Establishment Of Several Cell Models For Preliminary Screening Of Small Molecular Compounds

In view of their characteristics of efficiency, safety, low cost and steady, more and more attention was paid to construction of novel models with high efficiency and specificity for screening the small organic compounds, which has been an important point in the new drug development. In this present study, several cell-based models were established that targeted to FKBP12 or either D1 or D4 domain of CD4 molecular or NFAT signal transduction pathways respectively, for preliminary screening of relevant synthetic small organic compounds .The study consists of two parts as below. 一, Establishment of a cell-based model for preliminary screening of FK506-like small molecular organic compounds1 Establishment and identification of a chemically inducible dimerization model for preliminary screening of drug targeted to FKBP12Full-length Bax gene amplified from 3T3 cell lines was determined by sequence analysis followed by cloning into pC4FV1E.The resulting recombinant plasmid pC4Fv1E/BAX was then introduced into HEK293 by lipofectin.Cell clones stably expressing FKBP12-mBAX fusion proteins were characterized by RT-PCR and Western-blotting.The effect of inducible apoptosis on cell clones by AP20187 was analyzed by morphological and flow cytometric detection.At last, competition assays by FK506 proved the feasibility of such a cell model as a technique for high throughput screening of FK506-like small molecular compounds targeted to FKBP12.2 Establishment of a cell model targeted to NFAT signal transduction pathway for preliminary screening of FK506-like immunosuppressantTo screen NFAT antagonistic drugs and research signal transduction pathway related to NFAT. Four recombinant vectors were constructed that each consists of three tandem copies of the human IL-2 distal NFAT-AP1 binding site in the context of the minimal IL-2 enhancer, either the sequence from -326—+46 or the sequence from-89—+46(only containing the TATA box), which drives a luciferase reporter gene or a destabilized enhanced green fluorescence protein(d2EGFP) reporter gene. Transient transfection of Jurkat cells was achieved by electroporation with 5-10ug of the above plasmid and once pulse at 200v, 65ms.Plasmid pEFBos-mNFATl constitutively expresses murine full length NFATl protein was used for transient cotransfection. The results showed that neither of non-stimulation nor PMA or Ionomycin stimulation alone could activate the reporter gene but PMA and Ionomycin costimulation. Furthermore, overexpressed murine NFATl augment the activation of whether JL-2 promoter or NFAT-AP1 enhancer drived reporter gene compared to the endogenous did. Such NFAT-dependent reporter gene activation was nearly completely inhibited by FK506 at 5ug/ml with pretreatment for lhr and then stimulation for 6-12hr in the presence of the drugs. These findings indicated that such a transient Jurkat cell model as a potential strategy for preliminary screening of FK506 or CsA-like immunosuppressants. ~ -. Establishment of a cell-based model for preliminary screening of CD4 inhibitors as potential novel immunosuppressants1 Establishment of a cell adhesion model based on CD4 and MHC-II interaction for screening of peptide-based and non-peptidic organic CD4 inhibitorsA recombinant plasmid containing CD4-GFP fusion gene was constructed, in which CD4 was deleted the short cytoplasmic tail. Stable cell clones as named 293/CD4 were obtained by transfecting the recombinant vector pEGFP-Nl/CD4 into HEK293, which was then selected by G418. CD4 expression was observed on cell membrane surface under Confocal Laser Scanning Microscopy. Rosset was formed after incubation of 293/CD4 with MHC-II positive Raji, that is, three or more Raji cells attached to one 293/CD4 cell surface.However,CD4 antibody can obviously inhibit such Rosset formation. This cell adhesion model offers a plausible applicable platform for screeningof peptide-based and non-peptidic organic CD4 inhibitors.2 Establishment of a tet-on HEK293 cell line regulated by Doxycycline with apoptosis effects due to CD4 self-association(1) Several indirect methods for analysis of CD4 self-association and its function instable CD4-MHC-II bindingTo examine the self-association of CD4 molecules and preliminary studies on its biological function by several indirect methods.A series of CD4 chimeras were generated including truncated CD4 lacking the short cytoplasmic tail, deleted mutants -D1/D2 devoid of D3 and D4 and D3/D4 devoid of Dl and D2 by PCR techniques , as well as another three CD4 chimeric genes by fused human Fas cytoplasmic death domain to the downstream of the above chimeras respectively. All these molecules were subcloned into pEGFP-Nl, forming the corresponding expression vectors. After introducing into HEK293 cells, gene-modified cell morphological changes and target protein subcellular localization were observed and analyzed by a confocal microscopy. Moreover, stable 293/CD4 clones were obtained by transfecting the truncated CD4 recombinant plasmid into the HEK293 cell line and selected by G418. The fluorescene intensity and rosette formation of different clones was each analyzed by a confocal microscopy and cell adhesive assays. It's seen that CD4-Fas fusion gene could induce approximately 80% cell apoptosis of transfected HEK293 cells, compared to FKBP12-Fas is about 30% and CD4 gene only is 7%. Furthermore, both Dl/D2-Fas and D3/D4-Fas chimeras could trigger nearly all transfected HEK293 cells to death. Cell adhesion assays showed that neither the D1/D2 nor D3/D4 chimeras when expression in HEK293 cells binds to MHC class IT Raji B cells. Interestedly, there were two type stable clones among 293/CD4. Fluorescence intensity analysis displayed that one' mean fluorescence intensity value is about twice of the other while cell-cell binding examination showed that the former is capable of forming rosette with Raji cells but the latter. All these results suggest that CD4 molecules most likely could exist as a dimer or even an oligomer on transfected HEK293 cell surface, which constitute a functional form for stable binding to MHC class II molecules.(2) Establishment of a tet-on cell model for preliminary screening of CD4 inhibitors targeted to CD4 dimerization sites: either Dl or D4To establish a stable HEK293 Tet-on cell line for doxycycline regulated target gene expression. First, pTet-on was transfected into HEK293 by lipofectamine and about 50 clones were obtained after G418 selection. Two among these 50 clones was...