Targeting RAF Isoforms and Tumor Microenvironments in RAS or BRAF Mutant Colorectal Cancers with SJ-C1044 for Anti-Tumor Activity

Round 1

Reviewer 1 Report

This is a very interesting study with systematic activity analysis of compound named SJ-C1044.

Authors need to provide docking methodology and source of compound 3D structure

While this paper has a good method to identify the mechanism of this BRAF inhibitor. But this work is far from novel. Authors seem to be misleading authors by using a unique name SJ-C1044 for an already well known compound N-[2-fluoro-5-[[3-(7H-purin-6-yl)pyridin-2-yl]amino]phenyl]-3,5-bis(trifluoromethyl)benzamide Pubmed ID: US10844062, Example 65. AKA: SCHEMBL19233403 /BDBM473629 / NSC791136 / NSC-791136

This compound is patented US Patent US10844062 

Authors need to use a universal identifier instead of an in-house one so that the data is comparable. The source of compounds, purity etc. has to be disclosed. The reference given is incorrect (Arora, R.; Di Michele, M.; Stes, E.; Vandermarliere, E.; Martens, L.; Gevaert, K.; Van Heerde, E.; Linders, J.T.; 515, Brehmer, D.; Jacoby, E. Structural investigation of B-Raf paradox breaker and inducer inhibitors. Journal of 516, medicinal chemistry 2015, 58, 1818-1831.) citation number 20 does not describe this compound. 

Authors need to review the reported bio-assays and discuss their findings. Data regarding colon lines and colorectal cancer seems to be available. Authors need to review and discuss the same with their results. 

Given this is a well carried out systematic study, the revised manuscript should be more acceptable though less novel

Authors need to change language matching their conference abstract https://doi.org/10.1158/1538-7445.AM2017-5162

Author Response

Response to Reviewer 1 Comments

We thank the Reviewer #1 for insightful and positive comments.

Detailed point-by-point answers to comments are provided below.

Point 1. This is a very interesting study with systematic activity analysis of compound named SJ-C1044. Authors need to provide docking methodology and source of compound 3D structure

Response 1: According to the reviewer’s suggestion, we corrected the manuscript (Line 104 - 122).

Point 2: While this paper has a good method to identify the mechanism of this BRAF inhibitor. But this work is far from novel. Authors seem to be misleading authors by using a unique name SJ-C1044 for an already well known compound N-[2-fluoro-5-[[3-(7H-purin-6-yl)pyridin-2-yl]amino]phenyl]-3,5-bis(trifluoromethyl)benzamide Pubmed ID: US10844062, Example 65. AKA: SCHEMBL19233403 /BDBM473629 / NSC791136 / NSC-791136. This compound is patented US Patent US10844062. Authors need to use a universal identifier instead of an in-house one so that the data is comparable.

Response 2: It appears that Reviewer 2 has raised a valid point. The first author and corresponding author are inventors on US Patent US10844062. In this paper, we present the characterization of compound SJ-C1044, specifically Example 65, which was previously disclosed solely within the patent. As SJ-C1044 is currently undergoing preclinical testing, it does not possess a universal identifier. Consequently, we assigned it the name SJ-C1044 for the purpose of this publication, providing experimental data associated with this compound. Notably, SJ-C1044, an Example 65 compound, was first reported in this scientific paper, where it was rigorously validated by scientists to elucidate its biological properties.

Point 3 : The source of compounds, purity etc. has to be disclosed

Response 3: According to the reviewer’s suggestion, we corrected the manuscript (Line 260-262).

Point 4 : The reference given is incorrect (Arora, R.; Di Michele, M.; Stes, E.; Vandermarliere, E.; Martens, L.; Gevaert, K.; Van Heerde, E.; Linders, J.T.; 515, Brehmer, D.; Jacoby, E. Structural investigation of B-Raf paradox breaker and inducer inhibitors. Journal of 516, medicinal chemistry 2015, 58, 1818-1831.) citation number 20 does not describe this compound.

Response 4: According to the reviewer’s suggestion, we corrected the manuscript (Line 276).

The reference (Structural investigation of B-Raf paradox breaker and inducer inhibitors. Journal of 516, medicinal chemistry 2015, 58, 1818-1831) was deleted.

Point 5 : Authors need to review the reported bio-assays and discuss their findings. Data regarding colon lines and colorectal cancer seems to be available. Authors need to review and discuss the same with their results.

Response 5: According to the reviewer’s suggestion, we corrected the manuscript (Line 534-545).

Reviewer 2 Report

The authors proposed a novel molecule SJ-C1044 as a potent inhibitor of RAF isoforms. The study is intended to be both experimental and computational. Preumbelly SJ-C1044 inhibits both f pan-RAF and CSF1R

Overall the manuscript is well written; however, there are some issues.

1.       Line 87 ,, all ligands were drawn,, - who are those ligands? Moreover, how have they been chosen?

2.       The authors state that after docking, some energy was computed,. In the results section, those energies are not presented. Figure 1 only presents some docking with BRAF V6))E of 1 ligand. Also, some docking interaction diagrams should be added to clarify the interaction.

3.       Also, starting from Table 1, docking should also be performed with some kinase in this table in order to have a clear view and compare the results ( computational to experimental)

4.       Line 256, the authors should explain more clearly that VEGFR2, TIE2, or CSF1R have not been included.

5.       Line 264 -d SJ-C1044 inhibited 264 VEGFR2 activity at the cellular level – this should be better explained in a few lines.

6.       Line 363 - The tolerability of SJ-C1044 was thoroughly assessed in all preclinical pharmacology studies ples detail these in a few lines .

7.       Structural similarities and distinct characteristics of SJ-C1044 in comparison with other molecules should be presented and discussed in a should be discussed ( vemurafenib and dabrafenib with more details) . Probably the authors s should perform a brief computational study ( docking of vemurafenib and dabrafenib  BRAF ), but this is only a suggestion.

Also, the data availability, funding, and conflict of interest lines should be completed.

Author Response

Response to Reviewer 2 Comments

We thank the Reviewer #2 for insightful and positive comments.

Detailed point-by-point answers to comments are provided below.

The authors proposed a novel molecule SJ-C1044 as a potent inhibitor of RAF isoforms. The study is intended to be both experimental and computational. Preumbelly SJ-C1044 inhibits both pan-RAF and CSF1R. Overall the manuscript is well written; however, there are some issues.

Point 1.       Line 87 ,, all ligands were drawn,, - who are those ligands? Moreover, how have they been chosen?

Response 1: Reviewer 2 points out a valid point. All ligands is incorrect. Only SJ-C1044 was used as a ligand. Therefore, the sentence is corrected (Line 105).

Point 2. The authors state that after docking, some energy was computed,. In the results section, those energies are not presented. Figure 1 only presents some docking with BRAF V6))E of 1 ligand. Also, some docking interaction diagrams should be added to clarify the interaction.

Response 2: We appreciate the valuable feedback provided by the reviewer. In response to the suggestion, we have made the necessary revisions to the manuscript. Firstly, we have included the computed binding energies resulting from the docking analysis in the Results section and supplementary Figure 1 (Line 278 - 281). This addition provides important quantitative information about the binding affinities of SJ-C1044. Secondly, we have incorporated docking interaction diagrams in Supplementary Figure 1 to provide a clearer representation of the interactions between SJ-C1044 and BRAF V600E. These diagrams highlight the specific molecular interactions, such as hydrogen bonds that contribute to the binding between SJ-C1044 and BRAF. We have identified three significant hydrogen bonds formed by SJ-C1044 with Glu501, Cys532, and Asp594 in all binding modes. These interactions further support the potential binding and stabilization of SJ-C1044 within the active site of BRAF V600E.

Point 3. Also, starting from Table 1, docking should also be performed with some kinase in this table in order to have a clear view and compare the results ( computational to experimental)

Response 3: We appreciate the reviewer's suggestion to expand our docking analysis to include additional kinases listed in Table 1. In response to this feedback, we have included the computed binding energies resulting from the docking analysis in the Results section and supplementary Figure 2, 3 (Line 314 - 316). The results of these additional docking simulations are presented in Supplementary Figure 2 and Supplementary Figure 3. These new docking analyses allow for a clearer view and facilitate the comparison between the computational results and experimental data. The low CDOCKER Energy and CDOCKER Interaction Energy observed between SJ-C1044 and VEGFR2 as well as CSF1 suggest a stable binding between the compound and these kinases. This implies that SJ-C1044 has the potential to effectively inhibit the kinase activity of VEGFR2 and CSF1.The CDOCKER Energy represents the overall stability of the protein-ligand complex, encompassing the ligand's strain energy and the protein-ligand interaction energy. The low CDOCKER Energy value indicates a favorable and stable binding between SJ-C1044 and VEGFR2 or CSF1R. Furthermore, the CDOCKER Interaction Energy specifically quantifies the non-bonding interactions, including van der Waals and electrostatic interactions, between the protein and the ligand. The low CDOCKER Interaction Energy suggests strong attractive forces between SJ-C1044 and VEGFR2 or CSF1R, contributing to the stability of the complex.

Point 4. Line 256, the authors should explain more clearly that VEGFR2, TIE2, or CSF1R have not been included.

Response 4: In response to the suggestion, we have made the necessary revisions to the manuscript (Line 327- 329).

Point 5. Line 264 - SJ-C1044 inhibited VEGFR2 activity at the cellular level – this should be better explained in a few lines.

Response 5: In response to the suggestion, we have made the necessary revisions to the manuscript (Line 340 - 343).

Point 6. Line 363 - The tolerability of SJ-C1044 was thoroughly assessed in all preclinical pharmacology studies ples detail these in a few lines.

Response 6: In response to the suggestion, we have made the necessary revisions to the manuscript (Line 465 -476).

Point 7.  Structural similarities and distinct characteristics of SJ-C1044 in comparison with other molecules should be presented and discussed in a should be discussed ( vemurafenib and dabrafenib with more details) . Probably the authors s should perform a brief computational study ( docking of vemurafenib and dabrafenib  BRAF ), but this is only a suggestion.

Response 7: In response to the suggestion, we have made the necessary revisions to the manuscript (Line507-520). We also added a sentense to the following in the paper's discussion. The presence of diverse structural features among compounds, such as vemurafenib and SJ-C1044, can contribute to the development of unique patterns of hydrogen bonding interactions with BRAF. These distinct patterns of hydrogen bonding can ultimately lead to a wide range of biological effects, including the potential for paradoxical activation of the MAPK pathway.

Point 8. Also, the data availability, funding, and conflict of interest lines should be completed.

Response 8: In response to the suggestion, we have made the necessary revisions to the manuscript (Line 615-622).

Round 2

Reviewer 1 Report

All answers are satisfactory. I still think authors should cite their own patents for interested readers

Reviewer 2 Report

The authors responded to all comments. The manuscript can be published.