Abstract

One third of current drug discovery pipelines are
focussed on enzyme drug targets and half of currently marketed drugs are enzyme
inhibitors.

During the initial drug development process, after
a high throughput screening (HTS) campaign of small molecules, hits of interest
are further characterised by performing concentration response curve (CRC)
studies whereby the potential for enzyme inhibition is determined by steady
state affinity measurements and quantified in terms of IC₅₀.
This IC₅₀ information together with structural analysis
defines the structural-activity relationship (SAR) of classes of hit chemical
matter.

Whilst important, the IC₅₀  value
in isolation is insufficient to describe the compound’s mechanism of action
(MOA), such as inhibitory mechanism, reversibility, or target residence time.
Therefore, MOA studies that historically tended to be positioned at the later
phases such as lead optimisation, over recent years have been brought into the
screening cascade much earlier.

Determining the mechanism of inhibition allows the
interplay between enzyme, substrate and inhibitor to be understood. Test
compounds may possess competitive, non-competitive or uncompetitive mechanisms
and defining this could help provide a way of extrapolating the activity seen
in biochemical assays to effects seen in whole cells.

To elucidate the mechanism of inhibition of inorganic phosphate-generating enzyme inhibitors, we have established a
fluorogenic, real time assay to perform substrate/inhibitor matrix experiments
Here, we present a study where we mechanistically characterised a discrete set
of ATPase inhibitors.