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Drug Discovery


Drug target mutation


Wild type drug target


Diseased cell proliferation CRISPR gene editing Phenotype rescue


Healthy cell proliferation


High drug sensitivity


Low drug sensitivity ON-target validation


Figure 1 Two applications of CRISPR- Cas9 technology in a phenotype rescue approach. Validation of a drug target can be performed by restoration of a disease-associated phenotype such as an excessively proliferating cancerous cell population upon correcting the putative disease-causing mutation. Correcting this mutation can also help confirming the mutation and on-target- specificity of a drug if sensitivity is abolished by reverting to the wild type state of the target.Thereby on-target effects on cell viability are discriminated from off-target effects resulting in non-specific loss of cellular fitness


advances in functional genomic screening, such as RNA interference and CRISPR-Cas9 systems, high-throughput phenotypic screens using libraries of small molecules, biologics (antibodies and enzymes) and TIDES (peptides and oligonu- cleotides) are key to the identification of moieties that induce a desired therapeutic effect in a specif- ic disease model. Also in silico analysis of drug- target interaction is valuable for tool compound prediction and lead optimisation. Altogether these target identification and hit finding approaches are the very first steps in a drug development pipeline. With the estimated probability for a novel target


to reach preclinical stage at only 3%, compared to 17% for a known target4, and more than half of 2018 FDA approvals targeting orphan diseases that are less well characterised5, the subsequent validation of newly-identified targets is fundamen- tal. The main objective in target validation is to confirm the involvement of the target in disease phenotype(s) and to select biologically plausible and relevant molecular targets for interference. Target validation can be broken down in two key steps: 1. Repetition of the target identification experi- ment to demonstrate reproducibility. 2. Introduction of changes in the experiment, such as a different cellular context or the use of different approaches to validate the functional role of the target in the disease phenotype. A variety of cell-based assays have been devel-


Drug DiscoveryWorld Summer 2019


oped in target validation using different strategies. The most popular approach modulates the cellular amount of the target by decreasing or disrupting the gene expression of a target using RNA interfer- ence or more recently by CRISPR-Cas9. The induced loss or gain-of-function allows for the determination of the physiological role of the tar- get in disease phenotype without the presence of a drug. Such methods for accurate target validation are critical to improve the downstream probability of success, but need to be executed carefully as out- lined below.


Pitfalls of poor target and hit validation Although early sifting by proper target and hit val- idation can have a tremendous positive impact on downstream success rates, candidates are some- times fed into downstream development pro- grammes without complete knowledge of the underlying physiology and even improper assump- tions on complex biology6. Common pitfalls in interpreting the relevance of a (novel) target for the disease are:


● Failing to account for multiple gene targets. Often it is hypothesised that a single target or driv- er mutation is associated with disease prognosis, whereas in practice it is much more common that multiple genes are contributing to a diseased state. These factors may even counteract each other or redundant genes may exist to safeguard a homeo- static and resistant environment. Also if a single


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