Baptiste Mouysset1, Jérémy Ariey-Bonnet1, Marion Le Grand1, Paul Brémond1, Luc Camoin2, Stéphane Audebert2, Maïlys Rossi3, Marie-Pierre Montéro3, Manon Carré3, Nicolas André3, Eddy Pasquier1
1. SCB, CRCM, Marseille, France
2. MAP, CRCM, Marseille, France
3. SMARTc, CRCM, Marseille, France
Drug repurposing, which consists in using already-approved drugs for new indications, has emerged as an effective strategy to develop novel treatments for unmet medical needs. Our lab has previously shown that anti-hypertensive drugs, beta-blockers, can increase the efficacy of certain chemotherapies in different refractory cancers1, 2, 3. This led to multiple ongoing clinical trials involving beta-blockers in various human cancers and to propranolol being granted orphan drug designation by the European Medicine Agency in 2016. The mechanism(s) involved in the chemo-sensitizing activity of beta-blockers remains however poorly understood.
Here, we showed that the enantiomers of propranolol (R and S) were equipotent at increasing the cytotoxic activity of vincristine in neuroblastoma and medulloblastoma cell lines using 2D and 3D models (between 3- and 25-fold, depending on the cell lines). Since only the (S)-enantiomer is able to bind and inhibits the beta-adrenergic receptors, we concluded that their canonical targets are not involved in their chemo-sensitizing activity. We therefore set out to identify their non-canonical targets in neuroblastoma and medulloblastoma using click chemistry – a class of biocompatible chemical reactions used to combine a specific molecule with a chosen substrate4.
Three clickable derivatives of beta-blockers have been synthetized by inserting an alkyne group in the parental compound: (R)-propra-click, (S)-propra-click and carvedilol-click. We first ensured that the clickable derivatives retained their chemo-sensitizing properties, thus providing useful tools to study the mechanism of action of beta-blockers. We then used azide-coupled fluorophores to detect the clickable derivatives in cancer cells using both flow cytometry and fluorescence microscopy.
Flow cytometry allowed us performing time-course and dose-response assays so as to define the optimal conditions for subsequent experiments, while fluorescence microscopy enabled the visua- lization of beta-blockers within cancer cells for the first time. Then, we used azide-coupled biotin and streptavidin magnetic beads to perform pull-down experiments with the clickable-derivatives in neuroblastoma and medulloblastoma cell lines. The pull-down interacting proteins were then identified using label-free quantitative tandem mass spectrometry. This led to the identification of
48 interacting proteins, common to all three clickable beta-blockers and all three tested cell lines. Pathway analysis revealed that some of these interactors are involved in metabolism regulation, suggesting that beta-blockers may increase the efficacy of chemotherapy agents by interfering with cancer cell metabolism.
Functional validation of the identified interactors of beta-blockers is currently underway using RNA interference. So far, we have silenced the gene expression of the top 8 interactors in neuroblastoma cells and found that 3 of them inhibited tumor spheroid growth. One interactor in particular, ABCE1, increased neuroblastoma cell sensitivity to vincristine when silenced. However, the synergism between beta-blockers and chemotherapy was still observed following gene silencing of indivi- dual interactors, suggesting that the chemo-sensitizing activity of beta-blockers involves multiple non-canonical targets at once.
In conclusion, our results show that click chemistry-based proteomics is a powerful tool to identify the non-canonical targets of repurposed drugs. Unravelling the multi-targeted mechanism under- lying the chemo-sensitizing activity of beta-blockers could open major therapeutic avenues for the development of biology-guided drug repurposing strategies.
Keywords: repositionnement de médicaments, cancers pédiatriques, Beta-bloquants, chimie-click.
References
Pasquier E, et al. Propranolol potentiates the anti-angiogenic effects and anti-tumor efficacy
of chemotherapy agents: implication in breast cancer treatment. Oncotarget 2011; 2: 797–809. Pasquier E, et al. ?-blockers increase response to chemotherapy via direct antitumour
and anti-angiogenic mechanisms in neuroblastoma. Br J Cancer 2013; 108: 2485–2494. Pasquier E, et al. Effective management of advanced angiosarcoma
by the synergistic combination of propranolol and vinblastine-based metronomic chemotherapy: a bench to bedside study. EBioMedicine 2016; 6: 87–95.
Tyler DS, et al. Click chemistry enables preclinical evaluation of targeted epigenetic therapies. Science 2017; 356: 1397–401.