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Title: Development and validation of a generic assay to detect compounds acting via an aggregation-based mechanism
Keywords: HTS, false positives, aggregators, detergent shift, target specificity, meniscus curvature,
Issue Date: 21-Dec-2010
Source: SUKRITI MALPANI (2010-12-21). Development and validation of a generic assay to detect compounds acting via an aggregation-based mechanism. ScholarBank@NUS Repository.
Abstract: High throughput screening (HTS) has emerged as a reliable component of the drug discovery process. It is now recognized that a large number of compounds inhibit their target enzyme via an aggregation-based binding mechanism leading to false positive results in HTS assays. Aggregate-forming compounds act non-competitively; show little relation between structure and activity; have steep dose-response curves and are reported to inhibit multiple unrelated enzymes (McGovern et al. 2002; McGovern et al. 2003; Feng et al. 2007). Removal of these compounds from screening hit lists is desirable as they are not good starting points to initiate medicinal chemistry programs. There are many techniques currently in use to identify aggregation-based inhibition such as dynamic light scattering (DLS), testing sensitivity of inhibition potency to detergent or enzyme concentration, and measurement of meniscus curvature changes in high density multi-well plates associated with colloidal changes in solution. To evaluate the feasibility of large-scale identification of aggregate-based inhibition, hits from three enzyme screens (?-Lactamase, DENV RdRp and Pantothenate kinase) were analysed for signs of aggregate-based inhibitions using various techniques. For a majority of non-specific hits, characteristic features of aggregate-based inhibition such as steep dose-response curves, presence of aggregate particles in solution and inhibition of unrelated enzymatic targets were not found to be associated with detergent or enzyme-concentration sensitive inhibition. Particle size measurements by DLS were inconsistent for many compounds. Steepness of dose response curves depended on buffer composition and assay format employed. Aggregate-based inhibitors displayed target specificity towards their respective target enzymes rather than `promiscuous? inhibition of multiple targets. Different detergents often yielded conflicting results and required derivation of new cut-offs for different enzyme systems or different assay conditions. For example, while the sensitivity of inhibition potency to detergent was not dependent on the nature of the detergent for hits of ?-Lactamase, this was not the case for hits of the DENV RdRp enzyme. The inhibition potencies of the hits of DENV RdRp were found to have different degrees of sensitivity to different detergents. Furthermore, the results of the enzyme-concentration sensitivity tests for the DENV RdRp hits did not seem to correlate with the detergent-sensitivity results. It was observed that the interaction between the enzyme and its substrate possibly confounded the effect of varying the enzyme concentration. The measurement of changes in meniscus curvature, as a means of identification of aggregate-forming small molecule compounds, has been used for the first time in an actual HTS campaign, as reported in this study. The meniscus measurements of hits from all screens correlated well with detection of aggregation-based inhibition based on measurement of changes in inhibition potency. A classi?cation scheme is presented that can be used to rapidly characterize the hits from high throughput screens and eliminate compounds with a non-speci?c mechanism of inhibition. In summary, the meniscus-based aggregation assay is simple, cost-effective, and a reliable method to identify and eliminate compounds that inhibit a specific target enzyme via an aggregation-based mechanism.
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