Crotamiton derivative JM03 extends lifespan and improves oxidative and hypertonic stress resistance in Caenorhabditis elegans via inhibiting OSM-9
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Evaluation Summary:
Bao and colleagues present a chemical genetics study to identify novel compounds extending lifespan in C. elegans, and they proceed to investigate the mechanisms of action of their most potent compound. Based on the known target TRPV4 of the screening hit, the authors provide evidence for the involvement of a C. elegans homolog (osm-9) in the observed phenomenon, although this evidence is not completely conclusive. This study will be of interest to researchers working on drug discovery, repurposing and lead optimization in the context of ageing.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
While screening our in-house 1072 marketed drugs for their ability to extend the lifespan using Caenorhabditis elegans ( C. elegans ) as an animal model, crotamiton ( N -ethyl-o-crotonotoluidide) showed anti-aging activity and was selected for further structural optimization. After replacing the ortho-methyl of crotamiton with ortho-fluoro, crotamiton derivative JM03 was obtained and showed better activity in terms of lifespan-extension and stress resistance than crotamiton. It was further explored that JM03 extended the lifespan of C. elegans through osmotic avoidance abnormal-9 (OSM-9). Besides, JM03 improves the ability of nematode to resist oxidative stress and hypertonic stress through OSM-9, but not osm-9/capsaicin receptor related-2 (OCR-2). Then the inhibition of OSM-9 by JM03 reduces the aggregation of Q35 in C. elegans via upregulating the genes associated with proteostasis. SKN-1 signaling was also found to be activated after JM03 treatment, which might contribute to proteostasis, stress resistance and lifespan extension. In summary, this study explored a new small molecule derived from crotamiton, which has efficient anti-oxidative, anti-hypertonic, and anti-aging effects, and could further lead to promising application prospects.
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Evaluation Summary:
Bao and colleagues present a chemical genetics study to identify novel compounds extending lifespan in C. elegans, and they proceed to investigate the mechanisms of action of their most potent compound. Based on the known target TRPV4 of the screening hit, the authors provide evidence for the involvement of a C. elegans homolog (osm-9) in the observed phenomenon, although this evidence is not completely conclusive. This study will be of interest to researchers working on drug discovery, repurposing and lead optimization in the context of ageing.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
The manuscript "Crotamiton derivative JM03 extends lifespan and improves oxidative and hypertonic stress resistance in Caenorhabditis elegans via inhibiting OSM" by Bao et al. details a drug screen and optimization of a lead compound. Overall I am positive about this manuscript. The screen is of substantial size and appears well conducted. The n-number per condition is relatively small, but this mainly means that the screen will have missed some hits as false positives would be caught by subsequent confirmation studies. The second part of the manuscript then details validation and optimization of one hit of the screen, crotamiton. The authors carry out confirmation, some validation of a likely target and some medicinal chemistry / drug optimization. While there are some limitations to this part of the study, …
Reviewer #1 (Public Review):
The manuscript "Crotamiton derivative JM03 extends lifespan and improves oxidative and hypertonic stress resistance in Caenorhabditis elegans via inhibiting OSM" by Bao et al. details a drug screen and optimization of a lead compound. Overall I am positive about this manuscript. The screen is of substantial size and appears well conducted. The n-number per condition is relatively small, but this mainly means that the screen will have missed some hits as false positives would be caught by subsequent confirmation studies. The second part of the manuscript then details validation and optimization of one hit of the screen, crotamiton. The authors carry out confirmation, some validation of a likely target and some medicinal chemistry / drug optimization. While there are some limitations to this part of the study, the study is valuable, both in terms of the specific drug investigated and as a template for future studies.
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Reviewer #2 (Public Review):
Bao and colleagues present a chemical genetics study to identify novel compounds extending lifespan in C. elegans and investigate the mechanisms of action of their most potent compound JM03 derived from their primary hit crotamiton. Among to strong parts of the paper are the reasonable size of the screen of over 1000 compounds. The screen covers a substantial sector of pharmacological activity. Following the screen, they conducted structure-activity relationship studies to improve the drug's potency and succeed in that attempt. Few anti-aging compound screens in the literature have followed a screen with a structure-activity relationship study to optimize the primary hit. The follow-up and improvement of the primary hit by medicinal chemistry are one of the stronger points of the paper. Their main compound, …
Reviewer #2 (Public Review):
Bao and colleagues present a chemical genetics study to identify novel compounds extending lifespan in C. elegans and investigate the mechanisms of action of their most potent compound JM03 derived from their primary hit crotamiton. Among to strong parts of the paper are the reasonable size of the screen of over 1000 compounds. The screen covers a substantial sector of pharmacological activity. Following the screen, they conducted structure-activity relationship studies to improve the drug's potency and succeed in that attempt. Few anti-aging compound screens in the literature have followed a screen with a structure-activity relationship study to optimize the primary hit. The follow-up and improvement of the primary hit by medicinal chemistry are one of the stronger points of the paper. Their main compound, JM03, extends lifespan and improves health span, pharyngeal pumping, locomotion, and does not seem to affect brood size, all signs of a drug increasing health span. Because the target for the original drug crotamiton is known (TRPV4), they investigate the C.elegans homologs osm-9 and ocr-2. They find osm-9 mutants to be resistant to the longevity effect of JM03. This finding makes osm-9 a likely mechanistic target of JM03. However, because JM03 is a compound that was optimized based on lifespan and because the original drug crotamiton has not been shown to bind osm-9 (only TRPV4), the evidence is not yet sufficient to claim osm-9 to be the mechanistic target of JM03. Any experiment that would show that JM03 directly interacts with osm-9 or modulates the activity in vitro would make this paper much stronger and allow the authors to claim having identified the mechanistic target of JM03. The lack of an experiment that demonstrates a direct interaction is not a weakness in the paper but more a missed opportunity to add real strength to the paper, given that all the evidence they have pointed to osm-9 as a target.
There are a few weaknesses too. The cohort size of 15 animals/drugs during the screen suggests that there must have been a substantial amount of false negatives. That cohort size only detects 50% of all drugs that extend lifespan by 20% and miss roughly 80% of the drugs that extend lifespan by 10%, the cut-off criteria for hits. There is nothing wrong with this strategy, but the text should clearly state that not having found the effect of a drug in a screen is not evidence for the absence of an effect. Similarly, the effect of the JM03 is relatively small, making it more difficult to show that there is no effect in a given strain. To show the absence of an effect in osm-9, the authors use cohorts of roughly 150 animals in each arm to ensure that they can detect a longevity effect if it is present. The source data show that they used enough animals for figure 3 (10 x15 animals) and should detect a longevity effect of JM03 if there were one in osm-9. However, all the animals combined amounts to essentially one experiment, and a second repeat of that size is necessary for a firm conclusion in the paper. A final potential weakness may be in the RNAseq data. From the cluster analysis shown in Fig. 4i, it seems that the three replicates of treated and untreated samples are not very similar to each other. A principal component (PCA) plot that shows that the three control and 3 JM03 treated samples cluster would strengthen the confidence in the data and provide more substantial support for the conclusions in Figure 4.
Overall, the authors aim to identify new anti-aging compounds as well as the underlying mechanism of action. They largely succeed in these aims. The relatively small effect of the lifespan extension makes testing and validating the underlying mechanisms of action more difficult as the ~15% increase requires them to test large cohorts, but overall this is done well. This paper is one of the few in which the authors conduct medicinal chemistry to improve upon the longevity effect of their primary hit one aspect that makes notable compared to other, similar papers.
Compounds targeting TRPV channels like JM03 to slow aging is an interesting concept, particularly since Riera et al. (Cell 2014) have shown that lowering signaling by TRPV channels can also extend lifespan in mice. Identifying a compound that achieves the same, found in an unbiased chemical screen, suggests that TRPV channels may make a druggable target for aging that is evolutionarily conserved.
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