Development of a novel biochemical assay for the identification of promiscuous compounds.
Loading...
Date
2019
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Abstract
Promiscuous compounds, specifically aggregate-based inhibitors that result in false positives in
biochemical activity assays present a serious and increasing interference to early-stage drug
discovery processes. Although under-used, a number of purpose-specific assays have been
developed to enable the identification of promiscuous inhibitors. To advance on these efforts, this
study aimed to develop and optimise a novel thermal shift assay to concurrently identify both true
and promiscuous inhibitors. In particular, stem bromelain selected as the model protein for this
study was successfully isolated from the bromelain mixture through molecular exclusion
chromatography and shown to be enzymatically active in the titrimetric assay (gelatin digestive
unit/gm enzyme of 2024.36-2085.5). The radial diffusion assay predicted that the promiscuous
compound Epigallocatechin gallate and the known aggregate based-promiscuous compound
Congo Red activated the digestion of gelatin by stem bromelain. In the thermal shift assay,
bromelain yielded a melting temperature of ~75-76 °C which then shifted by 9 °C in the presence
of a true inhibitor E-64. A similar shift was surprisingly observed in the presence of
Epigallocatechin gallate and both these compounds were similarly not affected by the presence
of a detergent (0.004% sodium dodecyl sulfate). The protein was aggregated in the presence of
Congo Red, however, the addition of the detergent effectively restored the protein to its original
melting temperature. Both Epigallocatechin gallate and Congo Red demonstrated cytotoxicity. As
a proof of concept, this study showed that in addition to identifying true inhibitors, the detergent
based thermal shift assay can be successfully employed to identify promiscuous inhibitors and to
determine different mechanisms.
Description
Masters Degree. University of KwaZulu-Natal, Pietermaritzburg.