Development of a novel biochemical assay for the identification of promiscuous compounds.
Ntombela, Nomfundo Praise.
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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.