**2.1 Experimental strategies in drug repurposing**

### *2.1.1 Binding assays to identify target candidate*

Techniques like chromatography and mass spectrometry are being used to find all possible binding targets of drug candidates. Brehmer et al. conducted a study using HeLa cell extract to identify possible protein targets of gefitinib. The results of mass spectrometry revealed that the drug can interact with 20 different protein kinases which may be treated as possible targets of gefitinib [14].

#### *2.1.2 Phenotyping screening*

In the world of drug discovery and development, the term phenotyping screening is used to describe the ways adopted to identify the biological effects of a drug which is either directly or indirectly linked to a disease. With the development of robotic and sensitive screening tools, the approach is used to screen thousands of chemical drug libraries in a single go. It involves the screening of target drug candidates either using cell-based screens (high throughput screening (HTS)) or even whole organism [15]. Cell based assays involve cell lines derived from human or animals, immortalized cell lines or induced pluripotent cells lines (iPSCs), etc. For example, Iljin et al. [16] performed HTS of approximately 5000 small molecules using prostate cancer epithelial cell lines. In this experiment, disulfiram was found to possess selective antineoplastic property which was validated later using genome wide gene expression studies [16]. Important cell-based assays that are being performed in drug discovery and repurposing include: cell viability assay, signaling pathways assay and disease related mechanistic assay. Other cell assays

#### *Drug Repurposing - Hypothesis, Molecular Aspects and Therapeutic Applications*

that are being performed routinely in drug development include cell apoptosis, infection, cell motility, cell cycle arrest and many more [17]. Cousin et al. [18] have used zebrafish model to evaluate the efficacy of compounds against tobacco dependence. In this study the authors have found that the agents like apomorphine may be useful in modifying nicotine or ethanol induced dependence behavior [18]. Over the decades in drug discovery, these approaches are getting shifted more toward molecular targets based on phenotypic methods, which rely mainly on molecular targets of drugs and diseases and related mechanisms for hypothesis generation.
