Phenylhydrazone-based Endoplasmic Reticulum Proteostasis Regulator Compounds with Enhanced Biological Activity

Reviewer #1 (Public review):

Summary:

This study builds off prior work that focused on the molecule AA147 and its role as an activator of the ATF6 arm of the unfolded protein response. In prior manuscripts, AA147 was shown to enter the ER, covalently modify a subset of protein disulfide isomerases (PDIs), and improve ER quality control for the disease-associated mutants of AAT and GABAA. Unsuccessful attempts to improve the potency of AA147 have led the authors to characterize a second hit from the screen in this study: the phenylhydrazone compound AA263. The focus of this study on enhancing the biological activity of the AA147 molecule is compelling, and overcomes a hurdle of the prior AA147 drug that proved difficult to modify. The study successfully identifies PDIs as a shared cellular target of AA263 and its analogs. The authors infer, based on the similar target hits previously characterized for AA147, that PDI modification accounts for a mechanism of action for AA263.

Strengths:

The authors are able to establish that, like AA147, AA263 covalently targets ER PDIs. The work establishes the ability to modify the AA263 molecule to create analogs with more potency and efficacy for ATF6 activation. The "next generation" analogs are able to enhance the levels of functional AAT and GABAA receptors in cellular models expressing the Z-variant of AAT or an epilepsy-associated variant of the GABAA receptor, outlining the therapeutic potential for this molecule and laying the foundation for future organism-based studies.

Weaknesses:

Arguably, the work does not fully support the statement provided in the abstract that the study "reveals a molecular mechanism for the activation of ATF6". The identification of targets of AA263 and its analogs is clear. However, it is a presumption that the overlap in PDIs as targets of both AA263 and AA147 means that AA263 works through the PDIs. While a likely mechanism, this conclusion would be bolstered by establishing that knockdown of the PDIs lessens drug impact with respect to ATF6 activation. Alternatively, it has previously been suggested that the cell-type dependent activity of AA263 may be traced to the presence of cell-type specific P450s that allow for the metabolic activation of AA263 or cell-type specific PDIs (Plate et al 2016; Paxman et al 2018). If the PDI target profile is distinct in different cell types, and these target difference correlates with ATF6-induced activity by AA263, that would also bolster the authors' conclusion.

Reviewer #2 (Public review):

Modulating the UPR by pharmacological targeting of its sensors (or regulators) provides mostly uncharted opportunities in diseases associated with protein misfolding in the secretory pathway. Spearheaded by the Kelly and Wiseman labs, ATF6 modulators were developed in previous years that act on ER PDIs as regulators of ATF6. However, hurdles in their medicinal chemistry have hampered further development. In this study, the authors provide evidence that the small molecule AA263 also targets and covalently modifies ER PDIs, with the effect of activating ATF6. Importantly, AA263 turned out to be amenable to chemical optimization while maintaining its desired activity. Building on this, the authors show that AA263 derivatives can improve the aggregation, trafficking, and function of two disease-associated mutants of secretory pathway proteins. Together, this study provides compelling evidence for AA263 (and its derivatives) being interesting modulators of ER proteostasis. Mechanistic details of its mode of action will need more attention in future studies that can now build on this.

In detail, the authors provide strong evidence that AA263 covalently binds to ER PDIs, which will inhibit the protein disulfide isomerase activity. ER PDIs regulate ATF6, and thus their finding provides a mechanistic interpretation of AA263 activating the UPR. It should be noted, however, that AA263 shows broad protein labeling (Figure 1G), which may suggest additional targets, beyond the ones defined as MS hits in this study. Also, a further direct analysis of the IRE1 and PERK pathways (activated or not by AA263) would have been a benefit, as e.g., PDIA1, a target of AA263, directly regulates IRE1 (Yu et al., EMBOJ, 2020), and other PDIs also act on PERK and IRE1. The authors interpret modest activation of IRE1/PERK target genes (Figure 2C) as an effect on target gene overlap, indeed the most likely explanation based on their selective analyses on IRE1 (ERdj4) and PERK (CHOP) downstream genes, but direct activation due to the targeting of their PDI regulators is also a possible explanation. Further key findings of this paper are the observed improvement of AAT behavior and GABAA trafficking and function. Further strength to the mechanistic conclusion that ATF6 activation causes this could be obtained by using ATF6 inhibitors/knockouts in the presence of AA263 (as the target PDIs may directly modulate the behavior of AAT and/or GABAA). Along the same line, it also warrants further investigation why the different compounds, even if all were used at concentrations above their EC50, had different rescuing capacities on the clients.

Together, the study now provides a strong basis for such in-depth mechanistic analyses.

Reviewer #3 (Public review):

Summary:

This study aims to develop and characterize phenylhydrazone-based small molecules that selectively activate the ATF6 arm of the unfolded protein response by covalently modifying a subset of ER-resident PDIs. The authors identify AA263 as a lead scaffold and optimize its structure to generate analogs with improved potency and ATF6 selectivity, notably AA263-20. These compounds are shown to restore proteostasis and functional expression of disease-associated misfolded proteins in cellular models involving both secretory (AAT-Z) and membrane (GABAA receptor) proteins. The findings provide valuable chemical tools for modulating ER proteostasis and may serve as promising leads for therapeutic development targeting protein misfolding diseases.

Strengths:

(1) The study presents a well-defined chemical biology framework integrating proteomics, transcriptomics, and disease-relevant functional assays.

(2) Identification and optimization of a new electrophilic scaffold (AA263) that selectively activates ATF6 represents a valuable advance in UPR-targeted pharmacology.

(3) SAR studies are comprehensive and logically drive the development of more potent and selective analogs such as AA263-20.

(4) Functional rescue is demonstrated in two mechanistically distinct disease models of protein misfolding-one involving a secretory protein and the other a membrane protein-underscoring the translational relevance of the approach.

Weaknesses:

(1) ATF6 activation is primarily inferred from reporter assays and transcriptional profiling; however, direct evidence of ATF6 cleavage is lacking.

(2) While the mechanism involving PDI modification and ATF6 activation is plausible, it remains incompletely characterized.

(3) No in vivo data are provided, leaving the pharmacological feasibility and bioavailability of these compounds in physiological systems unaddressed.