Identification of Potential Anti-TB Candidates: A Step-by-Step Guide to Synthesis, MIC Determination, and Cytotoxicity Assessment in Mammalian Cells.

Tuberculosis (TB) remains one of the leading causes of death from a single infectious agent worldwide, with rising rates of drug resistance making the development of novel therapeutic agents a global health priority. This protocol describes a phenotypic screening approach for identifying potential anti-TB drug candidates through three integrated stages. First, a compound from the 4-aminoquinoline class is synthesized via a three-step route and fully characterized by high-performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR) spectroscopy, and high-resolution mass spectrometry (HRMS). Second, the antimicrobial activity of the compound is evaluated against Mycobacterium tuberculosis using the colorimetric resazurin reduction microplate assay (REMA), which determines the minimum inhibitory concentration (MIC) through a simple visual color-change readout. Third, the compound's cytotoxicity is assessed in two mammalian cell lines, Vero (African green monkey kidney) and HepG2 (human hepatocellular carcinoma), using the MTT assay (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) and the neutral red uptake (NRU) assay. These two complementary assays measure distinct cellular parameters, enabling cross-validation of cytotoxicity results. Together, the MIC and cytotoxicity data allow calculation of a selectivity index (SI) to assess the therapeutic potential of each compound. This protocol provides a reproducible, step-by-step framework for early-stage anti-TB hit identification and preclinical evaluation.