A small molecule VDAC ligand inhibits ERAD and induces selective cancer cell death via disruption of calcium homeostasis

Domain: chemistry
Published: 2026-01-17
Editorial outcome: major_revision

Abstract
Endoplasmic reticulum-associated degradation (ERAD) is a critical protein quality control mechanism that also regulates lipid metabolism and calcium homeostasis. Dysregulation of ERAD and unfolded protein response underlies diseases including cancer, neurodegenerative disorders, and metabolic syndromes. Small molecule modulators of ERAD could enable mechanistic discovery and therapeutic intervention, but few have been identified. Using a high-content screening, we discovered several ERAD-modulating compounds, including NCATS-SM0225, an ERAD inhibitor that unexpectedly binds all three isoforms of VDAC, outer mitochondrial membrane proteins enriched at mitochondria-associated membranes. This led us to discover an essential role for VDACs in ERAD and ER-phagy. NCATS-SM0225 elevates cytosolic, ER, and mitochondrial calcium through calcium influx and IP3R–MCU activity. This calcium imbalance strengthens VDAC1–IP3R coupling and activates PERK, which phosphorylates STIM1 and drives degradation of key ERAD regulators. Loss of these components amplifies PERK signaling and selectively kills cancer cells while sparing normal cells. These findings uncover a cancer-specific role of VDACs in ERAD regulation and calcium signaling, highlighting a therapeutically actionable vulnerability.

Introduction
The endoplasmic reticulum (ER) is a central organelle responsible for protein folding, lipids synthesis, and calcium homeostasis, essential for maintaining cellular proteostasis. ER-associated degradation (ERAD) is a critical quality control mechanism in which misfolded proteins in the endoplasmic reticulum (ER) are recognized, retrotranslocated to the cytosolic surface for ubiquitination and dislocation, and subsequently delivered to the proteasome for degradation1–6. Beyond protein quality control, ERAD also regulates physiological processes such as lipid metabolism and calcium signaling by selectively degrading transcription factors, enzymes, and regulatory proteins often in a cell type-specific manner3,7,8. ERAD is closely coordinated with ER-phagy, a selective autophagy process that degrades ER fragments and misfolded proteins, compensating for ERAD deficiencies and protecting the ER from dysfunction3,9,10. Insufficient removal and subsequent accumulation of misfolded proteins lead to ER stress and activates the unfolded protein response (UPR)11,12. The UPR is mediated by three ER stress sensors—IRE1α, ATF6, and PERK—which work to restore proteostasis. However, severe or prolonged ER stress can shift the UPR toward pro-apoptotic signaling13–15. While the UPR is critical for maintaining the function of secretory cells like pancreatic β-cells and B lymphocytes, it is also implicated in diseases such as cancer, neurodegenerative disorders, and metabolic syndromes14,16–19.

In neurodegenerative diseases like Alzheimer’s, Huntington’s, Parkinson’s and amyotrophic lateral sclerosis, toxic protein aggregates disrupt ERAD, causing chronic ER stress and neuronal cell death via pro-apoptotic UPR signaling20–28. In contrast, cancer cells often experience ER stress due to disrupted protein homeostasis; in this context, the UPR is reprogrammed to promote survival, tumorigenesis, metastasis, drug resistance, and immune evasion16,18,29,30. UPR activation has been documented in many human cancers, including breast, pancreatic, lung, prostate, brain, and hematologic malignancies16,30. Small-molecule inhibitors targeting specific UPR branches have been developed to explore therapeutic potential but overlapping functions and compensatory mechanisms may limit their efficacies31,32.

Modulating ERAD activity represents a promising strategy to influence ER stress and UPR signaling, as misfolded protein accumulation is the primary driver of ER stress. Enhancing or inhibiting ERAD can either promote clearance or exacerbate accumulation of misfolded proteins, respectively, thereby shaping cellular stress responses31,33,34. The development of potent and selective small molecule ERAD modulators as chemical biology tools is an important step towards dissecting ERAD mechanisms and roles in physiology and disease. ERAD modulators also offer therapeutic potential by enhancing ERAD to alleviate ER stress in neurodegenerative diseases and diabetes or suppressing ERAD to sensitize cancer cells to stress-induced apoptosis33–35. However, only a limited number of ERAD modulators have been identified to date, and the field remains underdeveloped.

In this study, we present the development of a high-content phenotypic screening assay designed to identify small molecule modulators of ERAD. Among the compounds screened, NCATS-SM0225 (referred to as 225) emerged as a potent ERAD inhibitor, but unexpectedly, it was found to bind to voltage-dependent anion channels (VDACs). Binding of 225 to VDACs triggers IP3R-MCU-dependent disruption of calcium homeostasis, which subsequently activates a calcium-PERK-STIM1 signaling. This cascade leads to the degradation of key ERAD complex proteins (HERP1, OS9, and FAM8A1) and selectively induces cancer cell death while sparing normal cells. These findings provide critical insights into ERAD biology, its interplay with calcium signaling, and its potential as a cancer therapeutic target. This work highlights the utility of small molecule modulators in probing ERAD function and towards developing first-in-class cancer-selective therapies.

Results
High-content screening for small molecule modulators of ERAD
During ERAD, misfolded proteins are recognized and retained in the ER, where luminal chaperones and lectins facilitate their delivery to ERAD complexes for retrotranslocation to the cytosol1,6. This process is essential for ERAD, as it enables misfolded proteins to be ubiquitinated and subsequently degraded by the proteasome. We have previously developed the drGFP reporter assay to monitor the final step of retrotranslocation, the complete dislocation of substrate proteins from the ER into the cytosol, in live cells36. This assay utilizes split GFP37, which reconstitutes fluorescence upon successful dislocation in the presence of a proteasome inhibitor, which provides a direct, real-time readout of ERAD activity (Fig. 1a, and Supplementary Movies 1–3). The null Hong Kong variant of α−1-antitrypsin (NHK), an ER luminal misfolded protein, was selected as the ERAD substrate (NHK-drGFP) in the primary screen, where changes in GFP intensity were visualized and quantified to assess ERAD activity36. CD3δ, an ER membrane substrate, was used in the drGFP (CD3δ-drGFP) assay as a secondary screen to confirm primary hits. Enhanced substrate dislocation increases GFP fluorescence, while inhibition (e.g., NMS-873-ap97/VCP inhibitor, in Fig. 1a) suppresses it, making this a powerful tool for studying ERAD and identifying potential modulators.

We first miniaturized and optimized the drGFP assay in 1536-well format to enable high-throughput screening. Pilot screening indicated robust assay performance (Supplementary Fig. 1a) with reproducible dose-dependent inhibition observed for p97/VCP inhibitor NMS-873, known to inhibit dislocation (Fig. 1b). The optimized assay was used to perform a screen of 96,047 compounds, which exhibited a favorable signal window (Fig. 1c) and a robust mean Z’ factor of 0.59 (Supplementary Fig. 1b). The primary screen yielded 956 hits, comprised of approved drugs, bioactive tool compounds, and diversity-collection molecules (Fig. 1d, and Supplementary Fig. 1c). We focused on the 419 compounds that originated from the diversity collections and retested them at 11 concentrations spanning 37 nM to 38 μM. Sixty-six compounds were confirmed as inhibitors, using the criteria of AC50 < 20 μM and > 60% efficacy. The hits were then tested in a second model of ER dislocation, using a misfolded CD3δ-based drGFP, with 62 of the 66 compounds (94%) categorized as active using the same 20 μM and 60% inhibition criteria. The 62 compounds represented thirteen chemotypes. Following removal of acutely cytotoxic compounds (showing cytotoxicity after 4 h treatment) and compounds with covalent warheads, six compounds, representing six chemotypes, were prioritized for further analysis (Fig. 1e and Supplementary Figs. 1d, e).

We next tested the six potential dislocation inhibitors for their effects on the stability of ΔF508CFTR, a well-established ERAD substrate and the cystic fibrosis-causing protein38,39. Baby hamster kidney fibroblasts (BHK) stably expressing HA-ΔF508CFTR (HA-ΔF508CFTR-BHK cells)40,41 were treated with the putative inhibitors for 24 h. The results revealed that only 225 significantly increased the levels of HA-ΔF508CFTR in both the ER (core-glycosylated B form) and at the cell surface (fully glycosylated mature C form) (Fig. 1f). These increases are likely due to the inhibition of HA-ΔF508CFTR degradation, which promotes its forward trafficking (Fig. 1f). Notably, the non-glycosylated form of ΔF508CFTR (A form) was not observed in either control or 225-treated cells (Fig. 1f), suggesting that 225 affects the degradation and trafficking of the glycosylated forms of ΔF508CFTR40,41. Next, we resynthesized 225 (methods section) and tested the multiple batches in parallel. All the batches consistently showed reproducible activity in the dislocation assay (Supplementary Fig. 1f). To further validate and potentially improve the activity of 225, we designed and conducted a focused structure–activity relationship (SAR) study by synthesizing several structural analogs (Supplementary Fig. 2 and Supplementary Note 1). Smaller substituents on the phenyl ring were generally tolerated and, in some cases, slightly improved potency (Supplementary Fig. 2; compounds 1-5). However, all other structural changes investigated (a subset which is included here) resulted in a significant loss of activity, including: the addition of alkyl groups in the benzylic position, N-alkylation of the sulfonamide, lactone-opening to various amides, truncation of the benzyl-sulfonamide altogether to parent amine, and changing sulfonamide to the analogous amide (Supplementary Fig. 2; compounds 6-10, respectively).

Validation of 225 as a dislocation inhibitor
Time-lapse imaging of the NHK-drGFP reporter showed that 225 inhibited dislocation as demonstrated by a dose-dependent reduction in drGFP, with an IC50 of 1.02 μM (Fig. 2a–c, and Supplementary Movies 4–7). Since inhibition of dislocation is expected to block ERAD, we further evaluated the effects of 225 on the degradation of NHK and an ER membrane substrate CD3δ, using cycloheximide (CHX) chase. Consistent with its role as a dislocation inhibitor, 225 significantly inhibited the degradation of both substrates (Fig. 2d–g). For ER luminal substrates, dislocation is a prerequisite for ubiquitination, as ubiquitination occurs on the cytosolic surface of the ER. As expected, 225 effectively inhibited NHK ubiquitination (Fig. 2h). These findings collectively establish 225 as a potent inhibitor of ERAD dislocation.

225 induces selective degradation of HERP1, OS9, and FAM8A1 in HeLa cells
Inhibition of ERAD is expected to induce ER stress and activate the unfolded protein response (UPR). To explore the effects of 225 on UPR signaling and ERAD-related proteins in HeLa cells, we performed immunoblotting (IB) analyses, using tunicamycin (Tm) as a positive control for ER stress. Remarkably, treatment with 225 for 24-h resulted in a significant downregulation of HERP1, OS9, and FAM8A1, accompanied by a phosphorylation-induced mobility shift of PERK (Fig. 3a), with the effects being both dose- and time-dependent. In addition, there was a modest downregulation of Sel1L protein (Fig. 3a). To determine whether the reduction in HERP1, OS9, and FAM8A1 levels was due to post-translational degradation, HeLa cells were treated with 225 alone or in combination with either the proteasome inhibitor bortezomib (BTZ) or the lysosome inhibitor chloroquine (CQ). All three proteins were stabilized by BTZ but not CQ (Supplementary Fig. 3a), indicating that 225 promotes their proteasomal degradation. Consistently, 225 did not decrease HERP1, OS9, or SEL1L mRNA; only FAM8A1 showed a modest reduction (Supplementary Fig. 3b).

Given that p97/VCP facilitates the extraction of ubiquitinated misfolded proteins from the ER for proteasomal degradation42, we investigated whether 225 targets HERP1, OS9, and FAM8A1 through the ERAD pathway. Treatment with p97/VCP inhibitors DBeQ and NMS-87343,44 effectively blocked the 225-induced degradation of all three proteins (Supplementary Fig. 3c), confirming their degradation via the ERAD-proteasome pathway. Consistent with these findings, gene set enrichment analysis (GSEA) of transcriptome data from 225-treated HeLa cells revealed significant enrichment of UPR activation (Fig. 3b), which was further validated by IB (Fig. 3c). Notably, PERK signaling emerged as the most prominently activated pathway (Fig. 3b, d). Inhibition of PERK kinase activity partially rescued HeLa cell death induced by 225 (Fig. 3e). Together, these results demonstrate that 225 disrupts protein dislocation, induces the degradation of ERAD complex proteins, and activates PERK-mediated proapoptotic signaling.

225 selectively targets all three isoforms of VDAC (voltage-dependent anion channel)
To identify the molecular target of 225, a diazirine-based photoaffinity and clickable probe (225-cc, Supplementary Fig. 4c) was synthesized by incorporating 225 with a minimally interfering photo-affinity label (MI-PAL)45 to enable target identification studies (Fig. 4a, b). First, we confirmed that 225-cc retained dislocation inhibitory activity, with an IC50 of 9.8 μM in the NHK-drGFP assay (Supplementary Figs. 4a, b). Next, cells treated with 225-cc were irradiated with UV light (365 nm) to induce diazirine photolytic conversion into a reactive carbene, capable of covalent insertion into amino acid residues in close proximity. To identify 225-cc target proteins, a commercially available trifunctional probe, DdeTAMRA Biotin Azide, which contains a fluorescent dye and a chemically cleavable biotin pull-down handle (Supplementary Fig. 4c), was used to label 225-cc-bound proteins via copper-catalyzed azide-alkyne cycloaddition (CuAAC; ‘click’ reaction)46 (Supplementary Fig. 4d). The 225-cc-labeled proteins, now covalently bound to a fluorescent moiety, could be visualized on SDS-PAGE gels (Fig. 4a), and the biotin tag facilitated identification by mass spectrometry (MS) following affinity capture using streptavidin beads (Fig. 4b). These experiments indicated that 225-cc is quite selective, as a single protein band of approximately 35 kDa was observed in both total cell lysates and streptavidin pull down samples (Fig. 4a). MS analysis of the protein band pulled down by streptavidin beads revealed that the top three identified proteins were the three isoforms of VDAC, outer mitochondrial membrane proteins also localized in mitochondria-associated membranes (MAMs)47,48 (Fig. 4b). Knockout of each VDAC isoform reduced 225 labeling to an extent consistent with the reported expression levels in HeLa cells49, with VDAC1 showing the most significant reduction (Fig. 4c). These results indicate that 225 binds to all three isoforms of VDAC.

The ER membrane protein complex (EMC) is an ER insertase, and recent reports suggest VDAC1 can associate with EMC via EMC3, EMC4, and EMC750. To further probe target specificity, we tested whether EMC mediates 225’s effects. Degradation of ERAD components (HERP1, OS9, FAM8A1) persisted in 225-treated EMC3-, EMC4-, and EMC7-knockdown cells, similar to controls, and 225 did not affect the levels of these three EMC proteins (Supplementary Fig. 4e); EMC3 or EMC7 knockdown also did not alter 225-mediated dislocation inhibition in the drGFP assay (Supplementary Fig. 4f); and VDAC–EMC interactions were unchanged by 225 as demonstrated by VDAC1 co-IP with EMC3 or EMC4 (Supplementary Fig. 4g). These results do not support an EMC-dependent mechanism of 225 and further substantiate VDACs as the specific targets of 225.

As VDACs are not known to control ERAD but have been recently isolated as interacting proteins for SEL1L, a key cofactor for the HRD1 ERAD E3 ubiquitin ligase51, we next investigated the potential connection by knocking down each of the three VDAC isoforms and assessing the effects on NHK dislocation using NHK-drGFP reporter. Surprisingly, single knockdown of any VDAC isoform by itself markedly inhibited dislocation (Fig. 4d, e), supporting a role for VDACs in ERAD. Given the marked effects on dislocation, it was surprising that we succeeded in establishing viable VDAC KO cell lines (Fig. 4c), suggesting that a compensatory mechanism was activated to protect cells from the effects of dislocation inhibition. Since ER-phagy is a key compensatory response to ERAD inhibition9,52,53, we monitored ER-phagy activity using the well-established ss-RFP-GFP-KDEL reporter54 stably expressed in HeLa cells. Knockdown of each VDAC isoform activated ER-phagy, as indicated by a decrease in the GFP/RFP ratio (Fig. 4f) and the formation of RFP puncta accompanied (Fig. 4g). Similarly, knockdown of VDAC1 led to the relocalization of CD3δ-mCherry into puncta and a decrease in its cellular levels (Fig. 4h, i). These findings demonstrate that 225 selectively targets all three isoforms of VDAC and suggest that VDACs play a critical role in maintaining ERAD activity. The activation of ER-phagy following VDAC knockdown suggests that ER-phagy sequesters and degrades ERAD substrates, thereby reducing their dislocation from the ER.

225 directly interacts with VDAC1 and regulates its function in vitro
To validate the binding of 225 to the identified target proteins, we conducted microscale thermophoresis (MST) analysis. The equilibrium dissociation constant (Kd) between 225 and recombinant human VDAC1 was determined to be 3.13 μM, indicating direct binding (Fig. 5a). To further examine 225 binding to VDACs in cells, we employed the Cellular Thermal Shift Assay (CETSA), which is based on the principle that ligand binding increases the thermal stability of its target protein, allowing detection of the stabilized protein in the soluble cellular fraction by immunoblotting or mass spectrometry55,56. Treatment with 225 increased the thermal stability of all three VDAC isoforms but not the control protein ATP5A (Fig. 5b). Together, these results provide strong evidence for a direct interaction between 225 and VDACs.

To examine if 225 binding to VDAC1 affects its channel function, we employed a voltage-clamp electrophysiology approach on a system of the recombinant human VDAC1 reconstituted into a planar lipid membrane57,58. This method allowed us to perform highly sensitive measurements of functional interaction between 225 and VDAC1 at the single-molecule level and to investigate the impact of 225 on VDAC1 basic properties, such as its single-channel conductance and voltage gating. The planar lipid membranes were formed from a mixture of dioleoyl-phosphatidylcholine (DOPC):dioleoyl-phosphatidylethanolamine (DOPE) (1:1 w/w), two major phospholipids of the mitochondrial outer membrane59,60, in symmetrical 1 M KCl buffer solution. Under these conditions, VDAC1 typically forms ion channels of ~4.0 nS conductance, which characteristically gate, i.e., transition from the high conducting “open” state to a variety of low conducting or “closed” states in response to the applied voltages61,62. In control, 225-free, experiments, the application of +/− 60 mV was required to observe consistent voltage gating (Fig. 5c). The addition of 225 at 200 nM and 400 nM concentrations resulted in VDAC1 gating at much lower amplitudes of negative potentials, specifically at −40 mV and −30 mV (indicated by arrows in Fig. 5c). This resulted in a 20–30 mV shift of the voltage required for gating of a single VDAC1 channel, compared to the control conditions (Fig. 5c). Thus, 225 promotes VDAC1 closure at reduced voltages in a concentration-dependent manner (Fig. 5c).

Quantification of voltage-gating and obtaining a 225 binding curve requires a statistical analysis of the gating of many channels. The multichannel experiments were performed under the application of slow triangular voltage waves (5 mHz, +/− 60 mV), which enable a slow channel closure and fast reopening, and allow us to collect responses of 50-100 channels to the applied voltages63,64. Figure 5d shows the normalized conductance-voltage plots at negative potentials (G/Gmax vs voltage), where G is the conductance at a given voltage and Gmax is the maximum conductance at voltages close to 0 mV. The multichannel measurements were performed under the same experimental conditions as single-channel measurements (Fig. 5c). The addition of 225 to the cis side of the membrane, enhanced VDAC1 gating by decreasing the minimum conductance (Gmin), calculated at |V| > 50 mV, in a dose-dependent manner (Fig. 5d). Notably, the increased voltage-gating was observed in single- and multichannel experiments predominantly at potentials that are negative from the side of 225 addition, the cis side of the membrane. The gating response to 225 addition was quantified as normalized change in conductance, (Gmax - Gmin)/Gmin, and plotted versus 225 concentrations (Fig. 5e). These results point to a direct functional interaction between 225 and VDAC1. We can speculate that by promoting the VDAC1 closed state, 225 promotes calcium influx to mitochondria47,65, thus regulating calcium signaling through direct interaction with VDAC1.

225 induces calcium elevation in mitochondria, ER, and cytosol, leading to HERP1 and OS9 degradation
VDAC, the most abundant protein in the mitochondrial outer membrane, plays a critical role in regulating calcium influx, metabolism, and cell death47,66–69. To investigate the effects of 225 on intracellular calcium dynamics, we monitored calcium levels in mitochondria, cytosol, and ER using established fluorescent calcium sensors, including the cytosol calcium sensor GCaMP6F, the mitochondrial calcium sensor Mitycam, and the ER calcium sensor GCaMPer70. We observed that 225 induced a dose- and time-dependent increase in calcium levels across all three compartments (Fig. 6a–c). KO of individual VDAC isoforms or dual VDAC2/3 reduced 225-induced calcium elevation, while RNAi-mediated knockdown (KD) of VDAC1 in the context of VDAC2/3 KO (VDAC1KD/VDAC2-/-/VDAC3-/-) nearly abolished the effect of 225 on calcium levels across all compartments (Fig. 6d–f). Furthermore, VDACs were found to mediate 225-induced cell death, as measured by caspase-3/7 activation (Fig. 6g, h). Loss of all three VDAC isoforms significantly diminished 225-induced cell death (Fig. 6i), further supporting the conclusion that VDACs are the molecular targets through which 225 elevates intracellular calcium and triggers cell death. As a negative control, EMC knockdown did affect 225-induced increase in mitochondrial calcium (Supplementary Fig. 4h).

To determine whether calcium elevation mediates 225-induced cytotoxicity, we used the calcium chelator BAPTA-AM to sequester intracellular calcium during 225 treatment of HeLa cells. Calcium chelation completely abolished 225-induced cell death (Fig. 6j). Moreover, BAPTA-AM treatment prevented 225-induced degradation of HERP1 and OS9 (Fig. 6k). These findings suggest that 225 binds to VDACs, triggering a rise in intracellular calcium levels that subsequently induces the degradation of HERP1 and OS9 and ultimately leads to cell death.

Given reports that efsevin stabilizes the calcium-conductive state of VDAC71, we tested it alongside 225 in our ERAD assays. Under identical conditions, efsevin neither impaired dislocation nor reduced HERP1 or OS9 protein levels, whereas 225 robustly inhibited dislocation and decreased HERP1/OS9 (Supplementary Fig. 6a). These data indicate that efsevin does not inhibit ERAD in our system and support a mechanism for 225 that is distinct from efsevin.

225 preferentially targets a subgroup of cancer cells while sparing normal cells
To determine the potential cell type-dependent effects of 225, we randomly examined 16 different cancer cell lines derived from various cancers, including melanoma, glioma, breast cancer, liver cancer, pancreatic cancer, osteosarcoma, and mucoepidermoid carcinoma. Treatment with 225 induced the degradation of HERP1, OS9, and FAM8A1 in 11 out of the 16 cancer cell lines while showing minimal or no effects in the remaining 5 cell lines (Fig. 7a). Notably, 225 had no impact on the protein levels of HERP1, OS9, and FAM8A1 in three types of primary normal human cells, including fibroblasts from three individuals, epidermal melanocytes, and hepatocytes (Fig. 7a). Importantly, the cytotoxicity of 225 showed a strong positive correlation with the degradation of HERP1, OS9, and FAM8A1, with degradation-resistant cells showing limited cytotoxicity (Fig. 7a, b). The differential cytotoxicity of 225 was further validated by measuring caspase-3/7 activation (Supplementary Figs. 7a–d). Remarkably, the viability of normal cells was not impacted by 225 up to 10 μM concentrations that impacted cancer cells (Fig. 7b, Supplementary Fig. 7d) and concentrations up to 50 μM had no effect on primary human hepatocytes (Supplementary Fig. 7f). These results suggest that 225 selectively targets a subgroup of cancer cells while sparing normal cells, and this selectivity strongly correlates with its ability to induce the degradation of HERP1, OS9, and FAM8A1.

To obtain proof-of-concept evidence for the antitumor efficacy of 225, we evaluated its activity in vivo using A375 melanoma xenografts, a 225-sensitive cancer model. Daily intraperitoneal administration of 225 (20 mg/kg) markedly inhibited tumor growth without detectable toxicity (Fig. 7c–e). Treated mice exhibited no clinical signs of adverse effects, and histological examination of the liver, pancreas, and kidney revealed no abnormalities (Supplementary Figs. 7g–i). Collectively, these results identify VDAC as a promising cancer-selective therapeutic target.

225-induced intracellular calcium elevation is a defining feature of its selective cytotoxicity
225 induces elevation of calcium in cytosol, mitochondria, and ER and causes death in HeLa cells (Fig. 6). Next, we examined whether this calcium elevation differentiates the selective cytotoxicity using 225-sensitive MCF7ca, A375, and A253 cells, and 225-insensitive MDA-MB-231, T47D, MDA-MD-453 cells, as well as primary human fibroblasts (HDFa, GM05294) as examples. Calcium increases were observed only in 225-sensitive cancer cells, with no significant changes observed in 225-insentitive cancer cells and primary human fibroblasts (Fig. 8a–c). These findings suggest that 225 preferentially targets a subgroup of cancer cells by disrupting calcium homeostasis and inducing the degradation of ERAD complex proteins, while sparing normal cells and a subgroup of cancer cells.

VDAC1 expression is frequently overexpressed in various cancers compared to normal cells and is associated with poor prognosis and tumor progression72,73. Next, we examined whether VDAC1 expression contributes to the differential sensitivity of 225. We found that VDAC1 protein levels are generally higher in sensitive cells and lower in insensitive or normal cells (Fig. 8d, e); however, one insensitive cell line (MDA-MB-231) expresses high levels of VDAC1 protein (Fig. 8d, e). Although the underlying mechanism that confer resistance of MDA-MB-231 to 225 are not understood, we speculate that mutations may alter downstream death-signaling pathways. We also analyzed mRNA expression data from the Human Protein Atlas74,75 and found no correlation between VDAC1 transcript abundance and protein levels (Fig. 8d-e vs. Supplementary Fig. 8a). This suggests that steady-state VDAC1 protein abundance is regulated by translational efficiency or protein stability rather than transcription. Consistent with this possibility, treatment of HeLa cells (a sensitive line) with the proteasome inhibitor bortezomib markedly stabilized VDAC proteins (Supplementary Fig. 8b), indicating that VDACs are an unstable protein subject to proteasomal degradation, thereby affecting their steady-state protein levels. Taken together, these new findings suggest that VDAC1 protein abundance may contribute to the differential sensitivity to 225.

Calcium influx and IP3R activity mediate 225-induced intracellular calcium elevation and cell death
The 225-induced increase in intracellular calcium is likely driven by calcium influx from the extracellular space. This was confirmed by the failure of 225 to elevate intracellular calcium when HeLa cells were cultured in calcium-free medium (Fig. 9a–c). The simultaneous increase in calcium levels across the ER, mitochondria, and cytosol suggests that 225 broadly disrupts calcium homeostasis following VDAC binding. One potential mediator of these effects is the inositol 1,4,5-trisphosphate receptor (IP3R), as IP3R, grp75, and VDAC form a complex at mitochondria-associated membranes (MAMs) that facilitates calcium transport from the ER to mitochondria76. To test this hypothesis, cells were treated with the IP3R inhibitor 2-APB77. 2-APB effectively inhibited 225-induced calcium increases in all three subcellular compartments and reduced cytotoxicity (Fig. 9d, e), implicating IP3R as a mediator of changes in calcium homeostasis. Furthermore, 2-APB also blocked 225-induced PERK activation and the degradation of HERP1 and OS9 (Fig. 9f). Coimmunoprecipitation experiments revealed that 225 significantly enhances the interaction between VDAC1 and IP3R in calcium-dependent manner (Fig. 9g, h), consistent with 225 altering complexes at the MAMs that control calcium. Inhibiting the mitochondrial calcium uniporter (MCU), which is specifically responsible for the uptake of calcium into the mitochondrial matrix78, also diminished the cytotoxicity of 225 (Fig. 9i). These findings suggest that 225 binding to VDAC1 enhances the VDAC1-IP3R interaction, facilitates a rise in calcium levels across intracellular compartments, activates PERK, and triggers degradation of ERAD complex proteins, which ultimately culminates in cell death.

STIM1 regulates 225-induced calcium influx, and its phosphorylation mediates 225-induced ERAD complex degradation and cell death
The observations that 225-induced intracellular calcium dynamic changes require (1) extracellular calcium influx (Figs. 9a–c) and (2) ER contributions, mediated by IP3R (Fig. 9d, e), led us to hypothesize that STIM1, a key regulator of store-operated calcium entry (SOCE), might contribute to these events79,80. To investigate this possibility, we knocked down STIM1 and measured its effects on intracellular calcium levels. STIM1 knockdown disrupted cellular calcium homeostasis, as reflected by increased calcium levels in the ER, cytosol, and mitochondria to varying degrees (Fig. 10a). Notably, 225 failed to further elevate calcium levels in STIM1-KD cells (Fig. 10b), suggesting that STIM1 regulates 225-induced calcium elevation.

While elevated calcium is generally toxic to cells, STIM1 knockdown unexpectedly diminished 225-induced cytotoxicity and reduced PERK expression (Fig. 10c, d). This result is consistent with experiments identifying PERK as a crucial mediator of 225-induced cell death (Fig. 3e). Coimmunoprecipitation experiments revealed that STIM1 interacts with PERK, and 225 enhanced this interaction (Fig. 10e). Given that PERK is activated by 225, we investigated whether PERK phosphorylates STIM1. Indeed, 225 induced a mobility shift in STIM1, and the Phos-Tag assay confirmed that this shift was due to phosphorylation (Fig. 10f, g). The 225-induced phosphorylation of STIM1 was inhibited by the PERK inhibitor GSK2606414 (Fig. 10f). Furthermore, PERK inhibition, like STIM1 knockdown, diminished 225-induced degradation of HERP1 and OS9 (Fig. 10f). Consistently, PERK knockdown reduced 225-induced inhibition of NHK dislocation (Fig. 10h, i). These findings indicate that 225 activates PERK, which phosphorylates STIM1, resulting in the degradation of HERP1 and OS9 and inhibition of ERAD.

STIM1 and PERK have not been previously reported to interact with VDAC1. To determine how 225 binding to VDAC1 could affect their association, we measured potential interactions among these proteins using co-immunoprecipitation (coIP). VDAC1 was found to interact with STIM1 and PERK in addition to IP3R (Fig. 10j). Interestingly, treatment with 225 enhanced the VDAC1–IP3R interaction but reduced its association with STIM1 and PERK (Fig. 10j). Concomitantly, 225 increased STIM1–PERK complex formation, which was dependent on calcium but independent of PERK kinase activity (Fig. 10k, l). These results suggest that 225 binding to VDAC1 elevates intracellular calcium levels, thereby remodeling the interaction network among VDAC1, IP3R, STIM1, and PERK. The increased VDAC1–IP3R interaction further amplifies calcium influx, while enhanced PERK–STIM1 association and PERK-mediated STIM1 phosphorylation destabilize HERP1, OS9, and FAM8A1. Collectively, these events coincide with sustained calcium elevation, culminating in cell death (see model in Figur S9).

Methods
Cell culture
HeLa (CCL-2), HepG2 (HB-8065), A-375 (CRL-1619), U-2OS (HTB-96), A253 (HTB-41), PSN1 (CRL-3211), Primary Dermal Fibroblast (HDFa, PCS-201-012) and Primary Epidermal Melanocyte (HEMa, PCS-200-013) cell lines were obtained from the American Type Culture Collection (ATCC, USA). U-251MG (09063001) and U-87MG (89081402) were obtained from Millipore Sigma. Normal untransformed fibroblast GM05294 and GM04390 cell lines were purchased from Coriell Institute. MDA-MB-231 (CRM-HTB-26), MDA-MB-453 (HTB-131), SK-BR-3 (HTB-30), BT474 (HTB-20), T-47D (HTB-133), and MIAPaCa-2 (CRL-1420) cell lines were obtained from Dr. Mark J. Henderson (NCATS/NIH). MCF7ca (MCF7 human breast cancer cell lines transfected with aromatase gene) cells were obtained from Dr. Yun Qiu (University of Maryland, Baltimore). Hepatocyte and Huh7 cells were obtained from Dr. Hongbing Wang (University of Maryland, Baltimore). HeLa cells stably expressing SP-S11-NHK-HA and S1-10 were established previously36.

All cell lines were maintained at 37°C in a 5% CO₂ humidified incubator. GM05294 and GM04390 were cultured in Minimum Essential Medium (MEM). HDFa was maintained in Fibroblast Basal Medium (ATCC® PCS-201-030™) supplemented with Fibroblast Growth Kit-Serum-free (ATCC® PCS-201-040™), HEMa is maintained in Dermal Cell Basal Medium (ATCC PCS-200-030) supplemented with Adult Melanocyte Growth Kit (ATCC PCS-200-042). PSN1 and T-47D were cultured in RPMI-1640 Medium. MDA-MB-231 and MDA-MB-453 were cultured in Leibovitz’s L-15 Medium. Other cell lines were maintained in Dulbecco’s Modified Eagle Medium (DMEM). All normal cell lines were supplemented with 15% fetal bovine serum and 1% Penicillin-Streptomycin. All cancer cell lines were supplemented with 10% fetal bovine serum and 1% Penicillin-Streptomycin. For calcium-free conditions, cells were incubated in calcium-free DMEM (21068028, Invitrogen).

Reagents and antibodies
225, its structural analogs, and 225-cc were synthesized in-house. 2-Aminoethyldiphenylborinate (2-APB, HY-W009724) and BAPTA-AM (HY-100545) were purchased from MedChemExpress. Phos-tag Acrylamide AAL-107 was purchased from Fujifilm. Efsevin was purchased from A1 BioChem Labs (cas# 212070-75-2). All other chemicals and lab reagents were purchased from Millipore Sigma unless otherwise indicated. For experiments, cells were treated with 225 at the indicated concentrations for specified time periods. 225 was dissolved in DMSO (1 mM stock solution), and control experiments included an equivalent volume of DMSO (final concentration did not exceed 0.1%). Inhibitors used were added 1 h prior to treatment with 225 where indicated.

The sources of the antibodies are as follows: Anti-HA11 (ENZ-ABS120, clone 16B12, 1:500) was purchased from Enzo Lifesciences. Anti-VDAC1 (820704, clone N152B123, 1:1000) and anti-STIM1 (621702, clone W18094A, 1:1000) were obtained from Biolegend. Anti-PERK (3192S, clone C33E10, 1:1000), anti-HERP1 (26739S, 1:1000), anti-OS9 (12497S, 1:1000), anti-UBE2G2 (63182S, clone D8Z4G, 1:500), anti-HRD1 (14773S, clone D302A, 1:1000) were purchased from Cell Signaling Technology. Anti-IP3R (NBP1-21398, 1:1000) was purchased from NOVUS. Anti-FAM8A1 (24746-1-AP, 1:1000), anti-ATF6 (66563-1-1g, 1:1000), anti-STIM2 (21192-1-AP, 1:2000) and anti-p-PERK (82534-1-RR, 1:1000), anti-EMC3 (67205-1-IG, clone 2F8G5, 1:2000), anti-EMC4 (27708-1-AP, 1:2000) and anti-EMC7 (27550-1-AP, 1:2000) were from ProteinTech. Anti-VDAC2 (PA5-28106, 1:2000), anti-VDAC3 (PA5-100190, 1:200) and anti-YOD1 (PA5-70852, 1:1000) were purchased from Invitrogen. Anti-HA-peroxidase (3F10) antibody (12013819001, 1:500) was purchased from Roche. Anti-Sel1L (S3699, 1:1000), anti-DERLIN1 (D4443, 1:1000), anti-XTP3B (SAB4200062, 1:1000), anti-DERLIN2 (D1194, 1:1000), and β-Actin-Peroxidase (A3854, clone AC-15, 1:3000) antibodies were purchased from Millipore Sigma. Anti-BiP (610978, 1:1000) antibody was from BD Biosciences. ubiquitin-HRP (sc-8017 HRP, clone P4D1, 1:200), anti-IRE1α (sc-390960, clone B-12, 1:300), anti-GRP94 (sc-393402, clone H-10, 1:1000), anti-UBE2J1 (sc-377002, clone B-6, 1:500), anti-UBXD8 (sc-98899, clone H-300, 1:200), CHOP (sc-7351, clone B-3, 1:300) and anti-PARP (sc-8007, clone F-2, 1:500) were purchased from Santa Cruz Biotechnology. Anti-Calnexin (CNX, ab22595, 1:2000) was purchased from Abcam Inc. Anti-RFP was kindly provided by Mervyn J. Monteiro (University of Maryland, Baltimore).

Generation of plasmid constructs
STIM1-EGFP (210370), pcDNA3.1 VDAC1-Linker-GFP (211734), calcium-sensitive plasmids including GCaMP6F (cytosol, 163045), Mitycam (mitochondria, 163046) and GCaMPer (ER, 163047)70 were purchased from addgene. pCIneo-HERP1-HA was constructed by inserting the cDNA fragments encoding the ORF of human HERP1 into the NheI/NotI sites of pCIneo. The plasmid pLVX-ssRFP-GFP-KDEL was generated from pCW57-CMV-ssRFP-GFP-KEDL (Addgene #128257)54. The ssRFP-GFP-KEDL insert was excised using the EcoRI restriction site and ligated into the pLVX-EF1α-IRES-Puro vector (Takara Bio), which was digested with the same enzyme. Related primers are listed in Supplementary Table 1 in Extended data. All expression constructs were verified by DNA sequencing.

Dislocation-induced reconstituted GFP (drGFP) assay
HeLa cells stably expressing SP-S11-NHK-HA and S1-10 (NHK-drGFP)36 were plated in 96-well plates (2×10⁴ cells/well) and cultured overnight. After replacing the medium, cells were treated with indicated concentrations of 225 and/or BTZ (2 μM). GFP fluorescence was monitored in real-time using a 20× objective on the IncuCyte S3 live-cell analysis system.

drGFP-based high-content screening
Primary screening. To identify small molecule modulators of ER dislocation, NHK-drGFP reporter-expressing HeLa cells were seeded in black low base 1536-well assay plates (EBA141000A; Aurora) at a density of ~1000 cells per well in 5 μL of Fluorobrite DMEM media (A18967-01; Gibco) supplemented with 10% FBS (SH30071.03, Cytivia), 5 U/mL penicillin/streptomycin (10378-016; Gibco), 21 mM HEPES (15630-080; Gibco) and 2 mM Glutamine (25030-081; Gibco) using a Multidrop Combi reagent dispenser (5840330; Thermo Fisher Scientific) equipped with a small tube metal tip dispensing cassette (24073295; Thermo Fisher Scientific). Plates were cultured overnight (16 h) in incubators maintained at 37°C, with 5% CO2, and 95% humidity prior to the addition of 20 nL compound (dissolved in DMSO) using a Kalypsys pintool equipped with a 1536-pin array. To monitor ER dislocation, cells were incubated for 15 minutes, then treated with 2 μL of media containing 21 μM (6 μM final) Bortezomib (S1013; SelleckChem) to promote the accumulation of NHK-drGFP following the addition of compound. Plates were then incubated for 4 h prior to GFP quantification using an Acumen EX3 Microplate Cytometer (TTPLabtech). Data was normalized to multiple replicates (n=32) of cells treated with and without bortezomib, plus an equivalent volume of DMSO. To monitor assay performance, each plate included a 16 pt titration of the p97 inhibitor, NMS-873 (HY-15713; MedChemExpress), as a reference control. Library compounds demonstrating significant reduction of GFP signal (≤60% activity relative to DMSO) were replated in 11 pt titration and re-tested, as described, to confirm inhibitory activity, generate dose-response curves and calculate AC50 values.

Secondary Assay. Compounds confirmed in dose-response follow-up testing and exhibiting select hit parameters (≤60% activity relative to DMSO controls and AC50 ≤25 μM) were then re-tested in a secondary assay employing the same stable cell line. Briefly, cells were seeded at ~1000 cells per well in ultra-low base 1536-well, 188 μM film-bottom assay plates (EBC241000A; Aurora) and treated with compound and bortezomib, as described. Following 3.75 h of incubation, each well received 1 μL of pre-warmed media containing 25 ng/mL (3.125 ng/mL final) Hoechst (H3570; Invitrogen) using a Multidrop Combi reagent dispenser (Thermo Fisher Scientific). Plates were pulse centrifuged (30 s at 1000 rpm) using a benchtop centrifuge (5804R; Eppendorf) equipped with a microplate rotor and bucket (Eppendorf; EP-2DWPAT) then incubated 15 min at room temperature prior to quantification using an InCell Analyzer 2000 Imaging Plate Reader System or Opera Phenix Plus High-Content Screening System. Acquired images were processed using a Columbus Image Data Storage and Analysis System to quantify cellular GFP based on nuclear staining and corrected for background. Data was normalized to multiple replicates (n=32) of cells treated with and without bortezomib, plus an equivalent volume of DMSO. To monitor assay performance, each plate included a 16 pt titration of the p97 inhibitor, NMS-873, as a reference control.

Establishment of gene-modified cell lines
ssRFP-GFP-KDEL stable cell line. HeLa cells were transfected with pLVX-ssRFP-GFP-KDEL plasmid using Lipofectamine 3000 (L3000015, Invitrogen) following the manufacturer’s protocol. After 48 h, stable transfectants were selected with puromycin. Positive clones were maintained under puromycin selection and verified by fluorescence microscopy and Western blot analysis.

VDAC knockout cell lines. VDAC1, VDAC2, VDAC3, and VDAC2/VDAC3 knockout HeLa cell lines were generated using CRISPR-Cas9. Single-guide RNA expression plasmids (Addgene) targeting each VDAC isoform were transfected into HeLa cells cultured in DMEM with 10% FBS. Transfected cells were selected with puromycin (2 μg/mL) for 3 days. Successful knockout was confirmed by immunoblotting using isoform-specific anti-VDAC antibodies. (Primer sequences are provided in Supplementary Table 1.)

siRNA Knockdown. Negative Control siRNA was purchased from Ambion. PERK siRNA (9024S) was purchased from Cell Signaling Technology. All other siRNAs were synthesized by Millipore Sigma (Supplementary Table 2). All siRNAs were transfected with Lipofectamine® RNAiMAX transfection reagent (Invitrogen) using siRNA in Opti-MEM with no antibiotic. After 72 h of transfection, knockdown efficiency was confirmed by western blotting. Cells were then treated with 225, and downstream effects were assessed.

Cytoheximide (CHX) chase35. To measure the degradation rate and stability of NHK and CD3δ35, HeLa cells stably expressing HA-tagged NHK or CD3δ were plated and allowed to reach 70−80% confluence before initiating the chase experiment. CHX was added to the culture medium at a final concentration of 50 μg/mL to halt de novo protein synthesis. Cells were incubated with either CHX alone or along with 225 for the indicated time points. Then cells were harvested and processed for immunoblotting with anti-HA and anti-β-actin antibodies.

Immunoprecipitation (IP). Cells transfected with indicated plasmids or empty vector were treated with DMSO (control) or 225 for 3 h, then lysed in buffer (150 mM NaCl, 10 mM Tris/HCl pH 7.4, 1 mM EDTA, 1 mM EGTA, 0.2% NP-40) containing protease inhibitors. For each IP reaction, 700 μg of protein in 800 μL total volume was used. Lysates from cells expressing ectopic proteins were incubated with anti-GFP or anti-HA affinity beads for 2 h at 4°C with gentle shaking. Beads were washed three times with 700 μL lysis buffer before processing for immunoblotting (IB).

NHK ubiquitination was analyzed by re-immunoprecipitation (Re-IP)35. Briefly, HeLa cells expressing NHK-HA were treated with BTZ (2 μM) with or without 2 μM 225. Cells were then collected and lysed in (150 mM NaCl, 10 mM Tris/HCl pH 7.4, 1 mM EDTA, 1 mM EGTA, 0.2% NP-40) containing protease inhibitors, and NHK-HA was immunoprecipitated using an anti-HA affinity matrix. To eliminate non-specifically bound proteins, immunoprecipitates were denatured with 2% SDS, and beads were removed by centrifugation. The supernatants were diluted 20-fold in native lysis buffer, and NHK-HA was re-immunoprecipitated before immunoblotting for HA and ubiquitin.

Immunoblotting (IB). Total cell lysates were prepared in lysis buffer (10 mM Tris/HCl, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.2% NP-40, 0.5% Triton X-100, pH 7.5) containing protease and phosphatase inhibitors. Proteins were resolved by SDS-PAGE and transferred to PVDF membranes. After blocking nonspecific binding sites with 5% milk for 1 h at room temperature, membranes were probed with primary antibodies, followed by HRP-conjugated secondary antibodies. Following TBST washes, immunoreactive bands were detected using SuperSignal West Pico PLUS or Femto Maximum Sensitivity chemiluminescent substrates.

Caspase-3/7 activity assay. Apoptosis was assessed using the IncuCyte® Caspase-3/7 Green Apoptosis Assay. HeLa cells were treated with 225 at the indicated concentrations in the presence of the caspase-3/7 reagent (1:2000 dilution). Fluorescence intensity, reflecting caspase-3/7 activity, was measured in real-time using the IncuCyte® S3 system.

Target identification using photoaffinity and clickable 225 and Mass Spectrometry (MS). Cells were cultured to ~80% confluence and incubated with 5 μM of 225-cc in the presence or absence of 80 μM 225 for 30 min at 37°C to allow compound binding to target proteins. After incubation, cells were rinsed with PBS and irradiated with UV light (355 nm) for 20 minutes on ice to covalently crosslink probe 225-cc to its interacting proteins. Following crosslinking, cells were washed with cold PBS and lysed in 1% NP40 buffer (25 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10% Glycerol, 1% NP40) containing EDTA free protease inhibitor cocktail. The lysates were clarified by centrifugation at 14,000 × g for 15 minutes at 4°C, and protein concentration was determined. Urea was then added to cleared lysate (~680 mg urea to 1 mL lysate) to achieve a final urea concentration of approximately 8 M.

For pull-down assay, 1.3 mL of the urea-denatured lysate (~1.5 mg proteins) was mixed with the following reagents: 30 μL of 1 mM Dde-TAMRA-Biotin-Azide (Click Chemistry Tools, 1367-1), 70 μL of 100 mM THPTA, 14 μL of 100 mM CuSO4 and 70 μL of 300 mM sodium ascorbate. The click reaction mixture was incubated for 30 min at room temperature in the dark on a rotator. Following incubation, proteins were precipitated by cold methanol and chloroform. Precipitated proteins were resuspended in 1% SDS, 50 mM Tris-HCl (pH 7.5), and 150 mM NaCl, followed by sonication. Each sample was incubated with 50 μL Pierce™ Streptavidin Magnetic Beads (Thermo Fisher, 88816) for 1.5 h at room temperature. Beads were washed three times with the same resuspension buffer. After washing, beads were boiled in SDS loading buffer containing 5 mM biotin and proceeded to SDS-PAGE and immunoblotting if applicable. For mass spectrometry analysis, fluorescent bands at ~35 kDa (both 225-cc and with competition control by adding free 225) and ~18 kDa (only 225-cc) were excised (3 gel pieces in total, N=1 for each condition) and cut into 1 mm pieces. Gel pieces were washed twice with 50% methanol for 10 minutes each, then the gel pieces were stored at −20°C prior to LC-MS/MS analysis. The click reaction can be scale down to 50 μL total volume with 50 μg proteins when the sample is only used for fluorescent visualization of the probe-labeled proteins in the SDS-PAGE gel.

For gel band excision, clean and sterile razors were used to cut the desired band/slice in a process-protection hood. The slice was further cut into 1×1 mm pieces prior to de-staining, reduction and alkylation, tryptic digestion and peptide extraction81. In brief, 1×1 mm gel pieces were transferred into pre-rinsed 1.5 mL Eppendorf tubes and re-swelled in 50 µL of 20 mM ammonium bicarbonate (ABC), followed by dehydration with 50 µL of acetonitrile. After discarding the solvent, the gel pieces were re-swelled in 10 mM dithiothreitol (DTT) and incubated for 1 h at 56°C for reduction, followed by alkylation with 55 mM iodoacetamide (IAA) for 45 min at room temperature in the dark. The solution was discarded, and the gel pieces were washed with 50 mM ABC and dehydrated again with acetonitrile. After the final dehydration step, the gels were dried completely using a SpeedVac. The dried gel pieces were then re-swelled on ice for 45–60 min in 25 µL of 10 ng/µL trypsin prepared in 20 mM ABC. Excess trypsin solution was removed, and the gel pieces were covered with 25 µL of 20 mM ABC and incubated overnight at 37°C for digestion. Peptides were extracted once with 25 µL of 20 mM ABC and three times with 50 µL of 50% acetonitrile containing 5% formic acid. All supernatants were combined and lyophilized by speed-vac and resuspended in 2% acetonitrile, 97.9% water, and 0.1% formic acid buffer for liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis. The LC-MS/MS analysis of in-gel digested product was performed using an UltiMate 3000-nano LC system coupled to the Orbitrap Fusion Lumos Tribrid mass spectrometer equipped with the Nanospray Flex ion source (Thermo Fisher). Peptides were loaded onto the trap column (Acclaim PepMap 100 C18, 75 μm × 2 cm, particle size: 3 μm, 100 Å; Thermo Fisher) by autosampler using loading solvent (2% acetonitrile in MS-grade water, 0.1% FA) at a flow rate of 4 μL/min. Elution of peptides from the analytical column (with spray tip, 75 μm × 20 cm, 1.7 μm, 100 Å; CoAnn Technologies) was performed using a 60 min method (~45 min gradient) starting at 98% solution A (0.1% formic acid in MS-grade water) at a flow rate of 300 nL/min. The mobile phase was maintained at 2% solution B (80% acetonitrile, 19.9% water, 0.1% formic acid) for 5 min, 2–9% B for 2 min, 9–38% B for 33 min, 38–50% B for 2 min, 50–90% B for 3 min, and maintained at 90% B for 6 min, followed by re-equilibration of the column with 2% B for 7 min. Column oven temperature was set as 40°C. The mass spectrometer was operated in positive-ionization mode with spray voltage set at 1800 V, and ion transfer tube temperature set at 250°C. The MS scan was operated at data-dependent acquisition mode, with full MS scans over a mass range of m/z 375–1800 with detection in the Orbitrap (120K resolution) and with auto gain control (AGC) set to 1.0×106. The fragment ion spectra were acquired in Orbitrap (15K resolution) with a normalized collision energy of 28% at HCD activation mode. In each cycle of data-dependent acquisition analysis, the most intense ions were selected for the MS/MS analysis, and the cycle time for MS and MS/MS analysis was set as 2 s. The AGC for MS/MS was set as 5.0×104 and the maximum injection time was 22 ms. Precursor ions with charges of +2 to +7 were isolated for MS/MS sequencing. The MS/MS isolation window was 1.2 Da, and the dynamic exclusion time was set at 60 s with a mass tolerance of ±10 ppm.

Proteome Discoverer software suite (version 3.0, Thermo Fisher) with Sequest algorithm was used for peptide identification. The MS raw data were searched against a Swiss-Prot human database (version November 2022, reviewed database) consisting of 20,328 entries using the following parameters: precursor ion mass tolerance of 10 ppm and a fragment ion mass tolerance of 0.02 Da. Peptides were searched using fully tryptic cleavage constraints and up to two internal cleavage sites were allowed for tryptic digestion. Fixed modifications consisted of carbamidomethylation of cysteine. Variable modifications considered were oxidation of methionine residues. Peptide identification false discovery rates (FDR) were limited to a maximum of 0.01 using identifications from a concatenated database from the non-decoy and the decoy databases. Minimum number of peptide for protein identification was set as 1 and protein was ranked by number of PSMs.

Differential expression and gene set enrichment analysis (GSEA)82. Differential expression analysis between 225-treated and untreated samples (three biological replicates per group) was first performed using the DESeq2 R package (v1.14.1), which models digital gene expression data with a negative binomial distribution. Genes with a p value < 0.05 were designated as differentially expressed and were subsequently ranked based on their expression changes to serve as input for gene set enrichment analysis (GSEA). GSEA was then performed using the GSEA software (v4.4.0, available at http://www.gsea-msigdb.org/gsea/index.jsp) with default parameters to identify biological pathways and transcriptional programs regulated by compound 225. Pre-defined gene sets from the Molecular Signatures Database (MSigDB), including the Hallmark and KEGG collections, were evaluated, and pathways with a false discovery rate (FDR)–adjusted p-value < 0.05 were considered significantly enriched. The normalized enrichment score (NES) was used to quantify the degree of enrichment for each gene set82.

Microscale thermophoresis (MST). Recombinant human VDAC1 bearing an N-terminal His-tag (Proteogenix, Cat#: ARO-P13933) was equilibrated on ice for 1 h in PBS (pH 7.4) supplemented with 0.05% n-dodecyl-β-D-maltoside (DDM). His-tag–directed fluorescent labeling was performed using the RED-tris-NTA 2nd Generation dye (NanoTemper, Cat#: MO-L018) according to the manufacturer’s instructions. The high-affinity interaction between His-tags and tris-NTA (~1–10 nM) ensured selective labeling without additional purification. To determine the binding affinity of compound 225 for VDAC1, a two-fold serial dilution of 225 was prepared in PBS containing 0.05% DDM, starting at 100 µM to generate a twelve-point titration series. Fluorescently labeled protein (10 µL, final concentration 50 nM) was mixed with 10 µL of each 225 concentration, resulting in a maximal compound concentration of 50 µM. Samples were incubated for 30 min at room temperature and loaded into Monolith Premium Capillaries (NanoTemper, Cat#: MO-K025). Two independent replicate measurements were collected for each concentration.

MST measurements were acquired using a Monolith X instrument (NanoTemper Technologies GmbH) with high IR-laser power and automatic excitation settings. MST response values were automatically converted to “fraction bound” in MO. Control software (v2.7.1). Duplicate measurements at each concentration were averaged, and the averaged values were used to generate the binding curve. The dissociation constant (Kd) was determined by nonlinear regression using the MO. Control software.

Cellular thermal shift assay (CETSA)55,56. CETSA was employed to evaluate compound–protein engagement in cells55,56. HeLa cells were seeded and allowed to adhere, then treated with 2 μM compound 225 or an equivalent volume of dimethyl sulfoxide (DMSO) at 37°C for 1 h. After treatment, cells were harvested by scraping and lysed in buffer (150 mM NaCl, 10 mM Tris/HCl pH 7.4, 1 mM EDTA, 1 mM EGTA, 0.4% NP-40) supplemented with protease inhibitors. The lysates were clarified by centrifugation at 14,000 × g for 10 min, and protein concentrations of the resulting supernatants were determined and normalized across samples. Normalized aliquots of the lysates were heated at the indicated temperatures for 3 min, followed by cooling to room temperature (25°C) for 3 min. The samples were then centrifuged at 20,000 × g for 20 min to separate soluble proteins from precipitated aggregates. Soluble fractions were mixed with 4× SDS sample buffer and subjected to immunoblotting.

VDAC1 gating. Planar lipid bilayers were made by the apposition of two monolayers comprised of the 1:1 (w:w) mixture of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) lipids (Avanti Polar Lipids, Alabaster, AL) dissolved in pentane. These monolayers were formed across a ∼125 μm (in multichannel experiments) and 75 μm (in single channel experiments) diameter aperture within a 15-μm-thick Teflon film separating two ∼1.5-mL compartments filled with 1 M KCl solution buffered by 5 mM HEPES at pH 7.4, as previously described64. The recombinant human VDAC1 was a generous gift of Tsyr-Yan Dharma Yu (Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei, Taiwan). VDAC1 channel insertion was obtained after addition of 0.2–0.5 μl of VDAC1 diluted in 100 mM Tris, 50 mM KCl, 1 mM EDTA, 15% (v/v) DMSO, 2.5% (v/v) Triton X-100, pH 7.35 to the cis compartment of the experimental chamber while stirring. The procedure of VDAC reconstitution into lipid bilayers was described previously64,83. Potential is defined as positive when it is greater on the cis side, the side of compound 225 and VDAC1 addition.

Ion current recordings were acquired using an Axopatch 200B amplifier (Axon Instruments) in voltage clamp mode, following previously reported protocols83. Current signals were filtered at 10 kHz with a low-pass Bessel filter, digitized at a sampling frequency of 50 kHz, and analyzed using pClamp 10.7 software (Axon Instruments). For single-channel analyses in Clampfit 10.7, recordings were further digitally filtered with a 5 kHz 8-pole Bessel low-pass filter.

Voltage-dependence of VDAC gating was analyzed using a previously described protocol63,64 under the application of a slow, symmetric triangular voltage wave (±60 mV, 5 mHz) generated by an Arbitrary Waveform Generator 33220A (Agilent Technologies). Data were sampled at 2 Hz frequency and analyzed as detailed earlier63 using a custom in-house algorithm and pClamp 10.7 software. In each experiment, current records were collected from membranes containing 10-50 channels in response to 5–10 periods of voltage waves to ensure data collection from more than 50 channels per experiment. Only the part of the wave, during which the channels were reopening, was used for the subsequent analysis for the reasons described elsewhere63.

Xenograft. The A375 human melanoma cell line was maintained in high-glucose DMEM supplemented with 10% fetal bovine serum at 37°C in a humidified atmosphere containing 5% CO2. Compound 225 was formulated in a vehicle consisting of N-methyl-2-pyrrolidone, polyethyleneglycol 300, propylene glycol, and 20% hydroxypropyl-β-cyclodextrin. Male BALB/c-nu mice (7 weeks old) were housed in a specific pathogen-free facility under standard conditions. We fully acknowledge the journal’s policy on sex inclusion and recognize the importance of sex as a biological variable. In our initial in vivo proof-of-principle study, only male BALB/c-nu mice were used to minimize biological variability and obtain clear efficacy data. This design avoided estrous cycle–related hormonal fluctuations that can influence tumor growth and pharmacokinetics, following standard early-stage oncology practice to isolate treatment effects. Our approach aligns with the NIH Sex as a Biological Variable policy (NOT-OD-15-102), which permits single-sex studies when scientifically justified, and with the FDA Modernization Act 2.0 and FDA 2025 Roadmap for Reducing Animal Testing, which emphasize refinement and reduction of animal use when feasible. Future studies will include both sexes and incorporate non-animal models, consistent with evolving NIH and FDA guidance promoting rigor, reproducibility, and responsible animal research. Log-phase A375 cells (2×10⁶) were suspended in a 1:1 mixture with Matrigel and injected subcutaneously into the right flank (n=8 per group). After 7 days, mice received daily intraperitoneal injections of 225 (20 mg/kg) or vehicle. Tumor size was measured every two days using calipers, and tumor volume was calculated as (a×b²)/2, where a and b represent the longest and shortest perpendicular diameters, respectively. On day 21, mice were euthanized, and tumors and major organs were collected for histological analysis. All animal procedures were performed in accordance with institutional guidelines and approved by the Experimental Animal Ethics Committee of the Institute of Clinical Pharmacology, Anhui Medical University (Approval No. PZ-2021-016).

Statistical analysis. All quantitative data presented are representative of at least three independent experiments. Data are shown as means ± SD. Differences between two groups were analyzed by unpaired two-tailed Student’s t-test using GraphPad Prism 7.0. For all analyses, the p-value was considered significant as follows: **p < 0.01, ***p < 0.001, ****p < 0.0001. Data visualization was performed using GraphPad Prism and the ggplot2 package (version 3.5.1) in R (version 4.4.3).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "General Assessment:\nYan et al. describe the identification of NCATS-SM0225 as an ERAD inhibitor via a GFP-based HTS and elucidate its\nmechanism involving VDAC binding, dysregulation of calcium homeostasis, and selective cancer cell cytotoxicity. While the\nstudy identifies a novel compound with an intriguing mechanism and therapeutic potential, several critical conclusions lack\nsufficient experimental validation. The mechanistic claims require substantial reinforcement to meet the journal's standards.\nMajor revisions addressing the following points are essential prior to reconsideration.\nMajor Concerns:\n1. HTS Compound Prioritization Rationale: The manuscript states that 6 compounds were prioritized from 62 initial hits for\nfurther study. The specific criteria (e.g., potency, chemical tractability, structural diversity, lack of known liabilities) guiding\nthis selection must be explicitly defined. Without this justification, the rationale for focusing on SM0225 remains unclear and\npotentially arbitrary.\n2. Mechanism of STIM1-PERK Affinity Enhancement (Fig 8e): The observation that SM0225 enhances endogenous STIM1-\nPERK affinity is significant. However, as SM0225 is not proposed to directly bind either protein, the underlying mechanism\nfor this effect remains entirely speculative. The authors must propose and experimentally test a plausible mechanism (e.g.,\ninvolving Ca²⁺ flux, ER stress sensor activation, or intermediary adaptors) or significantly temper their interpretation of this\nresult.\n3. Clarity on ERAD Reporter Substrate (Fig 1e): The misfolded CD3δ-based drGFP reporter used for secondary HTS\nvalidation must be clearly defined upon its first mention in the main text (HTS section). Ensure consistent nomenclature\nbetween text and Fig 1e.\n4. Validation of VDAC1-3 Binding: The identification of VDAC1-3 as targets via photoaffinity labeling (PAL) and SDS-\nPAGE/MS is a key finding. However, confirmation using orthogonal, quantitative biophysical methods is crucial to establish\nbinding affinity, specificity, and stoichiometry. Strongly recommend performing Cellular Thermal Shift Assay (CETSA)\ncombined with either Isothermal Titration Calorimetry (ITC) or Surface Plasmon Resonance (SPR) to validate and quantify\nthe direct interaction between SM0225 and VDAC isoforms 1-3. Relying solely on PAL (inherently detecting covalent\ninteractions) is insufficient proof of the physiological, non-covalent binding implied in the proposed mechanism.\n5. Incomplete Exploration of Differential Cell Sensitivity: The differential sensitivity of cancer cell lines to SM0225 is a\ncompelling observation with translational relevance. However, the exploration of the proposed mechanism (involving\nERAD/ER-phagy crosstalk) is superficial. To validate the hypothesis that ER-phagy capacity dictates sensitivity, the authors\nshould inhibit ER-phagy (e.g., via genetic knockdown of key regulators like FAM134B, SEC62, or CCPG1, or using\npharmacological inhibitors like CQ/HCQ where appropriate) in resistant/low-sensitivity cell lines and assess whether this\nsensitizes them to SM0225-induced cytotoxicity. Demonstrating this functional link is essential to support the model in\nFigure 9.\n6. Lack of In Vivo Efficacy and Safety Data: Demonstrating potent and selective cancer cell killing in vitro is promising, but in\nvivo validation is a fundamental requirement for a mechanistic/therapeutic study of this nature in Nature Communications.\nThe authors should provide evidence of SM0225's efficacy in relevant tumor xenograft or syngeneic models, alongside\nassessment of toxicity in normal tissues. This is critical for establishing the compound's therapeutic window and translational\npotential.", "reviewer_2": "In this study by Yan et al., the authors developed a chemical screening platform based on an ERAD dislocation reporter. The\nscreen identified compound 225 as a small-molecule ERAD inhibitor. They further dissected the mechanism of action of 225\nby identifying the VDACs as the drug targets, demonstrating disruption of calcium homeostasis, and linking the\nPERK/STIM1 axis to degradation of ERAD complex proteins and cell death. Overall, this is an interesting study that provides\nnovel insights into the regulation of UPR, calcium homeostasis, ERAD, and ER-phagy.\nSeveral minor comments are listed below:\n1. Figure 3a: The authors mentioned the degradation of OS9, HERP1, and FAM8A1. However, the data also show a\nreduction in SEL1L at 24 hours post-treatment. This should also be noted in the text.\n2. Figure 3: The authors should examine transcriptomic changes in the RNA-seq data for OS9, HERP1, FAM8A1, and\nSEL1L mRNA levels to rule out mRNA downregulation as the cause of decreased protein levels.\n3. Figures 2d–g and 4i: Treatment with 225 significantly inhibited the degradation of both NHK and CD3, resulting in\nincreased protein levels. However, in Figure 4i, siVDAC1 treatment reduced CD3-mCherry levels. These findings seem\ninconsistent and should be clarified.\n4. The term “dislocation” should be defined earlier in the manuscript for clarity.", "reviewer_3": "Yan and colleagues present compelling evidence that stress-induced apoptosis in cancer cells can be increased by\nsuppressing the Endoplasmic Reticulum-Associated Degradation (ERAD) pathway using small molecules. Through an\nextensive drug screening campaign employing a clever designed readout, they identified novel ERAD inhibitors. One of the\nmost potent molecules identified, compound 225, not only inhibits ERAD but also binds the mitochondrial porin VDAC1.\nWhile a direct link between VDAC1 and the ERAD pathway has not been conclusively established before, the authors\nprovide strong evidence supporting VDAC1 involvement in ERAD regulation.\nThe manuscript is data-rich and covers several key aspects, including the drug screening process, interaction studies (IP\nexperiments), and mechanistic insights that collectively support the proposed conclusions. However, I have several\nimportant questions and concerns that should be addressed prior to publication:\nMajor Points:\n1. The authors demonstrate that compound 225 stabilizes the interaction between VDAC1 and the IP3 receptor. Do the\nauthors have mechanistic insights or supporting data explaining why this interaction is enhanced in the presence of\nmolecule 225? Is this effect direct, or could it result from broader changes in organelle physiology?\n2. Compound 225 appears to have dual effects: it activates PERK, resulting in STIM1 phosphorylation, HERP1 degradation,\nand ERAD inhibition; while also binding to VDAC1. However, UV-crosslinking identified VDACs as the only direct targets.\nHow do the authors reconcile this dual activity? Is there evidence that the STIM1–PERK interaction is influenced by the\nVDAC–IP3R complex? Could the authors attempt IP experiments to pull down VDAC using STIM1 or PERK in the presence\nof 225 to test for complex formation?\n3. The manuscript claims that molecule 225 selectively targets cancer cells without affecting healthy cells. Given that\nVDAC1 is overexpressed in many solid tumors, did the authors assess VDAC1 expression levels in 225-sensitive versus -\ninsensitive cell lines? Quantifying VDAC1 levels by RNA-seq and Western blot would help clarify whether sensitivity to 225\ncorrelates with VDAC1 expression and might explain the lack of toxicity in healthy cells.\n4. The authors propose that molecule 225 enhances VDAC1–IP3R interaction, promoting calcium transfer between the ER\nand mitochondria, which subsequently activates PERK and drives ERAD inhibition. However, this does not fully explain the\nobserved increase in cytosolic calcium (Fig. 7c). Could the authors comment on the source of the cytosolic calcium and\nclarify whether this increase is a direct or indirect consequence of 225 treatment?\n5. The small molecule efsevin has been shown to stabilize the calcium-conductive form of VDAC1 (PMID: 32059260). It\nwould be informative to compare the effects of efsevin and compound 225 on ERAD inhibition. Do both compounds share a\nsimilar mechanism of action? Such a comparison might strengthen the proposed model.\nMinor Points:\nScheme on Figure 8j should be revised to clearly depict relevant subcellular compartments, improve image quality, and\nremove the pink background. Additionally, the graph located at the bottom left appears to be an error and should be\nremoved.", "reviewer_4": "Key Results\nThis manuscript identifies a novel small molecule, NCATS-SM0225 (“225”), as a selective ERAD inhibitor that binds to\nvoltage-dependent anion channels (VDACs). Through a combination of high-content screening, biochemical validation, and\nmechanistic dissection, the authors suggest that 225 triggers an intracellular calcium imbalance via a VDAC-IP3R-PERK-\nSTIM1 signaling axis. This leads to the degradation of ERAD complex components (HERP1, OS9, FAM8A1) and induces\ncell death selectively in cancer cells while sparing normal cells. The work uncovers a previously unrecognized role for\nVDACs in ERAD regulation and presents a compelling chemical probe for further study of ER proteostasis and cancer-\nselective cytotoxicity.\nValidity\nThe data are of high quality, robust, and generally well-controlled. The experimental workflow is thorough, including\northogonal validation of dislocation inhibition (GFP reporters, CHX chase, ubiquitination assays), target identification\n(photoaffinity labeling and MS), and functional studies using gene knockouts and chemical inhibitors. However, the precise\nmechanism of 225 binding to VDACs and how this affects VDAC function at a structural level remains speculative. The\nauthors acknowledge this and propose it as future work.\nSignificance\nThe identification of a VDAC-targeting small molecule that selectively disrupts ERAD and kills cancer cells is novel and\npotentially impactful. This work opens new avenues in cancer biology, proteostasis, and chemical biology. It also proposes\nan alternative strategy to targeting UPR sensors directly — by modulating ERAD upstream. The selective toxicity of 225 to\ncertain cancer cell lines suggests translational relevance. While the mechanistic basis of this selectivity is not fully resolved,\nthe dataset lays a solid foundation for follow-up studies.\nData and Methodology\nThe methodology is state-of-the-art and spans a wide range of experimental techniques, including high-content screening,\nSAR chemistry, CRISPR knockouts, calcium imaging, and GSEA transcriptomic profiling. The description of materials and\nmethods is detailed and transparent. Use of the drGFP reporter for dislocation, combined with dose-responses, time\ncourses, and structure–activity relationships, makes the data solid. Photoaffinity labeling with mass spectrometry is well\nexecuted, and VDAC target identification is convincing. If available, raw calcium traces or proteomics data could be made\npublic in supplementary datasets.\nAnalytical Approach\nQuantitative results are consistently supported by replicates, appropriate statistical tests, and careful normalization. The use\nof dose-response curves, IC50 values, and multiple replicates is appropriate. GSEA is correctly applied and complements\nthe mechanistic data. The authors have been transparent about sample sizes and statistical significance thresholds.\nSuggested Improvements\nThe authors present a compelling study identifying VDAC1, VDAC2, and VDAC3 as direct binding partners of compound\n225. They propose that 225 perturbs calcium homeostasis at the ER-mitochondria interface via these outer mitochondrial\nmembrane proteins, thereby inhibiting ER-associated degradation (ERAD), activating the unfolded protein response (UPR),\nand triggering apoptosis. While the data are generally sound and well-presented, I believe an alternative, structurally\ngrounded interpretation offers a more parsimonious explanation of the experimental findings.\nRecent structural studies (Li et al., Aging 2023, DOI:10.18632/aging.205660) demonstrate that VDAC1 directly binds the ER\nmembrane protein complex (EMC) at ER-mitochondria contact sites (MAMs), locking the EMC in a non-insertase\nconformation.\nEarlier proteomic mapping\n(Christianson, J., Olzmann, J., Shaler, T. et al. Defining human ERAD networks through an integrative mapping strategy. Nat\nCell Biol 14, 93–105 (2012). https://doi.org/10.1038/ncb2383)\nshowed that multiple EMC subunits interact with Derlin-1 and Derlin-2, core components of the ERAD dislocation\nmachinery. This raises the possibility that compound 225 inhibits ERAD not by modulating VDAC channel activity directly,\nbut by stabilizing a non-functional EMC–VDAC complex that disrupts EMC-dependent membrane insertion of ERAD\ncomponents such as Derlin-1.\nBelow, I analyze the major data types presented by the authors in support of their model and offer the alternative EMC-\nmediated interpretation.\n1. Identification of VDAC1/2/3 as targets of 225:\nThe authors used a clickable photoaffinity analog of 225 to identify VDAC1, VDAC2, and VDAC3 as binding targets.\nThis result is compatible with both their interpretation (VDAC as a functional target for calcium flux regulation) and my alternative\nmodel (VDACs as structural partners of EMC, mediating 225 activity via this complex).\n2. Inhibition of ERAD and activation of UPR by 225:\n225 blocks degradation of NHK and HA-DeltaF508CFTR, canonical ERAD substrates. It also induces expression of BiP,\nCHOP, and ATF4. The authors ascribe this to ER calcium dysregulation. However, a simpler model is that 225-bound\nVDAC stabilizes EMC in a non-insertase state, blocking the membrane integration of Derlin-1 and other EMC-dependent\nERAD components, thereby directly impairing ERAD and triggering proteostatic stress. This hypothesis explains all\nobserved consequences (ERAD block, UPR, cell death) without invoking a poorly defined Ca2+ channeling role for VDACs.\n3. Triple VDAC1 KD / VDAC2⁻/⁻ / VDAC3⁻/⁻ cells resist 225 phenotypes:\nThe authors interpret this as evidence that VDACs are the functional conduits for calcium flux perturbed by 225.\nHowever, in the alternative model, the absence of VDACs prevents 225 from stabilizing the EMC–VDAC complex. This\nrestores EMC function and Derlin-1 insertion, preserving ERAD and preventing downstream UPR and Ca2+\ndysregulation. Thus, the triple KO phenotype is compatible with both models.\n4. ER calcium flux and 225-induced cell death:\nCalcium flux is clearly altered by 225, but the authors do not provide direct electrophysiological or structural evidence that\n225 opens or blocks VDAC pores. Given that VDAC is primarily known as a metabolite channel in the outer mitochondrial\nmembrane, and that Ca2+ flow between ER and mitochondria typically involves IP3R-GRP75-VDAC tethers, it remains\nuncertain whether VDAC alone mediates Ca2+ leak. In contrast, the EMC-centered model predicts that ER stress and UPR\nactivation are primary, and that calcium dysregulation is a downstream effect of ER proteostasis failure.\nConclusion:\nIn light of published structural and proteomic data, the model in which compound 225 exerts its effects by stabilizing a non-\ninsertase EMC–VDAC complex provides a mechanistically unified explanation of all observed phenotypes: ERAD\ninhibition, UPR activation, and calcium flux. It avoids invoking a direct gating effect of 225 on VDACs, for which no structural\nor biophysical support is currently provided. I suggest the authors consider this alternative mechanism in the discussion and\ntest whether EMC activity or Derlin-1 insertion is altered upon 225 treatment.\nThis model is not incompatible with the authors' data, but may better contextualize the role of VDACs as structural, rather\nthan purely channel, components at MAMs. It also makes distinct predictions about EMC dependency that could be\nexperimentally tested.\nSuggested experiments:\nAssess Derlin-1 levels and membrane insertion after 225 treatment\nTest EMC–VDAC complex stability or conformation changes in the presence of 225\nCompare 225 sensitivity in cells with EMC subunit knockdown or mutation\nClarity and Context\nThe manuscript is clearly written and accessible. Figures are well designed and support the main conclusions. The\ndiscussion appropriately contextualizes the findings within existing literature and discusses limitations transparently. The\nnovelty of VDACs in ERAD is well supported.\nReferences\nThe referencing is thorough and appropriate. Key prior work on ERAD, UPR, VDAC function, and chemical screening is\ncited. I did not note any major omissions.\nYour Expertise\nMy expertise includes ER proteostasis and chemical biology. I am confident in assessing the main findings and mechanistic\nclaims. I did not independently evaluate the full MS data or chemical synthesis protocols but found the presented validation\nto be sufficient.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Response: We thank the reviewers for their constructive comments. In response, we have included\nadditional mechanistic data to support the proposed model of action of 225 and in vivo evidence\ndemonstrating its potential for clinical development, which has significantly strengthened our\nwork.\nPoint-to-point response to reviewers\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nGeneral Assessment:\nYan et al. describe the identification of NCATS-225 as an ERAD inhibitor via a GFP-\nbased HTS and elucidate its mechanism involving VDAC binding, dysregulation of\ncalcium homeostasis, and selective cancer cell cytotoxicity. While the study identifies a\nnovel compound with an intriguing mechanism and therapeutic potential, several critical\nconclusions lack sufficient experimental validation. The mechanistic claims require\nsubstantial reinforcement to meet the journal's standards. Major revisions addressing\nthe following points are essential prior to reconsideration.\nMajor Concerns:\n1. HTS Compound Prioritization Rationale: The manuscript states that 6 compounds\nwere prioritized from 62 initial hits for further study. The specific criteria (e.g., potency,\nchemical tractability, structural diversity, lack of known liabilities) guiding this selection\nmust be explicitly defined. Without this justification, the rationale for focusing on 225\nremains unclear and potentially arbitrary.\nResponse: We thank the reviewer for this helpful comment. We have revised the text to clarify\nthe selection rationale. The six compounds were prioritized from 62 confirmed hits based on\npotency (AC50 < 20 μM), structural diversity, absence of covalent warheads or acute\ncytotoxicity, and chemical tractability for optimization. The revised text (Results section, page\n4) and updated Figs. 1d, e and S1d,e now explicitly describe this prioritization process.\n2. Mechanism of STIM1-PERK Affinity Enhancement (Fig 8e): The observation that\n225 enhances endogenous STIM1-PERK affinity is significant. However, as 225 is not\nproposed to directly bind either protein, the underlying mechanism for this effect\nremains entirely speculative. The authors must propose and experimentally test a\nplausible mechanism (e.g., involving Ca2 flux, ER stress sensor activation, or\n+\nintermediary adaptors) or significantly temper their interpretation of this result.\nResponse: We agree the Reviewer that the mechanism must be explicit. Our new data support\na Ca2+-driven model in which 225 acts at VDAC1 to remodel ER–mitochondria signaling and\nthereby increases STIM1–PERK affinity.\n1) Direct target and Ca2+ remodeling: Photoaffinity labeling/mass spectrometry and new\nresults from CETSA and MST identify VDAC1 as the direct target (Figs. 4–5). In in vitro\nreconstituted assay using recombinant VDAC1, 225 shifts VDAC1 to a lower-conductance\n(“closed”) state that favors mitochondrial Ca2+ uptake (Fig. 5c-e). This perturbs ER Ca2+ and\nelicits compensatory Ca2+ influx, resulting in elevation of both ER and cytosolic Ca2+.\n2) Complex remodeling requires Ca2+: Our data showed that under basal conditions, VDAC1 co-\nimmunoprecipitates with STIM1, PERK, and IP3R (Fig. 10j). Upon 225 treatment, VDAC1– IP3R\ninteraction increased, whereas VDAC1–STIM1 and VDAC1–PERK associations decreased (Fig.\n10j), coinciding with enhanced PERK–STIM1 interaction (Figs. 10e, 10k). Notably, the 225-\ninduced increases in VDAC1–IP3R and PERK–STIM1 interactions were abolished by Ca2+\nchelation (Figs. 9h, 10k). Furthermore, pharmacological inhibition of PERK did not prevent the\n225-induced PERK–STIM1 interaction (Fig. 10l), indicating that elevated intracellular Ca2+,\nrather than PERK catalytic activity, drives the enhanced PERK–STIM1 association.\nWorking model: Under resting conditions, a PERK–STIM1 complex associates with VDAC1 and\nlimits VDAC1–IP3R coupling. By altering VDAC1 gating, 225 promotes Ca2+ flux that\nstrengthens PERK–STIM1 association and releases them from VDAC1, enabling enhanced\nVDAC1–IP3R interaction. This explains the observed co-IP changes and the rise in PERK–\nSTIM1 interaction (Fig. 10e, j, k). We have revised the text and model (Fig. 11) to reflect this\nmechanism and anchored our interpretation to the new Ca2+-dependence and co-IP data.\n3. Clarity on ERAD Reporter Substrate (Fig 1e): The misfolded CD3δ-based drGFP\nreporter used for secondary HTS validation must be clearly defined upon its first\nmention in the main text (HTS section). Ensure consistent nomenclature between\ntext and Fig 1e.\nResponse: We thank the Reviewer for highlighting this point. We have now clearly defined\nthe misfolded CD3δ-based drGFP reporter upon its first mention in the HTS section (page 4)\nand ensured consistent nomenclature between the main text and Figure 1e.\n4. Validation of VDAC1-3 Binding: The identification of VDAC1-3 as targets via\nphotoaffinity labeling (PAL) and SDS-PAGE/MS is a key finding. However, confirmation\nusing orthogonal, quantitative biophysical methods is crucial to establish binding affinity,\nspecificity, and stoichiometry. Strongly recommend performing Cellular Thermal Shift\nAssay (CETSA) combined with either Isothermal Titration Calorimetry (ITC) or Surface\nPlasmon Resonance (SPR) to validate and quantify the direct interaction between 225\nand VDAC isoforms 1-3. Relying solely on PAL (inherently detecting covalent\ninteractions) is insufficient proof of the physiological, non-covalent binding implied in the\nproposed mechanism.\nResponse: We have validated 225 binding to all three VDAC isoforms using orthogonal biophysical\nand functional approaches. CETSA analysis confirmed engagement of VDAC1–3 in cells (Fig. 5b).\nIn an in vitro reconstitution system, 225 modulated the gating activity of recombinant VDAC1 (Figs.\n5c–e), providing functional evidence of direct interaction. Microscale thermophoresis (MST)\nfurther demonstrated a direct, high-affinity interaction between 225 and purified VDAC1 (Fig. 5a).\nAlthough recombinant VDAC2 and VDAC3 proteins were not available for MST analysis, the\nconsistent CETSA results across all isoforms, combined with functional and quantitative binding\ndata for VDAC1, provide strong evidence that 225 directly engages all three VDAC isoforms.\n5. Incomplete Exploration of Differential Cell Sensitivity: The differential sensitivity of\ncancer cell lines to 225 is a compelling observation with translational relevance. However,\nthe exploration of the proposed mechanism (involving ERAD/ER-phagy crosstalk) is\nsuperficial. To validate the hypothesis that ER-phagy capacity dictates sensitivity, the\nauthors should inhibit ER-phagy (e.g., via genetic knockdown of key regulators like\nFAM134B, SEC62, or CCPG1, or using pharmacological inhibitors like CQ/HCQ where\nappropriate) in resistant/low-sensitivity cell lines and assess whether this sensitizes them\nto 225-induced cytotoxicity. Demonstrating this functional link is essential to support the\nmodel in Figure 9.\nResponse: We appreciate the reviewer’s insightful comment regarding the mechanistic basis of\ndifferential cellular sensitivity to 225. We agree that this is an important question with clear\ntranslational relevance. In our original submission, we demonstrated that sensitivity to 225\ncorrelates with a prolonged and dysregulated increase in intracellular calcium. Given that\nVDAC1 is the primary target of 225 and is highly expressed in cancers, we investigated whether\nVDAC1 expression levels contribute to the observed differences in sensitivity. Notably, our new\ndata showed that VDAC1 protein levels were generally higher in 225-sensitive cells and lower in\ninsensitive or normal cells (Fig. 8d, e). However, one insensitive line (MDA-MB-231) displayed\nunexpectedly high VDAC1 expression (Fig. 8d, e), suggesting additional regulatory mechanisms\nmay be involved. We speculate that mutations affecting downstream death-signaling pathways\nmay account for this discrepancy. Collectively, these data suggest that VDAC1 abundance\ninfluences the extent of 225-induced calcium dysregulation, contributing to differential cellular\nsensitivity.\nWe would like to clarify that our working model does not posit that “ER-phagy capacity dictates\nsensitivity.” Rather, our data indicate that VDAC1 normally restrains ER-phagy activation, since\nVDAC1 knockdown leads to ER-phagy induction (Fig. 4f-g). This increase in ER-phagy, in turn,\naccelerates the clearance of ERAD substrates (Fig. 4h, i), which may reduce substrate\navailability for dislocation and thereby dampen downstream stress signaling.\nBased on our current data, ER-phagy capacity does not appear to drive the differential sensitivity\nto 225. Therefore, experiments such as genetic or pharmacologic inhibition of ER-phagy in\nresistant cell lines would not directly test our model at this stage. Instead, our findings\nconsistently implicate 225-induced disruption of calcium homeostasis as the key differentiating\nfactor and its target (VDAC1) expression levels are likely to contribute to the sensitivity\ndifferences among cancer cell lines. Thus, the lower levels of VDAC1 expression in normal cells\nmay explain why 225 is not toxic to normal cells. Overall, calcium dysregulation, not ER-phagy\ncapacity, remains the most plausible driver of sensitivity. Our new data on VDAC1 protein\ninteractions and 225s’ impact substantially advance our understanding of its mechanism of\naction and will guide future studies of differential toxicity. Because calcium regulation is highly\ncomplex, we believe that fully defining the mechanism lies beyond the scope of this study.\n6. Lack of In Vivo Efficacy and Safety Data: Demonstrating potent and selective cancer\ncell killing in vitro is promising, but in vivo validation is a fundamental requirement for a\nmechanistic/therapeutic study of this nature in Nature Communications. The authors\nshould provide evidence of 225's efficacy in relevant tumor xenograft or syngeneic\nmodels, alongside assessment of toxicity in normal tissues. This is critical for\nestablishing the compound's therapeutic window and translational potential.\nResponse: We thank the reviewer for highlighting the importance of in vivo validation to\nestablish the translational potential of 225. We have now added new in vivo data into the revised\nmanuscript (Fig. 7c-e and S7g-i). Specifically, 225 significantly inhibits tumor growth in the\nA375 xenograft mouse model (Fig. 7c-e). Importantly, treated animals showed no clinical signs\nof toxicity, and histopathological analysis of liver, pancreas, and kidney revealed no abnormality\n(Fig. S7g-i). These results demonstrate both the anti-tumor efficacy and favorable safety profile\nof 225 in vivo, thereby supporting its translational relevance.\nReviewer #2 (Remarks to the Author):\nIn this study by Yan et al., the authors developed a chemical screening platform based\non an ERAD dislocation reporter. The screen identified compound 225 as a small-\nmolecule ERAD inhibitor. They further dissected the mechanism of action of 225 by\nidentifying the VDACs as the drug targets, demonstrating disruption of calcium\nhomeostasis, and linking the PERK/STIM1 axis to degradation of ERAD complex\nproteins and cell death. Overall, this is an interesting study that provides novel insights\ninto the regulation of UPR, calcium homeostasis, ERAD, and ER-phagy.\nSeveral minor comments are listed below:\n1. Figure 3a: The authors mentioned the degradation of OS9, HERP1, and FAM8A1.\nHowever, the data also show a reduction in SEL1L at 24 hours post-treatment. This\nshould also be noted in the text.\nResponse: We thank the reviewer for pointing this out. We have updated the manuscript\nto include the observed reduction in SEL1L at 24 hours post-treatment (page 5)..\n2. Figure 3: The authors should examine transcriptomic changes in the RNA-seq data\nfor OS9, HERP1, FAM8A1, and SEL1L mRNA levels to rule out mRNA downregulation\nas the cause of decreased protein levels.\nResponse: We have added the RNA-seq data as requested by the Reviewer (Fig. S3b). 225 slightly\nincreased HERP1 and OS9 mRNA, slightly decreased FAM8A1 mRNA, and caused no changes in\nSEL1L mRNA. These data ruled out mRNA downregulation as the cause of decreased protein\nlevels. In addition, we have shown that HERP1, OS9, and FAM8A1 downregulation is due to\nproteasomal degradation likely via ERAD in our first submission (Fig. S3a, c).\n3. Figures 2d–g and 4i: Treatment with 225 significantly inhibited the degradation of\nboth NHK and CD3, resulting in increased protein levels. However, in Figure 4i,\nsiVDAC1 treatment reduced CD3-mCherry levels. These findings seem\ninconsistent and should be clarified.\nResponse: We thank the reviewer for raising this point. We would like to clarify that the effects\nof siVDAC1 on CD3δ-mCherry shown in Figure 4i are mechanistically distinct from those of\n225. Specifically, VDAC1 knockdown induces ER-phagy, which accelerates the clearance of\nERAD substrates such as CD3δ-mCherry. In contrast, treatment with 225 inhibits ERAD,\nleading to stabilization and accumulation of CD3δ-mCherry. Thus, the apparent discrepancy\nreflects two different mechanisms of action rather than inconsistent data.\n4. The term “dislocation” should be defined earlier in the manuscript for clarity.\nResponse: We thank the reviewer for this suggestion. We have now defined “dislocation” in\nthe first paragraph of the Introduction as part of our ERAD description: “ER-associated\ndegradation (ERAD) is a key quality-control pathway in which misfolded proteins in the\nendoplasmic reticulum (ER) are recognized, retrotranslocated to the cytosolic surface for\nubiquitination and dislocation, and subsequently delivered to the proteasome for\ndegradation.”(page 2). This introduces the term at first mention and aligns with its usage\nthroughout the manuscript.\nReviewer #3 (Remarks to the Author):\nYan and colleagues present compelling evidence that stress-induced apoptosis in\ncancer cells can be increased by suppressing the Endoplasmic Reticulum-Associated\nDegradation (ERAD) pathway using small molecules. Through an extensive drug\nscreening campaign employing a clever designed readout, they identified novel ERAD\ninhibitors. One of the most potent molecules identified, compound 225, not only inhibits\nERAD but also binds the mitochondrial porin VDAC1. While a direct link between VDAC1\nand the ERAD pathway has not been conclusively established before, the authors\nprovide strong evidence supporting VDAC1 involvement in ERAD regulation.\nThe manuscript is data-rich and covers several key aspects, including the drug\nscreening process, interaction studies (IP experiments), and mechanistic insights\nthat collectively support the proposed conclusions. However, I have several\nimportant questions and concerns that should be addressed prior to publication:\nMajor Points:\n1. The authors demonstrate that compound 225 stabilizes the interaction between\nVDAC1 and the IP3 receptor. Do the authors have mechanistic insights or supporting\ndata explaining why this interaction is enhanced in the presence of molecule 225? Is\nthis effect direct, or could it result from broader changes in organelle physiology?\nResponse: We agree that a mechanistic explanation is needed. Our new data support a VDAC1-\ncentric, Ca2+-dependent mechanism by which 225 enhances the VDAC1–IP3R interaction.\n1) Direct target and Ca2+ remodeling: Photoaffinity labeling/mass spectrometry and new results\nfrom CETSA and MST identify VDAC1 as the direct target (Figs. 4–5). In in vitro reconstituted\nassay using recombinant VDAC1, 225 shifts VDAC1 to a lower-conductance (“closed”) state\nthat favors mitochondrial Ca2+ uptake (Fig. 5c-e). This perturbs ER Ca2+ and elicits\ncompensatory Ca2+ influx, resulting in elevation of both ER and cytosolic Ca2+.\n2) Complex remodeling requires Ca2+: Our data showed that under basal conditions, VDAC1 co-\nimmunoprecipitates with STIM1, PERK, and IP3R (Fig. 10j). Upon 225 treatment, VDAC1– IP3R\ninteraction increased, whereas VDAC1–STIM1 and VDAC1–PERK associations decreased (Fig.\n10j), coinciding with enhanced PERK–STIM1 interaction (Figs. 10e, 10k). Notably, the 225-\ninduced increases in VDAC1–IP3R and PERK–STIM1 interactions were abolished by Ca2+\nchelation (Figs. 9h, 10k). Furthermore, pharmacological inhibition of PERK did not prevent the\n225-induced PERK–STIM1 interaction (Fig. 10l), indicating that elevated intracellular Ca2+,\nrather than PERK catalytic activity, drives the enhanced PERK–STIM1 association.\nWorking model: Under resting conditions, a PERK–STIM1 complex associates with VDAC1 and\nlimits VDAC1–IP3R coupling. By altering VDAC1 gating, 225 promotes Ca2+ flux that\nstrengthens PERK–STIM1 association and releases them from VDAC1, enabling enhanced\nVDAC1–IP3R interaction. This explains the observed co-IP changes and the rise in PERK–\nSTIM1 interaction (Fig. 10e, j, k). We have revised the text and model (Fig. 11) to reflect this\nmechanism and anchored our interpretation to the new Ca2+-dependence and co-IP data.\nFunctional model: This alteration in VDAC] protein interactions yields two modules: VDAC]–\nIP3R (facilitating mitochondrial Ca2+ transfer) and PERK–STIM] (ERAD inhibition), aligning\nwith our phenotypic readouts.\n2. Compound 225 appears to have dual effects: it activates PERK, resulting in STIM1\nphosphorylation, HERP1 degradation, and ERAD inhibition; while also binding to\nVDAC1. However, UV-crosslinking identified VDACs as the only direct targets. How\ndo the authors reconcile this dual activity? Is there evidence that the STIM1–PERK\ninteraction is influenced by the VDAC–IP3R complex? Could the authors attempt IP\nexperiments to pull down VDAC using STIM1 or PERK in the presence of 225 to test\nfor complex formation?\nResponse: We appreciate the reviewer’s question. We reconcile the apparent “dual activity”\nwith a single-target model: 225 directly engages VDACs, and the PERK/STIM] phenotypes arise\nsecondarily from VDAC-driven Ca2+ remodeling at ER–mitochondria contact sites and\nsubsequently disruption of calcium homeostasis. Consistent with this, photoaffinity labeling\nidentifies VDAC]–3 as the only direct targets (Fig. 4a-c); CETSA shows thermal stabilization of\nVDACs (Fig. 5b); MST confirms binding to recombinant VDAC] (Fig. 5a); and an in vitro\nreconstituted VDAC] channel assay demonstrates 225-dependent gating modulation (Fig. 5c-e).\nIn addition, we do not detect 225 binding to PERK or STIM] by photoaffinity labeling (Fig. 4a-\nc).\nTo determine whether the STIM]–PERK interaction is influenced by the VDAC–IP3R complex,\nwe performed co-immunoprecipitations using VDAC] as bait. VDAC] co-precipitated IP3R,\nSTIM], and PERK (Fig. ]0j). Treatment with 225 increased VDAC]–IP3R association while\ndecreasing VDAC] interactions with STIM] and PERK (Fig. ]0j). Interestingly, in first\nsubmission, we showed that 225 enhanced the STIM]–PERK interaction (Fig. ]0e). Now we\nshow that both the 225-induced VDAC]–IP3R association and STIM]–PERK interaction was\nattenuated by intracellular Ca2+ chelation (BAPTA-AM) (Fig. 9h, ]0k), indicating Ca2+\ndependence. Collectively, these data led us to propose the working model described earlier:\nunder resting conditions, a PERK–STIM] complex associates with VDAC] and limits VDAC]–\nIP3R coupling. By altering VDAC] gating, 225 promotes Ca2+ flux that strengthens PERK–\nSTIM] association and releases them from VDAC], enabling enhanced VDAC]–IP3R\ninteraction. Functionally, blocking IP3R or knocking down MCU reduces 225-associated\ncytotoxicity, consistent with a role for the VDAC–IP3R–MCU axis in propagating mitochondrial\nCa2+ uptake and downstream effects.\n3. The manuscript claims that molecule 225 selectively targets cancer cells without\naffecting healthy cells. Given that VDAC1 is overexpressed in many solid tumors, did\nthe authors assess VDAC1 expression levels in 225-sensitive versus -insensitive cell\nlines? Quantifying VDAC1 levels by RNA-seq and Western blot would help clarify\nwhether sensitivity to 225 correlates with VDAC1 expression and might explain the\nlack of toxicity in healthy cells.\nResponse: We agree this is an important question with clear translational relevance. As\nsuggested by the reviewer, we examined VDAC1 expression at both the mRNA and protein levels\nin 225-sensitive versus -insensitive cell lines. We found that VDAC1 protein levels are generally\nhigher in sensitive cells and lower in insensitive and normal cells (Fig. 8d,e), although one\ninsensitive line (MDA-MB-231) exhibited unexpectedly high VDAC1 protein levels. We observed\nno correlation between VDAC1 transcript abundance (analyzed public RNA-seq datasets\n(https://www.proteinatlas.org) and protein levels (Fig. 8d,e vs. Fig. S8a), indicating that steady-\nstate VDAC1 protein abundance is likely regulated by translational efficiency or protein\nstability rather than mRNA expression. Consistent with this, treatment of HeLa cells (a sensitive\nline) with the proteasome inhibitor bortezomib stabilized VDAC1 protein (Fig. S8b), suggesting\nthat VDAC1 is an unstable protein subject to proteasomal degradation. Taken together, these\nfindings indicate that VDAC1 protein abundance, rather than mRNA expression alone,\ncontributes to the differential sensitivity to 225.\n4. The authors propose that molecule 225 enhances VDAC1–IP3R interaction,\npromoting calcium transfer between the ER and mitochondria, which subsequently\nactivates PERK and drives ERAD inhibition. However, this does not fully explain the\nobserved increase in cytosolic calcium (Fig. 7c). Could the authors comment on the\nsource of the cytosolic calcium and clarify whether this increase is a direct or indirect\nconsequence of 225 treatment?\nResponse: We appreciate the reviewer’s question. We added new experiment using in vitro\nreconstituted VDAC1 gating assay and found that 225 shifts VDAC1 into a “closed” state\nfavoring mitochondrial Ca2+ uptake (Fig. 5), which is consistent with our observations that\n225 increases VDAC1-IP3R interaction and calcium influx to mitochondria. We propose that\nthe mitochondrial calcium influx transiently lowers ER Ca2+, triggering compensatory Ca2+\ninflux from the extracellular space into the cytosol. The resulting elevation of cytosolic calcium\nreplenishes ER stores and ultimately increases calcium across the ER, cytosol, and\nmitochondria, resulting in compartment-wide Ca2+ elevation, as shown in our first\nsubmission. This model of action for 225 are supported by new data\n5. The small molecule efsevin has been shown to stabilize the calcium-conductive\nform of VDAC1 (PMID: 32059260). It would be informative to compare the effects of\nefsevin and compound 225 on ERAD inhibition. Do both compounds share a similar\nmechanism of action? Such a comparison might strengthen the proposed model.\nResponse: As suggested by the reviewer, we compared efsevin with compound 225 for their\neffects on dislocation inhibition and on degradation of HERP1, OS9, and FAM8A1. Efsevin\nshowed no detectable effect on dislocation or on degradation of these ERAD complex proteins\n(Fig. S2a,b), indicating that its mechanism of action differs from that of compound 225.\nMinor Points:\nScheme on Figure 8j should be revised to clearly depict relevant subcellular\ncompartments, improve image quality, and remove the pink background. Additionally,\nthe graph located at the bottom left appears to be an error and should be removed.\nResponse: The scheme has been revised as requested to clearly depict the relevant subcellular\ncompartments, improve image quality, and remove the pink background. The erroneous graph\nat the bottom left has also been removed. The updated version now appears as Figure 11.\nReviewer #4 (Remarks to the Author):\nKey Results\nThis manuscript identifies a novel small molecule, NCATS-225 (“225”), as a selective\nERAD inhibitor that binds to voltage-dependent anion channels (VDACs). Through a\ncombination of high-content screening, biochemical validation, and mechanistic\ndissection, the authors suggest that 225 triggers an intracellular calcium imbalance via\na VDAC-IP3R-PERK-STIM1 signaling axis. This leads to the degradation of ERAD\ncomplex components (HERP1, OS9, FAM8A1) and induces cell death selectively in\ncancer cells while sparing normal cells. The work uncovers a previously unrecognized\nrole for VDACs in ERAD regulation and presents a compelling chemical probe for\nfurther study of ER proteostasis and cancer-selective cytotoxicity.\nValidity\nThe data are of high quality, robust, and generally well-controlled. The experimental\nworkflow is thorough, including orthogonal validation of dislocation inhibition (GFP\nreporters, CHX chase, ubiquitination assays), target identification (photoaffinity labeling\nand MS), and functional studies using gene knockouts and chemical inhibitors.\nHowever, the precise mechanism of 225 binding to VDACs and how this affects VDAC\nfunction at a structural level remains speculative. The authors acknowledge this and\npropose it as future work.\nResponse: Thank you for the positive assessment. In this revision, we add new evidence of direct\n225–VDAC engagement by CETSA and MST (Fig. 5), complementing the photoaffinity\nlabeling/MS from the initial submission (Fig. 4). We also show in a reconstituted VDAC1 assay\nthat 225 shifts channel gating toward a lower-conductance (“closed”) state that favors\nmitochondrial Ca2+ uptake (Fig. 5). We acknowledge that the atomic binding mode and\nstructural basis of this effect remain to be defined and outline these as future work.\nSignificance\nThe identification of a VDAC-targeting small molecule that selectively disrupts ERAD\nand kills cancer cells is novel and potentially impactful. This work opens new avenues\nin cancer biology, proteostasis, and chemical biology. It also proposes an alternative\nstrategy to targeting UPR sensors directly — by modulating ERAD upstream. The\nselective toxicity of 225 to certain cancer cell lines suggests translational relevance.\nWhile the mechanistic basis of this selectivity is not fully resolved, the dataset\nlays a solid foundation for follow-up studies.\nResponse: Thank you for the thoughtful comments. We agree the work is novel and impactful. In\nthis revision, we began to address the basis of selectivity by quantifying VDAC1 at both mRNA\nand protein levels in 225-sensitive versus -insensitive lines. Protein abundance was generally\nhigher in sensitive lines and lower in insensitive or normal cells (Fig. 8d,e), with one outlier\n(MDA-MB-231) showing high VDAC1 despite relative insensitivity. By contrast, VDAC1\ntranscripts from public RNA-seq (Human Protein Atlas) did not correlate with protein levels or\nsensitivity (Fig. S8a). In HeLa cells, the proteasome inhibitor bortezomib stabilized VDAC1\nprotein (Fig. S8b), consistent with proteasomal turnover. Together, these data suggest that\nVDAC1 protein abundance contributes to 225 selectivity, but additional factors must be\ninvolved and warrants future investigations.\nData and Methodology\nThe methodology is state-of-the-art and spans a wide range of experimental techniques,\nincluding high-content screening, SAR chemistry, CRISPR knockouts, calcium imaging,\nand GSEA transcriptomic profiling. The description of materials and methods is detailed\nand transparent. Use of the drGFP reporter for dislocation, combined with dose-\nresponses, time courses, and structure–activity relationships, makes the data solid.\nPhotoaffinity labeling with mass spectrometry is well executed, and VDAC target\nidentification is convincing. If available, raw calcium traces or proteomics data could be\nmade public in supplementary datasets.\nResponse: All source data for figures have been submitted in this resubmission. No\nadditional datasets need to be made available in public database.\nAnalytical Approach\nQuantitative results are consistently supported by replicates, appropriate statistical\ntests, and careful normalization. The use of dose-response curves, IC50 values, and\nmultiple replicates is appropriate. GSEA is correctly applied and complements the\nmechanistic data. The authors have been transparent about sample sizes and\nstatistical significance thresholds.\nSuggested Improvements\nThe authors present a compelling study identifying VDAC1, VDAC2, and VDAC3 as\ndirect binding partners of compound 225. They propose that 225 perturbs calcium\nhomeostasis at the ER-mitochondria interface via these outer mitochondrial membrane\nproteins, thereby inhibiting ER-associated degradation (ERAD), activating the unfolded\nprotein response (UPR), and triggering apoptosis. While the data are generally sound\nand well-presented, I believe an alternative, structurally grounded interpretation offers a\nmore parsimonious explanation of the experimental findings.\nRecent structural studies (Li et al., Aging 2023, DOI:10.18632/aging.205660) demonstrate that VDAC1 directly binds the ER\nmembrane protein complex (EMC) at ER-mitochondria contact sites (MAMs), locking the EMC in a non-insertase\nconformation.\nEarlier proteomic mapping\n(Christianson, J., Olzmann, J., Shaler, T. et al. Defining human ERAD networks through an integrative mapping strategy. Nat\nCell Biol 14, 93–105 (2012). https://doi.org/10.1038/ncb2383)\nshowed that multiple EMC subunits interact with Derlin-1 and Derlin-2, core components of the ERAD dislocation\nmachinery. This raises the possibility that compound 225 inhibits ERAD not by modulating VDAC channel activity directly,\nbut by stabilizing a non-functional EMC–VDAC complex that disrupts EMC-dependent membrane insertion of ERAD\ncomponents such as Derlin-1.\nBelow, I analyze the major data types presented by the authors in support of their model and offer the alternative EMC-mediated interpretation.\n1. Identification of VDAC1/2/3 as targets of 225:\nThe authors used a clickable photoaffinity analog of 225 to identify VDAC1, VDAC2, and VDAC3 as binding targets. This result is compatible with both their interpretation (VDAC as a functional target for calcium flux regulation) and my alternative model (VDACs as structural partners of EMC, mediating 225 activity via this complex).\n2. Inhibition of ERAD and activation of UPR by 225:\n225 blocks degradation of NHK and HA-DeltaF508CFTR, canonical ERAD substrates. It also induces expression of BiP, CHOP, and ATF4. The authors ascribe this to ER calcium dysregulation. However, a simpler model is that 225-bound VDAC stabilizes EMC in a non-insertase state, blocking the membrane integration of Derlin-1 and other EMC-dependent ERAD components, thereby directly impairing ERAD and triggering proteostatic stress. This hypothesis explains all observed consequences (ERAD block, UPR, cell death) without invoking a poorly defined Ca2+ channeling role for VDACs.\n3. Triple VDAC1 KD / VDAC2-/- / VDAC3-/- cells resist 225 phenotypes:\nThe authors interpret this as evidence that VDACs are the functional conduits for calcium flux perturbed by 225. However, in the alternative model, the absence of VDACs prevents 225 from stabilizing the EMC–VDAC complex. This restores EMC function and Derlin-1 insertion, preserving ERAD and preventing downstream UPR and Ca2+ dysregulation. Thus, the triple KO phenotype is compatible with both models.\nResponses to comment #1-3: We thank the reviewer for the thoughtful comments. To test possible\nEMC involvement in 225’s action, we performed a series of experiments. However, our\ndata do not support an EMC-mediated mechanism: (1) ERAD complex degradation persists with\nEMC knockdown, 225 still caused degradation of HERP1, OS9, and FAM8A1 in EMC3, EMC4,\nand EMC7 knockdown cells, similar to controls (Fig. S4e); (2) the mitochondrial Ca2+ rise\ninduced by 225 is unchanged by EMC3/EMC4/EMC7 knockdown (Fig. S4h); (3) the dislocation\nreporter is unaffected by EMC knockdown, EMC3 or EMC7 knockdown did not alter 225’s\ninhibition in the drGFP assay (Fig. S4f); and (4) VDAC–EMC interactions are unchanged by\n225, VDAC1 co-IP with EMC3 or EMC4 did not increase (Fig. S4g). Together, these results do\nnot support EMC as the mediator of 225 activity and instead align with direct VDAC targeting\nand Ca2+-dependent remodeling at ER–mitochondria contact sites.\n4. ER calcium flux and 225-induced cell death:\nCalcium flux is clearly altered by 225, but the authors do not provide direct\nelectrophysiological or structural evidence that 225 opens or blocks VDAC pores. Given\nthat VDAC is primarily known as a metabolite channel in the outer mitochondrial\nmembrane, and that Ca2+ flow between ER and mitochondria typically involves IP3R-\nGRP75-VDAC tethers, it remains uncertain whether VDAC alone mediates Ca2+ leak. In\ncontrast, the EMC-centered model predicts that ER stress and UPR activation are\nprimary, and that calcium dysregulation is a downstream effect of ER proteostasis\nfailure.\nResponse: We agree that direct functional evidence is essential. As noted above, EMC is not\ninvolved in 225’s action. We provide orthogonal target engagement (photoaffinity labeling/MS,\nCETSA, MST) showing that 225 binds VDAC1 (Figs. 4-5), and we add new data using a\nreconstituted VDAC1 gating assay, demonstrating that 225 shifts the channel toward a lower-\nconductance (“closed”) state that favors mitochondrial Ca2+ uptake (Fig. 5d-e). Consistent with\nER–mitochondria tethers, 225 enhances VDAC1–IP3R association, and this remodeling is lost\nwith Ca2+ chelation (Figs. 9g–h, 10k). Taken together, these data support a VDAC-centered,\nCa2+-dependent mechanism for the observed flux changes and cell death, while single-channel\nelectrophysiology and structural mapping of the binding site remain future work.\nConclusion:\nIn light of published structural and proteomic data, the model in which compound 225\nexerts its effects by stabilizing a non-insertase EMC–VDAC complex provides a\nmechanistically unified explanation of all observed phenotypes: ERAD inhibition, UPR\nactivation, and calcium flux. It avoids invoking a direct gating effect of 225 on VDACs,\nfor which no structural or biophysical support is currently provided. I suggest the\nauthors consider this alternative mechanism in the discussion and test whether EMC\nactivity or Derlin-1 insertion is altered upon 225 treatment.\nResponse: Please refer Response to # 1-3 above.\nThis model is not incompatible with the authors' data, but may better contextualize the\nrole of VDACs as structural, rather than purely channel, components at MAMs. It also\nmakes distinct predictions about EMC dependency that could be experimentally tested.\nSuggested experiments:\nAssess Derlin-1 levels and membrane insertion after 225 treatment\nResponse: As noted above, EMC does not appear to mediate 225’s effects. Derlin-1 protein\nlevels were also not affected by 225 treatment (Fig. 3a); therefore, we did not assess Derlin-1\nmembrane insertion.\nTest EMC–VDAC complex stability or conformation changes in the presence of 225\nResponse: We tested whether 225 alters EMC–VDAC complex stability or conformation. VDAC1\nco-immunoprecipitation with EMC3 or EMC4 was unchanged in the presence of 225, indicating\nno detectable effect on EMC–VDAC complex stability (Fig. S4g).\nCompare 225 sensitivity in cells with EMC subunit knockdown or mutation\nResponse: We tested EMC involvement directly. Knockdown of EMC3, EMC4, or EMC7 did not\nchange (i) 225-induced degradation of ERAD complex proteins (HERP1, OS9, FAM8A1), (ii) the\nmitochondrial Ca2+ rise, or (iii) dislocation inhibition in the drGFP assay, and 225 did not\nincrease VDAC1 co-IP with EMC3/4 (Fig. S4). Because these activity-linked readouts were\nunchanged, EMC loss did not appear to modulate 225 sensitivity; therefore, we did not pursue\nadditional viability assays in EMC-mutant backgrounds.\nClarity and Context\nThe manuscript is clearly written and accessible. Figures are well designed and support\nthe main conclusions. The discussion appropriately contextualizes the findings within\nexisting literature and discusses limitations transparently. The novelty of VDACs in\nERAD is well supported.\nReferences\nThe referencing is thorough and appropriate. Key prior work on ERAD, UPR, VDAC\nfunction, and chemical screening is cited. I did not note any major omissions.\nYour Expertise\nMy expertise includes ER proteostasis and chemical biology. I am confident in\nassessing the main findings and mechanistic claims. I did not independently evaluate\nthe full MS data or chemical synthesis protocols but found the presented validation\nto be sufficient.\nReviewer #5 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed\nreports. This is part of the Nature Communications initiative to facilitate training in\npeer review and to provide appropriate recognition for Early Career Researchers\nwho co-review manuscripts.\nResponse: Thank you for co-reviewing our manuscript as part of Nature Communications ’\ninitiative to train and recognize Early Career Researchers. We appreciate the time and\nthoughtful evaluation from both you and your co-reviewer.\nPoint-to-point response to the Reviewers\nWe are grateful for the opportunity to resubmit our revised manuscript and sincerely thank\nReviewers #1, #2, #4, and #5 for their positive recommendations for acceptance in Nature\nCommunications. We also appreciate the constructive comments from Reviewer #3 and have\nrevised the data accordingly. We believe that the thoughtful feedback from all reviewers has\nsubstantially improved the quality of the manuscript.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nI would like to thank the authors for their comprehensive response to my previous\ncomments. The additional data and revisions have significantly improved the\nmanuscript, and I am impressed with the work. I am happy to recommend the\nmanuscript for acceptance. Congratulations to the team on a fine piece of research!\nResponse:\nWe sincerely thank Reviewer #1 for the positive evaluation and encouraging comments. We\ngreatly appreciate your support and are pleased that the revisions have strengthened the\nmanuscript.\nReviewer #2 (Remarks to the Author):\nThe authors have successfully addressed my comments. No additional questions.\nResponse:\nWe thank Reviewer #2 for acknowledging that our revisions have addressed all prior comments.\nWe appreciate your time and consideration in reviewing our manuscript.\nReviewer #3 (Remarks to the Author):\nThe authors have satisfactorily addressed all my previous concerns, many of which\nwere also raised by the other reviewers. In particular, the additional experiments now\nprovide stronger support for their proposed working model. The revised data establish a\nclear mechanism of action for compound 225, which stabilizes the VDAC1–IP3R\ninteraction at the expense of STIM1 and PERK association.\nI appreciate the authors’ efforts to confirm the direct binding of 225 to VDAC1 using\northogonal approaches, including electrophysiological assays that demonstrate an\neffect on VDAC1 gating consistent with stabilization of its closed state. However, I have\none remaining point of clarification. The reported dissociation constant (Kd) of ~3 μM for\n225–VDAC1 interaction (as determined by MST) suggests a moderate affinity. Given\nthis, why were the bilayer experiments performed using only 400 nM compound? At\nsuch a concentration—well below the K_D—binding would be expected to be minimal.\nIndeed, the dose–response curve in Figure 5a suggests little to no effect at this\nconcentration, yet the electrophysiological recordings show a measurable change in\nG/G_max. While binding and functional efficacy are not necessarily identical processes,\nbinding must occur for an effect to be observed. How do the authors reconcile this\napparent discrepancy between binding affinity and electrophysiological response at sub-\nKd concentrations (200–400 nM)?\nAlong similar lines, in Figure 5c, the representative traces appear to have been acquired\nat different voltages across the conditions (±60 mV for control, ±40 mV for 200 nM 225,\nand ±30 mV for 400 nM 225). Could the authors clarify the rationale behind using\ndifferent potentials for each condition? For better comparison, I suggest presenting\nrepresentative traces recorded at the same voltages for all experimental conditions, or\nalternatively including a supplemental figure showing the full voltage range. As currently\npresented, it is difficult to directly appreciate the effect of compound 225 across\nconditions in Figure 5c.\nResponse:\nOur orthogonal validation demonstrates that compound 225 exhibits a highly favorable, context-\ndependent mechanism of target engagement. We confirmed direct target engagement with a 3\nµM binding affinity using Microscale Thermophoresis (MST). However, when we assessed the\ncompound in a physiologically relevant functional assay, using VDAC1 reconstituted into its\nnative membrane environment, we observed a significantly enhanced functional potency of 400\nnM. This 7.5-fold increase in potency is a key finding. It indicates that the compound's activity is\noptimized in the correct biological context. The most plausible explanation is a membrane-\ninduced conformational change: VDAC1 likely adopts its specific, functional conformation only\nwithin a true lipid bilayer. 225 appears to have a much higher affinity for this active, membrane-\nembedded state. The MST assay, which measures an average affinity across all conformations in\ndetergent, reports a weaker value. This model is strongly supported by our cellular data, which\nshows an IC50 of 1 µM in inhibiting dislocation in cells, a context where VDAC1 is naturally\nanchored in the lipid membrane. Therefore, we have high confidence that the sub-micromolar\npotencies (400 nM - 1 µM) are the biologically and therapeutically relevant metrics,\ndemonstrating that our compound is a potent and functionally active inhibitor of VDAC1.\nFollowing the Reviewer's helpful suggestion, we modified Figure 5c and present the single-\nchannel current traces at the same applied voltages from -30 to 60 mV for each condition. The\nlowest voltages at which VDAC1 gating was observed in control and in the presence of 225 are\nindicated by arrows.The corresponding discussion has been added to the revised manuscript\n(page 12 in red).\nReviewer #4 (Remarks to the Author):\nI am happy with (and impressed by!) the new data gathered and presented and I\nrecommend the manuscript for publication.\nResponse:\nWe thank Reviewer #4 for the positive and encouraging comments. We greatly appreciate your\nrecognition of the new data and are pleased that our revisions meet your expectations.\nReviewer #5 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nResponse:\nWe thank Reviewer #5 for the co-review and appreciate Nature Communications’ initiative to\nsupport and recognize Early Career Researchers in the peer review process.\nREVIEWERS' COMMENTS\nReviewer #3 (Remarks to the Author):\nI thank the authors for their thorough and thoughtful response to my previous\ncomments. The additional data and revisions have substantially strengthened the\nmanuscript, and I am pleased with the improvements made. However, I noticed that the\nscale bars in Fig. 5c are now missing, whereas they were present in the previous\nversion of the manuscript. Aside from this minor issue, I am happy to recommend the\nmanuscript for acceptance.\nResponse:\nWe sincerely thank the reviewer for the thoughtful and positive evaluation of our work, as well\nas for the helpful comments that have strengthened the manuscript. We apologize for the\noversight regarding Fig. 5c, and the missing scale bar has now been added in the revised version."}, {"round": 2, "reviewer_comments": {"reviewer_1": "I would like to thank the authors for their comprehensive response to my previous comments. The additional data and revisions have significantly improved the manuscript, and I am impressed with the work. I am happy to recommend the manuscript for acceptance. Congratulations to the team on a fine piece of research!", "reviewer_2": "The authors have successfully addressed my comments. No additional questions.", "reviewer_3": "The authors have satisfactorily addressed all my previous concerns, many of which were also raised by the other reviewers. In particular, the additional experiments now provide stronger support for their proposed working model. The revised data establish a clear mechanism of action for compound 225, which stabilizes the VDAC1–IP3R interaction at the expense of STIM1 and PERK association.\nI appreciate the authors’ efforts to confirm the direct binding of 225 to VDAC1 using orthogonal approaches, including electrophysiological assays that demonstrate an effect on VDAC1 gating consistent with stabilization of its closed state. However, I have one remaining point of clarification. The reported dissociation constant (Kd) of ~3 μM for 225–VDAC1 interaction (as determined by MST) suggests a moderate affinity. Given this, why were the bilayer experiments performed using only 400 nM compound? At such a concentration—well below the K_D—binding would be expected to be minimal. Indeed, the dose–response curve in Figure 5a suggests little to no effect at this concentration, yet the electrophysiological recordings show a measurable change in G/G_max. While binding and functional efficacy are not necessarily identical processes, binding must occur for an effect to be observed. How do the authors reconcile this apparent discrepancy between binding affinity and electrophysiological response at sub-Kd concentrations (200–400 nM)?\nAlong similar lines, in Figure 5c, the representative traces appear to have been acquired at different voltages across the conditions (±60 mV for control, ±40 mV for 200 nM 225, and ±30 mV for 400 nM 225). Could the authors clarify the rationale behind using different potentials for each condition? For better comparison, I suggest presenting representative traces recorded at the same voltages for all experimental conditions, or alternatively including a supplemental figure showing the full voltage range. As currently presented, it is difficult to directly appreciate the effect of compound 225 across conditions in Figure 5c.", "reviewer_4": "I am happy with (and impressed by!) the new data gathered and presented and I recommend the manuscript for publication.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Response:\nOur orthogonal validation demonstrates that compound 225 exhibits a highly favorable, context-dependent mechanism of target engagement. We confirmed direct target engagement with a 3 µM binding affinity using Microscale Thermophoresis (MST). However, when we assessed the compound in a physiologically relevant functional assay, using VDAC1 reconstituted into its native membrane environment, we observed a significantly enhanced functional potency of 400 nM. This 7.5-fold increase in potency is a key finding. It indicates that the compound's activity is optimized in the correct biological context. The most plausible explanation is a membrane-induced conformational change: VDAC1 likely adopts its specific, functional conformation only within a true lipid bilayer. 225 appears to have a much higher affinity for this active, membrane-embedded state. The MST assay, which measures an average affinity across all conformations in detergent, reports a weaker value. This model is strongly supported by our cellular data, which shows an IC50 of 1 µM in inhibiting dislocation in cells, a context where VDAC1 is naturally anchored in the lipid membrane. Therefore, we have high confidence that the sub-micromolar potencies (400 nM - 1 µM) are the biologically and therapeutically relevant metrics, demonstrating that our compound is a potent and functionally active inhibitor of VDAC1.\nFollowing the Reviewer's helpful suggestion, we modified Figure 5c and present the single-channel current traces at the same applied voltages from -30 to 60 mV for each condition. The lowest voltages at which VDAC1 gating was observed in control and in the presence of 225 are indicated by arrows.The corresponding discussion has been added to the revised manuscript (page 12 in red)."}, {"round": 3, "reviewer_comments": {"reviewer_3": "I thank the authors for their thorough and thoughtful response to my previous comments. The additional data and revisions have substantially strengthened the manuscript, and I am pleased with the improvements made. However, I noticed that the scale bars in Fig. 5c are now missing, whereas they were present in the previous version of the manuscript. Aside from this minor issue, I am happy to recommend the manuscript for acceptance."}, "author_response": "Response:\nWe sincerely thank the reviewer for the thoughtful and positive evaluation of our work, as well as for the helpful comments that have strengthened the manuscript. We apologize for the oversight regarding Fig. 5c, and the missing scale bar has now been added in the revised version."}]

Decision letter