Reporter Enzyme Inhibitor Study To Aid Assembly of Orthogonal Reporter Gene Assays

Reporter
gene assays (RGAs) are commonly used to measure biological
pathway modulation by small molecules. Understanding how such compounds
interact with the reporter enzyme is critical to accurately interpret
RGA results. To improve our understanding of reporter enzymes and
to develop optimal RGA systems, we investigated eight reporter enzymes
differing in brightness, emission spectrum, stability, and substrate
requirements. These included common reporter enzymes such as firefly
luciferase (Photinus pyralis), Renilla reniformis luciferase, and β-lactamase, as well as mutated forms of R. reniformis luciferase emitting either blue- or green-shifted
luminescence, a red-light emitting form of Luciola cruciata firefly luciferase, a mutated form of Gaussia princeps luciferase, and a proprietary luciferase termed “NanoLuc”
derived from the luminescent sea shrimp Oplophorus gracilirostris. To determine hit rates and structure–activity relationships,
we screened a collection of 42,460 PubChem compounds at 10 μM
using purified enzyme preparations. We then compared hit rates and
chemotypes of actives for each enzyme. The hit rates ranged from <0.1%
for β-lactamase to as high as 10% for mutated forms of Renilla luciferase. Related luciferases such as Renilla luciferase mutants showed high degrees of inhibitor
overlap (40–70%), while unrelated luciferases such as firefly
luciferases, Gaussia luciferase, and NanoLuc showed
<10% overlap. Examination of representative inhibitors in cell-based
assays revealed that inhibitor-based enzyme stabilization can lead
to increases in bioluminescent signal for firefly luciferase, Renilla luciferase, and NanoLuc, with shorter half-life
reporters showing increased activation responses. From this study
we suggest strategies to improve the construction and interpretation
of assays employing these reporter enzymes.