On 2021-03-10 20:05:11, user Joseph Ong wrote:
Hadjikyriacou et al. present a novel method of drug screening across three different model research systems: Drosophila larva, C. elegans, and human cell culture (patient-derived fibroblasts). In particular, the authors devise a series of imaging-based assays to determine the effect of their library compounds against lysosomal disorders in these model systems with the goal of identifying novel drugs or drug targets against lysosomal disorders such as mucolipidosis type IV. More broadly, the authors claim that a multi-species method with the goal of identifying conserved biological pathways may lead to more viable treatment of diseases.
The Drosophila system uses larva heterozygous or null for trmpl, an endolysosomal ion channel. In this system, trmpl-compromised pupae should not eclose and result in no living animals. Tracking movement (that is, of a properly hatched adult fly) within a 96-well plate informs whether or not a particular drug rescued the phenotype. For the C. elegans system, a strain with a mutation in cup-5, was used. The mutation resulted in the sequestering of GFP into specialized cells, so visualizing the relative area of green cells to the total cell body served as a measure of lysosomal storage disfunction. In a similar manner, the fibroblast system (with a mutation in TRMPL1) used the dye LysoTracker, and also assessed the total fluorescence signal from the dye (suggesting lysosome accumulation) as a readout of lysosomal storage disfunction.
Screening small molecules (4,185 compounds targeting ~2000 mammalian genes) through this system, the authors find few small molecules that are "hits" among the different species: 3 between human and worm; 3 between human and fly; and 7 between fly and worm; with the addition of one compound shared among all three species [though what these drugs are is not clear; perhaps it is in the supplemental information that was not uploaded to bioRxiv]. Similarly, the shared gene targets of these drugs fell into four main categories: Cdk and Cdk-associated proteins (human and fly), nitric oxide synthesis (fly and worm), and Abl-kinase pathway proteins (human and worm). One gene, ILK, a kinase with diverse functions, was found to be a gene target of all three species. The Cdk and ILK targets were further validated via other chemical and genetic methods, and the authors conclude
I found the multi-species approach delightfully insightful. As the authors point out, despite how similar humans and mice are, studies in humans have consistently failed to recapitulate the results seen in murine models. The approach, then, to cast a broader/wider net to analyze pathways broadly conserved between invertebrate and mammalian tissue culture seems appropriate. Of course, there is a great difference between flys/worms and humans, so care must be taken when interpreting and applying various results across species [but this is the same critique used when we analyze mouse models, so perhaps we should focus on this aspect of the approach as a novelty rather than a fault].
However, the results of the screen were more confusing than what I would have hoped. While it is true that one target, ILK, was a target of all three systems, the Cdk and Cdk-associated proteins were only detected as "hits" within the human-fly collection and not within worms. While there may be many reasons for this discrepancy -- with species differences or using mammalian protein-targeting drugs in a worm being the most obvious -- this observation casts some doubt on the approach. Moreover, save for ILK, the targeted gene pathways between each species seemed to vary widely. Ideally, the most important pathways would have shown as "hits" across all the species tested; however, the pathways seem to have no obvious relationship to each other, except that they are all major signaling/growth pathways. Perhaps it is not surprising that the major hits are all broad signaling pathways -- but I would have hoped that this assay would have allowed convergence on a particular pathway.
Together (that the gene pathways have poor correlation across species and that the Cdks were missed in worms), this suggests that a multi-species approach may be too wide/broad an approach -- particularly, given how well-conserved and well-studied the Cdk system is (and all the ink that has been spilled over palbociclib and its analogues). Perhaps more care in choosing model organisms (say, zebrafish instead of worms) may strengthen the screening approach and result in more physiologically relevant "hits" (as the zebrafish system might be closer to the human system such that the drugs tested may have a better efficacy in a vertebrate rather than worm system; and the "hit" pathways may more closely align with humans/fly in a vertebrate rather than worm system). Nonetheless, to the author's credit, I acknowledge that the Cdks were "hits" in both the human and Drosophila system and validated as targets in subsequent analysis. Perhaps I am too naive in believing that the Cdks should have been hits in all three systems tested -- that would make biology too easy.
Briefer comments:<br /> There is no guarantee that the mammalian target of Drug A is actually Protein X in another organism; for example, while the authors assume that palbociclib is a Cdk4/6 inhibitor in mammalian cells, is palbo also a Cdk4/6 inhibitor in Drosophila and worms? Clarifying the protein targets of drugs in other species is particularly important to address off-target or gain-of-function-type effects of drugs.
The decision to quantify the ratio of LC3-II (the lower band?) to LC3-I + LC3-II (lower + higher band?) is not clear to me. Also, in Figure 4A, marking the higher and lower band as LC3-I/-II would be helpful.
Figure 5A: While I understand it is annoying to do so, the authors should demonstrate the specificity of their siRNAs and demonstrate the knockdown of the proteins of interest at least via qPCR, but preferrably via western blotting. Besides this, I find the results in Figure 5 altogether confusing. Loss of Cdk6 seems to be the only protein here that results in accumulation of LC3-II. Do the authors then suggest that between palbociclib targets Cdk4 and Cdk6, it is only Cdk6 that is involved in the lysosomal phenotype? Moreover, if drugging the kinase ILK leads to a rescue, why does knockdown of the kinase ILK not lead to a rescue? Given that ILK is a top hit in the screen, this result is starkly inconsistent. While a knockdown and inhibition are different, this discrepancy should be resolved. Some molecular biology here (for example, CRISPR to create a kinase-defective version of ILK or generating a Shokat gatekeeper mutation of ILK) may help.
Given that some of the drug targets were B-type cyclins, the omission of Cdk1 is strange. Did the authors test loss or siRNAs against Cdk1? I imagine it would lead to some kind of death, but at least a comment on this is necessary.