Phenotypic screening is gaining new momentum in drug discovery with the hope that this approach will improve the success rate of drug approval.1 In this article we look at some of the latest screening tools and their applications.

This is illustrated by their recent study with Dr Ayman Zen where the team developed a high-content imaging screen using the endothelial tube formation assay, miniaturized to a 384-well plate format. Screening with an annotated chemical library of 1,280 bioactive small molecules identified a retinoid agonist, Tazarotene, that enhanced in vitro angiogenesis and wound healing in vivo. This high content screen identified an already FDA-approved small molecule that could be potentially exploited in regenerative medicine.3

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Ebner is currently working in collaboration with recent Nobel-Prize winner Peter Ratcliffe, alongside scientists at Edinburgh University and MIT, to model hypoxia in glioblastoma. Hypoxia is a problem with some glioblastomas as it protects cells from radiotherapy treatment. Our aim is to use Peters expertise to help us set up an assay that mimics real tumor hypoxia. Then if we can identify small compounds that alter that hypoxic condition we can make the glioma cells more susceptible to either radiotherapy or temozolomide or some other treatment combination.

The labs main readout is high-content imaging, using fluorescent microscopy that can take many thousands of pictures. This approach utilizes different labels and harnesses software that automates the image analysis. The image analysis is set by the biologists but then it's applied across the entire screen. Its lower throughput than plate-based readout, but you get a lot more information out of the images, says Ebner. Increasingly, high content imaging is moving towards using AI and deep learning where you're trying to draw out even more information than the primary phenotype that you were looking at.

Indeed, a recent study using CRISPR-Cas9 mutagenesis showed that the proteins targeted by many cancer drugs currently in clinical development are non-essential for tumor growth, despite evidence to the contrary from previous studies using RNAi and small molecule inhibitors.4 In addition, the efficacy of the drugs tested was unaffected when CRISPR was used to knockout its assumed target suggesting that many are eliciting their anticancer activity through off-target effects.

The other benefit of CRISPR is that its extremely flexible, says Pettitt. This means you can expand the range of cell line models, for example, that you can screen in. The key reason why RNAi was such a popular technology, and now CRISPR is, is that you can basically knock out a gene by synthesizing just a short piece of RNA, he explains. CRISPR guides are very easy to synthesize, you can do it in a very high throughput setting, and you can design customized libraries to knock out every gene in the genome or a particular set of genes. As long as you can get the CRISPR machinery into your cells, it works very reliably.

The classic CRISPR (CRISPR-Cas9) system comprises a nuclease called Cas9 which you can program with a short RNA (20 nucleotides). The RNA will direct the nuclease to a certain site in the genome that matches and the nuclease will cleave the genome at that point. Repair of that double-strand break results in small insertions and deletions that result in knock out of a gene. But theres now more evolved applications of the technology emerging.

I think it's possible to be very creative with CRISPR in a way that it isnt with RNAi, says Pettitt. With RNAi you can really only shut genes off, but with CRISPR as well as making random mutations to knock out genes - you can also precisely edit genes if you provide a template region with a mutation with it. This can be incorporated into the target site for CRISPR so you can introduce the specific mutation youre interested in.

One such example is the problem with BRCA1 mutations: its important to be able to functionally classify whether these mutations are benign or pathogenic. A recent study used CRISPR to test 96.5% of all possible single-nucleotide variants (SNVs) in exons that encode functionally critical domains of BRCA1 and found over 400 non-functional missense SNVs were identified, as well as around 300 SNVs that disrupt expression. This knowledge will immediately aid clinical interpretation of BRCA1 genetic test results.5 In another study,6 Pettitt and colleagues used genome-wide CRISPR-Cas9 mutagenesis screens to identify the mutated forms of PARP that cause in vitro and in vivo PARP inhibitor resistance, and found that these mutations are also tolerated in cells with a pathogenic BRCA1 mutation resulting in a different profile of sensitivity to chemotherapy drugs compared with other types of PARP inhibitor resistance.

You couldnt screen at that level of detail using RNAi, where you design custom CRISPR that targets many different regions of the same gene and you can figure out which domains of the protein are important for your phenotype of interest, says Pettitt.

There are other evolutions of CRISPR now being developed as screens. For example, if you mutate the nuclease activity of Cas9, it still retains its ability to localize to the target site, so you can fuse Cas9 to transcriptional activators or repressors, and screen for transcriptional repression with CRISPR, as well as knock-out screens, says Pettitt. Theres also a whole range of CRISPR tools being developed that will edit bases by causing missense mutations rather than insertions or deletions, or causing methylation of DNA, or bringing in fluorescent proteins so you can visualize where the DNA sequences in the cells are. Its a measure of how flexible and useful CRISPR is in comparison to RNAi.

So will CRISPR be the one technology that everyone turns to for phenotypic screening in future? Im a firm believer that no technology answers every question, says Ebner. CRISPR is amazing, its use as a therapeutic or biologic is the stuff of science fiction. But as a tool for target identification, it comes with one important caveat. CRISPR knockout means exactly that it removes the potential protein that would otherwise be in the mix. Thats very different from a small compound inhibiting a protein that is still able to form a complex or that is just not active. Its the perfect example of a brilliant technology that is transformative, but it's not perfect. No technology is perfect.

References

1. Zheng W, Thorne N and McKew JC. Phenotypic screens as a renewed approach for drug discovery. Drug Discov. Today 2013; 18: 1067-1073.

2. Horvath P, Aulner N, Bickle M, et al. Screening out irrelevant cell-based models of disease. Nat Rev Drug Discov. 2016 Nov;15(11):751-769. doi: 10.1038/nrd.2016.175. Epub 2016 Sep 12.

3. Al Haj Zen A, Nawrot DA, Howarth A, et al. The Retinoid Agonist Tazarotene Promotes Angiogenesis and Wound Healing. Mol Ther. 2016 Oct;24(10):1745-1759. doi: 10.1038/mt.2016.153.

4.Lin et al. Off-target toxicity is a common mechanism of action of cancer drugs undergoing clinical trials. Science Translat Med. 2019; 11: (509). doi: 10.1126/scitranslmed.aaw8412

5.Findlay GM, Daza RM, Martin B et al. Accurate classification of BRCA1 variants with saturation genome editing. Nature. 2018 Oct; 562(7726): 217222. doi: 10.1038/s41586-018-0461-z

6.Pettitt et al. Genome-wide and high-density CRISPRCas9 screens identify point mutations in PARP1 causing PARP inhibitor resistance. Nat Commun. 2018 May 10;9(1):1849. doi: 10.1038/s41467-018-03917-2.

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Phenotypic Screening Advances in Technologies and Techniques - Technology Networks

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