Abstract
For a newly synthesized compound, identifying its target protein is a slow but pivotal step toward understand its pharmacologic mechanism. In this study, we systemically synthesized novel manzamine derivatives and chose 1-(9′-methyl-3′-carbazole)-3, 4-dihydro-β-carboline (MCDC) as an example to identify its target protein and function. MCDC had potent toxicity against several cancer cells. To identify its target protein, we first used a docking screen to predict macrophage migration inhibitory factor (MIF) as the potential target. Biochemical experiments, including mutation analysis and hydrogen-deuterium exchange assays, validated the binding of MCDC to MIF. Furthermore, MCDC was shown by microarrays to interfere with the cell cycle of breast cancer MCF7 cells. The activated signaling pathways included AKT phosphorylation and S phase-related proteins. Our results showed MIF as a potential direct target of a newly synthesized manzamine derivative, MCDC, and its pharmacologic mechanisms.
Highlights
There are two main approaches to identify the pairing between small molecules and their target proteins
Previous studies showed that elongation of the alkyl chain of 1-substituted carbazolyl-1, 2, 3, 4-tetrahydro-β-carboline and carbazolyl-3, 4-dihydro-β-carboline, both manzamine A-derived compounds, resulted in decreased anticancer activity[8]
Identifying the direct target protein of a newly discovered or synthesized compound is often effort-intensive when using the typical methods of small molecule affinity chromatography[4] and activity-based protein profiling[25]
Summary
There are two main approaches to identify the pairing between small molecules and their target proteins. We chose the manzamine-derived compound 1-(9′-methyl-3′-carbazole)-3, 4-dihydro-β-carboline (MCDC) as an example to demonstrate the possibility of rapid identification of the target protein(s) of a newly synthesized compound. To examine the cytotoxic effects of our 12 newly synthesized derivatives, we performed Calcein AM viability assays on four cancer cell lines from lung (A549, H1299), liver (HepG2), and breast (MCF7) with the dose range of each compound from 0, 1, 10 and 100 μM (Supplementary Fig. S1).
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