In vitro profiling of pesticides within the Tox21 10K compound library for bioactivity and potential toxicity.

Pesticides include a diverse class of toxic chemicals, often having numerous modes of actions when used in agriculture against targeted organisms to control insect infestation, halt unwanted vegetation, and prevent the spread of disease. In this study, the in vitro assay activity of pesticides within the Tox21 10K compound library were examined. The assays in which pesticides showed significantly more activities than non-pesticide chemicals revealed potential targets and mechanisms of action for pesticides. Furthermore, pesticides that showed promiscuous activity against many targets and cytotoxicity were identified, which warrant further toxicological evaluation. Several pesticides were shown to require metabolic activation, demonstrating the importance of introducing metabolic capacity to in vitro assays. Overall, the activity profiles of pesticides highlighted in this study can contribute to the knowledge gaps surrounding pesticide mechanisms and to the better understanding of the on- and off-target organismal effects of pesticides.

Lestaurtinib induces DNA damage that is related to estrogen receptor activation.

A number of chemicals in the environment pose a threat to human health. Recent studies indicate estradiol induces DNA damage through the activation of the estrogen receptor alpha (ERα). Given that many environmental chemical compounds act like hormones once they enter the human body, it is possible that they induce DNA damage in the same way as estradiol, which is of great concern to females with the BRCA1 mutation. In this study, we developed an antibody-based high content method measuring γH2AX, a biomarker for DNA damage, to test a subset of 907 chemical compounds in MCF7 cells. The assay was optimized for a 1536 well plate format and had a satisfactory assay performance with Z-factor of 0.67. From the screening, we identified 128 compounds that induce γH2AX expression in the cells. These compounds were further examined for their γH2AX induction in the presence of an ER inhibitor, tamoxifen. After tamoxifen treatment, four compounds induced less γH2AX expression compared to those without tamoxifen treatment, suggesting these compounds induced γH2AX that is related to ERα activation. These four compounds were chosen for further studies to assess their ERα activating capability and c-MYC induction. Only lestaurtinib, a selective tyrosine kinase inhibitor, induced ERα activation, which was confirmed by both ERα beta-lactamase reporter gene assay and molecular docking analysis. Lestaurtinib also increased c-MYC expression, a target gene of ERα signaling, measured by the quantitative PCR method. This data suggests that lestaurtinib acts as a DNA damage inducer that is related to ERα activation.

Screening Method for the Identification of Compounds That Activate Pregnane X Receptor.

The pregnane X receptor (PXR) is a nuclear receptor found mainly in the liver and intestine, whose main function is to regulate the expression of drug-metabolizing enzymes and transporters. Recently, it has been noted that PXR plays critical roles in energy homeostasis, immune response, and cancer. Therefore, identifying chemicals or compounds that can modulate PXR is of great interest, as these can result in downstream toxicity or, alternatively, may have therapeutic potential. Testing one compound at a time for PXR activity would be inefficient and take thousands of hours for large compound libraries. Here, we describe a high-throughput screening method that encompasses plating and treating HepG2-CYP3A4-hPXR cells in a 1536-well plate, as well as reading and interpreting assay (e.g., luciferase reporter gene activity) endpoints. These cells are stably transfected with a human PXR expression vector and CYP3A4-promoter-driven luciferase reporter vector, allowing the identification of compounds that activate PXR through cytochrome 450 3A4. We also describe how to analyze the data from each assay and explain follow-up steps, namely pharmacological characterization and quantitative polymerase chain reaction (qPCR) assays, which can be performed to confirm results from the original screen. These methods can be used to identify and confirm hPXR activators after completion of a compound screening. Published 2022. This article is a U.S. Government work and is in the public domain in the USA. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Establishment of a high-throughput assay to identify hPXR activators Basic Protocol 2: Quantitative high-throughput screening a compound library to classify hPXR activators Basic Protocol 3: Performing pharmacological characterization and qPCR assays to confirm hPXR activators.

The SARS-CoV-2 spike protein binds and modulates estrogen receptors.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein binds angiotensin-converting enzyme 2 as its primary infection mechanism. Interactions between S and endogenous proteins occur after infection but are not well understood. We profiled binding of S against >9000 human proteins and found an interaction between S and human estrogen receptor α (ERα). Using bioinformatics, supercomputing, and experimental assays, we identified a highly conserved and functional nuclear receptor coregulator (NRC) LXD-like motif on the S2 subunit. In cultured cells, S DNA transfection increased ERα cytoplasmic accumulation, and S treatment induced ER-dependent biological effects. Non-invasive imaging in SARS-CoV-2-infected hamsters localized lung pathology with increased ERα lung levels. Postmortem lung experiments from infected hamsters and humans confirmed an increase in cytoplasmic ERα and its colocalization with S in alveolar macrophages. These findings describe the discovery of a S-ERα interaction, imply a role for S as an NRC, and advance knowledge of SARS-CoV-2 biology and coronavirus disease 2019 pathology.

Identification of nonmonotonic concentration-responses in Tox21 high-throughput screening estrogen receptor assays.

Environmental endocrine-disrupting chemicals (EDCs) interfere with the metabolism and actions of endogenous hormones. It has been well documented in numerous in vivo and in vitro studies that EDCs can exhibit nonmonotonic dose response (NMDR) behaviors. Not conforming to the conventional linear or linear-no-threshold response paradigm, these NMDR relationships pose practical challenges to the risk assessment of EDCs. In the meantime, the endocrine signaling pathways and biological mechanisms underpinning NMDR remain incompletely understood. The US Tox21 program has conducted in vitro cell-based high-throughput screening assays for estrogen receptors (ER), androgen receptors, and other nuclear receptors, and screened the 10 K-compound library for potential endocrine activities. Using 15 concentrations across several orders of magnitude of concentration range and run in both agonist and antagonist modes, these Tox21 assay datasets contain valuable quantitative information that can be explored to evaluate the nonlinear effects of EDCs and may infer potential mechanisms. In this study we analyzed the concentration-response curves (CRCs) in all 8 Tox21 ERα and ERß assays by developing clustering and classification algorithms customized to the datasets to identify various shapes of CRCs. After excluding NMDR curves likely caused by cytotoxicity, luciferase inhibition, or autofluorescence, hundreds of compounds were identified to exhibit Bell or U-shaped CRCs. Bell-shaped CRCs are about 7 times more frequent than U-shaped ones in the Tox21 ER assays. Many compounds exhibit NMDR in at least one assay, and some EDCs well-known for their NMDRs in the literature were also identified, suggesting their nonmonotonic effects may originate at cellular levels involving transcriptional ER signaling. The developed computational methods for NMDR identification in ER assays can be adapted and applied to other high-throughput bioassays.

Identification of Selective CYP3A7 and CYP3A4 Substrates and Inhibitors Using a High-Throughput Screening Platform.

Cytochrome P450 (CYP) 3A7 is one of the major xenobiotic metabolizing enzymes in human embryonic, fetal, and newborn liver. CYP3A7 expression has also been observed in a subset of the adult population, including pregnant women, as well as in various cancer patients. The characterization of CYP3A7 is not as extensive as other CYPs, and health authorities have yet to provide guidance towards DDI assessment. To identify potential CYP3A7-specific molecules, we used a P450-Glo CYP3A7 enzyme assay to screen a library of ∼5,000 compounds, including FDA-approved drugs and drug-like molecules, and compared these screening data with that from a P450-Glo CYP3A4 assay. Additionally, a subset of 1,000 randomly selected compounds were tested in a metabolic stability assay. By combining the data from the qHTS P450-Glo and metabolic stability assays, we identified several chemical features important for CYP3A7 selectivity. Halometasone was chosen for further evaluation as a potential CYP3A7-selective inhibitor using molecular docking. From the metabolic stability assay, we identified twenty-two CYP3A7-selective substrates over CYP3A4 in supersome setting. Our data shows that CYP3A7 has ligand promiscuity, much like CYP3A4. Furthermore, we have established a large, high-quality dataset that can be used in predictive modeling for future drug metabolism and interaction studies.

Improving the solubility and antileukemia activity of Wnt/ß-catenin signaling inhibitors by disrupting molecular planarity.

Leukemia cells depend on the Wnt/ß-catenin signaling pathway for their growth. Pyrvinium, a known Wnt signaling inhibitor, has demonstrated promising efficacy in the treatment of the aggressive blast phase chronic myeloid leukemia (BP-CML). We previously developed potent inhibitors 1-2 for the Wnt/ß-catenin signaling pathway. However, the further application of these compounds as anti-leukemia agents is limited by their modest anti-leukemia activity in cells and poor aqueous solubility, due to the high molecular planarity of the chemical scaffold. Here, we reported our efforts in the synthesis and in vitro evaluation of 18 new compounds (4a-r) that have been designed to disrupt the molecular planarity of the chemical scaffold. Several compounds of the series showed significantly improved anti-leukemia activity and aqueous solubility. As a highlight, compounds 4c not only maintained excellent inhibitory potency (IC50 = 1.3 nM) for Wnt signaling but also demonstrated good anti-leukemia potency (IC50 = 0.9 µM) in the CML K562 cells. Moreover, compound 4c had an aqueous solubility of 5.9 µg/mL, which is over 50-fold enhanced compared to its parents 1-2.

The SARS-CoV-2 spike protein binds and modulates estrogen receptors.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein binds angiotensin-converting enzyme 2 (ACE2) at the cell surface, which constitutes the primary mechanism driving SARS-CoV-2 infection. Molecular interactions between the transduced S and endogenous proteins likely occur post-infection, but such interactions are not well understood. We used an unbiased primary screen to profile the binding of full-length S against >9,000 human proteins and found significant S-host protein interactions, including one between S and human estrogen receptor alpha (ERα). After confirming this interaction in a secondary assay, we used bioinformatics, supercomputing, and experimental assays to identify a highly conserved and functional nuclear receptor coregulator (NRC) LXD-like motif on the S2 subunit and an S-ERα binding mode. In cultured cells, S DNA transfection increased ERα cytoplasmic accumulation, and S treatment induced ER-dependent biological effects and ACE2 expression. Noninvasive multimodal PET/CT imaging in SARS-CoV-2-infected hamsters using [ 18 F]fluoroestradiol (FES) localized lung pathology with increased ERα lung levels. Postmortem experiments in lung tissues from SARS-CoV-2-infected hamsters and humans confirmed an increase in cytoplasmic ERα expression and its colocalization with S protein in alveolar macrophages. These findings describe the discovery and characterization of a novel S-ERα interaction, imply a role for S as an NRC, and are poised to advance knowledge of SARS-CoV-2 biology, COVID-19 pathology, and mechanisms of sex differences in the pathology of infectious disease.

Targeting CAR and Nrf2 improves cyclophosphamide bioactivation while reducing doxorubicin-induced cardiotoxicity in triple-negative breast cancer treatment.

Cyclophosphamide (CPA) and doxorubicin (DOX) are key components of chemotherapy for triple-negative breast cancer (TNBC), although suboptimal outcomes are commonly associated with drug resistance and/or intolerable side effects. Through an approach combining high-throughput screening and chemical modification, we developed CN06 as a dual activator of the constitutive androstane receptor (CAR) and nuclear factor erythroid 2-related factor 2 (Nrf2). CN06 enhances CAR-induced bioactivation of CPA (a prodrug) by provoking hepatic expression of CYP2B6, while repressing DOX-induced cytotoxicity in cardiomyocytes in vitro via stimulating Nrf2-antioxidant signaling. Utilizing a multicellular coculture model incorporating human primary hepatocytes, TNBC cells, and cardiomyocytes, we show that CN06 increased CPA/DOX-mediated TNBC cell death via CAR-dependent CYP2B6 induction and subsequent conversion of CPA to its active metabolite 4-hydroxy-CPA, while protecting against DOX-induced cardiotoxicity by selectively activating Nrf2-antioxidant signaling in cardiomyocytes but not in TNBC cells. Furthermore, CN06 preserves the viability and function of human iPSC-derived cardiomyocytes by modulating antioxidant defenses, decreasing apoptosis, and enhancing the kinetics of contraction and relaxation. Collectively, our findings identify CAR and Nrf2 as potentially novel combined therapeutic targets whereby CN06 holds the potential to improve the efficacy/toxicity ratio of CPA/DOX-containing chemotherapy.

Identification of environmental chemicals that activate p53 signaling after in vitro metabolic activation.

Currently, approximately 80,000 chemicals are used in commerce. Most have little-to-no toxicity information. The U.S. Toxicology in the 21st Century (Tox21) program has conducted a battery of in vitro assays using a quantitative high-throughput screening (qHTS) platform to gain toxicity information on environmental chemicals. Due to technical challenges, standard methods for providing xenobiotic metabolism could not be applied to qHTS assays. To address this limitation, we screened the Tox21 10,000-compound (10K) library, with concentrations ranging from 2.8 nM to 92 µM, using a p53 beta-lactamase reporter gene assay (p53-bla) alone or with rat liver microsomes (RLM) or human liver microsomes (HLM) supplemented with NADPH, to identify compounds that induce p53 signaling after biotransformation. Two hundred and seventy-eight compounds were identified as active under any of these three conditions. Of these 278 compounds, 73 gave more potent responses in the p53-bla assay with RLM, and 2 were more potent in the p53-bla assay with HLM compared with the responses they generated in the p53-bla assay without microsomes. To confirm the role of metabolism in the differential responses, we re-tested these 75 compounds in the absence of NADPH or with heat-attenuated microsomes. Forty-four compounds treated with RLM, but none with HLM, became less potent under these conditions, confirming the role of RLM in metabolic activation. Further evidence of biotransformation was obtained by measuring the half-life of the parent compounds in the presence of microsomes. Together, the data support the use of RLM in qHTS for identifying chemicals requiring biotransformation to induce biological responses.

Cell-Based Assays to Identify ERR and ERR/PGC Modulators.

The estrogen-related receptor alpha (ERRα, NR3B1) is an orphan nuclear receptor which plays a role in endocrine disruption, energy homeostasis, and cancer prognosis. One of the unique features of this transcription factor is the interplay with its cofactors. For instance, certain modulators require the presence of proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) alongside ERRα. Therefore, identification of ERRα agonists and antagonists require examination of this nuclear receptor alone and together with PGC-1α. In this book chapter, we describe the step-by-step protocol of a multiplex luciferase assay designed to identify ERRα agonists, antagonists, and toxicity in one quantitative high-throughput screening assay using two different stable cell lines.

Cell-Based hERG Channel Inhibition Assay in High-Throughput Format.

Human ether-a-go-go-related gene (hERG) channel plays an essential role in the repolarization of the cardiac action potential. Genetic mutations and some chemicals/drugs interfere with hERG channel activity, which may prolong the QT interval and potentially cause long QT syndrome. The FluxOR™ thallium flux assay performed in two cell lines, U2OS and HEK293, with stable hERG expression can be used to identify compounds that inhibit hERG channel activity. This chapter describes a cell-based hERG channel inhibition assay that has been optimized and performed in a 1536-well plate format. The homogeneous and robust assay can be used to identify compounds that inhibit hERG channel activity.

Acetylcholinesterase Inhibition Assays for High-Throughput Screening.

Acetylcholinesterase (AChE) hydrolyzes acetylcholine (ACh), a vital neurotransmitter that regulates muscle movement and brain function, including memory, attention, and learning. Inhibition of AChE activity can cause a variety of adverse health effects and toxicity. Identifying AChE inhibitors quickly and efficiently warrants developing AChE inhibition assays in a quantitative, high-throughput screening (qHTS) platform. In this chapter, protocols for multiple homogenous AChE inhibition assays used in a qHTS system are provided. These AChE inhibition assays include a (1) human neuroblastoma (SH-SY5Y) cell-based assay with fluorescence or colorimetric detection; (2) human recombinant AChE with fluorescence or colorimetric detection; and (3) combination of human recombinant AChE and liver microsomes with colorimetric detection, which enables detection of test compounds requiring metabolic activation to become AChE inhibitors. Together, these AChE assays can help identify, prioritize, and predict chemical hazards in large compound libraries using qHTS systems.

GFP-LC3 High-Content Assay for Screening Autophagy Modulators.

Autophagy plays an important role in maintaining cellular homeostasis. Defects in autophagy have been linked to various human diseases, such as cancer, neurodegenerative diseases, and cardiovascular diseases. Therefore, it is useful to develop an assay that can measure the functions of autophagy and also be used to identify autophagy modulators by screening a large number of compounds. This chapter describes a cell-based high content green fluorescent protein (GFP)-LC3 assay using mouse embryonic fibroblasts (MEF) stably expressing GFP-LC3.

A gene expression biomarker for predictive toxicology to identify chemical modulators of NF-κB.

The nuclear factor-kappa B (NF-κB) is a transcription factor with important roles in inflammation, immune response, and oncogenesis. Dysregulation of NF-κB signaling is associated with inflammation and certain cancers. We developed a gene expression biomarker predictive of NF-κB modulation and used the biomarker to screen a large compendia of gene expression data. The biomarker consists of 108 genes responsive to tumor necrosis factor α in the absence but not the presence of IκB, an inhibitor of NF-κB. Using a set of 450 profiles from cells treated with immunomodulatory factors with known NF-κB activity, the balanced accuracy for prediction of NF-κB activation was > 90%. The biomarker was used to screen a microarray compendium consisting of 12,061 microarray comparisons from human cells exposed to 2,672 individual chemicals to identify chemicals that could cause toxic effects through NF-κB. There were 215 and 49 chemicals that were identified as putative or known NF-κB activators or suppressors, respectively. NF-κB activators were also identified using two high-throughput screening assays; 165 out of the ~3,800 chemicals (ToxCast assay) and 55 out of ~7,500 unique compounds (Tox21 assay) were identified as potential activators. A set of 32 chemicals not previously associated with NF-κB activation and which partially overlapped between the different screens were selected for validation in wild-type and NFKB1-null HeLa cells. Using RT-qPCR and targeted RNA-Seq, 31 of the 32 chemicals were confirmed to be NF-κB activators. These results comprehensively identify a set of chemicals that could cause toxic effects through NF-κB.

Discovery and characterization of potent And-1 inhibitors for cancer treatment.

Acidic nucleoplasmic DNA-binding protein 1 (And-1), an important factor for deoxyribonucleic acid (DNA) replication and repair, is overexpressed in many types of cancer but not in normal tissues. Although multiple independent studies have elucidated And-1 as a promising target gene for cancer therapy, an And-1 inhibitor has yet to be identified. Using an And-1 luciferase reporter assay to screen the Library of Pharmacologically Active Compounds (LOPAC) in a high throughput screening (HTS) platform, and then further screen the compound analog collection, we identified two potent And-1 inhibitors, bazedoxifene acetate (BZA) and an uncharacterized compound [(E)-5-(3,4-dichlorostyryl)benzo[c][1,2]oxaborol-1(3H)-ol] (CH3), which specifically inhibit And-1 by promoting its degradation. Specifically, through direct interaction with And-1 WD40 domain, CH3 interrupts the polymerization of And-1. Depolymerization of And-1 promotes its interaction with E3 ligase Cullin 4B (CUL4B), resulting in its ubiquitination and subsequent degradation. Furthermore, CH3 suppresses the growth of a broad range of cancers. Moreover, And-1 inhibitors re-sensitize platinum-resistant ovarian cancer cells to platinum drugs in vitro and in vivo. Since BZA is an FDA approved drug, we expect a clinical trial of BZA-mediated cancer therapy in the near future. Taken together, our findings suggest that targeting And-1 by its inhibitors is a potential broad-spectrum anti-cancer chemotherapy regimen.

AZD8055 enhances in vivo efficacy of afatinib in chordomas.

Chordomas are primary bone tumors that arise in the cranial base, mobile spine, and sacrococcygeal region, affecting patients of all ages. Currently, there are no approved agents for chordoma patients. Here, we evaluated the anti-tumor efficacy of small molecule inhibitors that target oncogenic pathways in chordoma, as single agents and in combination, to identify novel therapeutic approaches with the greatest translational potential. A panel of small molecule compounds was screened in vivo against patient-derived xenograft (PDX) models of chordoma, and potentially synergistic combinations were further evaluated using chordoma cell lines and xenograft models. Among the tested agents, inhibitors of EGFR (BIBX 1382, erlotinib, and afatinib), c-MET (crizotinib), and mTOR (AZD8055) significantly inhibited tumor growth in vivo but did not induce tumor regression. Co-inhibition of EGFR and c-MET using erlotinib and crizotinib synergistically reduced cell viability in chordoma cell lines but did not result in enhanced in vivo activity. Co-inhibition of EGFR and mTOR pathways using afatinib and AZD8055 synergistically reduced cell viability in chordoma cell lines. Importantly, this dual inhibition completely suppressed tumor growth in vivo, showing improved tumor control. Together, these data demonstrate that individual inhibitors of EGFR, c-MET, and mTOR pathways suppress chordoma growth both in vitro and in vivo. mTOR inhibition increased the efficacy of EGFR inhibition on chordoma growth in several preclinical models. The insights gained from our study potentially provide a novel combination therapeutic strategy for patients with chordoma. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Characterization of human pregnane X receptor activators identified from a screening of the Tox21 compound library.

The pregnane X receptor (PXR; NR1I2) is an important nuclear receptor whose main function is to regulate enzymes within drug metabolism. The main drug metabolizing enzyme regulated by PXR, cytochrome P450 (CYP) 3A4, accounts for the metabolism of nearly 50% of all marketed drugs. Recently, PXR has also been identified as playing a role in energy homeostasis, immune response, and cancer. Due to its interaction with these important roles, alongside its drug-drug interaction function, it is imperative to identify compounds which can modulate PXR. In this study, we screened the Tox21 10,000 compound collection to identify hPXR agonists using a stable hPXR-Luc HepG2 cell line. A pharmacological study in the presence of a PXR antagonist was performed to confirm the activity of the chosen potential hPXR agonists in the same cells. Finally, metabolically competent cell lines - HepaRG and HepaRG-PXR-Knockout (KO) - were used to further confirm the potential PXR activators. We identified a group of structural clusters and singleton compounds which included potentially novel hPXR agonists. Of the 21 selected compounds, 11 potential PXR activators significantly induced CYP3A4 mRNA expression in HepaRG cells. All of these compounds lost their induction when treating HepaRG-PXR-KO cells, confirming their PXR activation. Etomidoline presented as a potentially selective agonist of PXR. In conclusion, the current study has identified 11 compounds as potentially novel or not well-characterized PXR activators. These compounds should further be studied for their potential effects on drug metabolism and drug-drug interactions due to the immense implications of being a PXR agonist.

Systematic Identification of Molecular Targets and Pathways Related to Human Organ Level Toxicity.

The mechanisms leading to organ level toxicities are poorly understood. In this study, we applied an integrated approach to deduce the molecular targets and biological pathways involved in chemically induced toxicity for eight common human organ level toxicity end points (carcinogenicity, cardiotoxicity, developmental toxicity, hepatotoxicity, nephrotoxicity, neurotoxicity, reproductive toxicity, and skin toxicity). Integrated analysis of in vitro assay data, molecular targets and pathway annotations from the literature, and toxicity-molecular target associations derived from text mining, combined with machine learning techniques, were used to generate molecular targets for each of the organ level toxicity end points. A total of 1516 toxicity-related genes were identified and subsequently analyzed for biological pathway coverage, resulting in 206 significant pathways (p-value <0.05), ranging from 3 (e.g., developmental toxicity) to 101 (e.g., skin toxicity) for each toxicity end point. This study presents a systematic and comprehensive analysis of molecular targets and pathways related to various in vivo toxicity end points. These molecular targets and pathways could aid in understanding the biological mechanisms of toxicity and serve as a guide for the design of suitable in vitro assays for more efficient toxicity testing. In addition, these results are complementary to the existing adverse outcome pathway (AOP) framework and can be used to aid in the development of novel AOPs. Our results provide abundant testable hypotheses for further experimental validation.

A direct peptide reactivity assay using a high-throughput mass spectrometry screening platform for detection of skin sensitizers.

Chemical-peptide conjugation is the molecular initiating event in skin sensitization. The OECD test guideline uses a high-performance liquid chromatography/ultraviolet (HPLC/UV) detection method to quantify chemical-peptide conjugation in a direct peptide reactivity assay (DPRA), which measures the depletion of two synthetic peptides containing lysine or cysteine residues. To improve assay throughput, sensitivity and accuracy, an automated 384-well plate-based RapidFire solid-phase extraction (SPE) system coupled with tandem mass spectrometry (MS/MS) DPRA was developed and validated in the presence of a newly designed internal standard. Compared to the HPLC/UV-based DPRA, the automated SPE-MS/MS-based DPRA improved throughput from 16 min to 10 s per sample, and substrate peptides usage was reduced from 100 mM to 5 µM. When implementing the SPE-MS/MS-based DPRA into a high-throughput platform, we found 10 compounds that depleted lysine peptide and 24 compounds that depleted cysteine peptide (including 7 unreported chemicals from 55 compounds we tested) in a concentration-response manner. The adduct formation between cysteine and cinnamic aldehyde and ethylene glycol dimethacrylate were further analyzed using high-performance liquid chromatography time-of-flight mass spectrometry (HPLC-TOF-MS) to confirm the conjugation. Overall, the automated SPE-MS/MS-based platform is an efficient, economic, and accurate way to detect skin sensitizers.