LAUSR

Simple Summary To identify inhibitors of mitochondrial respiration as potential anticancer drugs is not an easy matter since cancer cells cultured as monolayers may escape by shifting their metabolic preference toward the use of glycolysis. Here we propose to capitalize on this apparent weakness to exploit the associated increase in L-lactate release as a primary screening assay to identify and optimize the development of inhibitors of mitochondrial oxidative phosphorylation. As a secondary assay, we used a protocol based on O2 consumption rate in permeabilized cancer cells to get further insights on a possible direct or indirect inhibition of mitochondrial electron transport chain. Finally, 3D tumor spheroids helped us to further select drug candidates endowed with the capacity to exert growth inhibitory effects in tumor-mimicking conditions but also to act as potent radiosensitizers by promoting reoxygenation. Abstract Cancer cells may stimulate glycolytic flux when O2 becomes insufficient. Increase in L-lactate release therefore appears as an escape mechanism to drugs targeting mitochondrial respiration but also represents a response that may be exploited to screen for compounds blocking either mitochondrial carriers of oxidizable substrates or the electron transport chain. Here, we developed a screening procedure based on the capacity of cancer cells to release L-lactate to gain insights on the development of mitochondrial complex I inhibitors. For this purpose, we synthesized derivatives of carboxyamidotriazole, a compound previously described as a potential OXPHOS inhibitor. Two series of derivatives were generated by cycloaddition between benzylazide and either cyanoacetamides or alkynes. A primary assay measuring L-lactate release as a compensatory mechanism upon OXPHOS inhibition led us to identify 15 hits among 28 derivatives. A secondary assay measuring O2 consumption in permeabilized cancer cells confirmed that 12 compounds among the hits exhibited reversible complex I inhibitory activity. Anticancer effects of a short list of 5 compounds identified to induce more L-lactate release than reference compound were then evaluated on cancer cells and tumor-mimicking 3D spheroids. Human and mouse cancer cell monolayers exhibiting high level of respiration in basal conditions were up to 3-fold more sensitive than less oxidative cancer cells. 3D tumor spheroids further revealed potency differences between selected compounds in terms of cytotoxicity but also radiosensitizing activity resulting from local reoxygenation. In conclusion, this study documents the feasibility to efficiently screen in 96-well plate format for mitochondrial complex I inhibitors based on the capacity of drug candidates to induce L-lactate release.