Glucose-regulated protein 94 (Grp94) is an isoform of the heat shock protein 90 kDa (Hsp90) family of molecular chaperones. Inhibiting Grp94 has been implicated for many diseases. Co-crystal structures of two generations of Grp94 inhibitors revealed the importance of investigating the ester group, which is projected into the site 2 pocket unique to Grp94. Therefore, a series of KUNG65 benzamide analogs was designed and synthesized to evaluate their impact on the affinity and selectivity for Grp94. The data demonstrated that substituents with small and saturated ring systems that contain hydrogen bond acceptors exhibited increased affinity for Grp94, whereas larger saturated ring system manifested increased selectivity for Grp94 over Hsp90α.

Background: Aggressive mature T-cell lymphoma (TCL) is a disease that carries a poor prognosis. Methods: We analyzed the expression of 22 tumor cell functional proteins in 16 randomly selected patients with TCL. Immunohistochemistry was performed in paraffin-embedded tumor tissue sections to determine the protein expression statuses in tumor cells. Results: Glucose-regulated protein 94 (GRP94), a protein that serves as a pro-survival component under endoplasmic reticulum (ER) stress in the tumor microenvironment, was significantly associated with a shortened survival. Furthermore, significant differences were observed when GRP94 was combined with six other factors. The six factors were (1) programmed cell death-ligand 1 (PD-L1); (2) programmed cell death 1 (PD-1); (3) aldo-keto reductase family 1 member C3 (AKR1C3); (4) P53, a tumor suppressor; (5) glucose-regulated protein 78 (GRP78), an ER stress protein; and (6) thymidine phosphorylase (TP). Based on the combination of GRP94 and the six other factors expressed in the tumors, we propose a new prognostic classification system for TCL (TCL Urayasu classification). Group 1 (relatively good prognosis): GRP94-negative (n = 6; median OS, 88 months; p < 0.01); Group 2 (poor prognosis): GRP94-positive, plus expression of two of the six factors mentioned above (n = 5; median OS, 25 months; p > 0.05); and Group 3 (very poor prognosis): GRP94-positive, plus expression of at least three of the six factors mentioned above (n = 5; median OS, 10 months; p < 0.01). Conclusions: Thus, the TCL Urayasu prognostic classification may be a simple, useful, and innovative classification that also explains the mechanism of resistance to treatment for each functional protein. If validated in a larger number of patients, the TCL Urayasu classification will enable a targeted treatment using selected inhibitors acting on the abnormal protein found in each patient.

A recent study by Amankwah et al. reports how co-chaperone proteins and ATP hydrolysis fine-tune the function of endoplasmic reticulum (ER)-resident Hsp90 paralog Grp94.

Plasmodium falciparum parasites export more than 400 proteins to remodel the host cell environment and increase its chances of surviving and reproducing. The endoplasmic reticulum (ER) plays a central role in protein export by facilitating protein sorting and folding. The ER resident member of the Hsp90 family, glucose-regulated protein 94 (Grp94), is a molecular chaperone that facilitates the proper folding of client proteins in the ER lumen. In P. falciparum, Grp94 (PfGrp94) is essential for parasite survival, rendering it a promising anti-malarial drug target. Despite this, its druggability has not been fully explored. Consequently, this study sought to identify small molecule inhibitors targeting the PfGrp94. Potential small molecule inhibitors of PfGrp94 were designed and screened using in silico studies. Molecular docking studies indicate that two novel compounds, Compound S and Compound Z selectively bind to PfGrp94 over its human homologues. Comparatively, Compound Z had a higher affinity for PfGrp94 than Compound S. Further interrogation of the inhibitor binding using molecular dynamics (MD) analysis confirmed that Compound Z formed stable binding poses within the ATP-binding pocket of the PfGrp94 N-terminal domain (NTD) during the 250 ns simulation run. PfGrp94 interacted with Compound Z through hydrogen bonding and hydrophobic interactions with residues Asp 148, Asn 106, Gly 152, Ile 151 and Lys 113. Based on the findings of this study, Compound Z could serve as a competitive and selective inhibitor of PfGrp94 and may be useful as a starting point for the development of a potential drug for malaria.Communicated by Ramaswamy H. Sarma.

Hsp90s are ATP-dependent chaperones that collaborate with co-chaperones and Hsp70s to remodel client proteins. Grp94 is the ER Hsp90 homolog essential for folding multiple secretory and membrane proteins. Grp94 interacts with the ER Hsp70, BiP, although the collaboration of the ER chaperones in protein remodeling is not well understood. Grp94 undergoes large-scale conformational changes that are coupled to chaperone activity. Within Grp94, a region called the pre-N domain suppresses ATP hydrolysis and conformational transitions to the active chaperone conformation. In this work, we combined in vivo and in vitro functional assays and structural studies to characterize the chaperone mechanism of Grp94. We show that Grp94 directly collaborates with the BiP chaperone system to fold clients. Grp94\'s pre-N domain is not necessary for Grp94-client interactions. The folding of some Grp94 clients does not require direct interactions between Grp94 and BiP in vivo, suggesting that the canonical collaboration may not be a general chaperone mechanism for Grp94. The BiP co-chaperone DnaJB11 promotes the interaction between Grp94 and BiP, relieving the pre-N domain suppression of Grp94\'s ATP hydrolysis activity. In structural studies, we find that ATP binding by Grp94 alters the ATP lid conformation, while BiP binding stabilizes a partially closed Grp94 intermediate. Together, BiP and ATP push Grp94 into the active closed conformation for client folding. We also find that nucleotide binding reduces Grp94\'s affinity for clients, which is important for productive client folding. Alteration of client affinity by nucleotide binding may be a conserved chaperone mechanism for a subset of ER chaperones.

The present study was designed to investigate potential biomarkers of depression and targets of antidepressants from the perspective of hippocampal endoplasmic reticulum stress (ERS) based on cerebrospinal fluid (CSF) proteomics. Firstly, a six-week depression model was established and treated with fluoxetine (FLX). We found antidepressant-FLX could ameliorate depression-like behaviors and cognition in depressed rats caused by chronic unpredictable mild stress (CUMS). FLX significantly increased neuronal numbers in dentate gyrus (DG) and CA3 regions of hippocampus. CSF proteome data revealed thirty-seven differentially expressed proteins (DEPs) co-regulated by CUMS and FLX, including GRP94 and EIF2α. Results of Gene Oncology (GO) annotation and KEGG pathway enrichment for DEPs mainly included PERK-mediated unfolded protein response, endoplasmic reticulum, and translational initiation. The expression levels of GRP94, p-PERK, p-EIF2α, CHOP and Caspase-12 were increased in hippocampus of CUMS rats, and FLX worked the opposite way. FLX had strong affinity and binding activity with GRP94 protein, and four key proteins on the PERK pathway (PERK, EIF2α, p-EIF2α, CHOP). We proposed that FLX may exert antidepressant effects and neuroprotective action by alleviating excessive activation of the hippocampal PERK pathway and reducing neuronal deficits in depressed rats. PERK, EIF2α, p-EIF2α, and CHOP may be potential targets for antidepressant-FLX. GRP94 in CSF may be a potential biomarker of depression and the therapeutic effects of antidepressants.

The pursuit of single drugs targeting multiple targets has become a prominent trend in modern cancer therapeutics. Natural products, known for their multi-targeting capabilities, accessibility, and cost-effectiveness, hold great potential for the development of multi-target drugs. However, their therapeutic efficacy is often hindered by complex structural modifications and limited anti-tumor activity. In this study, we present a novel approach using celastrol (CST)-based Proteolysis Targeting Chimeras (PROTACs) for breast cancer therapy. Through rational design, we have successfully developed compound 6a, a potent multiple protein degrader capable of selectively degrading GRP94 and CDK1/4 in tumor cells via the endogenous ubiquitin-proteasome system. Furthermore, compound 6a has demonstrated remarkable inhibitory effects on cell proliferation and migration, and induction of apoptosis in 4T1 cells through cell cycle arrest and activation of the Bcl-2/Bax/cleaved Caspase-3 apoptotic pathway. In vivo administration of compound 6a has effectively suppressed tumor growth with an acceptable safety profile. Our findings suggest that the CST-based PROTACs described herein can be readily extended to other natural products, offering a potential avenue for the development of natural product-based PROTACs for cancer treatment.

BACKGROUND: Glucoregulatory protein 94 (Grp94) is necessary for the post-viral life cycle and plays a quality control role in viral proteins, but the role of Grp94 in regulating viral replication in host cells is not well known. Therefore, finding a compound that can regulate Grp94 will help us to study the mechanism of viral replication. Previously, we synthesized a coumarin pyrazoline derivative HCP1 that is an effective inhibitor of Grp94. We suppose that HCP1 may inhibit viral replication.
OBJECTIVE: This study aimed to investigate the effect of HCP1 on the replication ability of Senecavirus A (SVA), so as to provide a target and a leading compound for revealing the pathogenic mechanism of the virus and developing antiviral drugs.
METHODS: Rat cell lines BHK-21 and porcine cell lines PK-15 were infected with SVA, and the infected cells were treated with different concentrations of HCP1. The cell viability (CCK-8), virus titer (TCID50), autophagy level, and Grp94 expression were measured.
RESULTS: The results showed that a low concentration of HCP1 decreased viral titer and viral load in BHK-21 and PK-15 cells, and 5μM HCP1 significantly decreased the expression of SVA VP2 protein. In addition, SVA infection can lead to an increased level of autophagy, and HCP1 can inhibit host cell autophagy caused by SVA infection, thereby inhibiting viral replication and infection.
CONCLUSIONS: These findings reveal that Grp94 is a key factor in controlling SVA replication, and its inhibitor HCP1 suppresses SVA replication by inhibiting the increase of Grp94 protein level and autophagy induced by SVA. This study will contribute to the development of a new class of small-molecule antiviral drugs.

Glucose regulatory protein 94 (GRP94) is an endoplasmic reticulum (ER)-resident member of the heat shock protein 90 (HSP90) family, that plays an important role in secreted protein folding. Bombyx mori nuclear polyhedrosis virus (BmNPV) is one of the main pathogens in sericulture, causing serious economic losses every year. Previous studies showed that HSP90 members promote BmNPV replication in silkworm, but the function of BmGRP94 in BmNPV infection and proliferation is still not understood. In this study, we investigated the interplay between BmGRP94 and BmNPV infection in silkworm. We first identified a single gene of BmGRP94 in the Bombyx mori genome, which encodes a polypeptide with 810 amino acids in length. Spatio-temporal expression profiles showed that BmGRP94 was highly expressed in hemocytes and midgut, and was significantly induced by BmNPV infection. Furthermore, overexpression of BmGRP94 facilitates viral proliferation, while BmGRP94 inhibition evidently decreased BmNPV proliferation in BmN cells and in silkworm midgut. Mechanistically, BmGRP94 inhibition triggers ER stress, as judged by increased expression of PERK/ATF4/ERO1, H2O2 production, and ER calcium efflux, which promotes cell apoptosis to restrict BmNPV replication in silkworm. These results suggest that BmGRP94 plays an important role in facilitating BmNPV proliferation, and provides a potential molecular target for BmNPV prevention.

Grp94 is the endoplasmic reticulum paralog of the hsp90 family of chaperones, which have been targeted for therapeutic intervention via their highly conserved ATP binding sites. The design of paralog-selective inhibitors relies on understanding the structural elements that mediate each paralog\'s response to inhibitor binding. Here, we determined the structures of Grp94 and Hsp90 in complex with the Grp94-selective inhibitor PU-H36, and of Grp94 with the non-selective inhibitor PU-H71. In Grp94, the 8-aryl moiety of PU-H36 is inserted into Site 2, a conditionally available side pocket, but in Hsp90 it occupies Site 1, a non-selective side pocket that is accessible in all hsp90 paralogs. The structure of Grp94 in complex with the non-selective PU-H71 shows only Site 1 binding. Large conformational shifts involving helices 1, 4 and 5 of the N-terminal domain of Grp94 are associated with the engagement of the Site 2 pocket for ligand binding. To understand the origins of Site 2 pocket engagement, we tested the binding of Grp94-selective ligands to chimeric Grp94/Hsp90 constructs. These studies show that helix 1 of the Grp94 N-terminal domain is the discriminating element that allows for remodeling of the ATP binding pocket and exposure of the Site 2 selective pocket.