A pull-down and slot blot-based screening system for inhibitor compounds of the podoplanin-CLEC-2 interaction.
Nobuo WatanabeMasako KidokoroYusuke SuzukiMakiko TanakaShigeaki InoueHideo TsukamotoNoriaki HirayamaPei-Wen HsiehChing-Ping TsengYoshihide NakagawaSadaki InokuchiPublished in: PloS one (2019)
Podoplanin, a transmembrane glycoprotein, is overexpressed in certain types of tumors and induces platelet aggregation by binding to C-type lectin-like receptor 2 (CLEC-2) on the platelet membrane. Activated platelets release granule components, which in turn, trigger epithelial-mesenchymal transition and confer invasive capacity to the tumor cells. Therefore, blocking the podoplanin-CLEC-2 interaction by a small-molecule compound is a potential therapeutic strategy to prevent cancer metastasis and invasion. To effectively identify such inhibitory compounds, we have developed a pull-down-based inhibitory compound screening system. An immunoglobulin Fc domain-CLEC-2 fusion protein was used as a bait to capture podoplanin derived from podoplanin-overexpressing HeLa cells in the presence and absence of the test compound. The protein complex was then pulled down using protein A beads. To shorten the turnaround time, increase throughput, and decrease the workload for the operators, centrifugal filter units were employed to separate free and bound podoplanin, instead of using customary aspiration-centrifugation washing cycles. Slot blotting was also utilized in lieu of gel electrophoresis and electrical transfer. Thus, the use of our pull down screening system could facilitate the effective selection of potential inhibitor compounds of the podoplanin-CLEC-2 interaction for cancer therapy. Importantly, our methodology is also applicable to targeting other protein-protein interactions.
Keyphrases
- small molecule
- cancer therapy
- epithelial mesenchymal transition
- protein protein
- induced apoptosis
- drug delivery
- cell cycle arrest
- squamous cell carcinoma
- risk assessment
- binding protein
- amino acid
- transforming growth factor
- signaling pathway
- oxidative stress
- young adults
- human health
- endoplasmic reticulum stress
- low density lipoprotein