Name: Identification of Potential Anti-TB Candidates: A Step-by-Step Guide to Synthesis, MIC Determination, and Cytotoxicity Assessment in Mammalian Cells (Video) | JoVE Journal
Uploaded: 2026-05-22T12:45:00.000+00:00
Duration: 10 min 24 s
Description: Learn a phenotypic screening pipeline for anti-TB drug discovery using synthesis, MIC determination via REMA, and cytotoxicity assessment in mammalian cells. This method enables early identification of selective therapeutic candidates.

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This work was supported by the National Institute of Science and Technology on Tuberculosis (MCTI/CNPq/SECTICS/MS/CAPES/FAPERGS, Grant No. 404838/2024-0), CNPq/MCTI N&#176; 44/2024 &#8211; Universal (Grant N&#176; 420934/2025-1) and FAPERGS 06/2025 &#8211; PqG (Grant N&#176; 25/2551-00002734-9). C.V.B (CNPq, Grant No. 311949/2019-3), L.A.B. (CNPq, Grant No. 303499/2021-4), and P.M. (CNPq, Grant No. 310888/2022-0) are Research Career Awardees of CNPq. This study was financed in part by the Coordenac&#808;&#227;o de Aperfeic&#808;oamento de Pessoal de N&#237;vel Superior &#8211; Brasil (CAPES), Finance Code 001.

<ol><li>Pai, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tuberculosis%5BTitle%5D)+AND+%22Nat+Rev+Dis+Primers%22%5BJournal%5D)">Tuberculosis.</a> Nat Rev Dis Primers. 2, 16076(2016).</li>
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<li>Larkins-Ford, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Systematic+measurement+of+combination-drug+landscapes+to+predict%C2%A0in+vivo%C2%A0treatment+outcomes+for+tuberculosis%5BTitle%5D)+AND+%22Cell+Syst%22%5BJournal%5D)">Systematic measurement of combination-drug landscapes to predict in vivo treatment outcomes for tuberculosis.</a> Cell Syst. 12 (11), 1046-1063.e7 (2021).</li>
<li>Khawbung, J. L., Nath, D., Chakraborty, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Drug+resistant+Tuberculosis%3A+A+review%5BTitle%5D)+AND+%22Comp+Immunol+Microbiol+Infect+Dis%22%5BJournal%5D)">Drug resistant Tuberculosis: A review.</a> Comp Immunol Microbiol Infect Dis. 24 (6), 101574(2021).</li>
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<li>Pauli, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Discovery+of+new+inhibitors+of+Mycobacterium+tuberculosis+InhA+enzyme+using+virtual+screening+and+a+3D-pharmacophore-based+approach%5BTitle%5D)+AND+%22J+Chem+Inf+Model%22%5BJournal%5D)">Discovery of new inhibitors of Mycobacterium tuberculosis InhA enzyme using virtual screening and a 3D-pharmacophore-based approach.</a> J Chem Inf Model. 53 (9), 2390-2401 (2013).</li>
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<li>Parish,, Tanya,, Roberts,, David, M. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aParish%2C++%22Mycobacteria+Protocols%22">Mycobacteria Protocols</a>. , Third edition, Humana Press. New York. (2015).</li>
<li>Franzblau, S. G., DeGroote, M. A., Cho, S. H., Andries, K., Nuermberger, E., Orme, I. M., Mdluli, K., Angulo-Barturen, I., Dick, T., Dartois, V., Lenaerts, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comprehensive+analysis+of+methods+used+for+the+evaluation+of+compounds+against+Mycobacterium+tuberculosis%5BTitle%5D)+AND+%22Tuberculosis+%28Edinburgh%2C+Scotland%29%22%5BJournal%5D)">Comprehensive analysis of methods used for the evaluation of compounds against Mycobacterium tuberculosis.</a> Tuberculosis (Edinburgh, Scotland). 92 (6), 453-488 (2012).</li>
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No potential conflict of interest was reported by the author(s).

A&#160;phenotypic screening&#160;approach, which relies on whole-cell activity rather than isolated enzymes, is an effective strategy for identifying active compounds against M. tuberculosis. Here, we described the initial steps of this approach, encompassing the chemical synthesis of a candidate compound, the evaluation of its inhibitory activity via minimum inhibitory concentration (MIC) determination, and the assessment of its cytotoxicity in mammalian cells (Vero and HepG2) using MTT and NRU assays.
The synthetic route consisted of three main steps (Figure 1), that provides the target compound in high purity using operationally straightforward transformations. Several critical steps were identified as determinant for reproducibility and efficiency. The initial condensation requires precise stoichiometric control and sustained reflux to ensure complete intermediate formation. Incomplete solvent removal prior to thermal cyclization may negatively affect ring closure efficiency. The cyclization step itself demands strict temperature control, as insufficient heating results in incomplete aromatization, whereas excessive or prolonged heating may promote byproduct formation. During chlorination, careful control of reaction temperature and gradual addition of phosphoryl chloride are recommended to minimize side reactions and ensure selective activation at the 4-position. During method optimization, minor procedural adjustments improved robustness. In cases of incomplete precipitation after cyclization, gradual cooling rather than rapid quenching enhanced solid recovery. If reduced yields were observed in the nucleophilic substitution step, extending reaction time or verifying amine purity proved beneficial.
The method&#39;s main limitations are the need for high-temperature cyclization and corrosive chlorinating reagents like phosphoryl chloride, requiring specific lab infrastructure and safety. Substituent electronic effects may necessitate re-optimization for different anilines or amines, limiting broad scaffold diversification without adaptation. However, the Conrad&#8211;Limpach approach is operationally simpler than Skraup-type syntheses (harsh oxidizers) or transition-metal-catalyzed protocols (specialized catalysts/inert atmospheres). This metal-free strategy efficiently yields functionalized quinoline intermediates for medicinal chemistry.
The protocol used to determine the MIC is a colorimetric assay that relies on visual inspection. A potential challenge with this method is incomplete color change, resulting in a purple well instead of distinct blue (inhibition) or pink (growth). If this occurs, it should be interpreted as a failure to inhibit mycobacterial growth. Additionally, as the compound is assessed through serial dilution, the maximum testable concentration is dictated by the compound&#8217;s solubility. As a result, in some cases, the outcome may be inconclusive if no inhibition is observed at the maximum soluble concentration. Moreover, It is important to acknowledge that other laboratories may adapt certain steps of the protocol to their specific conditions, which may lead to variations in the results obtained. To increase throughput, this protocol can be adapted for automation. Implementing liquid handling systems for 96-well plate preparation would reduce pipetting errors, thereby improving accuracy and reproducibility.
The cytotoxicity evaluation protocol utilizes two distinct mammalian cell lines (Vero and HepG2) and two complementary viability assays (MTT and NRU). Several steps within this protocol are critical for ensuring data reproducibility and accuracy, and each laboratory should perform its own validation. For instance, the initial cell seeding density must be carefully optimized, as over-confluence can lead to contact inhibition and altered metabolic states, confounding the results, while under-seeding can make cells more susceptible to minor insults, potentially overestimating toxicity15. Furthermore, the highest tested concentration is often limited by compound solubility, and precipitation can lead to inaccurate results. Also, the suggested plate layout (Figure 3A) is designed for primary screening, maximizing the number of tested compounds by forgoing technical replicates and ignoring edge effects in favor of validation through independent biological replicates.
A key strength of this protocol is the parallel implementation of both MTT and NRU assays. While both are well-established, they measure different cellular parameters. The MTT assay quantifies the metabolic activity of mitochondrial dehydrogenases, which generally correlates with cell viability16. However, this assay is susceptible to interference from compounds with reducing or oxidizing properties or those that perturb mitochondrial respiration, leading to potential false results15. Running the NRU assay in parallel helps troubleshoot such artifacts. The NRU assay measures the ability of viable cells to maintain lysosomal integrity and pH gradients required for dye uptake17. Discrepancies between MTT and NRU results can reveal specific mechanisms of toxicity, such as mitochondrial dysfunction, warranting further investigation.
In summary, this protocol outlines the fundamental steps for identifying potential anti-tubercular drug candidates. Compounds demonstrating promising selectivity and potency can be subsequently advanced to more complex infection models, such as in vivo studies.

Caused predominantly by the bacillus Mycobacterium tuberculosis (Mtb), tuberculosis (TB) is an infectious respiratory disease in humans that primarily targets the lungs, although extrapulmonary variants can also occur. TB spreads through airborne Mtb bacilli released by individuals with active disease1. Depending on disease progression, a person infected with TB may immediately acquire active tuberculosis. Usually, however, a newly infected individual will develop latent tuberculosis, where Mtb cells with low metabolic activity survive inside granulomas1,2. A person with latent tuberculosis may eventually develop the active form of the disease, manifesting clinical symptoms3. Currently, it is estimated that one-fourth of the world&#8217;s population has latent tuberculosis infection4.
Despite many advances, TB remains a serious public health problem worldwide. Data from the most recent Global Tuberculosis Report show that 10.7 million people developed the disease in 2024. During the same year, 1.23 million deaths from TB were registered worldwide, making TB the leading cause of death by a single infectious agent5. Besides the pathology and complex biology of Mtb, socio-economic aspects such as poverty and malnutrition, along with health-related factors such as smoking, diabetes, and HIV co-infection, make disease control even more challenging5. This contributes to TB&#8217;s long-standing permanence as a global health issue.
Furthermore, the current TB treatments have limitations that complicate the fight against the disease. Against susceptible strains of Mtb, the standard treatment consists of a combination therapy of 4 drugs: isoniazid (INH), rifampicin (RIF), ethambutol (EMB), and pyrazinamide (PZA). These antibiotics are administered for a minimum of 6 months, requiring strict adherence throughout the treatment. This therapeutic regimen, despite being efficient for most cases and having a success rate of around 85%, still faces many challenges5,6. Among the biggest issues are the side effects and prolonged duration, which often lead to premature interruption by the patient. This discontinuation, along with incorrect administration of the antibiotics, favors the selection of resistant Mtb strains, which require more toxic, expensive and extensive therapeutic regimens7,8.
Considering this scenario, the development of new, potent anti-TB drugs is urgent. This protocol outlines the early stages of researching new drug candidates using phenotypic screening. To illustrate the application of this protocol, we selected a 4-aminoquinoline compound derived from LABIO-17 for evaluation. LABIO-17 was previously identified by our research group as an inhibitor of the enoyl-ACP-reductase (InhA) enzyme in Mtb, which is the same molecular target as isoniazid (INH)9,10. InhA is involved in mycolic acid biosynthesis in M. tuberculosis and is essential for the survival of the bacillus, highlighting its relevance as a promising molecular target11

<table><tbody><tr><td>&lt;strong&gt;CELL LINES&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>HepG2 (human hepatocellular carcinoma)</td><td>ATCC</td><td>HB-8065</td><td>Human liver carcinoma cell line</td></tr><tr><td>Mycobacterium tuberculosis H37Ra</td><td>ATCC</td><td>25177</td><td></td></tr><tr><td>Mycobacterium tuberculosis H37Rv</td><td>ATCC</td><td>27294</td><td></td></tr><tr><td>Vero (African green monkey kidney)</td><td>ATCC</td><td>CCL-81</td><td>Primate kidney epithelial cell line</td></tr><tr><td>&lt;strong&gt;CHEMICALS &amp;ndash; SYNTHESIS&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>4-(Piperidin-1-yl)aniline</td><td>Sigma-Aldrich</td><td>TCI: P0887</td><td>Amine precursor for compound 5/6</td></tr><tr><td>4-Bromoaniline</td><td>Sigma-Aldrich</td><td>108-67-8</td><td>Starting material for quinoline synthesis</td></tr><tr><td>Acetic acid (glacial)</td><td>Sigma-Aldrich</td><td>695092</td><td>Reagent in synthesis and mobile phase preparation</td></tr><tr><td>Acetonitrile (HPLC grade)</td><td>Sigma-Aldrich</td><td>34967</td><td>HPLC mobile phase component and NMR solvent</td></tr><tr><td>Chloroform</td><td>Sigma-Aldrich</td><td>C2432</td><td>Solvent for product extraction</td></tr><tr><td>DMSO-d6 (deuterated)</td><td>Sigma-Aldrich</td><td>175641</td><td>NMR solvent</td></tr><tr><td>Dowtherm A (heat transfer fluid)</td><td>Sigma-Aldrich / Solutia</td><td>TBD</td><td>High-boiling solvent for thermal cyclization (230&amp;ndash;250 &amp;deg;C); formerly listed as Dowtherm&amp;reg; A</td></tr><tr><td>Ethanol (absolute)</td><td>Sigma-Aldrich</td><td>459836</td><td>Reaction solvent for Conrad&amp;ndash;Limpach step</td></tr><tr><td>Ethyl acetate (HPLC grade)</td><td>Sigma-Aldrich</td><td>270989</td><td>Column eluent component</td></tr><tr><td>Ethyl acetoacetate</td><td>Sigma-Aldrich</td><td>E12806</td><td>&amp;beta;-ketoester reagent for quinoline ring formation</td></tr><tr><td>Formic acid (HPLC grade)</td><td>Sigma-Aldrich</td><td>F0507</td><td>0.1% additive in mobile phase for HPLC and HRMS</td></tr><tr><td>Glass beads (1 mm, sterile)</td><td>Sigma-Aldrich</td><td>G8772</td><td>For homogenization of M. tuberculosis culture</td></tr><tr><td>HCl (hydrochloric acid, 37%)</td><td>Sigma-Aldrich</td><td>320331</td><td>Acid catalyst in nucleophilic substitution</td></tr><tr><td>Hexane (HPLC grade)</td><td>Sigma-Aldrich</td><td>34859</td><td>Non-polar eluent for column chromatography</td></tr><tr><td>Isopropanol</td><td>Sigma-Aldrich</td><td>278475</td><td>Reaction solvent for synthesis of compound 6</td></tr><tr><td>Magnesium sulfate (MgSO4, anhydrous)</td><td>Sigma-Aldrich</td><td>M7506</td><td>Drying agent and reaction catalyst</td></tr><tr><td>Methanol (HPLC grade)</td><td>Sigma-Aldrich</td><td>34860</td><td>HPLC mobile phase and NMR solvent component</td></tr><tr><td>Phosphorus(V) oxychloride (POCl3)</td><td>Sigma-Aldrich</td><td>201170</td><td>Chlorination reagent for intermediate 4</td></tr><tr><td>Pyridine</td><td>Sigma-Aldrich</td><td>270970</td><td>Base catalyst in nucleophilic substitution</td></tr><tr><td>Silica gel (35&amp;ndash;70 mesh) for flash chromatography</td><td>Macherey-Nagel</td><td>815349</td><td>Stationary phase for flash chromatography steps</td></tr><tr><td>Silica gel 60 &amp;Aring; (70&amp;ndash;230 mesh) for column chromatography</td><td>Macherey-Nagel</td><td>815381</td><td>Stationary phase for column chromatography</td></tr><tr><td>Sodium sulfate (Na2SO4, anhydrous)</td><td>Sigma-Aldrich</td><td>239313</td><td>Drying agent for organic phase</td></tr><tr><td>TLC Silica gel 60 F254 plates</td><td>Merck</td><td>1.05554</td><td>Thin-layer chromatography monitoring</td></tr><tr><td>Toluene</td><td>Sigma-Aldrich</td><td>244511</td><td>Solvent for chlorination step</td></tr><tr><td>Ultrapure water</td><td>Millipore / equivalent</td><td>N/A</td><td>Used in mobile phase preparation; formerly referenced as Milli-Q water in text</td></tr><tr><td>&lt;strong&gt;EQUIPMENT &amp;amp; INSTRUMENTS&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>0.22 &amp;micro;m syringe filter</td><td>Millipore / any</td><td>SLGP033RS</td><td>Sterilization of resazurin solution (2.1.4); filtration of MTT working solution (3.3); filtration of Neutral Red working solution (3.4)</td></tr><tr><td>0.45 &amp;micro;m syringe filter</td><td>Millipore / any</td><td>SLHA033SS</td><td>Filtration of mobile phase solutions</td></tr><tr><td>15 mL conical tube (sterile)</td><td>Falcon / any</td><td>352097 or equivalent</td><td>Collection and centrifugation of cell suspension after trypsinization</td></tr><tr><td>96-well plate (flat bottom, tissue culture treated)</td><td>Corning / any</td><td>3596</td><td>For cytotoxicity and MIC assays</td></tr><tr><td>Aluminum foil</td><td>Any</td><td>&amp;mdash;</td><td>Light protection of plates during MTT and NRU incubation and shaking steps</td></tr><tr><td>Autoclave</td><td>Any</td><td>&amp;mdash;</td><td>Sterilization of media (121 &amp;deg;C, 98.07 kPa, 10 min)</td></tr><tr><td>Biosafety cabinet (Class II, certified)</td><td>Any (BSL-3 certified)</td><td>&amp;mdash;</td><td>Required for M. tuberculosis manipulations (BSL-3)</td></tr><tr><td>Cell counter (automated) or hemocytometer</td><td>e.g., Bio-Rad TC20 / Neubauer</td><td>TBD</td><td>For cell concentration and viability determination</td></tr><tr><td>Centrifuge (capable of 200 &amp;times; g)</td><td>Any</td><td>&amp;mdash;</td><td>Cell pelleting step</td></tr><tr><td>CO2 incubator (37 &amp;deg;C, 5% CO2, humidified)</td><td>Any</td><td>&amp;mdash;</td><td>Cell culture and treatment incubation</td></tr><tr><td>Heating mantle</td><td>Fisotom&amp;nbsp;</td><td>12M</td><td>Temperature-controlled heating mantle with Dowtherm&amp;reg; A</td></tr><tr><td>HPLC system (Dionex UltiMate 3000)</td><td>Thermo Fisher Scientific</td><td>5035.9200</td><td>Dual pump, automatic injector, UV detector; for purity analysis of compound 6</td></tr><tr><td>Inverted microscope (or equivalent)</td><td>Any</td><td>&amp;mdash;</td><td>Visual inspection of cells at attachment (3.2), during treatment checkpoint (3.3, 3.4), and for assay status verification</td></tr><tr><td>Magnetic stirrer / hotplate</td><td>Any</td><td>&amp;mdash;</td><td>Solution preparation and reaction mixing</td></tr><tr><td>Mass spectrometer (LTQ Orbitrap Discovery)</td><td>Thermo Fisher Scientific</td><td>&amp;mdash;</td><td>High-resolution Orbitrap combined with LTQ XL; for HRMS of compound 6</td></tr><tr><td>Melting point apparatus</td><td>Any (e.g., Stuart SMP10)</td><td>&amp;mdash;</td><td>Melting point determination of compound 6</td></tr><tr><td>Microplate reader (absorbance)</td><td>Any (e.g., BioTek Epoch)</td><td>&amp;mdash;</td><td>Reads absorbance at 570 nm (MTT) and 540 nm (NRU)</td></tr><tr><td>NMR spectrometer (Avance III HD, 400 MHz)</td><td>Bruker Corporation</td><td>N/A</td><td>1H and 13C NMR acquisition; located at PUCRS facility</td></tr><tr><td>Orbital shaker</td><td>Any</td><td>&amp;mdash;</td><td>100 rpm (MTT crystal dissolution); 150 rpm (NRU extraction)</td></tr><tr><td>Parafilm M</td><td>Bemis / Sigma-Aldrich</td><td>P7793</td><td>Sealing of 96-well plates during incubation</td></tr><tr><td>Qualitative filter paper</td><td>UNIFIL</td><td>50,11,250</td><td>Whatman Grade, cellulose; 11 &amp;micro;m retention; ~80 g/m&amp;sup2;</td></tr><tr><td>Reverse-phase C18 HPLC column (250 &amp;times; 4.6 mm, 5 &amp;micro;m)</td><td>Macherey-Nagel (Nucleodur C18)</td><td>760201.46</td><td>Analytical column for HPLC purity analysis</td></tr><tr><td>Rotary evaporator</td><td>IKA</td><td>&amp;mdash;</td><td>Evaporate the solvent under reduced pressure</td></tr><tr><td>Ultrasonic Cleaner</td><td>Branson</td><td>Model 2510</td><td>Ulltrasonic tank for degassing solutions</td></tr><tr><td>Vortex mixer</td><td>Any</td><td>&amp;mdash;</td><td>Homogenization of bacterial culture with glass beads</td></tr><tr><td>&lt;strong&gt;GROWTH MEDIA &amp;amp; SUPPLEMENTS&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>ADC (Albumin-Dextrose-Catalase) enrichment</td><td>BD Biosciences</td><td>212352</td><td>10% supplement for 7H9 broth (assay wells)</td></tr><tr><td>Amphotericin B solution (250 &amp;micro;g/mL)</td><td>&lt;br/&gt; Gibco/Thermo Fisher</td><td>&lt;br/&gt; 15290026</td><td>Antifungal supplement, 0.1% in complete medium</td></tr><tr><td>DMEM (Dulbecco&#039;s Modified Eagle Medium) low glucose</td><td>Gibco/Thermo Fisher</td><td>&lt;br/&gt; 31600034</td><td>Basal medium for HepG2 and Vero cells</td></tr><tr><td>FBS (Fetal Bovine Serum)</td><td>Gibco/Thermo Fisher</td><td>&lt;br/&gt; 12657029</td><td>10% supplement in complete medium</td></tr><tr><td>Middlebrook 7H9 broth powder</td><td>BD Biosciences</td><td>271310</td><td>Base medium for M. tuberculosis culture</td></tr><tr><td>OADC (Oleic Acid-Albumin-Dextrose-Catalase) enrichment</td><td>BD Biosciences</td><td>212240</td><td>10% supplement for 7H9 broth (culture)</td></tr><tr><td>Penicillin/streptomycin solution (10,000 U/mL)</td><td>Gibco/Thermo Fisher</td><td>15140122</td><td>1% in complete medium</td></tr><tr><td>Sodium bicarbonate</td><td>Sigma-Aldrich</td><td>S5761</td><td>1.8 g/L supplement in DMEM; also used for NaHCO3 saturated solution</td></tr><tr><td>Trypsin-EDTA solution (0.25%)</td><td>Gibco/Thermo Fisher</td><td>&lt;br/&gt; 25200072</td><td>For cell detachment from culture flasks</td></tr><tr><td>Tween-80 (Polysorbate 80), 10% solution</td><td>Sigma-Aldrich</td><td>P4780</td><td>0.05% supplement for 7H9-OADC broth</td></tr><tr><td>&lt;strong&gt;REAGENTS &amp;amp; ASSAY COMPONENTS&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DMSO (dimethyl sulfoxide)</td><td>Sigma-Aldrich</td><td>D8418</td><td>Solvent for compound stock solutions</td></tr><tr><td>DPBS (Dulbecco&#039;s Phosphate Buffered Saline)</td><td>Gibco/Thermo Fisher</td><td>&lt;br/&gt; D565210X1L</td><td>For compound dilution (3.2) and PBS wash in NRU assay (3.4)</td></tr><tr><td>MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide)</td><td>Sigma-Aldrich</td><td>M2128</td><td>Cell viability reagent for MTT assay</td></tr><tr><td>Neutral Red (NR) dye</td><td>Sigma-Aldrich</td><td>N2889</td><td>Cell viability reagent for NRU assay</td></tr><tr><td>Resazurin sodium salt</td><td>Sigma-Aldrich</td><td>R7017</td><td>Colorimetric indicator for REMA assay</td></tr><tr><td>Trypan blue solution (0.4%)</td><td>Gibco/Thermo Fisher</td><td>15250061</td><td>For cell viability determination by exclusion</td></tr><tr><td>&lt;strong&gt;SOFTWARE&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Chromeleon 6.80 SR11 (chromatography data acquisition)</td><td>Thermo Fisher Scientific</td><td>Build 3160 (183147)</td><td>HPLC data acquisition and processing</td></tr><tr><td>Xcalibur 2.0.7 (mass spectrometry data analysis)</td><td>Thermo Fisher Scientific</td><td>Release 2.0.7</td><td>Elemental composition determination (Qual Browser module)</td></tr></tbody></table>

identification of potential anti-tb candidates: a step-by-step guide to synthesis, mic determination, and cytotoxicity assessment in mammalian cells

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&#39;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.

The synthesis yielded the target compound 6 as a light green solid (0.53 g, 67% yield). The compound exhibits a melting point of 227–229 °C and a UHPLC purity of 99% (tR = 14.11 min) (Figure 4). The chemical structure was confirmed by nuclear magnetic resonance (NMR) spectroscopy and high-resolution mass spectrometry (HRMS). The 1H NMR spectrum (Figure 5) showed signals corresponding to the piperidine moiety between 1.50 and 3.13 ppm. The methyl group at the 2-position appeared as a typical singlet at 2.38 ppm. The hydrogen at the 3-position appeared as a singlet at 6.56 ppm. The phenyl ring displayed two doublets at 6.97 and 7.17 ppm, with a coupling constant of 8.9 Hz. The hydrogens at the 7- and 8-positions generated a multiplet at 7.63–7.75 ppm, while the hydrogen at the 5-position of the quinoline core produced a doublet at 8.59 ppm (J = 2.0 Hz). The amine hydrogen was observed as a singlet at 8.66 ppm. The 13C NMR spectrum (Figure 6) exhibited a signal at 23.81 ppm attributed to the methyl group at the 2-position, while signals between 24.85 and 49.70 ppm corresponded to the piperidine moiety. The carbon at the 3-position appeared at 100.52 ppm. Remaining downfield signals were attributed to the quinoline core and phenyl ring carbons. HRMS confirmed the molecular formula of compound 6, showing an [M+H]+ ion at m/z 396.1063 (calcd for C21H23BrN3: 396.1070).
<img alt="High-performance liquid chromatography result; UV-VIS absorption spectrum; peak analysis data." class="xfigimg" src="/files/ftp_upload/70693/70693fig4.jpg" data-altupdatedat="1787025648000" data-alt="Figure 4"> 
Figure 4. HPLC chromatogram and UV spectra of compound 6. <a href="https://www.jove.com/files/ftp_upload/70693/70693fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="NMR spectroscopy graph displaying chemical shift values and peak assignments for molecular analysis." class="xfigimg" src="/files/ftp_upload/70693/70693fig5.jpg" data-altupdatedat="1787025648000" data-alt="Figure 5"> 
Figure 5. 1H NMR (400 MHz, DMSO-d6) spectra of compound 6. <a href="https://www.jove.com/files/ftp_upload/70693/70693fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="X-ray diffraction pattern graph; crystalline structure analysis; peak intensity data; material science." class="xfigimg" src="/files/ftp_upload/70693/70693fig6.jpg" data-altupdatedat="1787025648000" data-alt="Figure 6"> 
Figure 6. 13C NMR (101 MHz, DMSO-d6) spectra of compound 6. <a href="https://www.jove.com/files/ftp_upload/70693/70693fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The inhibitory activity of the compound against M. tuberculosis was evaluated by determining the MIC, defined as the lowest concentration of a compound that completely prevents visible bacterial growth. In the REMA assay, a lack of color change (remaining blue) indicates growth inhibition, while a shift to pink indicates bacterial growth. Isoniazid (INH) and rifampicin (RIF) served as drug controls. The results are presented in Figure 7. Each plate included a growth control (column 11, without drugs) and a sterility control (column 12, without mycobacteria). Compounds were serially diluted from column 10 (highest concentration) to column 1 (lowest concentration). As shown in Figure 7, the MIC value for the compound in row A (isoniazid) is observed in column 4, while the MIC values for the compounds in rows B (rifampicin) and C (synthesized compound) are observed in column 5. In our hands, MIC values for INH are 0.31 µg/mL for H37Rv strain and 0.16 µg/mL for H37Ra, while for RIF we obtain 0.08 µg/mL for H37Rv and 0.01 µg/mL for H37Ra strain.
<img alt="Microplate colorimetric assay results; rows A-C, columns 1-12; concentration gradient analysis." class="xfigimg" src="/files/ftp_upload/70693/70693fig7.jpg" data-altupdatedat="1787025648000" data-alt="Figure 7"> 
Figure 7. Determination of the minimum inhibitory concentration (MIC) in Mycobacterium tuberculosis using the resazurin microplate assay (REMA). Rows A–C, columns 1–10: Two-fold serial dilutions of isoniazid (row A), rifampicin (row B), and the synthesized compound (row C). Column 11: Viability control (medium containing 2.5% DMSO + bacteria). Column 12: Sterility control (medium + compound). A blue color indicates inhibition of bacterial growth, whereas a pink color indicates bacterial viability due to the reduction of resazurin to resorufin. <a href="https://www.jove.com/files/ftp_upload/70693/70693fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
A representative 96-well plate for both the NRU (Vero, top) and MTT (Vero, bottom) assays is shown in Figure 3B. In the MTT plate, a clear color gradient is visible, shifting from translucent (no viable cells) at high compound concentrations to a deep purple (high cell viability) at low concentrations. Similarly, the NRU plate shows a gradient from translucent to deep pink, indicating the uptake of the neutral red dye into the lysosomes of viable cells.
Microscopy provides a visual confirmation of the assay status after the 48 h incubation time (Figure 8). Figure 8A compares healthy, untreated control cells (Vero and HepG2) with cells treated with a cytotoxic concentration of the compound. The treated cells appear rounded, detached, and non-viable. Figure 8B provides a critical visual checkpoint prior to the final solubilization step. It shows the expected formation of dark purple formazan crystals inside viable cells (MTT assay) and the accumulation of red dye within the lysosomes (NRU assay) in both cell types.
Raw absorbance data were normalized to the vehicle control and analyzed via non-linear regression to generate dose-response curves as detailed in protocol step 3.5. For the target compound 6 (derived from LABIO-17), this analysis yielded a CC50 of 12.2 µM (Vero) and 18.3 µM (HepG2) in the NRU assay, and a CC50 &gt;20 µM for both cell lines in the MTT assay. This resulted in a Selectivity Index (SI) ranging from 30.5 to &gt;50, as previously reported10.
<img alt="Cell viability assay results; Vero, HepG2 cells, toxic compound; MTT, NRU methods, comparison chart." class="xfigimg" src="/files/ftp_upload/70693/70693fig8.jpg" data-altupdatedat="1787025648000" data-alt="Figure 8"> 
Figure 8. Representative microscopy images of cytotoxicity assays in Vero and HepG2 cells. All images were taken at 100x magnification. (A) Comparison of cell morphology after 48 h of treatment. Untreated control wells for Vero (top) and HepG2 (bottom) cells show a healthy, confluent monolayer. In contrast, cells treated with a cytotoxic concentration of a compound appear rounded, shrunken, and detached from the plate surface. (B) Critical visual checkpoints after reagent addition and prior to solubilization. In the MTT assay (left panels for each cell line), viable cells contain visible dark purple formazan crystals. In the NRU assay (right panels for each cell line), viable cells show an accumulation of red dye within intact lysosomes. <a href="https://www.jove.com/files/ftp_upload/70693/70693fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Here, we present a protocol to identify potential anti-tuberculosis drug candidates through phenotypic screening, encompassing the chemical synthesis of a 4-aminoquinoline compound, determination of its minimum inhibitory concentration against Mycobacterium tuberculosis, and assessment of its cytotoxicity in mammalian cell lines.

Tuberculosis (TB) remains one of the leading causes of death from a single infectious agent worldwide, with rising rates of drug resistance making the ...

The focus of our research is the development and validation of novel anti-tuberculosis drug candidates aimed at advancing therapeutic strategies. The preventive phenotypic screening pipeline can be used in the search of new pharmaceuticals against mycobacterium tuberculosis strains. To begin, gather the required reagents and proceed with preparing the reaction mixture using the presented proportions.Stir the reaction mixture at 85 degrees Celsius for 16 hours. Use a rotary evaporator to remove the solvent. Add approximately 20 milliliters of saturated sodium bicarbonate solution to the residue.Filter this suspension using an 11 micrometer cellulose qualitative filter paper to collect the residual solids. Dissolve the residual solids in 50 milliliters of chloroform. Use a separating funnel to rinse the chloroform solution with 15 milliliters of water three times.Removing the water layer each time. Add anhydrous sodium sulfate to the organic phase to remove the residual water and dry it. After evaporating the solvent, wash the resulting green solid four times with 10 milliliters of hot hexane at approximately 50 degrees Celsius.To one milligram of Compound 6, add one milliliter of a one-to-one mixture of acetonitrile and methanol to prepare a stock solution. Dilute the stock solution to 0.5 milligrams per milliliter and filter it prior to analysis. Load the sample into the HPLC system.Use chromatography data acquisition software to acquire and process the data. Set the chromatographic method to use a 250 by 4.6 millimeters reverse phase C18 column of particle size five micrometers. Set the flow rate to 1.5 milliliters per minute, and set UV detection at 254 nanometers.Set up a gradient as presented with programmed mobile phase composition changes. Transfer approximately 15 to 20 milligrams of Compound 6 into a nuclear magnetic resonance or NMR tube. Dissolve it in an appropriate volume of DMSOD6.Once the acquisition parameters are set as presented, acquire the proton NMR spectrum using a standard pulse sequence. Adjust acquisition parameters. Initiate the proton and carbon 13 NMR spectrum and report chemical shifts in parts per million.Visualize and process the NMR data with the free induction decay file obtained from the experiment. For chromatography-based purification of Compound 6, pack a column with silica gel. Load the crude Compound 6 onto the column.Elute the compound and collect fractions. Monitor the fractions using thin layer chromatography and combine the fractions containing the pure product. Add 100 microliters of 7H9 broth supplemented with 10%albumin dextrose catalase or ADC to column 12 of the 96-well plate.Then transfer 100 microliters of 7H9 broth containing 10%ADC and 5%DMSO to column 11. And then sequentially from column nine to one. Add 200 microliters of the compound solution to column 10, assigning one row per compound.Pipette 100 microliters of the compound solution at twice the highest test concentration to column 12 of the 96-well plate. Transfer 100 microliters from column 10 to column nine and mix well by pipetting to begin a two-fold cereal dilution. Continue the serial dilution sequentially till column one.Add 100 microliters of the bacterial suspension of mycobacterium tuberculosis sequentially from column 11 to column one and cover the plate. Seal the plate with parafilm and incubate at 37 degrees Celsius for seven days. Next, add 60 microliters of 0.01%resazurin solution to each well of the plate and cover it.Seal the plate with parafilm and incubate it at 37 degrees Celsius for 48 hours. Then check for the color change from blue to pink. Prepare a working solution of 0.5 milligrams per milliliter MTT in serum-free DMEM.Obtain Vero and HEPG2 cells treated with Compound 6 for 48 hours and inspect them to assess treatment effects. Aspirate the medium from the plates and add 50 microliters of the MTT working solution to each well under low ambient light conditions. Following a three hour incubation, add 100 microliters of DMSO to each well to dissolve the Formazan crystals under low ambient light conditions.Place the plate on an orbital shaker at 100 RPM for 15 minutes while keeping it protected from light. Inspect the control wells to confirm complete crystal dissolution. Put the plate in a microplate reader.Set the reference wavelength of 630 to 690 nanometers and check the absorbance at 570 nanometers. Prepare a 40 microgram per milliliter neutral red working solution in serum-free DMEM. Then prepare the neutral red desorption solution containing 1%glacial acetic acid, 49%ethanol, and 50%ultrapure water by volume.Once the effect of Compound 6 is assessed on Vero and HEPG2 cells, remove the media and add 100 microliters of the filtered neutral red working solution to each well. Incubate the plate for two hours at 37 degrees Celsius with 5%carbon dioxide. After removing the neutral red working solution, rinse each well once with 150 microliters of sterile PBS.Add 150 microliters of the neutral red desorption solution to each well. Place the plate on an orbital shaker at 150 RPM for 15 minutes to extract the dye. Check the control wells to confirm complete dye extraction.Read the absorbance at 540 nanometers with a microplate reader. The HPLC chromatogram showed that Compound 6 had a retention time of 14.11 minutes and a purity of 99%The proton NMR spectrum showed characteristic signals for the piperidine moiety between 1.50 and 3.13 parts per million. A methyl singlet at 2.38 parts per million.And aromatic signals including doublets at 6.97 and 7.17 parts per million and a multiplet at 7.63 to 7.75 parts per million. The carbon 13 NMR spectrum showed a methyl carbon signal at 23.81 parts per million, and piperidine related signals between 24.85 and 49.70 parts per million. The MTT plate showed a clear color gradient across compound concentrations, indicating that cytotoxicity was highest at high compound concentrations with translucent wells and lowest at low compound concentrations with deep purple wells.The neutral red uptake plate showed a clear color gradient across compound concentrations, indicating that cytotoxicity was highest at high compound concentrations with translucent wells and lowest at low compound concentrations with deep pink wells. This protocol represents a pipeline for the development of drug candidates against tuberculosis based on phenotypic screening. It is important to consider and evaluate the low solubility of the synthesized compounds while applying the protocol.This research can serve as a basis for future bioavailability, macrophage infection and in vivo studies.

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