The first step of the drug discovery workflow involves identifying and confirming a 'target' that plays a role in a disease condition. Targets can range from proteins and genes to entire pathways or disease-related metabolites. A target must be clinically, and commercially viable, and above all, druggable in an effective and safe way. 

“Druggability” refers to a target's accessibility to potential drug molecules and its ability to produce a measurable biological response upon interaction. Targets are identified through various methods such as loss and gain of function assays (e.g., RNAi, CRISPR-ko, CRISPRa, CRISPRi screens), bioinformatic analyses like genome-wide association studies, or hypothesis-driven approaches.

Yet, target identification using screens or data base mining provides only correlative information between the observed phenotype and the genotype. As an example, it remains difficult to understand the link between genetic variation and disease predisposition, phenotypic disease development and treatment response. In addition, traditional methods for studying the link between genotype and phenotype have limitations, such as the difficulty in obtaining matched samples and the time required to perform such studies.

To perform target validation, monoclonal cell lines were employed to clarify relationships between genotype and phenotype. The advent of CRISPR-Cas technology has revolutionized this area by facilitating the creation of isogenic cell lines. This technology allows for precise targeting of genes, splice variants, or even multiple genes within a pathway (multiplex editing), making it highly effective for studying diseases and potential drug targets. CRISPR-Cas can also be used to generate models for studying point mutations or single nucleotide polymorphisms (SNPs) in genes that are linked to specific diseases. These cellular models enable comprehensive studies using a variety of biochemical, molecular, and cellular assays, supplemented by more advanced models like organoids and transgenic animals. 

In compound screening, large libraries of small molecules or proteins are screened for their biological activity, specifically their impact on a validated target phenotype. Compounds that show promise in initial screenings are called 'hits.' Transgenic reporter cell lines, generated by CRISPR-Cas and HDR, are a cost-effective tool for this. These cell lines contain a genetic element that produces a detectable signal, such as fluorescence, when the target is affected. Alternatively, in vitro methods are used, especially when the target is a well-understood protein. Here, the protein is produced and purified, and its interaction with the compounds is quantified. Often, both approaches are combined for comprehensive screening.

These hits are then validated through further tests to confirm their potential as drug candidates. After multiple rounds of screening, a few hundred compounds undergo further validation, often using a different read-out or the previously used isogenic cell lines.  Compounds that significantly alter or reverse the phenotype are considered strong candidates. The 'hit' compounds are then chemically engineered and tested to improve their stability, solubility, selectivity, efficacy, and pharmacokinetic properties, resulting in 'lead' compounds. Lead compounds undergo rigorous testing in vitro (in test tubes - "in cellulo") and in vivo (in animal models) to evaluate their safety, efficacy, and pharmacokinetics. Ultimately, only a handful, often just 1-2, proceed to the next stage of drug discovery.

Prior to clinical trials are pre-clinical development. This stage of the drug discovery process consists mainly of in-depth phenotypic validation using cellular and animal models. Furthermore, the toxicity, the efficacy and the pharmacokinetics are assessed. At this stage of the drug discovery process, only one compound will enter the clinical phase. In the clinical phase, the toxicity is assessed again in healthy volunteers before the drug will be tested in broad clinical studies. Clinical trials are carried out in several phases (Phase I, II, III, and sometimes IV) to test the drug's safety, optimal dosage, and efficacy in humans.

• Phase I: Primarily focuses on safety in a small number of healthy volunteers.
• Phase II: Looks at the drug's efficacy and optimal dosage in a larger number of patients.
• Phase III: Further assesses efficacy and monitors adverse reactions in a large cohort of patients.
• Phase IV: Post-marketing studies after the drug is approved and released.

The final stages comprise FDA approval, drug production, and further monitoring.