*3.1.2.1 Cell-based assays*

Various unexplored targets and pathways lie within the components of cellular complexity which offers an excellent platform to identify antimicrobial lead molecules through the cell (or organism)-based HTS. Thus, multiple targets can be screened using cell-based assays in all the stages of drug discovery. In simple words, these assays are used when the desired cellular target is either unknown or the

**Figure 3.** *Ligand and structure-based drug designing process.*

*High-Throughput Screening for Drug Discovery toward Infectious Diseases: Options and Challenges DOI: http://dx.doi.org/10.5772/intechopen.102936*

phenotype cannot be separated from the cellular context. Nevertheless, these assays provide additional information which cannot be obtained from biochemical assays or vHTS, such as membrane permeability, pharmacodynamic (agonist, partial agonist, inverse agonist, and antagonist) status, cell proliferation (or viability), cytotoxicity, heterogeneity, protein expression, transcriptional readouts, and phenotypic biomarker readouts. Thus, cell-based assays may be classified depending on the methodologies used such as: (i) cell viability assays using (a.) dyes like Alamar blue, tetrazolium compounds (MTT assay, XTT assay, and MTS assay) which get converted to generate fluorescence or color indicating cell death or viability; (b.) luciferin-luciferase assay where ATP content is measured using luciferin-luciferase to generate bioluminescence; (c.) intercalation with membrane-permeant DNA dyes; (ii) reporter gene assay; (iii) secondary messenger assay; (iv) protein-fragment complementation assay; (v) protein–protein interaction assay; (vi) label-free methods; and (vii) phenotype biomarker assays. For anti-infective drug discovery, cell viability assays with different cell lines are utilized to screen and identify molecules that can kill or inhibit the growth of pathogens. These assays are further utilized to evaluate the safety issues of the organs such as the liver because the liver is the primary center for drug metabolism [29].

#### *3.1.2.2 Biochemical assays*

Biochemical assays involve screening of chemical libraries for *in vitro* inhibition of purified target protein (enzyme, receptor, and ion channels) in competition format where the known substrate bound to protein is replaced by the ligand or compound under study. The biological response is detected using optical methods such as fluorescence, luminescence, or absorbance [29].

#### **3.2 Biological response detection methods in HTS**

The detection of biological response in the cell-based and/or biochemical assay may be performed using different analytical technologies such as, fluorescencebased assays [FRET, HTRF, dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA), time-resolved FRET (TR-FRET), fluorescence polarization (FP), fluorescent lifetime (FLT)], luminescence-based assays [bioluminescence resonance energy transfer (BRET), amplified luminescent proximity homogeneous assay (ALPHA), electrochemiluminescence assay (ECL)], atomic absorption spectroscopy (AAS), high-throughput electrophysiology (HT electrophysiology), protein complementation assay (PCA), Scintillation proximity assay (SPA), and enzyme fragment complementation (EFC) [18], which are further modified with different variations. However, the detailed discussion on the usage of these variations in the design of HTS assays is beyond the scope of this chapter.

### **4. Applications and outcomes of HTS in anti-infective drug discovery**

HTS is being applied in a myriad of ways starting from the biology of infectious diseases to finding the lead molecules for anti-infective drug discovery. Few applications of HTS in infectious biology are the identification of pathogenic molecular mechanisms, evolutionary analysis of pathogens, and determination of the determinants required for survival and pathogenesis of the mutant strains of the microbial population [30]. Although, HTS is an early-stage drug development program, however, the anti-infective drug discovery efforts with HTS from the year 2000 to date have led to the approval of 38 new antibacterial drugs and 67 drug candidates

are in the clinical development stage for both Gram-negative and Gram-positive bacteria including *Mycobacterium tuberculosis*. Nevertheless, 19 different compounds with novel pharmacophore are in different stages of clinical development (6 compounds in Phase I, 9 compounds in Phase II, 4 compounds in Phase III) [31].
