Table 3.

Strengths and Weaknesses of Different Target Validation Methods (Modified From [11])

Method	Strengths	Weaknesses
Chemical validation	Addresses the key druggability issues of cell permeability (in vitro whole cell assays); selective toxicity and drug metabolism (in vivo animal models); safety and efficacy (clinical) Identifies non-protein targets Identifies pro-drugs and compounds acting by lethal synthesis	Highly specific inhibitors frequently not available Lack of specificity for target resulting in poor structure-activity-relationships (SAR) Variable cellular pharmacokinetics can cause poor SAR Correlation between target inhibition and predicted molecular or biochemical phenotype sometimes difficult to demonstrate in vitro or in vivo
All genetic validation	Many complete genomes available Suitable for genes of unknown or uncertain function	Cannot identify non-gene targets (e.g. haemozoin) Does not address key druggability issues Does not identify drugs acting via lethal synthesis Does not distinguish between structural and catalytic requirement
Knockout methods	Definitive, “clean” phenotype Few or no off-target effects	Laborious (usually requires multiple transfections in diploid organisms) Null mutants for essential genes require genetic or nutritional rescue Multicopy genes can be problematic Compensatory (suppressor) mutations can occur
RNA interference (RNAi)	Rapid and easy to perform Suitable for multicopy gene families	Not possible in many parasite species No phenotype due to insufficient silencing Off-target effects due to unintentional silencing “Escape” mutants with essential genes