High Throughput Enzymology Studies to Determine Inhibitors with Differing Mechanisms


Nikki Carter1 Derek Barratt2 and Claire McWhirter2
1Discovery Sciences – HTS, AstraZeneca, UK; 2Discovery Sciences – SAR, AstraZeneca, UK.

Reversible enzyme inhibitors can work by a variety of mechanisms: binding to the free enzyme (competitive inhibition), binding to the enzyme substrate complex (uncompetitive inhibition) or binding to both free enzyme and the enzyme substrate complex (mixed and non-competitive inhibition). In practise, many of the inhibitors that are found during screening campaigns work by a substrate competitive mechanism of inhibition. This can lead to a large drop-off in potency between enzyme and cell assays.

As it is a low throughput, labour intensive process, detailed mechanistic characterization of compounds tends to be performed on a limited number of compounds during lead optimization (LO). Identification of compounds with alternative mechanism of action or long residence times earlier in the drug discovery process would allow series with differing kinetics to be progressed into LO.

During a recent HTS campaign we ran hit confirmation assays at KM and high substrate concentrations and with a pre-incubation step which allowed the identification of series that showed non-competitive kinetics and long target residence times to be identified for detailed mechanistic characterization.

This work has lead to a decision tree to be employed during HTS campaigns, where possible, to highlight any mechanistic differences between hit compounds.

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