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Generation of a large batch of cells for assay validation

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1 Generation of a large batch of cells for assay validation
Development of a Cell Based Assay to Screen for Inhibition of MALT1 Protease Activity Helena Peilot Sjögren1, Linda Roth1, Pia Hansson1, Elisabeth Bäck1, Tyrrell Norris1, Robert Roth1 and Helen Boyd1 1Discovery Sciences iMed, AstraZeneca Mölndal, Sweden Aim Generation of Transient Assay Ready Cells using MaxCyte Flow Electroporation MALT1 is Crucial for Proteolytic Activity of the CBM Complex Assay Validation Development of a robust cell-based assay to determine inhibition of MALT1 proteolytic activity. A. No DNA B. CBM Complex C. no MALT1 A B Activity (%) Log Concentration (M) Optimisation: DNA concentration, DNA ratio, if needed. Time needed for cell recovery and transcript expression Feasibility test Determine the signal in the assay and that the signal is specific for the target. Optimisation of transfection conditions against the following parameters: Cell viability, %recovery of cells after transfection and S/B and %CV in assay Generation of a large batch of cells for assay validation Assay validation: Determination of inter and intra assay variation Typical Work Flow: Background - MALT1 and NFkB Mucosa associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is scaffolding protein and a cysteine protease that specifically cleaves a number of substrates including, Bcl10, CYLD, RelB and A20. Thereby MALT1 trigger intracellular responses including NFkB and JNK signaling and T-cell activation. Fig 4. MALT1 activity was measured using the LRSR-Rh110 peptide substrate alone or in the presence of 10mM of a peptide inhibitor, Z-VRPR-fmk (5). A. no DNA control, B. CBM, C. CB, no MALT1. Fig 7. A. Max and Min data of 384 well assay. B. Concentration response curve of one out of six tool compounds used for validation. Data represents average from four independent experiments run in triplicate. Average IC50 = 0.65 mM. Optimization of Peptide Cleavage Sequence to Improve Assay Performance “Use of Transient Assay Ready Cells is a Straight Forward Way to Generate Cells for Assay Development and Screening” Bcl10 Modified from Gewies et. al. T-cell activation CBM Complex Following optimization of the assay, the signal to background was 5 and Z’ was determined to 0,5. Further statistical validation using six different tool compounds returned a confidence interval ratio (CIR) value of 2,1, given the assay is run in duplicates at one occasion. Furthermore, early attempts to miniaturize the assay to a 1536 are promising, with an assay window of 3. Wt CBM Wt CBM Wt CBM Wt CBM LRSR ALVSR LVSR LVSRR Fig 5. MALT1 activity was measured using the Rh110 peptide substrate alone or the presence of 10mM of a peptide inhibitor, Z-VRPR-fmk (5) in CBM expressing cells and non-transfected control cells (wt). ALVSR substrate was chosen for further optimization and assay development Bcl10(1) RelB(1) Cells: Peptide: Assay Principle Intact substrate (Quenched Rh110 signal) Rh110 Malt1 proteolytic activity results in a stripped Rh110 (Rh110 Emission at 520 nm) MALT1 Proteolytic activity MALT1 Specific peptide Time (min) MALT1 Activity RFU 485/520 “This Assay Correlates Well to Other Internal Cell Assays Using Full Length Protein Substrates and an NFkB Reporter Gene Assay” Fig 1: The formation of the CBM complex is crucial for MALT1 dependent NFkB and JNK signaling. Optimization of DNA Concentration and Incubation Time Prior to Cryopreservation Canonical NFkB pathway Fig 2: NFkB signalling is important for inflammation, immunity and cell proliferation. Inhibition of MALT1 proteolytic activity therefore represents a promising therapeutic target for autoimmune diseases and cancer. A B Fig 3. Cells expressing the CBM complex are incubated with a cell permeable Rh110 peptide substrate. The Rh110 signal is a measure of MALT1 proteolytic activity. References Wiesmann et.al. J Mol Biol: 419 (2012), pp. 4-21 Gewies et. al. Cell Reports: 9, (2014) pp.1292–1305 Lenz et. al. Science: 319, (2008), pp. 1676–1679 Qiao et. al. Mol Cell: 51, (2013), pp Rebeaud et al. Nat Immunol: 9, (2008), pp Plasmid constructs containing full length MALT1 and Bcl10 (1) and a full length and modified CARMA1 were used for assay development. CARMA1 was modified to obtain expression of a constitutively active CBM complex (3). A ratio of 45% MALT1, 45% BCL10 and 10% CARMA1 was used for assay development, (4). Fig 6. MALT1 activity was measured using the ALVSR Rh110 peptide substrate alone or the presence of 10mM of a peptide inhibitor, Z-VRPR-fmk (5). A. Cells Transfected with 50 or 150ug total DNA. B. Comparison of cell recovery time, 20min, 24h or 48h after transfection prior to cropreservation. DNA concentration of 150ug and 24h recovery time were choosen for further optimisation and assay development. Supported by Presented at the ELRIG - Advances in Cell Based Screening in Drug Discovery , AstraZeneca Mölndal, Sweden, June, 2015


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