Screening GLP-1 Receptor Ligands from Natural Products in Herbs through High-Content Technique

Aim and Objective: Screening of active components from a natural product, especially from a crude extract, is a great challenge. To avoid potential activity interference of the N-terminus modification in the most common constructs based on GCPRs labeled with GFP technology, a Cterminus tGFP-labeled hGLP-1 receptor containing recombinant cell line hGLP-1R-tGFP was constructed and tried to be used in the screening of natural products from Chinese herb. Materials and Methods: The GLP1 receptor gene was amplified and the inserts pCMV6-AC-tGFP and tGFP were fused at the C-terminus of GLP1 receptor to construct a recombinant plasmid. The recombinant was transfected into U2OS cell and selected with antibiotics and flow cytometry. The constructed cell line was named as hGLP-1R-tGFP cell line. The expression levels of GLP-1R-tGFP protein were confirmed by western-blot. The fluorescence imaging of re-distribution from diffusing to aggregate spots inside the cells was quantitated and analyzed by High Content Screening (HCS) assay. Meanwhile, the specificity, stability and C-terminus function of hGLP-1R-tGFP cell line were characterized. In order to allow the recombinant cell line of hGLP-1R-tGFP to be suitable in highcontent system of Arrayscan-infinity-700 in screening mode, several conditions have also been optimized. In the end, a total of 100 crude extract samples provided by the Yunnan Institute of Materia Medica have been screened with this method. Results: Upon the activation of GLP-1 receptors by Exendin 4, fluorescent patches appeared on the cell membrane and subsequently internalized to form fluorescent aggregates inside the cells under fluorescent microscopy examination. The agonistic activity, sensitivity and specificity of the formation of fluorescent aggregate spot in hGLP-1R-tGFP cells have been confirmed by the activation of GLP-1R using the GLP-1analogues. The agonistic effects of GLP-1 analogues are blocked by a GLP-1R antagonist, Exendin9-39. The downstream of GLP-1 pathway, the activation of adenylate cyclase and the raising of cellular cAMP levels, remained intact in these tGFP modified C-terminus GLP-1 receptor cells. Meanwhile, a total of 100 crude extract samples from Chinese herbs have been screened by this method to find new active ingredients. Conclusion: Combined with High Content Screening image and data automatic acquisition processing, a new screening assay based on a recombinant U2OS cell line which GFP labeled at the C terminus of GLP1 receptor has been developed. GLP-1R agonist activity in extracts of Astragalus propinquus and Panax notoginseng from Chinese herbs has been determined by this method.

Kinase inhibition of G2019S-LRRK2 restores autolysosome formation and function to reduce endogenous alpha-synuclein intracellular inclusions

The Parkinson’s disease (PD)-associated kinase Leucine-Rich Repeat Kinase 2 (LRRK2) is a potent modulator of autophagy and impacts on lysosome biology and function, but unclarity exists on the precise mechanics of its role and the direction of this modulation. LRRK2 is also involved in the degradation of pathological alpha-synuclein, with pathogenic mutations precipitating neuropathology in cellular and animal models of PD, and most LRRK2 familial cases manifesting with Lewy neuropathology. Defects in autophagic processing and lysosomal degradation of alpha-synuclein have been postulated to underlie its accumulation and onset of neuropathology. Thus, it is critical to reconcile these independent pieces of information to obtain a comprehensive knowledge on LRRK2-associated pathology that could also be generalized to the idiopathic disease.Here, we report a focused investigation on the role of PD-causing G2019S-LRRK2 in the autophagy-lysosome pathway in a recombinant cell line model. Initially, we evaluated the effect of LRRK2 expression on autophagy-related transcriptome. Then, we found that G2019S-LRRK2 leads to accumulation of autophagosomes with no net effect on autophagy induction. This is linked to abnormalities in lysosome morphology and proteolytic activity that are associated with a decrease in the successful formation of autolysosomes. Despite some of these features being shared by WT-LRRK2, alpha-synuclein intracellular inclusions are specifically found in G2019S-LRRK2 cells. Pharmacological kinase inhibition is capable of rescuing defects in the autophagy-lysosome pathway and reducing the number of inclusions. Notably, this effect is prevented by upstream blockade of autophagosome-lysosome fusion events, highlighting this step of the process as critical for alpha-synuclein clearance.

Development of Recombinant Cell Line Co-expressing Mutated Nav1.5, Kir2.1, and hERG for the Safety Assay of Drug Candidates

To provide a high-throughput screening method for human ether-a-go-go-gene–related gene (hERG) K+ channel inhibition, a new recombinant cell line, in which single action potential (AP)–induced cell death was produced by gene transfection. Mutated human cardiac Na+ channel Nav1.5 (IFM/Q3), which shows extremely slow inactivation, and wild-type inward rectifier K+ channel, Kir2.1, were stably co-expressed in HEK293 cells (IFM/Q3+Kir2.1). In IFM/Q3+Kir2.1, application of single electrical stimulation (ES) elicited a long AP lasting more than 30 s and led cells to die by more than 70%, whereas HEK293 co-transfected with wild-type Nav1.5 and Kir2.1 fully survived. The additional expression of hERG K+ channels in IFM/Q3+Kir2.1 shortened the duration of evoked AP and thereby markedly reduced the cell death. The treatment of the cells with hERG channel inhibitors such as nifekalant, E-4031, cisapride, terfenadine, and verapamil, recovered the prolonged AP and dose-dependently facilitated cell death upon ES. The EC50 values to induce the cell death were 3 µM, 19 nM, 17 nM, 74 nM, and 3 µM, respectively, whereas 10 µM nifedipine did not induce cell death. Results indicate the high utility of this cell system for hERG K+ channel safety assay.