Pharmacologic ATF6 activating compounds are metabolically activated to selectively modify endoplasmic reticulum proteins

Thank you for submitting your article "A Small Molecule that Preferentially Activates the ATF6 Pathway of the Unfolded Protein Response is Metabolically Activated to Selectively Modify Endoplasmic Reticulum Proteins" for consideration by eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Randy Schekman as the Senior Editor. The reviewers have opted to remain anonymous.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

The authors present an excellent follow up study to their previous eLife report (Plate et al., 2016) of a novel small molecule compound that specifically upregulates the ATF6 pathway, one of the three arms of the unfolded protein response. Because pharmacological modulation of stress pathways is a promising avenue of intervention for numerous diseases, the molecule called 147, was proposed to have potential therapeutic value. However, the mechanism of activation was unclear in the previous study. In the current study, the authors took an exhaustive approach to understand how 147 activates the pathway. The mechanism involves the modification of 147 via enzymatic oxidation by cytochrome P450. Quite surprisingly, activated 147 then preferentially reacts with ER proteins, particularly protein disulfide isomerases (PDIs). Because PDIs are targets of the UPR and can play a role in stress regulation, the finding was intriguing but potentially troubling if 147 or a similar derivative is to be used as a therapeutic. However, previous work suggests that 147 does not cause detrimental effects to the assembly and secretion of molecules examined.

In summary, it is the consensus view of reviewers that this manuscript represents an important advancement to the previous study. The experimental designs are well conceived and experiments well performed and presented. The text is well written and remarkably approachable for a topic that might otherwise be challenging to the broad readership of eLife. The reviewer recommendation is accept with minor revisions.

Essential revisions:

1) One of the big remaining mysteries is why 147 labels primarily ER proteins. This is interesting because other efforts have generated molecules that localize to the mitochondria (from P. Wipf, S. Kelley, etc.), but there is less known about ER localization. A few quick experiments might be warranted here, using existing tools. First, does 147-20 actually localize to the ER or is it just preferentially activated there? The experiment is a centrifugation separation of the ER compartment and a comparison of the levels of 147-20 in the ER and cytoplasm to measure relative enrichment. If this experiment was performed early after treatment, little covalent modification will have taken place and the ratio of enrichment might be determined. The other experiment is to understand if the P450-mediated activation to the quinone takes place under reducing conditions in vitro? One simple model is that the 147reactivity is quenched by other reducing agents (glutathione) in other compartments (cytoplasm, nucleus), but the unique oxidizing environment of the ER (Birk et al., 2013, J. Cell Sci. 126:1604) allows reactivity.

2) The other interesting question is whether 147 treatment elevates PDIA levels through ATF6 activation over time? In other words, does the ATF6 activation lead to a shut-off of the compound activity by increasing the receptor level? In the author's previous work, I think they found that ATF6 activates PDIA4 and PDIA6 (Shoulders et al., 2013). This part of the mechanism is potentially important for the use of 147 in longer term treatment regimes and it might explain the relatively low toxicity.

3) The evidence for apparent selectivity of ER proteins and specifically 6 PDIs by 147 is convincing, and further supported by the genetic evidence using shRNA knockdown that PDIA3, PDIA4, and PDIA5 affect the 147-dependent regulation of ATF6. The basis for specificity of these oxidized PDIs and perhaps other identified targets on regulation of ATF6 is not examined or much discussed. Presumably the effect of 147 is not due to conformational effects, i.e. causing protein damage and generation of misfolded proteins in the ER, however some of these PDIs are known to interact with calreticulin and other components of the lumen chaperone machinery. This needs to be addressed, either experimentally or minimally by a more thorough discussion as the stress signals for XBP1 and HSF1 are directly linked to increased flux of misfolded proteins. Alternatively, these data might suggest a regulatory process involving electrophile stress similar to Nrf2/Keap1.

Reviewer #1:

This work is an extension of studies to identify arm-selective activators of the UPR. In the prior work, a series of high throughout screens and secondary assays was used to identify compounds, including 147, that activate the ATF6 pathway. Here, the authors take the next step – identifying the target and mechanism-of-action (MoA) of compound 147. This is an important step because any phenotypic screen will provide active molecules, but knowledge of the target(s) and MoA is critical to utility and advancement as a chemical probe.

In this work, the authors start with an analog effort that is designed to reveal the pharmacophore. Through ~20 molecules, they quickly find that the 2-amino-p-cresol is absolutely required for activity in an ATF6 reporter cell line. The NMR and full chemical characterization is included in supporting information, and the purity and identity look appropriate. One of the key findings of the SAR study was the requirement for the methyl group opposite the 2-amino phenol, which strongly suggests a quinone tautamerization (as in eugenol and acetaminophen, Figure 2—figure supplement.1). Indeed, many pieces of evidence supported this idea, including co-treatment with a P450 inhibitor or BME (Figure 2). The authors take advantage of this mechanism and the prior SAR to create an alkyne-containing Click probe (147-20) that could be used to covalently modify the relevant cellular targets (Figure 3). The authors use all the right controls in this tricky proteomics experiment, including using 5-fold excess of 147 as a competitor and 147-4 as a negative control. This effort pointed to PDIs as likely targets, among a handful of other proteins (Table 1). The enrichment of ER proteins was a big surprise to this reviewer, as there did not seem to be any structural feature of 147 that might cause it to localize in the ER (see below). Next, the authors perform important studies using knockdown of PDIs to show that PDIA3, PDIA4 and PDIA5 seems to be especially important in ATF6 activation (Figure 5D) and for 147 activity (Figure 5E), suggesting how 147 works through these proteins. Again, these studies are comprehensive, with transcriptional support for the mechanism.

This is an interesting study that represents a mountain of difficult work. It advances the first well-characterized inhibitors of PDIs and some of the first ATF6 activators. Finally, it is the logical next step from the previous eLife manuscript. I support publication with a handful of clarifications (below).

1) One of the big remaining mysteries is why 147 labels primarily ER proteins. This is interesting because other efforts have generated molecules that localize to the mitochondria (from P. Wipf, S. Kelley, etc.), but there is less known about ER localization. A few quick experiments might be warranted here, using existing tools. First, does 147-20 actually localize to the ER or is it just preferentially activated there? The experiment is a centrifugation separation of the ER compartment and a comparison of the levels of 147-20 in the ER and cytoplasm to measure relative enrichment. If this experiment was performed early after treatment, little covalent modification will have taken place and the ratio of enrichment might be determined. The other experiment is to understand if the P450-mediated activation to the quinone takes place under reducing conditions in vitro? One simple model is that the 147reactivity is quenched by other reducing agents (glutathione) in other compartments (cytoplasm, nucleus), but the unique oxidizing environment of the ER (Birk et al., 2013, J. Cell Sci. 126:1604) allows reactivity.

2) The other interesting question is whether 147 treatment elevates PDIA levels through ATF6 activation over time? In other words, does the ATF6 activation lead to a shut-off of the compound activity by increasing the receptor level? In the author's previous work, I think they found that ATF6 activates PDIA4 and PDIA6 (Shoulders et al., 2013). This part of the mechanism is potentially important for the use of 147 in longer term treatment regimes and it might explain the relatively low toxicity.

Reviewer #2:

This paper examines the mechanism by which N-(2-hydroxy-5-methylphenyl)-3-phenylpropanamide (hereafter 147), previously identified by the Kelly lab, activates the ATF6 arm of the UPR. The authors show that 147 is oxidized by cytochrome P450s to generate an ER localized electrophile that reacts preferentially with a set of mostly ER-localized proteins including protein disulfide isomerases that are proposed to regulate ATF6 activity.

This is a clearly presented paper and the chemistry for specificity of 147 on activation of ATF6 appears thorough and well executed. The underlying chemistry of 147 and its use as a chemical probe is intriguing. The data show that the effects of 147 are essentially ER compartment specific; that other cell stress responses such as the HSR are not induced and from the proteomics data that mostly ER localized proteins are targets of 147. While the logic makes sense, there is a gap in the story to understand how oxidation of specific PDIs by 147 leads to activation ATF6. Nevertheless, there are many aspects of this paper that are worthy of publication.

The evidence for apparent selectivity of ER proteins and specifically 6 PDIs by 147 is convincing, and further supported by the genetic evidence using shRNA knockdown that PDIA3, PDIA4, and PDIA5 affect the 147-dependent regulation of ATF6. The basis for specificity of these oxidized PDIs and perhaps other identified targets on regulation of ATF6 is not examined or much discussed. Presumably the effect of 147 is not due to conformational effects, i.e. causing protein damage and generation of misfolded proteins in the ER, however some of these PDIs are known to interact with calreticulin and other components of the lumen chaperone machinery. This needs to be addressed, either experimentally or minimally by a more thorough discussion as the stress signals for XBP1 and HSF1 are directly linked to increased flux of misfolded proteins. Alternatively, these data might suggest a regulatory process involving electrophile stress similar to Nrf2/Keap1.