Abstract
ABSTRACTHigh attrition rates in tuberculosis (TB) drug development have been largely attributed to safety, which is likely due to the use of endpoint assays measuring cell viability to detect drug cytotoxicity. In drug development for cancer, metabolic, and neurological disorders and for antibiotics, cytotoxicity is increasingly being assessed using extracellular flux (XF) analysis, which measures cellular bioenergetic metabolism in real time. Here, we adopt the XF platform to investigate the cytotoxicity of drugs currently used in TB treatment on the bioenergetic metabolism of HepG2 cells, THP-1 macrophages, and human monocyte-derived macrophages (hMDMs). We found that the XF analysis reveals earlier drug-induced effects on the cells’ bioenergetic metabolism prior to cell death, measured by conventional viability assays. Furthermore, each cell type has a distinct response to drug treatment, suggesting that more than one cell type should be considered to examine cytotoxicity in TB drug development. Interestingly, chemically unrelated drugs with different modes of action on Mycobacterium tuberculosis have similar effects on the bioenergetic parameters of the cells, thus discouraging the prediction of potential cytotoxicity based on chemical structure and mode of action of new chemical entities. The clustering of the drug-induced effects on the hMDM bioenergetic parameters are reflected in the clustering of the effects of the drugs on cytokine production in hMDMs, demonstrating concurrence between the effects of the drugs on the metabolism and functioning of the macrophages. These findings can be used as a benchmark to establish XF analysis as a new tool to assay cytotoxicity in TB drug development.
Highlights
High attrition rates in tuberculosis (TB) drug development have been largely attributed to safety, which is likely due to the use of endpoint assays measuring cell viability to detect drug cytotoxicity
We adopted a multiwell noninvasive extracellular flux analysis platform that rapidly detects the modulation of bioenergetic metabolism of cells induced by anti-TB drugs in real time prior to cell death that is measured by conventional viability assays
The major drawback of viability assays is that they only focus on the effects of the drugs on one aspect of metabolism, such as the generation of oxidized reducing equivalents contributing to the viability of the cells and are not sensitive enough to detect alterations to the health of the cell that would impact the functioning of the cell in the absence of cell death
Summary
High attrition rates in tuberculosis (TB) drug development have been largely attributed to safety, which is likely due to the use of endpoint assays measuring cell viability to detect drug cytotoxicity. The response of OCR and ECAR to the consecutive addition of known mitochondrial and electron transport chain (ETC) modulators or stressors is used to calculate bioenergetic parameters associated with oxidative phosphorylation (OXPHOS) and metabolism of the cells, namely, basal respiration, basal ECAR, ATP-linked OCR, compensatory ECAR, maximal respiration, spare respiratory capacity, proton leak, and nonmitochondrial respiration [35] In some cases, these parameters can reveal drug cytotoxicity that is not detected in measurements of oxygen consumption rate (OCR) or extracellular acidification rate (ECAR) alone [36]. This assay measures the ability of NAD(P)H-dependent oxidoreductase enzymes to reduce a tetrazolium salt to formazan and is considered a measure of metabolism [56] These modulations of the bioenergetic parameters were compared with the effects of the anti-TB drugs on the functions of the hMDMs by measuring the cytokine levels in the supernatants of the hMDMs following treatment with the anti-TB drugs
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