Quantitative analyses of aggregation, autofluorescence, and reactivity artifacts in a screen for inhibitors of a thiol protease

Abstract

The perceived and actual burden of false positives in high-throughput screening has received considerable attention; however, few studies exist on the contributions of distinct mechanisms of nonspecific effects like chemical reactivity, assay signal interference, and colloidal aggregation. Here, we analyze the outcome of a screen of 197861 diverse compounds in a concentration-response format against the cysteine protease cruzain, a target expected to be particularly sensitive to reactive compounds, and using an assay format with light detection in the short-wavelength region where significant compound autofluorescence is typically encountered. Approximately 1.9% of all compounds screened were detergent-sensitive inhibitors. The contribution from autofluorescence and compounds bearing reactive functionalities was dramatically lower: of all hits, only 1.8% were autofluorescent and 1.5% contained reactive or undesired functional groups. The distribution of false positives was relatively constant across library sources. The simple step of including detergent in the assay buffer suppressed the nonspecific effect of approximately 93% of the original hits.

Figure 1

Composition of the 197,861-sample library screened against cruzain (A) and similarity between the distribution of major screening hit categories across compound sources (artifacts [aggregators (B), autofluorescent (C)], reactive (D) and conclusive inhibitors (E) as defined in the text and Figure 4) and the library makeup.

Figure 2

Z′ trend of 1,106 plates screened in the detergent-free (small rhombs) and detergent-present (large circles) assays. The average Z′ of screen without detergent was 0.78 and improved to 0.93 with addition of detergent.

Figure 3

Cruzain concentration-response data of control titrations and all library samples tested in the detergent-free (A) and detergent-present (B) assays. Control titrations (in green) were included on each 1,536-well plate screened. Apparent inhibitors are shown in blue, apparent activators are in red, and inactives are in black. A total of 14% of samples gave signal increase or signal decrease in the detergent-free while only 1% of samples resulted in any activity response in the detergent-present assay.

Figure 4

Categorization of hits. Signal-increase compounds lead to false ‘activation’ artifact (Category A) which disappeared completely upon inclusion of detergent. Among the signal decrease-compounds, a majority of the apparent inhibition was due to aggregation (category B and C). Kinetic reads during screening helped identify fluorescent false positives (Category D), while reactive and problematic functional group filtering eliminated additional promiscuous inhibitors (Category E). The remaining weak (Category F) or potent (Category G) inhibitors represented 3.6% of the total actives found in the detergent-free qHTS.

Figure 5

Fluorescence Interference Analysis. A) Plate heatmaps associated with one 7-concentration compound series in 1,536-well format. Shown are the first and last fluorescent reads and the activity calculated from the 60-second initial rates. Subtracting out the higher-than-average but steady fluorescence of some compounds (red dots on heatmaps associated with first and last reads) leads to the significant reduction of interference (small number of blue dots in the activity heatmap). However, for compound 42 which is highly fluorescent (left plots in B and C), the drift in inherent fluorescence within the reaction time course (indicated within panel B) can lead to the erroneous calculation of concentration-response effect (C, left plot). A non-fluorescent inactive compound 43 (right plots in B and C) displays nearly overlapping reaction time courses (B, right plot) and as a consequence the corresponding concentration-response trend is relatively flat, resulting in the correct assignment of inactive phenotype (C, right plot).

Figure 6

(A) Hill slopes of 3,844 Category C aggregators vs. 228 of Category E reactive or problematic inhibitors vs. 493 Category G top detergent sensitive inhibitors. (B) qHTS detergent-free curve of an aggregator SID 17508642 (44) with a Hill slope of 4. (C) Detergent resistant inhibition curves of two reactive compounds, SID 50107106 (45) (steep Hill slope) and SID 14739064 (46) (Hill slope of 0.5). (D) Detergent resistant cruzain specific inhibitor SID 24406445 (47) with Hill slope of 1.