Drug Discovery

target has multiple roles in various pathways7 it may not be favourable to challenge them all or it may be considered for drug repurposing8. ● Cloudy forecasts. Despite great advancements in genome-wide association and expression profiling, it is still hard to predict whether altered gene or protein expression is a consequence or a cause of the disease. ● Misinterpreting target expression and knock- down levels. Assessment of target expression levels by RNA interference also poses confounding fac- tors that need to be considered. Besides practical concerns such as residual expression by incomplete mRNA knockdown, high variation of knockdown efficiency between cell lines and a high incidence of confounding off-target effects9, data could be mis- interpreted by biological issues. For example, a gene may be processed at transcription or transla- tion level by which genetic disruption would not decrease expression of all isoforms. Also, a gene could be expressed in excess, by which incomplete knock-down is not sufficient to establish the func- tion of the target.

Besides validation of the relationship between

the target and the phenotype, the drug-target inter- action and its predicted biological effect should also be confirmed. Key points that should not be overlooked herein are:

●Off-target effects. In complex biological assays it is not always evident whether the resulting drug- induced phenotype is (entirely) due to the antici- pated response at the desired target or if off-target effects should be taken into account. For example, cellular fitness is governed by many different pro- cesses in parallel. Unintentionally disrupting any of these pathways may impact cell viability indepen- dent of the intended on-target effect of an anti-can- cer drug candidate. ● Choose your model wisely. Another important consideration for accurate drug-target validation is the model system used. A simple cell line will likely react differently to a stressor than a 3D cell model or primary patient material that may be a better reflection of the in vivo situation. Even more so, individual background mutations differ- ing between patients may interfere with the effica- cy of a compound. For this reason, drug-target interaction and phenotype modulation need to be confirmed in relevant cellular and disease con- texts. All in all, this highlights the importance of not

disregarding comprehensive target and hit valida- tion and carefully confirming the hypothesis before


proceeding to the next stages of development for a novel compound.

Phenotypic rescue approach: a gold standard in target validation Many of the considerations described above can be tackled by a phenotypic rescue approach. In such experiments, the role of a target and modifying drug in the disease phenotype is investigated by manipulating the target in the cellular context, such as:

● The modification or deletion of the active site of the target; ● The restoration from the disease-associated vari- ant of the target to thewild type variant of the target; ● The precise deletion of the region of interaction with the drug.

Such approaches can confirm that the target is

causative for the disease and putative off-target effects can be distinguished from on-target effects without confounding factors such as different cel- lular backgrounds. Currently, phenotype rescue is often performed by decreasing or disrupting other pathways to compensate for the mutant pheno- type, by transient expression or integration of the original or mutated form of the target elsewhere in the genome. Each of these strategies has drawbacks of its own, however. Indirect rescue via another pathway is often hard

to interpret given the complexity of interacting cel- lular systems. Re-expression systems may lead to overexpression artefacts by disrupting the natural balance of genes expressed in a homeostatic state. Also for transient expression systems the timing may not coincide with the disease-causing mecha- nisms and integration elsewhere in the genome may disrupt the function of the locus where it is integrated. It may thus be clear that improvement of the methods commonly used for phenotypic res- cue is highly desired.

Added value of CRISPR-Cas9 technology in phenotypic rescue experiments There is no doubt that the CRISPR-Cas9 technol- ogy has revolutionised many facets of science and target identification and validation approaches for drug discovery are no exception to that. It is now relatively easy to edit a specific target gene of interest and genome-wide knockout libraries are commercially available, making it a popular tool for phenotypic screens. Besides this well-known application in target discovery, the CRISPR-Cas9

Drug DiscoveryWorld Summer 2019

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