High-throughput screening identified selective inhibitors of exosome biogenesis and secretion: A drug repurposing strategy for advanced cancer

Amrita Datta,Madhu Lal,Adedoyin E Johnson,Asim B Abdel-Mageed,Sudha Talwar,Juan Marugan,Xin Hu,Marc Ferrer,Hogyoung Kim,Debasis Mondal,Lauren Mcgee,Noel Southall

doi:10.1038/s41598-018-26411-7

Abstract

Targeting exosome biogenesis and release may have potential clinical implications for cancer therapy. Herein, we have optimized a quantitative high throughput screen (qHTS) assay to identify compounds that modulate exosome biogenesis and/or release by aggressive prostate cancer (PCa) CD63-GFP-expressing C4-2B cells. A total of 4,580 compounds were screened from the LOPAC library (a collection of 1,280 pharmacologically active compounds) and the NPC library (NCGC collection of 3,300 compounds approved for clinical use). Twenty-two compounds were found to be either potent activators or inhibitors of intracellular GFP signal in the CD63-GFP-expressing C4-2B cells. The activity of lead compounds in modulating the secretion of exosomes was validated by a tunable resistive pulse sensing (TRPS) system (qNano-IZON) and flow cytometry. The mechanism of action of the lead compounds in modulating exosome biogenesis and/or secretion were delineated by immunoblot analysis of protein markers of the endosomal sorting complex required for transport (ESCRT)-dependent and ESCRT-independent pathways. The lead compounds tipifarnib, neticonazole, climbazole, ketoconazole, and triademenol were validated as potent inhibitors and sitafloxacin, forskolin, SB218795, fenoterol, nitrefazole and pentetrazol as activators of exosome biogenesis and/or secretion in PC cells. Our findings implicate the potential utility of drug-repurposing as novel adjunct therapeutic strategies in advanced cancer.

Highlights

Targeting exosome biogenesis and release may have potential clinical implications for cancer therapy
Optimized assay conditions included the coating of the plates with different coating solutions, seeding 2,000 cells/ well, using low serum media, and treatment of the cells with Manumycin A (MA), a farnesyl transferase inhibitor for 96 hrs (Supplementary Fig. S1C)
The effect of MA, a previously reported inhibitor of exosome biogenesis and secretion was used as a control (REF)

Summary

Results

Development and optimization of the qHTS-compatible exosome biogenesis assay. The pCMV-CD63-GFP expression plasmid was used to generate a stable C4-2B cell line expressing the exosomal marker CD63 fused with GFP. An additional compound GW4869 (a neutral sphingomyelinase inhibitor) known to interfere with exosome secretion and vesicle trafficking, in general, was used to validate the assay (Supplementary Fig. S1D). (Table 1) produced a robust and reproducible CRC score on CD63-GFP signal (Fig. 1B) and, were subjected to further validation to determining whether they affected exosome biogenesis or secretion. Based on our recent study[20], we employed a modified workflow for the isolation of exosomes (Supplementary Fig. S3) and their detection using the qNano IZON system (NP100 nanopore) to validate the ability of the 22 qHTS assay hits to modulate the particle concentration, the mean diameter (nm) and diameter mode (nm) secreted by the C4-2B-CD63-GFP cells (Supplementary Table S1). We selected compounds that showed increased GFP signal in our qHTS assays as activators of exosome secretion. Additional biological studies are required to further validate the activators of biogenesis/secretion exosomes

CRC values

Discussion

Therapeutic Information

Materials and Methods

Additional Information