A whole-animal phenotypic drug screen identifies suppressors of atherogenic lipoproteins

Reviewer #1 (Public review):

Summary:

A whole-organism drug screen was performed to identify molecules that decrease Apolipoprotein B (ApoB) as a target for agents to reduce atherosclerosis. Kelpsch et al. used a zebrafish reporter line, LipoGlo, which is a fusion of the Nano-luciferase protein to the ApoB protein as a proxy for the presence of ApoB-containing lipoproteins (B-lps) in larval stages. The LipoGlo line was screened against a well-characterized drug library and identified 49 hits from their primary screen. Follow-up studies further refined this list to 19 molecules that reproducibly reduced B-lps significantly. The authors focused their studies on enoxolone, a licorice root extract, and showed that larvae treated with this agent can reduce the production of B-lps. As enoxolone has been reported to suppress Hepatocyte Nuclear factor 4a (HNF4a), the authors investigated whether loss-of-hnf4a or pharmacological inhibition of hnf4a in zebrafish also produced similar phenotypes as enoxolone treatment. Their studies showed that this was the case. Transcriptomic studies after enoxolone treatment resulted in altered expression of genes involved in cholesterol biosynthesis and in glucose/insulin signaling pathways. This study highlights the utility of a zebrafish whole-organism chemical screen for modifiers of B-lps production and/or its clearance. A significant finding is that enoxolone inhibits hnf4a in zebrafish to reduce B-lps production and supports targeting HNF4a as a therapeutic means to reduce the emergence of atherosclerosis.

Strengths:

The authors performed a whole-organism chemical screen with over 3000 agents. Such screens are challenging, and the authors used strict criteria for determining hits. The conclusions of this study are well supported by the presented data.

Weaknesses:

There are areas within the study and writing that can be improved and extended, specifically within the gene expression studies.

Reviewer #2 (Public review):

Summary:

The authors aimed to develop a large-scale drug screen to identify B-lp modulators in a vertebrate whole-animal system. Using the zebrafish LipoGlo system that the authors had previously published and validated, the authors screened 2762 drug candidates to generate 49 hits and ultimately validated 19 drugs as genuine ApoB-lowering drugs. Using LipoGlo-Electrophoresis, the authors are able to obtain insights into the ApoB-lipoprotein size/subclass distribution. The authors further validate and study the mechanism of a strong hit, Enoxolone, known as also known as 18β-Glycyrrhetinic acid, which has previously been reported to modulate lipid metabolism. The authors also show that Enoxolone effects are mediated through HNF4⍺, which has been previously shown in the mouse system, but this is the first time it has been shown in the zebrafish.

Strengths:

The study was methodical and robust, using a published and well-validated zebrafish LipoGlo model. The authors validated the hits from the screen independently and considered the possibility that some drugs may have been detected as false positive results due to effects on the enzymatic activity of NanoLuciferase; only one hit, verteporfin, was shown to be a false positive. Using LipoGlo-Electrophoresis, the authors are able to obtain extra insights into the ApoB-lipoprotein size/subclass distribution. They showed that while enoxolone treatment reduces total B-lps, there are no overt changes in B-lp size distribution compared to vehicle-treated animals, other than a slight increase in the zero mobility (ZM) fraction, which contains very large particles and/or tissue aggregates. In contrast, the positive control, lomitapide, does show a change in B-lp size distribution compared to vehicle-treated animals - an increase in frequency of LDLs (low-density lipoprotein), but a decrease in VLDLs (very low-density lipoprotein). This study also assesses the LipoGlo-Electrophoresis profile of HNF4⍺ inhibitors. Work in the zebrafish larvae means that the effect on overall development and an entire vertebrate organism can also be assessed. Finally, the authors applied a thorough statistical measure to define a hit, using the Strictly Standardized Mean Difference (SSMD) method.

Weaknesses:

While the screen was thorough and well-validated, the authors missed a chance to provide a lot of extra significance to a wide range of readership. While the hits were thoroughly validated and displayed, the authors could have also presented the LipoGlo-Electrophoresis for all validated hits or at least a number of them. This would hugely increase the insights into these compounds. Also, the authors chose to validate and follow up a mechanism for Enoxolone, yet this hit was already known to modulate lipid metabolism through HNF4⍺, therefore, hugely limiting the impact of the paper. So what the authors have shown that is novel is only subtly added to this - consistent in vertebrate models, RNA sequencing of pathways, further validation of the HNF4⍺ pathway, and a profile of resulting B-lp size distribution. It seemed an easy way out to pick such a candidate, and they could have followed up by validating more thoroughly a completely novel drug. Also, the authors' prior paper showing the methodology also depicted complementary EM and LipoGlo-microscopy approaches. The microscopy especially, would have been an easy complementary add-on to the screen to really give extra insights into B-lp metabolism in a whole organism for all candidates. This felt like a missed opportunity.

Reviewer #3 (Public review):

Summary:

In "A‬‭ whole-animal‬‭ phenotypic‬‭ drug‬‭ screen‬‭ identifies‬‭ suppressors‬‭ of‬‭ atherogenic‬ lipoproteins", Kelpsch et al seek to identify new, chemically targetable pathways that regulate ApoB function and could ultimately serve as treatments for elevated lipid disorders and/or cardiovascular disease. Given the interconnected nature of lipid regulation in the whole organism with interdependent organs and secreted components (i.e. lipoproteins), they use the vertebrate model zebrafish to screen a large library of ~3000 compounds for their ability to lower the important ApoB-containing lipoproteins. They find 49 hits with 19 compounds passing a higher level of scrutiny, and focus on the role of enoxolone in modulating B-Ip levels at least partly through the HNF4alpha transcription factor and, putatively, through downstream cholesterol/lipid biosynthetic pathways.

Strengths:

The study uses a well-validated in vivo stain (LipoGlo) for measuring lipoproteins in the context of a developing whole organism with a quantitative read-out on a high-throughput platform, allowing for screening of thousands of compounds altering the complex metabolic/physiologic functions necessary for lipoprotein production.

The use of genetic mutant HNF4alpha to assign the mechanism of action to the prime candidate compound studied (enoxolone) is a powerful approach for this challenging aspect of chemical genetics studies. See caveats in weaknesses.

Weaknesses:

As shown in Figure 5A, the HNF4alpha mutant homozygous -/- already lowers lipoproteins. Is it just that the mutant level is already at a minimum in this homozygous mutant (and thus enoxolone can not induce even lower lipoprotein levels), or is it true that the enoxolone molecule is primarily acting through this TF (i.e. HNF4alpha homozygous mutant is truly epistatic to enoxolone function) as favored in the text.

While it is definitely interesting to study enoxolone effects during whole embryo development, the link to HNF4alpha had previously been described in the literature, as pointed out by the authors. The generalizability of the approach to identify truly novel pathways remains to be fully realized, but sharing this available screen data to date will invite further inquiry and be very valuable to the community.

Figure 5 - The same allele of HNF4alpha loss of function/hypomorph (rdu14) is used in both 5A and 5B, but labeled differently in each subpanel. This is explained in the figure legend, but could be updated to use the same nomenclature in both panels to clarify the Figure presentation.

Author response:

We would like to thank the editors and reviewers for their time and their helpful feedback. We largely agree with the reviewer recommendations and comments, which we will address for the next Version on Record of this manuscript. We plan to address reviewer comments in the following ways.

Reviewers requested a more comprehensive analysis of our RNA-seq experiment comparing vehicle treatment to enoxolone treatment over time. We will improve our analysis by providing clear, accessible, and organized tables defining differentially expressed genes at each time point, gene set lists that comprise our gene ontology analysis, and the lists of shared differentially expressed genes from enoxolone treatment and HNF4⍺ knockout. While some of this data was provided in the supplementary files, we recognize that it should be more accessible for the reader. Furthermore, as suggested by the Reviewer, we will enhance our transcriptomic analysis by utilizing bioinformatic tools such as Enrichr.

The Reviewers noted that we identified a number of lipoprotein-lowering compounds through our drug screen, but limited the impact of our manuscript by focusing on enoxolone, a known inhibitor of HNF4⍺ and modulator of lipid metabolism. While we understand with the sentiment that other novel compounds would be interesting to study, we aimed to demonstrate proof of concept in this manuscript. We view the characterization of novel compounds as beyond the direct scope of this manuscript. We did not perform LipoGlo imaging and electrophoresis experiments on each drug because these experiments are low-throughput given the number of drugs and doses we examined. In light of the Reviewer’s comments, we will add some additional characterizations of our validated hits with LipoGlo imaging and electrophoresis studies.

The reviewers also identified a number of typos in text and figures that will be addressed in the next Version on Record. We believe that the recommended changes will strengthen our manuscript and broaden its appeal. We are grateful for the opportunity to improve our work based on the reviewers’ valuable suggestions.