Ceapins are a new class of unfolded protein response inhibitors, selectively targeting the ATF6α branch

Minor points:

1) Introduction, second paragraph: "principle" should be "principal"; "co-operatively" should be corrected.

Done.

2) Figure 1. Some images appear out of focus.

These images were acquired at low magnification using a high-throughput microscope. The panels in the original submission were cropped from these images and enlarged for the figure, leading to loss of resolution. We have replaced these panels with a crop of a larger area that clearly illustrates the point without the loss in resolution.

3) Figure 2A. There appears to be far less GFP-ATF6 signal in panels C and D compared to A and B. Do the inhibitors affect steady state levels?

These images were acquired using a high-throughput microscope engineered to record qualitative differences and as such may be deceiving. In particular, the instrument performs automatic exposure adjustments, which explains the apparent discrepancies. For this reason, images were normalized internally prior to comparison between wells by calculating the ratio of nuclear to ER GFP signal (nuc:ER ratio) for each cell.

4) Figure 2E. Consider changing one of the colors in the plot. It's difficult to discern dark grey and black.

Thank you for pointing this out. Unstressed has been changed from grey solid line to grey dashed line in the histogram and a grey-checkered box in the bar graph.

5) Figure 2F. It is unclear what is meant in the Y-axis label "relative to unstressed controls". Isn't the unstressed control represented by the first bar, at under 20%?

We agree the axis labeling was confusing and have relabeled it “Percent activated cells”.

6) In the second paragraph of the subsection “Isolation of small molecule inhibitors of ER stress-induced nuclear translocation of GFP-ATF6 α”: The distributions showed a wide range of responses within the population of cells in each well. Does this apply to Ceapin-A1? If so, please indicate the range. In other words, are a fraction of cells entirely unresponsive?

We have now included these data as Figure 2—figure supplement 3. The range for Ceapin-A1 is similar to the other treatments – the difference between treatments is not the range per se but the position of the histogram on the X-axis. In the secondary screen, a fraction of the cells are indeed unresponsive to Ceapin-A1 (see where the threshold crosses the histogram for Ceapin-A1). This concentration of this aliquot of Ceapin-A1 inhibits nuclear translocation of GFP-ATF6 to 35.07 ± 18.15%.

This low activity is because the concentration of active Ceapin-A1 tested in the secondary screen is below its IC₅₀. Ceapin-A1 in the library is less active than repurchased stocks (IC₅₀ of 8.49 μM compared to 4.9 ± 1.2 μM in ERSE-luciferase assays). Additionally, for the secondary screen all compounds were tested at a single concentration of 6.6 μM, which is below the IC₅₀ of the library aliquot of Ceapin-A1. For this reason, these data were not included in the original version of the manuscript as they report on the potency of Ceapin-A1 in the library stock and not the potency of fresh stocks.

7) Figure 3—figure supplement 1A: The authors should comment on why the compounds that passed through multiple filters should act so poorly on activation of endogenous genes.

We do not know the reason for this discrepancy. We exclusively focused on the Ceapin series after obtaining the results in Figure 3—figure supplement 1, which validated the effects of the compound on endogenous gene expression and additionally had an increased stringency filter applied. We have added this explanation to the figure legend of Figure 3—figure supplement 1.

8) Figure 3—figure supplement 4: While the mobility of ATF6α-M's mobility is obvious in Figure 3D, it is not clear in this panel. Although I don't feel it necessary to re-do the experiment (given the other supporting data), either a better gel system or re-ordering of the lanes (ATF6α-N and ATF6α-M in adjacent lanes) to better show the difference is recommended in future experiments.

Thank you, point taken.

9) Figure 5B. Ceapin-A5 appears to augment induction of BiP under both forms of stress. Is that correct?

No. We have done this experiment three times each with two different antibodies and augmented induction of BiP by the inactive Ceapin analog A5 was only observed in this one particular blot. We have replaced the figure with data from a representative experiment.

10) Figure 5—figure supplement 1 is missing.

We have included the missing supplement in the revised manuscript.

11) Ceapin does not inhibit ATF6β processing, but the positive control of S1P inhibitor has little effect. Presumably this is due to the issues discussed around ATF6-M, but these data are not convincing as presented since the positive control does not work well. A parallel experiment with cell fractionation as in Figure 3D would be more convincing.

We have added new data to address this question. We performed differential centrifugation on U2-OS cells and looked for endogenous ATF6α and ATF6β. We detected both ATF6α-N and ATF6β-N in the nuclear extract of cells subjected to ER stress.

Although both ATF6α and ATF6β were cleaved in ER stressed cells treated with the site-1 protease inhibitor, analogous to the full-length proteins, neither cleavage product was detected in the nuclear fraction, indicating that neither ATF6α-N nor ATF6β-N were generated. These data suggest that the cleavage product detected by the anti-ATF6β antibody is ATF6β-M.

In ER stressed cells treated with Ceapin-A7 we reproducibly found that while ATF6α is not cleaved and there is no ATF6α-N in the nuclear fraction, ATF6β is cleaved and ATF6β-N is present in the nuclear fraction. These data strengthen the argument that Ceapins specifically inhibit processing of ATF6α but not ATF6β.

12) Abstract, the phrase "detrimental in cancer models" is confusing. Perhaps rephrasing as "Activation of ATF6 promotes cell survival in cancer models."

Thank you for the suggestion – we changed it.

13) In the second sentence of the subsection “Quantitative PCR analysis of mRNA expression to measure ATF6α target gene induction”, remove "at".

Done.

14) Figure 2E/F would be improved if gray was lighter for "unstressed cells" to better differentiate from "stressed".

Done – see point 4 above.

15) Shouldn't A5 be described as "the inactive ceapin analog A5" as "inactive ceapin" is confusing.

We agree and have changed this call-out in both manuscripts.

16) I assume the HEK293T cells used for screening are a stable cell line, if so please indicate in the text.

This fact was already included in the methods. We have now added a statement to the Results section also.

17) It would be a plus if Figures 2A-D were increased in size and much better described in the text.

We have increased Figures 2A-D in size. To facilitate this change and keep Figure 2 on a single page panel G from the original figure has been moved to Figure 2—figure supplement 2. We have edited the text to clarify why this assay was performed and the results interpreted from the images.