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EBNA1-targeted probe for the imaging and growth inhibition of tumours associated with the Epstein–Barr virus

Abstract

Epstein–Barr nuclear antigen 1 (EBNA1), a dimeric oncoprotein of the Epstein–Barr virus (EBV), is essential for both viral-genome maintenance and the survival of infected cells. Despite EBNA1’s potential as a therapeutic target, tools for the direct monitoring of EBNA1 in vitro and in vivo are lacking. Here, we show that a peptide-based inhibitor that luminesces when bound to EBNA1 inside the nucleus of EBV+ cells can regulate EBNA1 homodimer formation and selectively inhibit the growth of EBV+ tumours of nasopharyngeal carcinoma cells (C666-1 and NPC43) and Burkitt’s lymphoma Raji cells. We also show that the peptide-based probe leads to 93% growth inhibition of EBV+ tumours in mice. Our findings support the hypothesis that selective inhibition of EBNA1 dimerization can be used to afford better EBV-related cancer differentiation, and highlight the potential application of the probe as a new generation of biotracers for investigating the fundamental biological function of EBNA1 and for exploring its application as a therapeutic target.

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Figure 1: Rational design of probes and MD simulations of their binding to EBNA1.
Figure 2: Selectivity and emission of L2P4 on addition of EBNA1.
Figure 3: In vitro imaging of probes in EBV-positive and EBV-negative cells.
Figure 4: Homodimerization and cytotoxicity assays.
Figure 5: In vivo studies of P4 and L2P4 as EBV-specific anti-cancer agents.

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Acknowledgements

This work is funded by the Hong Kong Baptist University (FRG2/14-15/013013), Hong Kong Polytechnic University (HKPolyU), Hong Kong Research Grants Council (HKBU 20301615), Hong Kong Polytechnic University Central Research Grant (G-UC08), Hong Kong Research Grants Council (PolyU 153012/15P), University Research Facility for Chemical and Environmental Analysis (UCEA) and Area of Excellent Grants (1-ZVGG) of Hong Kong Polytechnic University, ECS-Grant - RGC (PolyU 253002/14P), HK PolyU (PolyU 5096/13P), HKBU and HKPolyU Joint Research Programme (RC-ICRS/15-16/02F-WKL02F-WKL), the EPSRC (Durham University, DTA award) and Research Grants Council of the Hong Kong SAR for the NPC Area of Excellence (AoE/M 06/08 Center for Nasopharyngeal Carcinoma Research).

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Authors and Affiliations

Authors

Contributions

N.-K.M. and K.-L.W. conceived and supervised the project. L.J. performed the synthesis, characterization, spectroscopic properties measurements and cytotoxicity assays in normal and some EBV-negative cells; H.L. contributed the photophysical data analysis. R.L. purified the WT-EBNA1 and EBNA1 mutants, and conducted the dimerization assay; T.H., Z.-X.B. and W.-T.W. contributed to the MD simulations; C.-F.C. carried out the confocal imaging, co-localization and some cytotoxicity assays; S.L., J.Z. and S.L.C. performed the peptide synthesis and purified the peptide conjugates; W.-Y.W., M.M.-L.L., B.D.C. and W.C.S.T. carried out the in vivo inhibition. M.L.L., H.L.L., S.W.T. and G.S.T. contributed to the in vitro cytotoxicity in EBV-positive and EBV-negative pairs of cancer cell lines; G.-L.L. participated in the cell work and molecular design; L.J., W.-L.C., W.-S.L., G.-L.L., S.L.C. and K.-L.W. wrote the manuscript.

Corresponding authors

Correspondence to Nai-Ki Mak, Ga-Lai Law, Wing-Tak Wong, Steven L. Cobb or Ka-Leung Wong.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary methods, figures, tables and references. (PDF 11131 kb)

Supplementary Video 1

Dynamic distribution of L2P4 in EBV-negative HeLa cells. (MOV 2419 kb)

Supplementary Video 2

Dynamic distribution of L2P4 in EBV-positive C666-1 cells. (MOV 2723 kb)

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Jiang, L., Lan, R., Huang, T. et al. EBNA1-targeted probe for the imaging and growth inhibition of tumours associated with the Epstein–Barr virus. Nat Biomed Eng 1, 0042 (2017). https://doi.org/10.1038/s41551-017-0042

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