Getting Back to Nature: Using in vitro Assays that Mimic Physiological Systems, and Label-free Assays in Small Molecule Discovery
Jim Kranz,
Senior Scientist
Johnson & Johnson
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Abstract: The benefits of designing assays to mimic the physiologically relevant state are obvious, yet often artificial systems are employed for technical reasons, including the ability to miniaturize the assay for High Throughput Analysis. Assays used in HTS campaigns are often highly optimized due to demands on throughput and cost; labeled substrates and indirect detection methods are often employed. These shortcuts, however, are employed at the expense of physiological relevance. This is particularly true of protease substrates which often resemble short peptides with an absorbance- or fluorescence-based signal change upon proteolysis. Chymase and Cathepsin-G are serine proteases released from leukocytes that sustain inflammatory processes, and are targets to treat asthma and chronic obstructive pulmonary disease. We describe how traditional assay development approaches led to Chymase-selective compounds, while selectivity for Cathepsin-G was difficult to obtain. An alternative approach has been employed that utilizes native peptide-based substrates and label-free assays to explore novel inhibitors that bind outside the space defined by Chymase-selective inhibitors. The results show mixed patterns of overlapping inhibition comparing different assay reagents, requiring a thorough exploration of inhibitor mechanism of action in classifying progressible leads.
Presenter Biography: James Kranz is currently a Senior Scientist in Johnson & Johnson Pharmaceutical Research & Development, LLC (Springhouse, PA) in the Lead Generation Biology group, which encompasses HTS, Biophysics, Cell Biology, & Enzymology. Their approach to understanding the mechanism of action of small molecule inhibitors of protein function relies on both traditional and novel technologies. Dr. Kranz joined J&J PRD in 2003 as a part of the acquisition of 3-Dimensional Pharmaceuticals, a drug discovery company with an emphasis on structure-based drug design. During his Ph.D. (Washington University) and postdoctoral work (University of Pennsylvania), he developed novel biophysical approaches to explore the mechanisms of intermolecular complexation in control of biological processes using a combination of thermodynamic and NMR-based biophysical approaches.
Online Presentation: http://lifescience.planetconnect.com/ppt/LSPrinceton/wednesday/JIM_KRANZ.ppt
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