ABSTRACT
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 ß-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 ß-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.
Subject(s)
Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Enzymes/genetics , Genes, Reporter/drug effects , Luminescent Measurements/methods , Animals , Cell Line , Drug Evaluation, Preclinical/methods , Enzyme Stability , Enzymes/metabolism , Humans , Luciferases/antagonists & inhibitors , Luciferases/genetics , Luciferases, Firefly/antagonists & inhibitors , Luciferases, Firefly/genetics , Luciferases, Renilla/antagonists & inhibitors , Luciferases, Renilla/genetics , Luminescence , Mutation , Small Molecule Libraries/pharmacology , Structure-Activity Relationship , beta-Lactamase Inhibitors , beta-Lactamases/geneticsABSTRACT
More than 300 human clinical trials utilize recombinant adenoviruses (rAds) as a gene transfer vector, confirming that rAds continue to be of high clinical interest. A primary weakness of rAds is their known propensity to trigger an innate, proinflammatory immune response rapidly after high-dose, systemic administration. In this study, we investigated what affects that pre-emptive treatment with anti-inflammatory glucocorticoids might have upon Ad vector-triggered inflammatory immune responses. We found that a simple pretreatment regimen with Dexamethasone (DEX) can significantly reduce most Ad-induced innate immune responses. DEX prevented rAd induction of systemic cytokine/chemokine releases in a dose-dependent fashion, with higher dosages preventing rAd induction of acute thrombocytopenia, endothelial cell activation, proinflammatory gene induction, and leukocyte infiltration into transduced organs. Transient glucocorticoid pretreatment also significantly reduced rAd-induced adaptive immune responses, including a decreased induction of Ad-neutralizing antibodies (NAbs). Importantly, use of DEX did not reduce the efficacy of rAd-mediated gene transduction nor rAd-derived transgene expression. Our results demonstrate that a simple, pre-emptive and transient glucocorticoid pretreatment is a viable approach to reduce rAd-associated acute toxicities that currently limit the use of Ad vectors in systemic clinical applications.
Subject(s)
Adenoviridae/genetics , Dexamethasone/pharmacology , Genetic Vectors/adverse effects , Immunity, Innate/drug effects , Animals , Gene Expression/drug effects , Inflammation Mediators/metabolism , Kupffer Cells/cytology , Liver/cytology , Liver/drug effects , Mice , Mice, Inbred C57BL , Transduction, GeneticABSTRACT
Transcription factor IIE (TFIIE) is a general initiation and promoter escape factor for RNA polymerase II composed of p56 (TFIIE-alpha) and p34 (TFIIE-beta) subunits. Our laboratories experienced difficulty producing adequate quantities of recombinant human TFIIE-alpha for in vitro studies using available clones. We therefore re-engineered the TFIIE subunit production vectors and tested various Escherichia coli host strains to optimize expression. We report a much-improved system for production of pure, soluble, and active TFIIE complex for in vitro studies.