drugs that exhibit selective inhibition of the SARS-CoV-2 viral 3C-like protease.

Abstract P6-10-18: Developing novel lysyl oxidase (LOX) inhibitors to overcome chemotherapy resistance in triple negative breast cancer

Abstract Triple negative breast cancer (TNBC) is the most aggressive breast cancer subtype. It accounts for ~15% of all breast cancer patients yet is responsible for 30% of breast cancer deaths. TNBC is treated primarily with conventional chemotherapy; however, resistance to therapy is common, leading to high mortality rates. Importantly, the benefit of current therapeutic strategies used in chemoresistant TNBC, i.e., immunotherapy and antibody-drug conjugates, is confined to only a fraction of patients, and survival benefit is limited. Therefore, there is an urgent need to identify novel and effective treatment strategies to overcome chemoresistance. Recently, we identified hypoxia-induced ECM re-modeler, lysyl oxidase (LOX), a member of LOX family, as a key mediator of chemoresistance in TNBC. However, currently available LOX inhibitors are either non-selective and/or show toxicity. Here, we performed a high-throughput cell-based LOX activity screen (HTS) with more than 5,000 molecules selected from a diversified compound library and identified the bi-thiazole derivatives as novel potent LOX inhibitors. Our structure activity relationship (SAR) analysis resulted in two lead compounds 6403, a relatively LOX-specific inhibitor, and 6415, a more LOX/LOXL2 inhibitor. Both compounds reduced collagen cross-linking (canonical function of LOX) and led to chemosensitization in TNBC cell lines and in chemoresistant TNBC PDX organoids. In addition, 6403 and 6415 reduced the TGF-beta induced fibrosis and inhibited migration capacity of the breast cancer cell lines. Importantly, 6403 showed excellent pharmacokinetic profile and did not lead to any observable toxicity in mice. Notably, 6403 overcame doxorubicin resistance in LOX-expressing 4T1 syngeneic model with no apparent toxicity. Furthermore, our novel LOX and LOX/LOXL2 dual inhibitors show superior inhibition of LOX activity compared to the recently developed and clinically tested LOX family or LOXL2/LOXL3 inhibitors. Overall, we identified novel potent LOX inhibitors with no observable toxicity for further preclinical development and future clinical testing to overcome chemoresistance in TNBC. Citation Format: Metin Cetin, Ozge Saatci, Ozge Akbulut, Chad Beneker, Abdol-Hossein Rezaeian, Mikhail Chernov, Campbell McInnes, Ozgur Sahin. Developing novel lysyl oxidase (LOX) inhibitors to overcome chemotherapy resistance in triple negative breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-10-18.

Inhibition of Neuromyelitis Optica Immunoglobulin G Binding to Aquaporin-4 by the Small Molecule Blocker Melanthioidine.

Neuromyelitis optica (NMO) is a severe neurological demyelinating autoimmune disease affecting the optic nerves and spinal cord. The binding of neuromyelitis optica immunoglobulin G (NMO-IgG) and aquaporin-4 (AQP4) on the surface of astrocytes in the serum and cerebrospinal fluid is the main pathogenesis of NMO. Currently, therapeutic strategies for NMO include a reduction of the secondary inflammation response and the number of NMO-IgG, which can only alleviate clinical symptoms rather than fundamentally preventing a series of pathological processes caused by NMO-IgG binding to AQP4. The purpose of this study was to investigate the blocking effect of melanthioidine on the binding of NMO-IgG to AQP4 and its potential cytotoxicity. The current study developed a cell-based high-throughput screening approach to identify a molecular blocker of NMO-IgG binding to AQP4 using the Chinese hamster lung fibroblast (V79) cells expressing M23-AQP4. By screening ~400 small molecules, we identified melanthioidine with blocking effects without affecting AQP4 expression or its water permeability. Melanthioidine effectively blocked the binding of NMO-IgG to AQP4 in immunofluorescence assays and reduced complement-dependent cytotoxicity against both NMO-IgG/complement-treated Fischer rat thyroid-AQP4 cells and primary astrocytes. The docking computations identified the putative sites of blocker binding at the extracellular surface of AQP4. This study serves as proof of a potential NMO therapy by using a small-molecule blocker to target NMO pathogenesis.

High-Throughput Screening for Epigenetic Compounds That Induce Human β-Defensin 1 Synthesis

Antimicrobial host defense peptides (HDPs) are critically important for innate immunity. Small-molecule compounds with the ability to induce HDP synthesis are being actively explored for antimicrobial therapy. To facilitate the discovery of the compounds that specifically activate human β-defensin 1 (DEFB1) gene transcription, we established a cell-based high-throughput screening assay that employs HT-29/DEFB1-luc, a stable reporter cell line expressing the luciferase gene driven by a 3-Kb DEFB1 gene promoter. A screening of a library of 148 small-molecule epigenetic compounds led to the identification of 28 hits, with a minimum strictly standardized mean difference of 3.0. Fourteen compounds were further selected and confirmed to be capable of inducing DEFB1 mRNA expression in human HT-29 colonic epithelial cells. Desirably, the human cathelicidin antimicrobial peptide (CAMP) gene was also induced by these epigenetic compounds. Benzamide-containing histone deacetylase inhibitors (HDACi) were among the most potent HDP inducers identified in this study. Additionally, several major genes involved in intestinal barrier function, such as claudin-1, claudin-2, tight junction protein 1, and mucin 2, were differentially regulated by HDP inducers. These findings suggest the potential for the development of benzamide-based HDACi as host-directed antimicrobials for infectious disease control and prevention.

Toxicity prediction: An application of alternative testing and computational toxicology in contaminated groundwater sites in Taiwan

Groundwater contamination remains a global threat due to its toxic effects to humans and the environment. The remediation of contaminated groundwater sites can be costly, thus, identifying the priority areas of concern is important to reduce money spent on resources. In this study, we aimed to identify and rank the priority groundwater sites in a contaminated petrochemical district by combining alternative, non-animal approaches – chemical analysis, cell-based high throughput screening (HTS), and Toxicological Priority Index (ToxPi) computational toxicology tool. Groundwater samples collected from ten different sites in a contaminated district showed pollutant levels below the detection limit, however, hepatotoxic bioactivity was demonstrated in human hepatoma HepaRG cells. Integrating the pollutants information (i.e., pollutant characteristics and concentration data) with the bioactivity data of the groundwater samples, an evidence-based ranking of the groundwater sites for future remediation was established using ToxPi analysis. The currently presented combinatorial approach of screening groundwater sites for remediation purposes can further be refined by including relevant parameters, which can boost the utility of this approach for groundwater screening and future remediation.

Berbamine Reduces Chloroquine-Induced Itch in Mice through Inhibition of MrgprX1.

Chloroquine (CQ) is an antimalaria drug that has been widely used for decades. However, CQ-induced pruritus remains one of the major obstacles in CQ treatment for uncomplicated malaria. Recent studies have revealed that MrgprX1 plays an essential role in CQ-induced itch. To date, a few MrgprX1 antagonists have been discovered, but they are clinically unavailable or lack selectivity. Here, a cell-based high-throughput screening was performed to identify novel antagonists of MrgprX1, and the screening of 2543 compounds revealed two novel MrgprX1 inhibitors, berbamine and closantel. Notably, berbamine potently inhibited CQ-mediated MrgprX1 activation (IC50 = 1.6 μM) but did not alter the activity of other pruritogenic GPCRs. In addition, berbamine suppressed the CQ-mediated phosphorylation of ERK1/2. Interestingly, CQ-induced pruritus was significantly reduced by berbamine in a dose-dependent manner, but berbamine had no effect on histamine-induced, protease-activated receptors 2-activating peptide-induced, and deoxycholic acid-induced itch in mice. These results suggest that berbamine is a novel, potent, and selective antagonist of MrgprX1 and may be a potential drug candidate for the development of therapeutic agents to treat CQ-induced pruritus.

308 (PB088) - Develop selective inhibitors of drug-metabolizing enzymes CYP3A4/5 to improve cancer drug efficacy and reduce drug toxicity and resistance

Background: The human cytochrome P450 (CYP) CYP3A4 and CYP3A5 enzymes metabolize more than half of marketed drugs. They share high structural and substrate similarity and are often studied together as CYP3A4/ 5. However, they preferentially metabolize different clinically prescribed drugs. Moreover, the differential distribution and expression levels of CYP3A4 and CYP3A5 in both normal and diseased tissues can aggravate toxicity and induce resistance during treatment. Therefore, selective inhibitors of CYP3A4 and CYP3A5 are needed to distinguish their roles and serve as starting points for potential therapeutic development. Materials and methods: We used biochemical and cell-based assays and high-throughput screening to discover and characterize selective inhibitors. We used X-ray crystallography to unravel the structural basis of selective inhibition. Results: We discovered clobetasol propionate as the first selective inhibitor of CYP3A5 and reported the crystal structure of CYP3A5 in complex with clobetasol propionate. Supported by structure-guided mutagenesis analyses, the CYP3A5-clobetasol propionate structure showed that a unique conformation of the F–F′ loop in CYP3A5 enables selective binding of clobetasol propionate to CYP3A5, thus proving the structural basis for the selective inhibition. Based on these results we performed large-scale high-throughput screening to identify additional selective inhibitors of both CYP3A4 and CYP3A5 that are suitable for chemical optimization. Conclusions: It is feasible to selectively inhibit the highly homologous CYP3A4 and CYP3A5 enzymes, thus chemically validating CYP3A4 and CYP3A5 as targets for development of therapeutics to improve drug efficacy while reducing drug toxicity and resistance during treatment. No conflict of interest.