Methods and Compositions for Re-activating Epstein-Barr Virus and Screening Compounds Therefor
a technology of epstein-barr virus and composition, which is applied in the field of compositions for reactivating epstein-barr virus and screening compounds therefor, can solve the problems of unsuitable pharmacokinetics of butyrate (nab), and limitations of viral “oncolytic therapy”
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Assay Development and Methods Used
[0132]To develop a high-throughput, cell-based assay for identification of small molecule activators of EBV lytic cycle gene expression, stable cell lines containing reporter genes under the control of EBV lytic cycle promoters for BZLF1 (Zp) or BHLF1 (Hp) were generated. BZLF1 is the immediate early gene encoding the ZTA lytic activator. BHLF1 is an early lytic gene highly responsive to ZTA transcriptional activation. To create reporter constructs that can be maintained as chromatinized episomes similar to latent EBV genome, the OriP-containing, hygromycin resistant vector pHEBO (Yates, 1985, Nature 313:812-815) was utilized. EBV promoters Hp and Zp were cloned upstream of the luciferase reporter gene in pHEBO to generate pHEBO-Hp-Luc and pHEBO-Zp-Luc. These plasmids were then used to generate stable episomal reporters in EBV positive cell lines LCLs, Mutul, and Raji.
[0133]A. Cell Culture
[0134]Akata (EBV+ BL), BJAB, Akata EBV-negative (EBV− BL), JS...
example 2
High-Throughput Screening Process
[0150]Assay plates were prepared by seeding 25,000 Mutu-Hp-Luc cells in 50 μL of RPMI supplemented with 15% FBS in white opaque 384 well tissue culture plates (Corning), using a Biotek Microflo and a 5 μL cassette. Fifty nanoliters of test compound or DMSO was transferred to assay plates using a Janus MDT® NanoHead system (Perkin Elmer). Columns 1, 2, 23, and 24 received DMSO (0.2% final concentration). The remaining 320 wells in columns 3-22 received test compounds at a final concentration of 10 μM cumulative concentration for the 5 compounds / 2 μM individual compound concentration / 0.2% final DMSO concentration. Cells were incubated with compounds for 48 h at 37° C. / 5% CO2. Twenty microliters of Steady-Glo® Luciferase Reagent (Promega) was added to assay plates and incubated for 15 minutes at room temperature. Plates were centrifuged and luminescence was measured on an EnVision® Excite multilabel microplate reader, using the ultrasensitive luminescen...
example 3
Structure Studies
[0174]All EBV activators 1-26 share similar structure belonging to the tetrahydrocarboline structural class of compounds. Tetrahydrocarbolines are an interesting class of molecules that are known to possess diverse biological activity such as kinesin spindle protein, topoisomerase II, protein tyrosine phosphatase, phosphodiesterase inhibition, and sst3 receptor antagonists (Pasternak, 2012, ACS Med. Chem. Lett. 3:289-293 and Daugan, 2003, J. Med. Chem., 46:4525-4532). The active molecules 1-5 provide some initial structure activity relationships (SAR) required for EBV reactivation. All five have substitution on the 6 position of the indole ring with either a chloro or a methoxy group. Also all five are linked through the piperidine nitrogen with a urea linkage terminating with a substituted aromatic ring. The initial lead chemical series was chemically tractable and readily amenable to analog synthesis to flesh out the SAR to improve potency and drug-like properties...
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