Synthesis and Pharmacological Characterization of 2-Aminoethyl Diphenylborinate (2-APB) Derivatives for Inhibition of Store-Operated Calcium Entry (SOCE) in MDA-MB-231 Breast Cancer Cells

Round 1

Reviewer 1 Report

This is a very nice study that describes how modifications of 2-APB impact on CRAC channel inhibition, calcium release from stores and cell viability. The authors systematically add halogens and other small substituents on each position of a phenyl ring. In this way, they have synthesized an impressive arsenal of halogen- and para-substituted 2-APB analogues. The experiments are well done, and the paper is nicely written and easy to follow.The findings are important and enhance our understanding of drug-CRAC channel interaction and I support publication. I have a few only minor comments.

Figure 4 shows the effects of various 2-APB analogues on calcium release. In panel 4a, m-Br-2-APB releases calcium and the subsequent thapsigargin response looks suppressed. It is similar for the p-Br analogue. In the concluding remarks, the authors state: 2-APB-type SOCE inhibitors (DPB162-AE), are able to mobilize calcium from intracellular stores of MDA-MB-231 cells at high concentration. This thapsigargin-independent store release does not activate plasma membrane Orai channels for calcium influx.

However, it seems to me that the loss of the thapsigargin response indicates the analogues do indeed target the Thap-sensitive store. I think the authors mean the analogues release calcium independent of thapsigargin? Regardless, the sentence needs making clearer. I assume the lack of SOCE in the presence of 2-APB analogues that release calcium from the stores is simply because these analogues also block CRAC channels over a similar concentration range.

The relatively weak effects of synta 66 are surprising, since this inhibitor is relatively potent in other cell types in calcium assays. The reduced efficacy of Synta could be due to the cell types used. Alternatively, the incubation time might be a factor. The authors mention they increased the time for the GSK compound, they saw more block. Did they check this with Synta66?

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 2 Report

Summary

In this manuscript Schild and co-workers synthesize over a dozen analogues of 2-APB, a “parent” compound known to both activate and inhibit SOCE channels, depending on the dose. The authors assess the ability of these new compounds to drive SOCE in the MDA-MB-231 breast cancer cell line primarily using fluorescence based Ca2+ plate reader assay; however, they also perform some electrophysiology assessments on two of the compounds, and they assess morphological changes and cytotoxicity to MDA-MB-231 cancerous and MCF-10A “normal” breast cancer cell lines. It should be noted that the authors also provide crystal structure data for some of their own newly synthesized compounds as well as previously published compounds, in addition to NMR characterization.

Overall, the manuscript presents a lot of chemistry, synthesis and chemical structure characterization data (in the supplementary information), which will be of interest to chemists. The compound assessments will likely be of interest to the calcium signaling and cancer research fields. But, in terms of the IJMS audience, I would have liked to see the authors include some of the cell biology into the main text and simplify some of the chemistry/synthesis description so that it is more amenable to a broad audience. Nevertheless, I do think the work warrants publications after my major and minor concerns are addressed below:

Major

1. Scheme 1 – This is an important schematic since it shows the structures of the compounds; however, the structures and how they compare to 2-APB is not clear. At least one full structure of 2-APB should be shown, and the locations of “Ar” and “Ph” on that 2-APB structure should be indicated. Clarifying the presentation is important for non-chemists. Does panel B represent “Ar” positions in the compounds shown in the figure?
2. Similarly, Scheme 2 should show the full parent compound structure with Ph groups labeled, and then the subsequent adducts.
3. Figure 1c – I think the data points on this bar graph should be shown so the reader can get an idea of the data spread. This figure panel would also benefit from some statistical comparisons. The data points should be shown for all of the bar graphs in the manuscript.
4. The rationale for synthesizing, characterizing and testing the previously published compounds in this system is unclear and needs clarification. What was the point of doing all of that work, when the focus of the study is on the new 2-APB derivatives?
5. Figure 2c – Statistical analysis is needed here; particularly since you are claiming some of the compounds enhance SOCE.
6. Figure 4 – Why use slopes to assess the Ca2+ release response rather than AUC? Rationale is needed for this.
7. Figure S8 and S9 are important additions to the paper, which should be moved into the main text. Additionally, it is not clear why some of the new 2-APB compounds were more potent inhibitors than the previously published DPB-162AE, yet the cytotoxic affects in the breast cancer cell lines were less robust. More discussion is needed i) pointing out these discrepancies and ii) explaining possible reasons why they exist.

Minor

1. Intro – make sure all your acronyms are defined on first usage (see PIP2 for example).
2. Intro – you talk about the role of SOCE in refilling intracellular Ca2+ stores; however, another important role of SOCE is to signal physiological and pathophysiological processes.
3. Lines 65-80 – A table summarizing the concentrations, cell types, protein target (Orai versus STIM), etc and the inhibition versus activation effects of 2-APB would be beneficial to the reader.

Author Response

Please see the attachment

Author Response File: Author Response.pdf