Synthetic Lethal Targeting of Cyclin Dependent Kinase-12-Deficient Prostate Cancer with PARP Inhibitors.

PURPOSE: CDK12 is a cyclin-dependent kinase (CDK) that is mutated or amplified in multiple cancers. We previously described a subtype of prostate cancer (PC) characterized predominantly by frameshift, loss-of-function mutations in CDK12. This subtype exhibits aggressive clinical features. EXPERIMENTAL DESIGN: Using isogenic PC models generated by CRISPR/Cas9-mediated inactivation of CDK12, we conducted a chemical library screen of ~1800 FDA-approved drugs. We inhibited cyclin K and CDK13 and evaluated the effects on poly ADP-ribose polymerase inhibitor (PARPi) sensitivity. CDK12 truncation and kinase domain mutations were expressed in cell lines to determine effects on PARPi sensitivity. Mice bearing control and CDK12 mutant prostate tumors were treated with rucaparib. Finally, we evaluated prostate specific antigen (PSA) responses in patients with CDK12 mutations treated with rucaparib on the TRITON2 trial. RESULTS: Cancer cells lacking CDK12 are more sensitive to PARPi than isogenic wild-type cells, and sensitivity depends on the degree of CDK12 inhibition. Inhibiting cyclin K, but not CDK13, also led to PARPi sensitivity and suppressed homologous recombination. CDK12 truncation mutants remained sensitive to PARPi, whereas kinase domain mutants exhibited intermediate sensitivity. The PARPi rucaparib suppressed tumor growth in mice bearing CDK12-mutated tumors. Finally, 6 of 11 (55%) PC patients with biallelic CDK12 mutations had reductions in serum PSA levels when treated with rucaparib on the TRITON2 clinical trial. CONCLUSIONS: In PC, sensitivity to PARPi is dependent on the specific type and zygosity of the CDK12 mutation. PARPi monotherapy may have some activity in PC patients with biallelic inactivating CDK12 alterations.

Development of a NanoBRET assay for evaluation of 14-3-3σ molecular glues.

We report the development of a 384-well formatted NanoBRET assay to characterize molecular glues of 14-3-3/client interactions in living cells. The seven isoforms of 14-3-3 are dimeric hub proteins with diverse roles including transcription factor regulation and signal transduction. 14-3-3 interacts with hundreds of client proteins to regulate their function and is therefore an ideal therapeutic target when client selectivity can be achieved. We have developed the NanoBRET system for three 14-3-3σ client proteins CRAF, TAZ, and estrogen receptor α (ERα), which represent three specific binding modes. We have measured stabilization of 14-3-3σ/client complexes by molecular glues with EC50 values between 100 nM and 1 µM in cells, which align with the EC50 values calculated by fluorescence anisotropy in vitro. Developing this NanoBRET system for the hub protein 14-3-3σ allows for a streamlined approach, bypassing multiple optimization steps in the assay development process for other 14-3-3σ clients. The NanoBRET system allows for an assessment of PPI stabilization in a more physiologically relevant, cell-based environment using full-length proteins. The method is applicable to diverse protein-protein interactions (PPIs) and offers a robust platform to explore libraries of compounds for both PPI stabilizers and inhibitors.

Identifying ligands for the PHD1 finger of KDM5A through high-throughput screening.

PHD fingers are a type of chromatin reader that primarily recognize chromatin as a function of lysine methylation state. Dysregulated PHD fingers are implicated in various human diseases, including acute myeloid leukemia. Targeting PHD fingers with small molecules is considered challenging as their histone tail binding pockets are often shallow and surface-exposed. The KDM5A PHD1 finger regulates the catalytic activity of KDM5A, an epigenetic enzyme often misregulated in cancers. To identify ligands that disrupt the PHD1-histone peptide interaction, we conducted a high-throughput screen and validated hits by orthogonal methods. We further elucidated structure-activity relationships in two classes of compounds to identify features important for binding. Our investigation offers a starting point for further optimization of small molecule PHD1 ligands.

A Top-Down Proteomic Assay to Evaluate KRAS4B-Compound Engagement.

Development of new targeted inhibitors for oncogenic KRAS mutants may benefit from insight into how a given mutation influences the accessibility of protein residues and how compounds interact with mutant or wild-type KRAS proteins. Targeted proteomic analysis, a key validation step in the KRAS inhibitor development process, typically involves both intact mass- and peptide-based methods to confirm compound localization or quantify binding. However, these methods may not always provide a clear picture of the compound binding affinity for KRAS, how specific the compound is to the target KRAS residue, and how experimental conditions may impact these factors. To address this, we have developed a novel top-down proteomic assay to evaluate in vitro KRAS4B-compound engagement while assessing relative quantitation in parallel. We present two applications to demonstrate the capabilities of our assay: maleimide-biotin labeling of a KRAS4BG12D cysteine mutant panel and treatment of three KRAS4B proteins (WT, G12C, and G13C) with small molecule compounds. Our results show the time- or concentration-dependence of KRAS4B-compound engagement in context of the intact protein molecule while directly mapping the compound binding site.

An in vivo screening platform identifies senolytic compounds that target p16INK4a+ fibroblasts in lung fibrosis.

The appearance of senescent cells in age-related diseases has spurred the search for compounds that can target senescent cells in tissues, termed senolytics. However, a major caveat with current senolytic screens is the use of cell lines as targets where senescence is induced in vitro, which does not necessarily reflect the identity and function of pathogenic senescent cells in vivo. Here, we developed a new pipeline leveraging a fluorescent murine reporter that allows for isolation and quantification of p16Ink4a+ cells in diseased tissues. By high-throughput screening in vitro, precision-cut lung slice (PCLS) screening ex vivo, and phenotypic screening in vivo, we identified a HSP90 inhibitor, XL888, as a potent senolytic in tissue fibrosis. XL888 treatment eliminated pathogenic p16Ink4a+ fibroblasts in a murine model of lung fibrosis and reduced fibrotic burden. Finally, XL888 preferentially targeted p16INK4a-hi human lung fibroblasts isolated from patients with idiopathic pulmonary fibrosis (IPF), and reduced p16INK4a+ fibroblasts from IPF PCLS ex vivo. This study provides proof of concept for a platform where p16INK4a+ cells are directly isolated from diseased tissues to identify compounds with in vivo and ex vivo efficacy in mice and humans, respectively, and provides a senolytic screening platform for other age-related diseases.

The p97/VCP adaptor UBXD1 drives AAA+ remodeling and ring opening through multi-domain tethered interactions.

p97, also known as valosin-containing protein, is an essential cytosolic AAA+ (ATPases associated with diverse cellular activities) hexamer that unfolds substrate polypeptides to support protein homeostasis and macromolecular disassembly. Distinct sets of p97 adaptors guide cellular functions but their roles in direct control of the hexamer are unclear. The UBXD1 adaptor localizes with p97 in critical mitochondria and lysosome clearance pathways and contains multiple p97-interacting domains. Here we identify UBXD1 as a potent p97 ATPase inhibitor and report structures of intact human p97-UBXD1 complexes that reveal extensive UBXD1 contacts across p97 and an asymmetric remodeling of the hexamer. Conserved VIM, UBX and PUB domains tether adjacent protomers while a connecting strand forms an N-terminal domain lariat with a helix wedged at the interprotomer interface. An additional VIM-connecting helix binds along the second (D2) AAA+ domain. Together, these contacts split the hexamer into a ring-open conformation. Structures, mutagenesis and comparisons to other adaptors further reveal how adaptors containing conserved p97-remodeling motifs regulate p97 ATPase activity and structure.

Structure-Based Optimization of Covalent, Small-Molecule Stabilizers of the 14-3-3σ/ERα Protein-Protein Interaction from Nonselective Fragments.

The stabilization of protein-protein interactions (PPIs) has emerged as a promising strategy in chemical biology and drug discovery. The identification of suitable starting points for stabilizing native PPIs and their subsequent elaboration into selective and potent molecular glues lacks structure-guided optimization strategies. We have previously identified a disulfide fragment that stabilized the hub protein 14-3-3σ bound to several of its clients, including ERα and C-RAF. Here, we show the structure-based optimization of the nonselective fragment toward selective and highly potent small-molecule stabilizers of the 14-3-3σ/ERα complex. The more elaborated molecular glues, for example, show no stabilization of 14-3-3σ/C-RAF up to 150 µM compound. Orthogonal biophysical assays, including mass spectrometry and fluorescence anisotropy, were used to establish structure-activity relationships. The binding modes of 37 compounds were elucidated with X-ray crystallography, which further assisted the concomitant structure-guided optimization. By targeting specific amino acids in the 14-3-3σ/ERα interface and locking the conformation with a spirocycle, the optimized covalent stabilizer 181 achieved potency, cooperativity, and selectivity similar to the natural product Fusicoccin-A. This case study showcases the value of addressing the structure, kinetics, and cooperativity for molecular glue development.

From Tethered to Freestanding Stabilizers of 14-3-3 Protein-Protein Interactions through Fragment Linking.

Small-molecule stabilization of protein-protein interactions (PPIs) is a promising strategy in chemical biology and drug discovery. However, the systematic discovery of PPI stabilizers remains a largely unmet challenge. Herein we report a fragment-linking approach targeting the interface of 14-3-3 and a peptide derived from the estrogen receptor alpha (ERα) protein. Two classes of fragments-a covalent and a noncovalent fragment-were co-crystallized and subsequently linked, resulting in a noncovalent hybrid molecule in which the original fragment interactions were largely conserved. Supported by 20 crystal structures, this initial hybrid molecule was further optimized, resulting in selective, 25-fold stabilization of the 14-3-3/ERα interaction. The high-resolution structures of both the single fragments, their co-crystal structures and those of the linked fragments document a feasible strategy to develop orthosteric PPI stabilizers by linking to an initial tethered fragment.

The p97/VCP adapter UBXD1 drives AAA+ remodeling and ring opening through multi-domain tethered interactions.

p97/VCP is an essential cytosolic AAA+ ATPase hexamer that extracts and unfolds substrate polypeptides during protein homeostasis and degradation. Distinct sets of p97 adapters guide cellular functions but their roles in direct control of the hexamer are unclear. The UBXD1 adapter localizes with p97 in critical mitochondria and lysosome clearance pathways and contains multiple p97-interacting domains. We identify UBXD1 as a potent p97 ATPase inhibitor and report structures of intact p97:UBXD1 complexes that reveal extensive UBXD1 contacts across p97 and an asymmetric remodeling of the hexamer. Conserved VIM, UBX, and PUB domains tether adjacent protomers while a connecting strand forms an N-terminal domain lariat with a helix wedged at the interprotomer interface. An additional VIM-connecting helix binds along the second AAA+ domain. Together these contacts split the hexamer into a ring-open conformation. Structures, mutagenesis, and comparisons to other adapters further reveal how adapters containing conserved p97-remodeling motifs regulate p97 ATPase activity and structure.

IFNα primes cancer cells for Fusicoccin-induced cell death via 14-3-3 PPI stabilization.

The natural product family of the fusicoccanes (FCs) has been shown to display anti-cancer activity, especially when combined with established therapeutic agents. FCs stabilize 14-3-3 protein-protein interactions (PPIs). Here, we tested combinations of a small library of FCs with interferon α (IFNα) on different cancer cell lines and report a proteomics approach to identify the specific 14-3-3 PPIs that are induced by IFNα and stabilized by FCs in OVCAR-3 cells. Among the identified 14-3-3 target proteins are THEMIS2, receptor interacting protein kinase 2 (RIPK2), EIF2AK2, and several members of the LDB1 complex. Biophysical and structural biology studies confirm these 14-3-3 PPIs as physical targets of FC stabilization, and transcriptome as well as pathway analyses suggest possible explanations for the observed synergistic effect of IFNα/FC treatment on cancer cells. This study elucidates the polypharmacological effects of FCs in cancer cells and identifies potential targets from the vast interactome of 14-3-3s for therapeutic intervention in oncology.

Engaging a Non-catalytic Cysteine Residue Drives Potent and Selective Inhibition of Caspase-6.

Caspases are a family of cysteine-dependent proteases with important cellular functions in inflammation and apoptosis, while also implicated in human diseases. Classical chemical tools to study caspase functions lack selectivity for specific caspase family members due to highly conserved active sites and catalytic machinery. To overcome this limitation, we targeted a non-catalytic cysteine residue (C264) unique to caspase-6 (C6), an enigmatic and understudied caspase isoform. Starting from disulfide ligands identified in a cysteine trapping screen, we used a structure-informed covalent ligand design to produce potent, irreversible inhibitors (3a) and chemoproteomic probes (13-t) of C6 that exhibit unprecedented selectivity over other caspase family members and high proteome selectivity. This approach and the new tools described will enable rigorous interrogation of the role of caspase-6 in developmental biology and in inflammatory and neurodegenerative diseases.

Reversible Dual-Covalent Molecular Locking of the 14-3-3/ERRγ Protein-Protein Interaction as a Molecular Glue Drug Discovery Approach.

Molecules that stabilize protein-protein interactions (PPIs) are invaluable as tool compounds for biophysics and (structural) biology, and as starting points for molecular glue drug discovery. However, identifying initial starting points for PPI stabilizing matter is highly challenging, and chemical optimization is labor-intensive. Inspired by chemical crosslinking and reversible covalent fragment-based drug discovery, we developed an approach that we term "molecular locks" to rapidly access molecular glue-like tool compounds. These dual-covalent small molecules reversibly react with a nucleophilic amino acid on each of the partner proteins to dynamically crosslink the protein complex. The PPI between the hub protein 14-3-3 and estrogen-related receptor γ (ERRγ) was used as a pharmacologically relevant case study. Based on a focused library of dual-reactive small molecules, a molecular glue tool compound was rapidly developed. Biochemical assays and X-ray crystallographic studies validated the ternary covalent complex formation and overall PPI stabilization via dynamic covalent crosslinking. The molecular lock approach is highly selective for the specific 14-3-3/ERRγ complex, over other 14-3-3 complexes. This selectivity is driven by the interplay of molecular reactivity and molecular recognition of the composite PPI binding interface. The long lifetime of the dual-covalent locks enabled the selective stabilization of the 14-3-3/ERRγ complex even in the presence of several other competing 14-3-3 clients with higher intrinsic binding affinities. The molecular lock approach enables systematic, selective, and potent stabilization of protein complexes to support molecular glue drug discovery.

Venetoclax and dinaciclib elicit synergistic preclinical efficacy against hypodiploid acute lymphoblastic leukemia.

Hypodiploid acute lymphoblastic leukemia (ALL) is an aggressive blood cancer with a poor prognosis despite intensive chemotherapy or stem cell transplant. Children and adolescents with positive end-of-induction minimal residual disease have an overall survival lower than 30%. However, data regarding therapeutic alternatives for this disease is nearly nonexistent, emphasizing the critical need for new or adjunctive therapies that can improve outcomes. We previously reported on the therapeutic efficacy of venetoclax (ABT-199) in hypodiploid B-lineage ALL but with limitations as monotherapy. In this study, we set out to identify drugs enhancing the anti-leukemic effect of venetoclax in hypodiploid ALL. Using a highthroughput drug screen, we identified dinaciclib, a cyclin-dependent kinase inhibitor that worked synergistically with venetoclax to induce cell death in hypodiploid cell lines. This combination eradicated leukemic blasts within hypodiploid ALL patient-derived xenografts mice with low off-target toxicity. Our findings suggest that dual inhibition of BCL-2 (venetoclax) and CDK9/MCL-1 (dinaciclib) is a promising therapeutic approach in hypodiploid ALL, warranting further investigation to inform clinical trials in this high-risk patient population.

LC3B Binds to the Autophagy Protease ATG4b with High Affinity Using a Bipartite Interface.

Autophagy is a catabolic cellular process in which unwanted proteins and organelles are degraded by lysosomes. It is characterized by the formation of the double-membrane autophagosome decorated with LC3B, a protein that mediates autophagosomal fusion with lysosomes. The cysteine protease ATG4b acts at two stages in the life cycle of LC3B. We set out to characterize the protein-protein interaction between LC3B and ATG4b. Through biochemical and biophysical studies, we show that the ubiquitin-like core of LC3B (residues 1-115; "LC3B-115"), which lacks the C-terminal cleavage site (between residue 120 and 121), binds to full-length ATG4b with a surprisingly tight dissociation constant (KD) in the low nanomolar range; 10-30-fold tighter than that of the substrate pro-LC3B (residues 1-125) or the product LC3B-I (residues 1-120). Consequently, LC3B-115 is a potent inhibitor of the ATG4b-mediated cleavage of pro-LC3B (IC50 = 15 nM). Binding of the LC3B-115 has no effect on the conformation of the active site of ATG4b, as judged by the turnover of a peptide substrate ("substrate-33"), derived from LC3B-I residues 116-120. Conversely, truncations of ATG4b show that binding and proteolysis of LC3B critically depend on the C-terminal tail of ATG4b, whereas proteolysis of the peptide substrate-33 does not require the C-terminal tail of ATG4b. These results support a bipartite model for LC3B-ATG4b binding in which the core of LC3B binds to ATG4b and the C-terminal tail of pro-LC3B organizes the ATG4b active site; additionally, the C-terminal tail of ATG4b contributes at least 1000-fold higher binding affinity to the LC3B-ATG4b interaction and likely wraps around the LC3B-ubiquitin core. PPIs are often described as containing an energetic "hot spot" for binding; in the case of LC3B-ATG4b, however, the substrate-enzyme complex contains multiple, energetically relevant domains that differentially affect binding affinity and catalytic efficiency.

Adaptor-Specific Antibody Fragment Inhibitors for the Intracellular Modulation of p97 (VCP) Protein-Protein Interactions.

Protein-protein interactions (PPIs) form complex networks to drive cellular signaling and cellular functions. Precise modulation of a target PPI helps explain the role of the PPI in cellular events and possesses therapeutic potential. For example, valosin-containing protein (VCP/p97) is a hub protein that interacts with more than 30 adaptor proteins involved in various cellular functions. However, the role of each p97 PPI during the relevant cellular event is underexplored. The development of small-molecule PPI modulators remains challenging due to a lack of grooves and pockets in the relatively large PPI interface and the fact that a common binding groove in p97 binds to multiple adaptors. Here, we report an antibody fragment-based modulator for the PPI between p97 and its adaptor protein NSFL1C (p47). We engineered these antibody modulators by phage display against the p97-interacting domain of p47 and minimizing binding to other p97 adaptors. The selected antibody fragment modulators specifically disrupt the intracellular p97/p47 interaction. The potential of this antibody platform to develop PPI inhibitors in therapeutic applications was demonstrated through the inhibition of Golgi reassembly, which requires the p97/p47 interaction. This study presents a unique approach to modulate specific intracellular PPIs using engineered antibody fragments, demonstrating a method to dissect the function of a PPI within a convoluted PPI network.

Phenotypic Screening Using High-Content Imaging to Identify Lysosomal pH Modulators in a Neuronal Cell Model.

Lysosomes are intracellular organelles responsible for the degradation of diverse macromolecules in a cell. A highly acidic pH is required for the optimal functioning of lysosomal enzymes. Loss of lysosomal intralumenal acidity can disrupt cellular protein homeostasis and is linked to age-related diseases such as neurodegeneration. Using a new robust lysosomal pH biosensor (FIRE-pHLy), we developed a cell-based fluorescence assay for high-throughput screening (HTS) and applied it to differentiated SH-SY5Y neuroblastoma cells. The goal of this study was twofold: (1) to screen for small molecules that acidify lysosomal pH and (2) to identify molecular targets and pathways that regulate lysosomal pH. We conducted a screen of 1835 bioactive compounds with annotated target information to identify lysosomal pH modulators (both acidifiers and alkalinizers). Forty-five compounds passed the initial hit selection criteria, using a combined analysis approach of population-based and object-based data. Twenty-three compounds were retested in dose-response assays and two compounds, OSI-027 and PP242, were identified as top acidifying hits. Overall, data from this phenotypic HTS screen may be used to explore novel regulatory pathways of lysosomal pH regulation. Additionally, OSI-027 and PP242 may serve as useful tool compounds to enable mechanistic studies of autophagy activation and lysosomal acidification as potential therapeutic pathways for neurodegenerative diseases.

In Vivo Dopamine Neuron Imaging-Based Small Molecule Screen Identifies Novel Neuroprotective Compounds and Targets.

Parkinson's disease (PD) is the second most common neurodegenerative disorder with prominent dopamine (DA) neuron degeneration. PD affects millions of people worldwide, but currently available therapies are limited to temporary relief of symptoms. As an effort to discover disease-modifying therapeutics, we have conducted a screen of 1,403 bioactive small molecule compounds using an in vivo whole organism screening assay in transgenic larval zebrafish. The transgenic model expresses the bacterial enzyme nitroreductase (NTR) driven by the tyrosine hydroxylase (th) promotor. NTR converts the commonly used antibiotic pro-drug metronidazole (MTZ) to the toxic nitroso radical form to induce DA neuronal loss. 57 compounds were identified with a brain health score (BHS) that was significantly improved compared to the MTZ treatment alone after FDR adjustment (padj<0.05). Independently, we curated the high throughput screening (HTS) data by annotating each compound with pharmaceutical classification, known mechanism of action, indication, IC50, and target. Using the Reactome database, we performed pathway analysis, which uncovered previously unknown pathways in addition to validating previously known pathways associated with PD. Non-topology-based pathway analysis of the screening data further identified apoptosis, estrogen hormone, dipeptidyl-peptidase 4, and opioid receptor Mu1 to be potentially significant pathways and targets involved in neuroprotection. A total of 12 compounds were examined with a secondary assay that imaged DA neurons before and after compound treatment. The z'-factor of this secondary assay was determined to be 0.58, suggesting it is an excellent assay for screening. Etodolac, nepafenac, aloperine, protionamide, and olmesartan showed significant neuroprotection and was also validated by blinded manual DA neuronal counting. To determine whether these compounds are broadly relevant for neuroprotection, we tested them on a conduritol-b-epoxide (CBE)-induced Gaucher disease (GD) model, in which the activity of glucocerebrosidase (GBA), a commonly known genetic risk factor for PD, was inhibited. Aloperine, olmesartan, and nepafenac showed significant protection of DA neurons in this assay. Together, this work, which combines high content whole organism in vivo imaging-based screen and bioinformatic pathway analysis of the screening dataset, delineates a previously uncharted approach for identifying hit-to-lead candidates and for implicating previously unknown pathways and targets involved in DA neuron protection.

Inhibiting a dynamic viral protease by targeting a non-catalytic cysteine.

Viruses are responsible for some of the most deadly human diseases, yet available vaccines and antivirals address only a fraction of the potential viral human pathogens. Here, we provide a methodology for managing human herpesvirus (HHV) infection by covalently inactivating the HHV maturational protease via a conserved, non-catalytic cysteine (C161). Using human cytomegalovirus protease (HCMV Pr) as a model, we screened a library of disulfides to identify molecules that tether to C161 and inhibit proteolysis, then elaborated hits into irreversible HCMV Pr inhibitors that exhibit broad-spectrum inhibition of other HHV Pr homologs. We further developed an optimized tool compound targeted toward HCMV Pr and used an integrative structural biology and biochemical approach to demonstrate inhibitor stabilization of HCMV Pr homodimerization, exploiting a conformational equilibrium to block proteolysis. Irreversible HCMV Pr inhibition disrupts HCMV infectivity in cells, providing proof of principle for targeting proteolysis via a non-catalytic cysteine to manage viral infection.

Exploration of a 14-3-3 PPI Pocket by Covalent Fragments as Stabilizers.

The systematic discovery of functional fragments binding to the composite interface of protein complexes is a first critical step for the development of orthosteric stabilizers of protein-protein interactions (PPIs). We have previously shown that disulfide trapping successfully yielded covalent stabilizers for the PPI of 14-3-3 with the estrogen receptor ERα. Here we provide an assessment of the composite PPI target pocket and the molecular characteristics of various fragments binding to a specific subpocket. Evaluating structure-activity relationships highlights the basic principles for PPI stabilization by these covalent fragments that engage a relatively large and exposed binding pocket at the protein/peptide interface with a "molecular glue" mode of action.

Real-Time Assessment of Mitochondrial Toxicity in HepG2 Cells Using the Seahorse Extracellular Flux Analyzer.

The liver is the primary organ responsible for drug detoxification. Drug-induced liver injury (DILI) is a leading cause of attrition during drug development and is one of the main reasons that drugs are withdrawn from the market. Hence, the prevention of DILI plays a central role in the overall drug-discovery process. Most of the liver's energy supply comes in the form of adenosine triphosphate (ATP), which is largely generated by mitochondria. This article describes the evaluation of drug-induced mitochondrial dysfunction using the Seahorse Extracellular Flux Analyzer (Agilent). The described protocols detail the accurate measurement of ATP production rate in HepG2 cells after exposure to a panel of potentially toxic compounds. This assay measures changes in extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) as indicators of glycolysis and mitochondrial respiration-the two major energy-generating pathways in a cell. This assay provides a useful model to predict mitochondrial dysfunction-mediated DILI. © 2021 Wiley Periodicals LLC. Basic Protocol: Measurement of cellular ECAR, OCR, and ATP production in live HepG2 cells Support Protocol 1: Culturing and maintaining of HepG2 cells Support Protocol 2: Determining optimal cell density per well.