Construction Of Pregnane X Receptor Ligand Screening Model And Acetylcholinesterase Inhibitor Screening Model

I Construction of pregnane X receptor ligand screening modelThe nuclear pregnane X receptor [PXR; NR112, also known as SXR and PAR] is a member of the nuclear receptor (NR) family of ligand-dependent transcriptional factors and a key regulator of genes involved in xenobiotic and endobiotic metabolism. PXR is composed of four functional modules including the modulator domain, the DNA-binding domain (DBD), the hinge region, and the ligand-binding domain (LBD). Elucidation of the three-dimensional structure of the PXR ligand binding domain reveals that it has a large, spherical ligand binding cavity that allows it to interact with a wide range of endogenous and exogenous chemicals. PXR binds with coregulatory proteins in the absence of ligands to DNA response elements in the regulatory regions of its target genes to modulate transcription of its target genes including cytochrome P450 3A monooxygenase genes and a number of other genes involved in the metabolism and elimination of xenobiotics from the body. The interaction between PXR and drug provides a molecular basis for the reported induction of drug metabolism enzyme gene expression and interactions between various prescription drugs.In this study, pETDuet-1-SRC88-PXRLBD expression plasmid was constructed and transformed into Escherichia coli Rosetta (DE3) to coexpress PXRLBD and SRC88, and equilibrium dialysis model was constructed to study the interaction between PXR and drugs, which may be used in the screening of PXR ligands in vitro.1. Expression of PXRLBD protein 1) construction of the expression vector pGEX-4T-1-PXRLBDThe expression vector pGEX-4T-1-PXRLBD was generated by PCR amplification and subcloning of nucleotides 390-1302 of the hPXR into the BamHâ… and Xhoâ… sites of the pGEX-4T-1 expression vector. The expression vector was confirmed by electrophoretic analysis and sequencing.2) expression and identification of GST-PXRLBDThe pGEX-4T-1-PXRLBD plasmid was transformed into the Rosetta (DE3) strain of Escherichia coli and expressed with isopropyl-β-D-thiogalactopyranoside (50μmol/L) for 10h in shaker flasks at 20-25℃.Cell pellets were resuspended, lysed by French pressure cell press and sonication, and clarified by centrifugation. Total-cell lysated, soluble fraction of cell lysated and insoluble fraction of cell lysated were assessed by SDS-polyacrylamide gel electrophoresis (PAGE) with Coomassie blue staining. The result revealed the GST-PXRLBD was expressed as inclusion bodies.3) Construction of the expression vector pETDuet-1-SRC88-PXRLBDThe expression vector pETDuet-1-SRC88-PXRLBD was generated by insertion of nucleotides 1867-2130 of steroid receptor coactivator-1 (SRC88) by Ncoâ… and Hindâ…¢sites into the multiple cloning site 1 of pETDuet-1 expression vector, and nucleotides 388-1302 of hPXR with a polyhistidine-tagged C-terminal of PXRLBD by Ndeâ… and Xhoâ… sites into the multiple cloning site 2 of pETDuet-1 expression vector. The expression vector was confirmed by electrophoretic analysis and sequence analysis, and transformed into Escherichia coli Rosetta (DE3).4) Coexpression of PXRLBD and SRC88pETDuet-1-SRC88-PXRLBD Rosetta (DE3) were grown in 500mL LB with 1% glucose to an OD₆₀₀ of 0.6 at 37℃. The cells were harvested and grown in 2×YT with 50μmol/L IPTG for 10h at 20-25℃.5) Purification and identification of PXRLBDCell pellets were resuspended, lysed by French pressure cell press and sonication, and clarified by centrifugation. The cleared supernatant was loaded onto His·Bind Resin. purified protein was eluted by 8mL elute buffer and then analysed by SDS-PAGE and Western blot. Two single protein bands of PXRLBD and SRC88 were detected on gel which revealed that SRC88 binded to PXRLBD as complex, and the concentration of purified protein was (0.411±0.005) g/L measured by BCA Protein Assay Kit.2. Construction of PXR ligand screening modelThe PXRLBD/SRC88 complex and drugs (clotrimazole or dexamethasone) were respectively added to the inside and outside of the dialysis bag. PXRLBD interacted with drugs infiltrating into bag filter. After dialysis equilibrium the buffer inside and outside of the dialysis bag was digested with 10μg/mL protease K for 1h, methanol was then added to precipitate protein and drug concentration was measured by HPLC.The HPLC data, that A (defined as ratio of drug peak area between the inside and outside of dialysis bag) of clotrimazole group equaled to 1.382±0.086 and A of dexamethasone group equaled to 1.004±0.012 indicated that clotrimazole binded to PXRLBD, while dexamethasone did not bind to PXRLBD, which coincided with the property of PXR. The equilibrium dialysis model for studying the interaction between PXR and drugs was established successfully and may be used in the screening of PXR ligands in vitro. â…¡Construction of acetylcholinesterase inhibitor screening modelAcetylcholinesterase (AChE) hydrolyzes the neurotransmitter, acetylcholine at neuromuscular junctions and brain cholinergic synapses, and thus terminates signal transmission. AChE inhibitors can selectively inhibit the activity of AChE in the brain, which improved cholinergic nerve function of Alzheimer's disease (Alzheimer's Disease, AD) patients and reduced the decline in cognitive ability.FDA has approved five acetylcholinesterase inhibitors, including Tarcrine, Donepezil, Rivastigmine, Galanthamine and Huperzine A, which have been the main therapeutics of mild to moderate Alzheimer's disease. However, the clinical application of AChE inhibitors were limited because of shortcomings such as short half-life, serious peripheral cholinergic system side effects, and scientists focus on a new generation of AChE inhibitors with small side-effect but more effective.In this study, recombinant human acetylcholinesterase (rhAChE) was expessed by HEK293 cells, enzyme kinetics of the rhAChE was investigated, and AChE inhibitors screening model was established and applicated to assay the inhibition activity of new compounds.1. rhAChE was expressed by HEK293 cellsPlasmid of pCMV-AChE was kindly provided by Prof. Hermona Soreq in Hebrew University, and the DNA sequence of AChE was confirmed to be correct (GenBank Accession No: NM₀00665). HEK293 Cells were transfected with plasmid of pCMV-AChE, by utilizing the Cationic liposome lipofectamine^TM2000. rhAChE was secreted to cell culture medium and was collected for AChE activity assay.2. Assay of AChE Kinetic determinationsAChE activity was assayed according to modified Ellman method. The reaction mixture contained rhAChE, 5:5'-dithiobis (2- nitrobenzoic acid) (DTNB), acetylthiocholine iodide (ATCh). The assay was performed at 37℃and terminated by SDS. The reaction was monitored by recording the increase in absorbance at 412nm. Activity was monitored as OD/min.1) Kinetic parameters of the interaction between ATCh and rhAChE ATCh concentration was determined from the plot representing initial rhAChE enzyme velocity vs. ATCh concentration, rhAChE content was determined from the plot representing initial rhAChE enzyme velocity vs. rhAChE content, and incubation time was determined from the plot representing initial rhAChE enzyme velocity vs. incubation time.The reaction mixture contained rhAChE 4μL, 0.025%DTNB, different concentrations of ATCh (0.075, 0.1, 0.125, 0.175, 0.25, 0.3 mmol/L) and the assay was performed for 15 min at 37℃and terminated by SDS. K_m was calculated from the Lineweaver-Burk plot representing reciprocals of initial rhAChE enzyme velocity vs. reciprocal of ATCh concentration. K_m value is 151.9μmol/L.2) AChE activityThe reaction mixture contained rhAChE 32μL, 0.025%DTNB, 1.25mmol/L ATCh and the assay was performed at 37℃. The reaction was monitored by recording the increase in absorbance at 412nm every 0.5min for 6 times. The value of AChE activity is 0.698mU (μmol/min) according to the formula: U (μmol/min)=V×A/(ε×L).3) Kinetic parameters of the interaction between Donepezil and rhAChEThe reversible noncovalent inhibitor of AChE Donepezil (E2020) was shown to inhibit AChE with high affinity in a mixed competitive-non-competitive way. K_i was calculated by the plot representing slope rate of Lineweaver-Burk plot vs. different concentrations of Donepezil and K'_i was calculated by the plot representing Y-axis intercept of Lineweaver-Burk plot vs. different concentrations of Donepezil.The reaction mixture contained rhAChE 4μL, 0.025%DTNB, different concentrations of Donepezil (0, 2.5, 5.0,10, 20,40, 80nmol/L), different concentrations of ATCh (0.065, 0.125, 0.25, 0.5 mmol/L) and the assay was performed for 15min at 37℃. The value of K_i is 16.03nmol/L and K'_i is18.36nmol/L.3. Establishment and application of AChE inhibitor screening modelThe reaction mixture contained rhAChE 4μL, 0.025%DTNB, different concentrations of Donepezil (0,1.25,2.5, 5.0,10,20,40, 80nmol/L), 0.125mmol/L ATCh and the assay was performed for 15min at 37℃. The value of IC₅₀14.0nmol/L coinsistent with the report was obtained through linear regression of inhibition ratio vs. different concentrations of Donepezil.Novel AChE inhibitors were designed and synthesized through the condensation of 2-Bromo-5,6-dimethoxy-indan-l-one with various aminoalkyl phenols. IC₅₀values of the rhAChE inhibitors were obtained and the AChE inhibition activity was compared.