A specific mechanism for nonspecific activation in reporter-gene assays

Abstract

The importance of bioluminescence in enabling a broad range of high-throughput screening (HTS) assay formats is evidenced by widespread use in industry and academia. Therefore, understanding the mechanisms by which reporter enzyme activity can be modulated by small molecules is critical to the interpretation of HTS data. In this Perspective, we provide evidence for stabilization of luciferase by inhibitors in cell-based luciferase reporter-gene assays resulting in the counterintuitive phenomenon of signal activation. These data were derived from our analysis of luciferase inhibitor compound structures and their prevalence in the Molecular Libraries Small Molecule Repository using 100 HTS experiments available in PubChem. Accordingly, we found an enrichment of luciferase inhibitors in luciferase reporter-gene activation assays but not in assays using other reporters. In addition, for several luciferase inhibitor chemotypes, we measured reporter stabilization and signal activation in cells that paralleled the inhibition determined using purified luciferase to provide further experimental support for these contrasting effects.

Figure 1. The firefly luciferase sub-chemome

A hierarchical clustering algorithm based on maximum common substructures was used to group the structures. The dendrogram from the clustering hierarchy was automatically generated using an in-house graph layout algorithm. a) The reaction catalyzed by firefly luciferase is shown. b) Dendrogram representation of luciferase inhibitors identified in the cell-free luciferase qHTS (AID 411), and overlap with actives from a Steroidogenic Factor 1 (SF-1) receptor cell-based reporter-gene assay. SF-1 actives are designated as yellow circles (SF-1 activators) and blue circles (SF-1 inhibitors) for the primary cell-based reporter-gene screens (AIDs: 522 and 525 respectively). The right hemisphere of the dendrogram contains prominent chemical series previously identified either as competitive or noncompetitive inhibitors of firefly luciferase. c) Example compounds are highlighted that include known non-competitive luciferase inhibitors such as i and xii that cause apparent inhibition in the reporter-gene assay as well as compounds that mimic the luciferase substrate (see for example iv or x) that appear as activators in the SF-1 activation assay. A representative quinoline showing activation (ii) that we identified as a potent competitive inhibitor of firefly luciferase is also shown. Compounds inactive in the reporter-gene assay were more diverse and included compounds with probable low cell penetration due to the presence of charged groups (for example compound v).

Figure 2. PubChem assays associated with luciferase inhibitors

Sections of the chart are colored as: P. pyralis luciferase-based assays (green; 46.3%) consisting of cell-based reporter-gene assays for either activators or inhibitors (20.9% and 9.3% respectively) and biochemical luciferase-coupled assays (16.1%). Also shown are a) cytotoxicity assays (14.4%), b) biochemical cytochrome P450 assays (9.0%), and c) other assay formats (30.3%) consisting of fluorescent-based assays (i; 24.1%), β-lactamase-based assays (ii; 1.8%), Alphascreen^™ or chemiluminescence (iii; 1.3%), and absorbance-based assays (iv; 3.1%). A total of 1,879 luciferase inhibitors identified in the qHTS associated with high quality CRCs were used to query the PubChem assays using the BioAssay Summary feature. Only assays that covered at least 75% of the luciferase inhibitors are shown (64 total).

Figure 3. Percentage of luciferase inhibitors within hits from 100 PubChem assays

The PubChem active list from each assay was compared to the luciferase qHTS activity and all compounds showing inhibitory CRCs in the luciferase assay were used to calculate the percentage. Assays are grouped by assay type and are shown in order as: (1) P. pyralis luciferase-based biochemical assays; (2) cytotoxicity assays using P. pyralis (Perkin Elmer detection reagent; (3, yellow) cell-based reporter-gene assays scored for activation (the last assay in this group (*) is a cell-based luciferase reporter-gene assay scored for activation that used an unusually stringent cut-off to designate the actives, 200% of control. All other assays typically used a cutoff between 27% to 50% of control values); (4, blue) cell-based reporter-gene assays scored for inhibition; (5) cell-based luciferase reporter-gene assays scored for activation where a short compound exposure was used (2.5 hrs); (6) cell-based reporter-gene assays using β-lactamase; (7) FRET-based assays; (8) luminescent cytotoxicity assays using Photuris pennsylvanica luciferase (Promega, CellTiter-Glo); (9) absorbance-based assays; (10) fluorescent-based assays; (11) luminescent cytochrome P450 assays using P. pennsylvanica luciferase; and (12) other assays including AlphaScreen and cell-free chemiluminescence assays. The type of luciferase is important to consider in this analysis. Luminescent cytotoxicity assays using CellTiter-Glo and coupled assays for P450s utilized an optimized variant of P. pennsylvania firefly luciferase (available in formulations from Promega Corp as Ultra-Glo^™) and from our previous studies we have determined that P. pennsylvania is largely resistant to inhibitors of P. pyralis luciferase (3). Consistent with this finding we found little enrichment for luciferase inhibitors in assays using the P. pennsylvania luciferase while a set of cytotoxicity assays using P. pyralis luciferase (Perkin Elmer reagent, 2) showed large enrichments for luciferase inhibitors.

Figure 4. Stabilization of firefly luciferase by inhibitors

a–c) Top graphs show luciferase activity from HEK293 cells expressing luciferase in cells treated with compound for 24 hrs (open circles; black fitted line) or the remaining luciferase activity following 24 hr treatment with cycloheximide (open triangles, red fitted line). Bottom graphs depict the cell-free luciferase activity determined using purified luciferase assayed with a reporter gene detection cocktail (SteadyGlo; open squares) or using Km concentrations of luciferase substrates (filled circles). The grey shaded rectangle shows the typical concentration range used in HTS. The structures of the compounds assayed are also shown within each graph. d) Decay of luciferase activity following cycloheximide treatment in the absence (closed circles) or presence of 6.25 µm compound for the compound shown in a (open squares), b (filled squares), or c (open circles).