Today’s paper is a continuation of our discussion on screening compounds for drug discovery: Kokel et al, 2010 Rapid behavior-based identification of neuroactive small molecules in the zebrafish, Nature Chemical Biology [PMC]. Having just returned from the IASP meeting in Montreal I am really pressed for time today so this is going to be brief; however, don’t let my brevity stop you from delving into the details of the paper — its a really fascinating study with major implications for drug discovery.
The problem is a simple one: when screening compounds we typically work on the assumption that the target for the disease is known. This is required because a typical library screen is based on receptor or enzyme activity and not a relevant behavioral phenotype. While this is fine for disorders with well understood targets, this is often not the case for neurobiological disorders where we don’t necessarily know the target beforehand. Hence, a behavior-based screening mechanism that is done in a high throughput fashion/screen (HTS) would be optimal for identifying novel neuroactive compounds. The problem is that behavioral experiments and model organisms are generally not conducive to this sort of thing:
Unlike target-based approaches, phenotype-based screens can identify compounds that produce a desired phenotype without a priori assumptions about their targets. Phenotype-based screens in cultured cells and whole organisms have identified powerful new compounds with novel activities on unexpected targets in vivo. However, it has been difficult to combine chemical-screening paradigms with behavioral phenotyping, perhaps because many well studied behaviors are too variable or occur in animals that are too large for screening in multi-well format.
So how have the authors provided a solution to this problem?
Given the unmet need for novel psychotropic drugs, we sought to develop a small-molecule discovery process that combined the scale of modern high-throughput screening with the biological complexity of behavioral phenotyping in living animals. Here, we report development of a fully automated platform for analyzing the behavioral effects of small molecules on embryonic zebrafish. Using this platform, we have identified hundreds of behavior-modifying compounds. We further demonstrate that complex behavioral changes can be distilled into simple behavioral ‘barcodes’ to classify psychotropic drugs and determine their mechanisms of action.
The data presented in the paper shows a novel behavioral phenotype for zebrafish embryos. Essentially, shining a bright light on them induces about 5 seconds of brisk activity with a latency of about 1 sec. After this burst of activity, normal embryos become refractory to a second light stimulus. The authors go on to show that certain classes of drugs modify this behavior in stereotypical ways. Beta adrenergic agonists stimulate more activity and benzodiazepines decrease activity, for instance. Moreover, certain classes of drugs produce a behavioral “barcode” which may indicate mechanism of action (MOA) for drugs with no known target. A screen of several thousand compounds led to a set of barcodes that could then be used to test whether drugs without a known MOA share an MOA with compounds with the same barcode. The authors show that this has predictive power for at least two compounds with no known MOA. In my opinion this is very nice and provides a potential solution to a problem that has vexed neuropharmacologists for decades. Its not easy to figure out how many behaviorally active compounds work and the combination of this HTS approach with informatics has the potential to make this task much easier in the future.
There are obviously many caveats here. 1) Its just one simple behavioral output they have used but there are potentially many other behavioral outputs that could be utilized in these zebrafish embryos. Compounds with anti-pain activity immediately come to mind as it seems that this would be an easy behavioral assay to develop using this technology. 2) The other main issue, at least in my mind, is that these are embryos without a fully developed nervous system. This is obviously problematic but its a start and I would not discount the technique based purely on that criticism.
The authors wrap up with a fascinating paragraph:
Behavioral assays such as the photomotor response (PMR) may also be useful for screening chemicals for neurotoxicology or other undesirable behavioral effects. Testing drug candidates for neurotoxicity remains a significant challenge, and new regulations requiring the testing of thousands of compounds are expected to require millions of rodents and cost billions of dollars. Given these costs and the ethical implications of increased animal use, inexpensive, high-throughput means of testing for neurotoxicity are needed, especially those involving nonmammalian species. Of course, questions remain about the degree of conservation of nervous-system drug effects in zebrafish and humans. Although we found that many drugs with psychotropic effects in humans also caused reproducible behavioral effects on the PMR, many other psychotropic drugs did not produce any detectable change in the PMR screen. We do not yet know the degree to which these ‘false negatives’ are due to screening dose, failure of absorption or imperfect conservation between zebrafish and humans. Clearly, answering these questions will be an important step toward translating behavioral pharmacology findings from zebrafish to humans.
Neurotoxicity is a massive problem in drug discovery (perhaps one of the biggest ones) and there certainly seems to be lots of utility here (see Fig 5 for details). A simple screen like this could save tons of money, time and maybe even future lawsuits. False negatives would obviously be a problem but full dose-response curves in this assay would be simple and I can’t imagine that looking at absorption for individual compounds would be that difficult.
One last issue I would like to touch on here is disease models and drug screening. Often-times we become enamored of models that resemble a human phenotype in model organisms but these models have all too often been unsuccessful in finding new drugs (or new targets for that matter). Screening drug classes that are effective (even minimally) in human neurological disorders in an unbiased screen like this may produce behavioral barcodes that suggest novel mechanisms. With the large dataset that has already been developed in this paper it may be straightforward to do disease based screens of compounds to look at behavioral barcodes to identify novel targets that have not been identified for the disease of interest before. Then again, this idea may be all too much pie in the sky. However, based on the simplicity of the assay and the existing large dataset (Fig 3) I can’t imagine that its not worth a shot.
Kokel, D., Bryan, J., Laggner, C., White, R., Cheung, C., Mateus, R., Healey, D., Kim, S., Werdich, A., Haggarty, S., MacRae, C., Shoichet, B., & Peterson, R. (2010). Rapid behavior-based identification of neuroactive small molecules in the zebrafish Nature Chemical Biology, 6 (3), 231-237 DOI: 10.1038/nchembio.307