ATF6 is one of three sentinels of the unfolded protein response, a cellular quality-control system that monitors the endoplasmic reticulum, the organelle where newly made proteins fold into their functional shapes. Its two counterparts, PERK and IRE1α, respond to an accumulation of misfolded proteins by throttling back protein synthesis and degrading faulty transcripts. ATF6 takes a different route: under stress it travels from the endoplasmic reticulum to the Golgi apparatus, where enzymes known as site-1 and site-2 protease cleave it into an active fragment called ATF6p50. This fragment then enters the nucleus and switches on genes encoding molecular chaperones, including BiP and Grp94, which boost the cell’s protein-folding capacity and help restore order in the endoplasmic reticulum. Far from being a mere emergency brake, ATF6 has emerged in recent years as an active defender of endothelial barriers, the tight cellular linings that control what passes into and out of tissues, and it also displays pronounced anti-inflammatory activity.

The research team, led by investigators at the University of Louisiana Monroe’s College of Pharmacy, asked a specific question: does ATF6 mediate the beneficial effects of growth hormone-releasing hormone antagonists, a family of synthetic peptides known for their anti-inflammatory and cytoprotective properties across many tissues, in corneal endothelial cells? Earlier work from the same group had shown that one such antagonist, JV-1-36, shields endothelial corneal cells from inflammation triggered by lipopolysaccharide, a bacterial toxin. JV-1-36 treatment reduced endothelial hyperpermeability, dampened the generation of reactive oxygen species, suppressed inflammatory signaling through the MAPK and STAT pathways, and lowered the production of pro-inflammatory cytokines. But the mechanism connecting the drug to those outcomes remained incomplete, and the unfolded protein response had already been implicated in the endothelial effects of these antagonists in lung models, both in cultured cells and in living animals.

To manipulate ATF6 pharmacologically, the team used two well-characterized small molecules that act on opposite ends of the pathway. AA147 promotes ATF6 activation, while Ceapin-A7 blocks it. Before drawing any conclusions about barrier function, the researchers first verified that neither compound was toxic to the bovine corneal endothelial cells they were studying, a line known as BCE C/D-1b. Using the MTT assay, which measures the metabolic activity of living cells, they found that both AA147, tested across a range of 1 to 200 micromolar, and Ceapin-A7, tested from 1 to 100 micromolar, left cell viability untouched between 1 and 25 micromolar. That safety window allowed the team to probe ATF6 signaling without the confounding effect of cell death, an essential control in studies of barrier integrity where membrane damage could masquerade as permeability changes.

The next step was to confirm that the two modulators genuinely moved the ATF6 needle. Western blot analysis of protein expression showed that AA147 increased the abundance of Grp94 and BiP, both canonical downstream targets of the activated transcription factor, in a manner that varied with dose and exposure time across 8, 16, and 24 hours. Ceapin-A7 did the opposite, suppressing the expression of both chaperones and thereby demonstrating that ATF6 signaling had been effectively silenced. These complementary results established a clean pharmacological system: one drug to turn the pathway up, another to turn it down, with two protein readouts to verify that the dial was actually moving. It is the kind of rigor that distinguishes pathway studies from simple drug-response experiments.

With the toolkit validated, the team turned to the central functional question. Barrier integrity was assessed with a fluorescein isothiocyanate dextran permeability assay, in which a 70-kilodalton fluorescent tracer is added to the top of a transwell culture insert and its passage through the cell monolayer is quantified by fluorescence measurement. When cells were pretreated with AA147 and then exposed to the growth hormone-releasing hormone antagonist, paracellular permeability dropped further than with either compound alone, indicating that activating ATF6 amplified the barrier-protective effect of the peptide. Conversely, pretreatment with Ceapin-A7 undermined the antagonist’s protection, and permeability rose significantly compared with cells receiving the antagonist alone. In other words, the same drug could be made more effective or less effective simply by dialing ATF6 up or down.

Oxidative stress told the same story from a different angle. Reactive oxygen species were measured with a dichlorofluorescein diacetate probe, which fluoresces upon oxidation inside living cells. Both the ATF6 activator and the growth hormone-releasing hormone antagonist reduced ROS generation on their own, and combined ATF6 activation with antagonist treatment produced a larger decline in ROS than either intervention by itself. When ATF6 was inhibited with Ceapin-A7, the antioxidant benefit of the antagonist was blunted and ROS levels climbed. This matters because excessive ROS is a major driver of endothelial dysfunction: it promotes cytoskeletal contraction, disrupts intercellular junctions, and activates redox-sensitive inflammatory signaling cascades such as NF-κB and MAPK. Moreover, oxidative stress and endoplasmic reticulum stress feed each other in a vicious loop, with impaired protein folding fueling further oxidant production and vice versa, accelerating barrier breakdown.

The clinical context gives these cellular findings their urgency. Corneal endothelial cell density is roughly 3,000 to 4,000 cells per square millimeter in newborns and declines by about 0.6 percent per year, falling to around 2,500 in adulthood and 2,000 in old age. Fuchs dystrophy, intraocular surgery, and inflammatory conditions such as keratitis accelerate that loss. Keratitis, whether caused by bacteria, viruses, fungi, or parasites, remains a leading cause of corneal morbidity worldwide, and contact lens wear is a prominent risk factor through microtrauma, hypoxia, and lapses in hygiene that invite microbial colonization. Once pathogen-associated molecular patterns engage the innate immune machinery of corneal endothelial cells, inflammatory pathways fire, junctions loosen, the stroma swells, and clarity fades. Current pharmacological options, including antibiotics, antivirals, antifungals, corticosteroids, and nonsteroidal anti-inflammatory drugs, can control infection and inflammation but carry real drawbacks, from secondary infections and delayed wound healing to elevated intraocular pressure. Therapies that restore barrier stability while sidestepping those side effects are badly needed.

That is where the therapeutic logic of this study becomes compelling. Growth hormone-releasing hormone antagonists have previously been shown to alleviate experimental ocular inflammation and to induce apoptosis selectively in retinoblastoma cells, and the present work demonstrates for the first time that ATF6 participates, at least in part, in mediating their protective effects on corneal endothelial paracellular permeability. The authors note an interesting wrinkle: somatostatin analogs, several of which are already FDA-approved, have been reported to activate ATF6, though they are not targeted ATF6 activators. That raises the possibility that existing drugs could be repurposed to engage this pathway while more selective ATF6 modulators are developed. The finding also echoes earlier work showing that ATF6 inhibition suppresses the barrier-protective effects of somatostatin analogs such as octreotide and pasireotide in other endothelial contexts, suggesting ATF6 may be a common final mediator of peptide-driven endothelial protection.

Important caveats remain before this science can reach the clinic. The experiments were performed in bovine corneal endothelial cells, and the team plans to validate the findings in human corneal endothelial cells and in rabbit models of corneal dysfunction, while also directly measuring ATF6 cleavage to reinforce pathway specificity. Off-target effects must be examined as well, particularly any impact on the other unfolded protein response branches, PERK and IRE1α, since those pathways also influence barrier function. Still, the core message is striking: a single stress-sensing transcription factor helps determine whether a protective peptide can keep the cornea’s delicate pump-leak machinery intact. If pharmacological activation of ATF6 can be harnessed safely, it could open a new front against inflammatory corneal disease, offering hope to the millions whose sight depends on the quiet, tireless work of a monolayer of cells most people never think about until it fails.

Subject of Research: The role of the unfolded protein response sensor ATF6 in mediating the protective effects of growth hormone-releasing hormone antagonists in the corneal endothelium

Article Title: Activating transcription factor 6 is involved in the beneficial effects of growth hormone-releasing hormone antagonists in the corneal endothelium

Article References: Sarker, M. M. R., Fakir, S., Sigdel, M., & Barabutis, N. (2026). Activating transcription factor 6 is involved in the beneficial effects of growth hormone-releasing hormone antagonists in the corneal endothelium. Frontiers in Ophthalmology, 6, Article 1946815. https://doi.org/10.3389/fopht.2026.1946815

Image Credits: AI Generated

DOI: 10.3389/fopht.2026.1946815

Keywords: corneal endothelium, ATF6, unfolded protein response, GHRH antagonists, barrier integrity, reactive oxygen species, keratitis, endoplasmic reticulum stress, BiP, Grp94, ocular inflammation, drug repurposing

Hepatocellular carcinoma, the most common primary liver cancer, remains one of the world’s deadliest malignancies. It is the third leading cause of cancer-related death globally, and although viral hepatitis, alcohol consumption, aflatoxin exposure and poor diet all contribute to its development, the clinical picture is dominated by a single grim statistic: most patients are diagnosed at an advanced stage, when surgical options such as resection, transplantation or ablation are no longer viable. Approved drug therapies for advanced disease, including multikinase inhibitors such as sorafenib, lenvatinib and cabozantinib, and antibody combinations built around agents like bevacizumab and atezolizumab, have extended survival only modestly. The field’s persistent hunger for molecules with new structures and new mechanisms is what makes the KGA-1002 story notable.

The compound’s origin lies in the chemistry of Artemisia, the genus that gave the world artemisinin. Sesquiterpenoid dimers, molecules in which two sesquiterpene units are joined, have repeatedly shown stronger antitumor activity than their monomeric parents, but their scarcity in plants has historically blocked serious drug development. The Kunming group sidestepped that bottleneck through biomimetic synthesis: using a Diels-Alder reaction, they assembled KGA-1002 from dehydrocostus lactone, an abundant natural sesquiterpenoid, on a ten-gram scale. The resulting dimer inhibited the proliferation of three hepatocellular carcinoma cell lines with IC50 values of 5.9, 6.9 and 6.3 micromolar against HepG2, Huh7 and SK-Hep-1 cells respectively, outperforming sorafenib and beating the parent monomer dehydrocostus lactone by roughly five- to seven-fold.

Identifying what a small molecule actually touches inside a cell is the hardest part of natural product pharmacology, and the team attacked it with a battery of orthogonal techniques. A drug affinity responsive target stability assay, which exploits the fact that ligand-bound proteins resist enzymatic digestion, flagged a protein band that survived pronase treatment only in the presence of KGA-1002. Mass spectrometry of that band yielded 49 candidate proteins, which transcriptomic profiling and bioinformatics narrowed to six. The decisive experiment was genetic: when the researchers silenced each candidate in liver cancer cells, only knockdown of GRP94, the endoplasmic reticulum-resident member of the heat shock protein 90 family, blunted the drug’s antiproliferative and anti-migratory effects. Surface plasmon resonance then measured direct binding, with a dissociation constant of 454 nanomolar, and a cellular thermal shift assay confirmed that KGA-1002 stabilizes GRP94 inside cells.

The structural detail goes further than most target-identification studies dare. KGA-1002 carries an alpha, beta-unsaturated carbonyl motif, a well-known covalent warhead, and molecular dynamics simulations showed that this group is essential for stabilizing the GRP94-ligand complex. Mass spectrometric sequencing of the treated protein revealed a peptide whose mass had increased by exactly the molecular weight of the drug, and secondary fragmentation localized the covalent bond to serine 106, a hydroxyl-bearing residue ideally positioned for nucleophilic attack on the unsaturated carbonyl. When the team mutated serine 106 to alanine, binding affinity collapsed from 522 nanomolar to 461 micromolar, a nearly thousand-fold loss. Critically, KGA-1002 did not stabilize the cytosolic family members HSP90alpha or HSP90beta, whose indiscriminate inhibition has doomed pan-HSP90 drugs in clinical trials. That isoform selectivity, aided by an additional contact with GRP94-specific asparagine 276, is arguably the compound’s most clinically relevant property.

With the target pinned down, the downstream biology fell into place. GRP94’s day job is folding, assembling and trafficking client proteins inside the endoplasmic reticulum, and its inhibition floods the ER lumen with misfolded polypeptides. Transcriptomic analysis of treated cells showed enrichment of ER-related pathways, upregulation of stress genes such as ERN1, ATF4 and DDIT3, and downregulation of ER trafficking genes. The canonical unfolded protein response sensors PERK and IRE1, normally held inactive by the chaperone BiP, were activated, driving expression and nuclear translocation of the death-promoting transcription factors XBP1 and CHOP. Because GRP94 also buffers calcium in the ER, the drug produced a dose-dependent rise in intracellular calcium, climbing to nearly 98 percent calcium-positive cells at 7.5 micromolar in both SK-Hep-1 and Huh7 lines. The unfolded protein response, in other words, was not a side effect but the engine of tumor killing.

The most novel mechanistic finding concerns AKT, the kinase at the heart of the PI3K survival pathway. KGA-1002 lowered AKT protein levels without touching its mRNA, and cycloheximide chase experiments showed the drug accelerating AKT degradation; proteasome and lysosome inhibitors each partially rescued the protein, implicating both disposal routes. The explanation proved to be a chaperone-client relationship: GRP94 physically holds AKT, and pulldown assays demonstrated that KGA-1002 disrupts that interaction, both in cell lysates and with purified proteins. Destabilized AKT could no longer phosphorylate SKP2, the F-box substrate-recognition protein of an E3 ubiquitin ligase that normally tags the cell cycle brake P21 for destruction. Deprived of AKT’s stabilizing phosphorylation, SKP2 itself was degraded through the proteasome, P21 accumulated in the nucleus, and the cells arrested in G0/G1 and died. Clinical databases reinforced the story: both GRP94 and SKP2 are overexpressed in hepatocellular carcinoma tissues and correlate with poor survival.

The death program did not stop at apoptosis. With AKT signaling crippled, the antioxidant defenses of the cell, maintained through the AKT-mTOR axis, Nrf2 and GSK-3beta, eroded, and reactive oxygen species accumulated. Mitochondrial membrane potential collapsed, lipid peroxidation measured as malondialdehyde rose, the ferroptosis gatekeeper GPX4 fell, and labile iron climbed, all hallmarks of ferroptosis, the iron-dependent form of regulated cell death. The ROS scavenger N-acetylcysteine and the ferroptosis inhibitor liproxstatin-1 each blunted these effects, confirming causality. KGA-1002 thus kills liver cancer cells along two converging routes, ER stress-driven apoptosis and AKT-dependent ferroptosis, both traceable to a single upstream lesion at GRP94.

In vivo, the compound held up. In nude mice bearing subcutaneous SK-Hep-1 tumors, intraperitoneal KGA-1002 at 15, 30 and 60 milligrams per kilogram over 60 days shrank tumors by 44.1, 52.1 and 53.5 percent, with the top dose matching sorafenib; direct intratumoral injection performed even better, reaching 61.6 percent inhibition. Tumors from treated animals showed reduced Ki67 staining and elevated phosphorylated IRE1 and PERK, confirming that the ER stress mechanism operates in living tissue. The decisive target-validation experiment came from xenografts grown from GRP94-knockdown cells: with the target partially removed, the drug’s antitumor effect and its suppression of Ki67 were significantly weakened, establishing that KGA-1002’s efficacy is GRP94-dependent. Safety readings were reassuring, with no weight loss, behavioral changes, liver or kidney function abnormalities, and no histopathological organ damage even at 150 milligrams per kilogram in a separate subacute study.

The study’s authors are careful about scope, and so should readers be. The work rests on cell lines and subcutaneous mouse models, not orthotopic liver tumors or patients, and a selectivity index of roughly 1.5 to 1.7 over normal hepatocytes, while adequate, leaves room for optimization. Yet the conceptual payoff is substantial: GRP94 has been implicated in breast cancer metastasis and multiple myeloma, and the authors note it is also overexpressed in colon, esophageal and bladder cancers, so a validated, covalent, isoform-selective inhibitor scaffold is a tool the wider oncology community can build on. By showing exactly where the molecule docks, which residue it attacks, and how that single molecular event cascades through protein folding, calcium handling, AKT stability, SKP2 degradation and ferroptosis, the Kunming team has delivered something rarer than another cytotoxic natural product: a mechanistically coherent proof that GRP94 is a druggable vulnerability in liver cancer, and a lead compound engineered to exploit it.

Subject of Research: A selective GRP94 inhibitor derived from a guaianolide sesquiterpenoid dimer as a therapeutic agent against hepatocellular carcinoma

Article Title: Guaianolide dimer KGA-1002 targets GRP94 and triggers unfolded protein response-associated degradation of SKP2/AKT axis as a novel antihepatoma agent

Article References: Li, Q.-H., Li, T.-Z., Wang, Y.-C., Huang, X.-Y., Ma, W.-J., Li, F.-J., Huang, F.-D., Hu, M.-M., & Chen, J.-J. (2026). Guaianolide dimer KGA-1002 targets GRP94 and triggers unfolded protein response-associated degradation of SKP2/AKT axis as a novel antihepatoma agent. Journal of Advanced Research, 88, 1077-1093. https://doi.org/10.1016/j.jare.2026.01.010

Image Credits: AI Generated

DOI: 10.1016/j.jare.2026.01.010

Keywords: hepatocellular carcinoma, GRP94, KGA-1002, guaianolide dimer, unfolded protein response, AKT, SKP2, P21, ferroptosis, endoplasmic reticulum stress, natural products, drug discovery