ABSTRACT
The envelope protein of dengue virus (DENV) is a primary target of the humoral immune response. The domain III of the DENV envelope protein (EDIII) is known to be the target of multiple potently neutralizing antibodies. One such antibody is 3H5, a mouse antibody that binds strongly to EDIII and potently neutralizes DENV serotype 2 (DENV-2) with unusually minimal antibody-dependent enhancement (ADE). To selectively display the binding epitope of 3H5, we strategically modified DENV-2 EDIII by shielding other known epitopes with engineered N-glycosylation sites. The modifications resulted in a glycosylated EDIII antigen termed "EDIII mutant N". This antigen was successfully used to sift through a dengue-immune scFv-phage library to select for scFv antibodies that bind to or closely surround the 3H5 epitope. The selected scFv antibodies were expressed as full-length human antibodies and showed potent neutralization activity to DENV-2 with low or negligible ADE resembling 3H5. These findings not only demonstrate the capability of the N-glycosylated EDIII mutant N as a tool to drive an epitope-directed antibody selection campaign but also highlight its potential as a dengue immunogen. This glycosylated antigen shows promise in focusing the antibody response toward a potently neutralizing epitope while reducing the risk of antibody-dependent enhancement.
ABSTRACT
7Methoxyheptaphylline (7MH) is a carbazole extracted from Clausena harmandiana, a medicinal plant that is used to treat headaches and stomachaches. The aim of the present study was to examine the neuroprotective effects and anticancer activity of 7MH. Cell death was assessed using an MTT assay and flow cytometry. The expression of apoptosisrelated proteins was determined by western blot analysis. An animal model was used to test antimetastasis. The interactions between 7MH and the molecular target were observed using molecular docking. The results revealed that 7MH provided protection against hydrogen peroxide (H2O2)induced neuronal cell death. In cancer cells, 7MH induced SHSY5Y, 4T1, HT29, HepG2, and LNCaP cell death. 7MH inhibited metastasis of HT29 cells in vitro and 4T1Luc cells in vitro and in vivo. 7MH inhibited proteins, including Pglycogen synthase kinase (GSK)3, and cleaved caspase3, but it activated antiapoptotic proteins in H2O2induced SHSY5Y cell death. By contrast, 7MH activated the cleaving of caspase3 and GSK3, but it suppressed antiapoptotic proteins in SHSY5Y cells. 7MH reduced the levels of NFκB and STAT3 in 4T1 cells; phosphop65, Erk, and MAPK13 in LNCaP cells; and phosphoErk and matrix metalloproteinase9 in HT29 cells. Molecular docking analysis showed that 7MH targets TAK1 kinase. The present study indicated that 7MH induced apoptosis of cancer cells and provided protection against H2O2induced neuron cell death via TAK1 kinase.
