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CONLFUENCE DISCOVERY TECHNOLOGIES | CASE STUDY CASE STUDY: CUSTOM ASSAY DEVELOPMENT & VALIDATION Synopsis A multiplex cytokine production assay using human peripheral blood mononuclear cells (PBMC) was developed and validated for use in a compound screening testing funnel as a functional cellular readout for ‘Target X’ modulation. Background ‘Target X’ is involved in pro-inflammatory cytokine production. As part of a testing funnel to identify compounds that potently and selectively inhibit ‘Target X’, a cellular functional readout assay was required. Human PBMCs, previously shown to express ‘Target X’ and produce multiple pro-inflammatory cytokines in response to stimulation, were selected for use in the development of this assay. Approach Human PBMCs from multiple donors were isolated from Leukopaks using Histopaque 1077 and cryo-preserved in donor specific aliquots of 1x108 cells. Cells were thawed, DMSO was removed and cells plated in 96-well microtiter plates for initial studies and for compound analysis. Initial studies consisted of time course analysis of stimulus dependent cytokine production to select appropriate stimulation times. For compound analysis, cells were treated for one hour with various concentrations of compounds then stimulated with a selected agonist. Conditioned media (25uL) were removed at the selected time and analyzed for cytokine levels using MESO Scale Discovery multiplex pro-inflammatory cytokine plates. Results & Conclusion The compound tested inhibited the four cytokines in a concentration dependent manner with IC50 values in the 1-10 nM range. The human PBMC multiplex cytokine production assayed proved to be a useful and highly reproducible assay for the triage of compounds developed to inhibit ‘Target X”. Additionally, the analysis is very efficient with respect to time, reagents and required sample quantities. Compound (nM) Our Services To see how the scientists at CDT can support your assay needs please contact us for a free consultation CDTv1.6.13 Confluence Discovery Technologies, Inc. 4320 Forest Park Avenue Saint Louis, MO 63108 Phone: 314-932-4032 x305 Fax: 314-932-4038 www.confluencediscovery.com [email protected] CONLFUENCE DISCOVERY TECHNOLOGIES | CASE STUDY OUR CAPABILITIES Biochemistry & Enzymology • • • • steady state kinetics biacore® (SPR) radioligand binding absorbance fluorescence luminescence Isothermal calorimetry Drug:target interaction Enzyme inhibitor mechanisms Direct binding kinetics Compound mechanism of action Cell & Molecular Biology • Identification of biologically important signal transduction pathways • Quantitation of efficacy and selectivity • Understanding safety issues linked to pathways • De-convolution to isolate pathway modes • Evaluation of multiple targets within the pathway • Identification of key pathway point(s) for therapeutic intervention • Target validation, cloning, expression and purification • Cell pathway interrogation • Custom assay development • High-throughput screening Custom Assay Development • • • • • • assay development assay instrumentation data analysis specialty screens problem solving Format reproducibility Available controls Robustness Appropriateness Reagent availability Linearity Translational Research • Identify target, mechanism & disease biomarkers • Select the appropriate biomarkers to assess for a drug discovery program • Assist in biomarker assay design, development, validation & interpretation Computational & Medicinal Chemistry • • • • CDTv1.6.13 Schrödinger® enabled computational drug design High throughput hit prioritization Chemical series prioritization Structure-activity relationship generation 13 target validation kinase pathway analysis human/non-human cell lines primary cell types adherent /non adherent multiple species tissues multiplex assay technologies secreted /intracellular analytes DNA purification custom cloning cell & tissue RNA purification synthetic gene construction RT-PCR analysis assays across species biological matrices multi-formats multiplexing complete statistical evaluation structure based drug design leads from virtual screening synthetic route analysis compound synthesis lead optimization library generation competitor landscape analysis Confluence Discovery Technologies, Inc. 4320 Forest Park Avenue Saint Louis, MO 63108 Phone: 314-932-4032 x305 Fax: 314-932-4038 www.confluencediscovery.com [email protected]
