Ultra-HTS Using 3456-Well Plate and HTRF Technology in GPCR Drug Discovery
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1 Ultra-HTS Using 3456-Well Plate and HTRF Technology in GPCR Drug Discovery 29 th Sep 2016 Toyo Sakurai Hit Discovery and Cell Processing Research Group, Biological Research Department
2 Agenda 3456-well Plate Miniaturization of HTRF camp Assay HTS Campaign 2
3 How many wells are there in the plate? 3.7 mm 384-well plate 16 x mm 1536-well plate 32 x 48 ~25 μl/well ~ 5 μl/well 1.1 mm 3456-well plate 48 x 72 ~ 1 μl/well 3
4 Notes of 3456-well Plate Lid Evaporation control well Assay well Required; Plate supplier: Aurora (from Waken Btech) Liquid dispenser: Echo and BioRAPTR (non-contact) Plate reader: PHERAstar and ViewLux (high resolution) Only Homogenous Assay! 4
5 Issues of 3456-well Assay Evaporation Evaporation of liquid causes terrible edge effect. Dispensing & mixing It is extremely difficult to dispense <1 μl to very small wells with accuracy. It is necessary to consider the generation of bubbles and the mixing reagents. Stacker It is not possible to use the stacker of plate reader, because 3456-well plate has no flange (or skirt). 5
6 3456-well Plate Miniaturization of HTRF camp Assay HTS Campaign 6
7 Ratio (665 nm/620 nm x 10 4 ) HTRF camp Assay Competitive, homogeneous assay camp concentration (Log) HTRF camp kit enables a quantitative measure of camp homogeneously. 7
8 camp conc. Use of Suspension Cells for Homogenous Assay GPCR-X Low High Ligand Homogeneous assay was available to evaluate the activity of GPCR-X using HTRF camp HiRange kit. 8
9 R a t io R a t io Miniaturization from 1536 to 3456-Well GPCR-X GPCR-Y w e ll w e ll w e ll w e ll L ig a n d [ L o g ( M ) ] L ig a n d [ L o g ( M ) ] Although miniaturization to 3456-well affected the ratio of HTRF signal slightly, it was enough sensitivity for HTS. 9
10 Protocol of GPCR-X camp Assay Steps 1536-well 3456-well Dispense compounds (Echo) ~ 30 nl ~ 7.5 nl Seed cells (BioRAPTR) 37 C for 90 minutes 3 μl (1,500 cells/well) 0.8 μl (400 cells/well) Add d2 & cryptate (BioRAPTR) 1 μl each 0.4 μl each Room temperature for 120 minutes Measure HTRF signal (PHERAstar) 8 min/plate 17 min/plate 5 μl/well, total 1.6 μl/well, total We established robust protocol with < 2 μl/well total volume in 3456-well format. 10
11 Advantage of Miniaturization 384-well 1536-well 3456-well Total (reagent) volume / well 20 (5) μl 5 (1) μl 1.6 (0.4) μl Cells / well 6,000 1, Plates / batch 60 plates 30 plates 20 plates Compounds / batch 19,200 38,400 64,000 Required for HTS campaign (e.g. 200,000 compounds) Regent 1,200 ml 240 ml 87 ml Cells 1.4 x 10 9 cells 3.6 x 10 8 cells 8.7 x 10 7 cells Plates 625 plates 157 plates 63 plates Batches 11 times 6 times 4 times We achieved excellent performance by miniaturization to 3456-well format. 11
12 Index of Assay Stability Z = 1 3*(SD_high + SD_low) / (MEAN_high MEAN_low) Z = 1 3*(SD_high + SD_sample) / (MEAN_high MEAN_sample) Low High Mean SD S/B 3.1 Z'
13 Robust Performance in 3456-well Format GPCR-X Agonist Assay DMSO for control Ligand DMSO GPCR-X Z = 0.68 Z = well assay indicated high stability and flatness. 13
14 R a t io Robust Performance in 3456-well Format GPCR-Y Antagonist Assay Seed cells Add ligand Add d2& cryptate Measure 0.5 μl 0.25 μl 0.3 μl, each GPCR-Y DMSO Ligand (EC 80 ) with DMSO Ligand (E max ) Ligand (EC 80 ) with DMSO for control H ig h D a t a P o s it iv e c o n t r o l L o w c o n t r o l Z = 0.57 Z = C o lu m n Antagonist screening is also available. 14
15 About 3456-well Plate Miniaturization of HTRF camp Assay HTS Campaign 15
16 Inhibition (%), replicate 2 Evaluation of Test Screening GPCR-Y Antagonist Reproducibility = 76% Inhibition (%), replicate well format showed good reproducibility. 16
17 Stability of HTS Campaign GPCR-Y Antagonist Z = 0.60 ± 0.05 HTS campaign using 3456-well plate was performed with good stability and quality. 17
18 Hit Profiling of GPCR-Y Antagonist Dose-Response Test for 3 Target (species, selectivity) 3456-well 384-well much data point 3456-well format is also quite helpful at the hit profiling stage. 18
19 Conclusion Miniaturized ultra-hts using 3456-well plates showed sufficient stability, cost effectiveness and time saving. HTS campaigns for GPCR-X agonists and GPCR-Y antagonists were implemented with robustness. camp HTRF assay is applicable to 3456-well format. Now we apply other HTRF technologies to 3456-well format. 19
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