MitoToxy assay: A novel cell-based method for the assessment of metabolic toxicity in a multiwell plate format using a lactate FRET nanosensor, Laconic

Yasna Contreras-Baeza; Sebastián Ceballo; Robinson Arce-Molina; Pamela Y. Sandoval; Karin Alegría; Luis Felipe Barros; Alejandro San Martín

doi:10.1371/journal.pone.0224527

Peer Review History

Original SubmissionJuly 23, 2019
9 Sep 2019 Decision Letter - Mária A. Deli, Editor PONE-D-19-20790 MitoToxy Assay: a novel cell-based method for the assessment of metabolic toxicity in a multiwell plate format using a lactate FRET nanosensor, Laconic PLOS ONE Dear Dr. San Martin, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses all the points raised by the reviewers. We would appreciate receiving your revised manuscript by Oct 24 2019 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. 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Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: No ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: No ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The current paper, written by Contreras-Baeza et al. describes a novel cell-based method for the assessment of metabolic toxicity during high-throughput drug screenings using a lactate FRET nanosensor, so called Laconic method, a measure of rapid lactate accumulation. The manuscript is well-written. The Laconic assay provides a quick and sensitive measurement. Advantages and weaknesses of the method are well-detailed. Table 1. provides a valuable comparison among the latest metabolic methods. My only suggestion is that while the well-known mitochondrial inhibitors show a prompt response, not all compounds having such effects might show the response so rapidly. Therefore, I suggest to recommend using the assay at/after 30 mins (as it was done in Figure 8. and 9.) in order not to detect these compounds as false negatives. Reviewer #2: The authors of the manuscript present a novel assay to detect potential mitochondrial dysfunction based upon a sensor designed to detect changes in lactate accumulation as a marker of glycolytic activation. The authors designed and performed experiments on a range of cells to inform the choice of which cell was most OXPHOS active and thus most suitable for insertion of the nanosensor. Once produced the cell model was characterised for utility using a range of classic mitochondrial toxins, hepatotoxins and negative controls. Overall, the study describes a novel method which may find utility in the drug screening program. However, this reviewer has several comments upon the design of the study and interpretation of the results which the authors for the authors. 1) Today, the majority of drug companies use the glu/gal screen to detect mitochondrial toxicity (2 or 24 h), see PMID 25023361. However, this assay is only very briefly mentioned in the manuscript. This should be the main comparator for their work, as it is this that their assay would be replacing. However, as they are both based on the same physiological event, the switch to glycolysis, it is questionable of how much more useful this would be in terms of increasing specificity and selectivity. 2) The authors assess a panel of cells in order to select the candidate for sensor insertion. Their results show that MDA-MB-231 cells are more oxidative when assessed in galactose media. However, it does not appear that any of the other cells were assessed in galactose media, and so this is an unfair comparison. Importantly the aforementioned glu/gal assay is routinely done in HepG2 cells and is currently in place in most preclinical screens already as a screen for hepatic toxicity. Therefore, this reviewer believes that it is important to also assess WI index of HepG2 cells in galactose. 3) The figures lack any evidence of statistical analysis of whether changes in delta R are significant.. 4) During assay refinement the authors demonstrate that the use a broad-spectrum inhibitor of MCT4 improves assay variability and sensitivity. Can they comment on whether the inclusion of such an inhibitor could effect drug dynamics? 5) In their assessment of how good their assay is (pg 20), the authors should consider their results in light of the Cmax. This would give an indication of whether any effects seen on the mitochondria would translate to clinical use. Furthermore, The authors use the phrase “false positives” in term of drugs which cause MMP changes but are considered relatively safe (pg 20, 509 – 512). This is important, as they should not consider this assay as an indicator of drug safety, this is probably too ambitious – in the absence of further information, it is only able to report on whether a drug affects the bioenergetic phenotype of a cell due to interactions with the mitochondrial ETC. 6) Some of the author’s comments do not adequately reflect the literature on the subject of screens to detect mitochondrial toxicity. For example (pg 20, line 499), publications have demonstrated mitochondrial toxicity of troglitazone at lower concentrations and short time-points (PMID 30096366 and 25746382). 7) The authors may like to comment on limitations of the use of this cell system to assess drug-induced metabolic toxicity, for example a lack of Phase 1 and Phase 2 metabolising enzymes. In addition mitochondrial toxicity often manifests over extended periods which may not be detected by their system. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. 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Revision 1
11 Oct 2019 Author Response Response to Reviewers Reviewer #1: The current paper, written by Contreras-Baeza et al. describes a novel cell-based method for the assessment of metabolic toxicity during high-throughput drug screenings using a lactate FRET nanosensor, so called Laconic method, a measure of rapid lactate accumulation. The manuscript is well-written. The Laconic assay provides a quick and sensitive measurement. Advantages and weaknesses of the method are well-detailed. Table 1. provides a valuable comparison among the latest metabolic methods. Author: We thank the reviewer for his/her positive comments. My only suggestion is that while the well-known mitochondrial inhibitors show a prompt response, not all compounds having such effects might show the response so rapidly. Therefore, I suggest to recommend using the assay at/after 30 mins (as it was done in Figure 8. and 9.) in order not to detect these compounds as false negatives. Author: The point is well taken. The recommendation has been included in the Discussion section: Lines 530-533 ”Although well-known mitochondrial inhibitors elicited a prompt Laconic response (within 5 min), not all toxic compounds might show the response so rapidly. To minimize the frequency of false negatives results, we recommend measurements to be carried out at or after 30 minutes of exposure.” ------------------------------------------------------------------------------------------------------ Reviewer #2: The authors of the manuscript present a novel assay to detect potential mitochondrial dysfunction based upon a sensor designed to detect changes in lactate accumulation as a marker of glycolytic activation. The authors designed and performed experiments on a range of cells to inform the choice of which cell was most OXPHOS active and thus most suitable for insertion of the nanosensor. Once produced the cell model was characterized for utility using a range of classic mitochondrial toxins, hepatotoxins and negative controls. Overall, the study describes a novel method which may find utility in the drug screening program. Author: We thank the reviewer for his/her positive appraisal of the article and the potential of the technique. However, this reviewer has several comments upon the design of the study and interpretation of the results which the authors for the authors. 1) Today, the majority of drug companies use the glu/gal screen to detect mitochondrial toxicity (2 or 24 h), see PMID 25023361. However, this assay is only very briefly mentioned in the manuscript. This should be the main comparator for their work, as it is this that their assay would be replacing. However, as they are both based on the same physiological event, the switch to glycolysis, it is questionable of how much more useful this would be in terms of increasing specificity and selectivity. Author: We thank the reviewer for directing our attention to the use of the glu/gal screen for drug discovery. The revised manuscript explains the importance of gluc/gal screens, which are typically based on measurement of oxygen, membrane potential and ATP. We are now citing PMID:25023361 and PMID:23147640, PMID:22689143, PMID: 17361016 and 21818751, which we deem most relevant to this paper. As it also uses galactose, the present method may be considered a variation of the glu/gal assay in which the readout is not oxygen consumption or cytosolic ATP levels but intracellular lactate. Although the present methods has advantages in terms of sensitivity, speed of detection and cost, we concur that it is not obvious that is better than oxygen/extracellular acidification detection in terms of specificity and selectivity. This is now discussed between lines 580-587, with reference to Table 1. 2) The authors assess a panel of cells in order to select the candidate for sensor insertion. Their results show that MDA-MB-231 cells are more oxidative when assessed in galactose media. However, it does not appear that any of the other cells were assessed in galactose media, and so this is an unfair comparison. Importantly the aforementioned glu/gal assay is routinely done in HepG2 cells and is currently in place in most preclinical screens already as a screen for hepatic toxicity. Therefore, this reviewer believes that it is important to also assess WI index of HepG2 cells in galactose. Author: New experiments were carried to assess WI in HepG2 in galactose as suggested. Galactose-fed HepG2 cells showed a low WI, similar that measured in MDA cells (Fig. 2D). However, their response to azide in terms of lactate accumulation was much weaker (Fig. 2C). These results are now described in lines 291-296.Therefore, whereas MDA cells are still the best option for toxicity detection using lactate levels, HepG2 cells could be approached with the WI, a more cumbersome protocol. This is commented in the Discussion section (lines 484-490). 3) The figures lack any evidence of statistical analysis of whether changes in delta R are significant. Author: Data were found to pass normality distribution and therefore statistical significance of the changes in ΔR was assessed using ANOVA. This is now indicated in Figures 9 and 10, and in Material and Methods, lines 232-235. 4) During assay refinement the authors demonstrate that the use a broad-spectrum inhibitor of MCT4 improves assay variability and sensitivity. Can they comment on whether the inclusion of such an inhibitor could affect drug dynamics? Author: This is a valid point. A toxicant that does not have access to mitochondria because of interference by pCMBS may not be detected, generating a false negative. If hydrophilic compounds are tested, we recommend skipping pCMBS. To preserve sensitivity, the assay would have to be performed in triplicate. This is now discussed in lines 558-564. 5) In their assessment of how good their assay is (pg 20), the authors should consider their results in light of the Cmax. This would give an indication of whether any effects seen on the mitochondria would translate to clinical use. Furthermore, the authors use the phrase “false positives” in term of drugs which cause MMP changes but are considered relatively safe (pg 20, 509 – 512). This is important, as they should not consider this assay as an indicator of drug safety, this is probably too ambitious – in the absence of further information, it is only able to report on whether a drug affects the bioenergetic phenotype of a cell due to interactions with the mitochondrial ETC. Author: We have added a paragraph that relates our findings with the respective values of the Cmax, Lines 524-527 “Our pilot screening detected the toxicity of ciglitazone, troglitazone, rosiglitazone and camptothecin at 5 µM, whereas the mitochondrial effects of terfenadine and flutamide were detected at 10 µM. As a reference, the maximum concentration of these drugs in plasma (Cmax) lies in the range of 3 to 10 µM (PMID:22987451, PMID:8792418, PMID:3986303, PMID:12235921 and PMID:2306725)”. 6) Some of the author’s comments do not adequately reflect the literature on the subject of screens to detect mitochondrial toxicity. For example (pg 20, line 499), publications have demonstrated mitochondrial toxicity of troglitazone at lower concentrations and short time-points (PMID 30096366 and 25746382). Author: Thanks for bringing to our attention these papers. We have added PMID 30096366 and 25746382 to the reference section. Also, we have corrected the manuscript Lines 506-508 “The hitherto most sensitive detection of thiazolidinedione toxicity was obtained for troglitazone, which induced ATP depletion with an IC50 at 28 and 31,4 µM (PMID:30096366 and PMID:25746382).” 7) The authors may like to comment on limitations of the use of this cell system to assess drug-induced metabolic toxicity, for example a lack of Phase 1 and Phase 2 metabolizing enzymes. In addition mitochondrial toxicity often manifests over extended periods which may not be detected by their system. Author: We concur. An assay based on cultured cells like the present one risks overlooking toxicants that are tissue specific. Not much is known about inter tissue mitochondrial variability. Interestingly, MDA-MB-231 cells do express Phase 1 and Phase 2 metabolizing enzymes PMID: 23873331. This is now mentioned in Lines 484-490. We agree that mitochondrial toxicity could manifest over extended periods which may not be detected by our assay. This is now mentioned in Lines 533-535. We are planning to test a variation of the present method for its ability to detect compounds whose toxicity develops over extended periods. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0224527.r002
16 Oct 2019 Decision Letter - Mária A. Deli, Editor MitoToxy Assay: a novel cell-based method for the assessment of metabolic toxicity in a multiwell plate format using a lactate FRET nanosensor, Laconic PONE-D-19-20790R1 Dear Dr. San Martin, We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements. Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication. Shortly after the formal acceptance letter is sent, an invoice for payment will follow. 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The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: (No Response) ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: (No Response) Reviewer #2: I thank the authors for the additions that they have made to the manuscript based upon my comments. ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No https://doi.org/10.1371/journal.pone.0224527.r003
Formally Accepted
23 Oct 2019 Acceptance Letter - Mária A. Deli, Editor PONE-D-19-20790R1 MitoToxy Assay: a novel cell-based method for the assessment of metabolic toxicity in a multiwell plate format using a lactate FRET nanosensor, Laconic Dear Dr. San Martín: I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. For any other questions or concerns, please email plosone@plos.org. Thank you for submitting your work to PLOS ONE. With kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Mária A. Deli Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0224527.r004

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