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Small molecule mediated stabilization of p53 tumor suppressor protein through sumoylation is a promising new strategy for improving cancer chemotherapy. A molecular tool that monitors p53 sumoylation status and expedites screening for drugs that enhance p53 sumoylation would be beneficial. We report a molecularly engineered reporter fragment complementation biosensor based on optical imaging of Firefly luciferase (FLuc), to quantitatively image p53 sumoylation and desumoylation in cells and living mice. We initially characterized this biosensor by successfully imaging sumoylation of several target proteins, achieving significant FLuc complementation for ERα (p < 0.01), p53 (p < 0.005), FKBP12 (p < 0.03), ID (p < 0.03), and HDAC1 (p < 0.002). We then rigorously tested the sensitivity and specificity of the biosensor using several variants of p53 and SUMO1, including deletion mutants, and those with modified sequences containing the SUMO-acceptor site of target proteins. Next we evaluated the performance of the biosensor in HepG2 cells by treatment with ginkgolic acid, a drug that reduces p53 sumoylation, as well as trichostatin A, a potential inducer of p53 sumoylation by enhancement of its nuclear export. Lastly, we demonstrated the in vivo utility of this biosensor in monitoring and quantifying the effects of these drugs on p53 sumoylation in living mice using bioluminescence imaging. Adoption of this biosensor in future high throughput drug screening has the important potential to help identify new and repurposed small molecules that alter p53 sumoylation, and to preclinically evaluate candidate anticancer drugs in living animals.

Post-translational addition of methyl groups to the amino terminal tails of histone proteins regulates cellular gene expression at various stages of development and the pathogenesis of cellular diseases, including cancer. Several enzymes that modulate these post-translational modifications of histones are promising targets for development of small molecule drugs. However, there is no promising real-time histone methylation detection tool currently available to screen and validate potential small molecule histone methylation modulators in small animal models. With this in mind, we developed genetically encoded molecular biosensors based on the split-enzyme complementation approach for in vitro and in vivo imaging of lysine 9 (H3-K9 sensor) and lysine 27 (H3-K27 sensor) methylation marks of histone 3. These methylation sensors were validated in vitro in HEK293T, HepG2, and HeLa cells. The efficiency of the histone methylation sensor was assessed by employing methyltransferase inhibitors (Bix01294 and UNC0638), demethylase inhibitor (JIB-04), and siRNA silencing at the endogenous histone K9-methyltransferase enzyme level. Furthermore, noninvasive bioluminescence imaging of histone methylation sensors confirmed the potential of these sensors in monitoring histone methylation status in response to histone methyltransferase inhibitors in living animals. Experimental results confirmed that the developed H3-K9 and H3-K27 sensors are specific and sensitive to image the drug-induced histone methylation changes in living animals. These novel histone methylation sensors can facilitate the in vitro screening and in vivo characterization of new histone methyltransferase inhibitors and accelerate the pace of introduction of epigenetic therapies into the clinic.

NF-E2-related factor-2 (Nrf2) activators promote cellular defense mechanism and facilitate disease prevention associated with oxidative stress. In the present study, Nrf2 activators were identified using cell-based luciferase enzyme fragment complementation (EFC) assay, and the mechanism of Nrf2 activation was studied by molecular imaging. Among the various Nrf2 activators tested, pterostilbene (PTS) showed effective Nrf2 activation, as seen by luminometric screening, and validation in a high throughput-intact cell-imaging platform. Further, PTS increased the expression of Nrf2 downstream target genes, which was confirmed using luciferase reporter driven by ARE-NQO1 and ARE-GST1 promoters. Daily administration of PTS disturbed Nrf2/Keap1 interaction and reduced complemented luciferase signals in HEK293TNKS mouse tumor xenografts. This study reveals the potentials of Nrf2 activators as chemosensitizing agents' for therapeutic intervention in cancer treatment. Hence, the validated assay can be used to evaluate the identified activators preclinically in small animal models by noninvasive molecular imaging approach.