Dongping Ma

Luminogenic cytochrome P450 assays

Luminogenic cytochrome P450 (CYP) assays couple CYP enzyme activity to firefly luciferase luminescence in a technology called P450-GloTM (Promega). Luminogenic substrates are used in assays of human CYP1A1, -1A2, -1B1, -2C8, -2C9, -2C19, -2D6, -2J2, -3A4, -3A7, -4A11, -4F3B, -4F12 and -19. The assays detect dose-dependent CYP inhibition by test compounds against recombinant CYP enzymes or liver microsomes. Induction or inhibition of CYP activities in cultured hepatocytes is measured in a nonlytic approach that leaves cells intact for additional analysis. Luminogenic CYP assays offer advantages of speed and safety over HPLC and radiochemical-based methods. Compared with fluorogenic methods the approach offers advantages of improved sensitivity and decreased interference between optical properties of test compound and CYP substrate. These homogenous assays are sensitive and robust tools for high-throughput CYP screening in early drug discovery.

Bioluminescent assays for ADME evaluation: dialing in CYP selectivity with luminogenic substrates

Introduction: The cytochrome P450s (CYPs) are central to ADME studies because of their central role in drug metabolism. Proper CYP assay design and a correct understanding of CYP assay selectivity are critical for generating and interpreting biologically relevant data during drug development. Bioluminescent CYP assays use luminogenic probe substrates that have the unique property of producing photons in a second reaction with luciferase. Areas covered: This article presents the general design principles for in vitro CYP assays. Specifically, the article focuses on the bioluminescent approach that couples CYP activity with photon production. Expert opinion: Highly selective luminogenic substrates for CYP1A1, CYP1A2, CYP2C9, CYP3A4, CYP3A7, CYP4A and CYP4F have been developed with utility for interrogating the roles of these enzymes in biochemical and cell-based formats. These selective substrates are part of a larger collection of probes that deliver CYP inhibition and induction data that predict in vivo drug interactions. Furthermore, they support highly sensitive, rapid and scalable assays for cell-based and cell-free biochemical applications, which offer an alternative and often enabling option over conventional assay strategies.

Novel No-Wash Luminogenic Probes for the Detection of Transporter Uptake Activity.

Luminogenic probes were designed and synthesized for the detection of uptake transporter activity in a lytic cell-based assay. These probes rely on a self-cleavable trimethyl lock quinone-cyanobenzothiazole (TMQ-CNBT) or trimethyl lock quinone-luciferin (TMQ-Luc) linked to the anion transporter substrate fluorescein. Upon cellular transport, the TMQ is reduced by viable cells, resulting in the facile intramolecular lactonization and rapid release of the bioluminescent reporter molecule. The uptake transporter activity can then be detected without removing and washing off the extracellular substrates. Six probes were tested with OATP1B1*1a and OATP1B3 overexpressing HEK293 cells, and all compounds showed up to 10.2-fold enhancement in uptake when compared to control cells. Uptake of TMQ-luciferin compounds 2, 4, and 6 increased linearly over time up to 30 min at a concentration ranging from 40 nM to 20 μM. The apparent Km values obtained at different time intervals up to 30 min were nearly identical for a given compound, which validates the 30 min window as appropriate for uptake transporter assays. The average apparent Km values ranged from 0.3 to 0.8 μM and 0.2 to 1.3 μM for OATP1B1*1a and OATP1B3, respectively, indicating good affinities to these anion transporters. Furthermore, uptake of compound 2 was inhibited by two inhibitors of OATP1B1*1a and OATP1B3: rifampicin and ritonavir. The preliminary results obtained from compound 2 exhibited a time-dependent, saturatable, and inhibitable nature of uptake, indicating the feasibility of using the probe for the detection of a transporter-mediated process. This add-and-read homogeneous assay may provide a convenient, rapid, and facile way to detect changes in transporter activity in a high-throughput format, and this assay design strategy could create a new platform for a general cell uptake assay for biomaterials in the future.

Abstract 3312: A novel plate-based assay for screening autophagic activity in 2D and 3D cell culture models

The critical importance of autophagy in cell health and its proposed role in disease-relevant biology, including cancer, inflammation, and immunology, has increased the need for more effective assays to screen for agents that modulate autophagic activity. Here we utilize NanoLuc Binary Technology (NanoBiT) to develop a homogeneous plate-based assay to measure autophagic flux in cell culture models. In this approach, an exogenous LC3B (Atg8) fusion protein was tagged on its N-terminus with an 11 amino acid peptide (HiBiT) and stably expressed in mammalian cells, including U2OS and HEK293. After exposure to various treatment conditions, cellular levels of this novel autophagy reporter were determined by addition of a lytic detection reagent containing Large BiT (LgBiT). LgBiT rapidly associates with HiBiT in the cell lysate, producing a bright, luminescent enzyme in the presence of the furimazine substrate. The bright signal allows low levels of expression of the reporter, maximizing the assay response, and the signal is stable, allowing assay of multiple 96- or 384-well plates in the same experiment. In response to autophagic stimuli, including nutrient deprivation and various mTORC inhibitors (e.g., PP242 and rapamycin), autophagic degradation of expressed LC3 reporter was evident by reduced assay signal. In contrast, in response to both upstream (e.g., 3-MA and wortmannin) and downstream (e.g., bafilomycin A1 and chloroquine) inhibitors of the autophagy pathway, degradation of the autophagic reporter was effectively blocked and assay signal was consistently increased as predicted. Compound effects were time dependent and stratified according to expected potency and efficacy of the test agents employed. The use of a mutant reporter based on LC3G120A further demonstrated the specificity of the wild-type LC3 reporter for the detection of autophagic activity. When assayed in 384-well plates with automation, HEK293 autophagy reporter cells produced Z’ values of ~0.7 in response to autophagy induction with PP242, while subsequent blockade of autophagy with bafilomycin A1 resulted in Z’ values of ~0.8. This data, and subsequent LOPAC library screening, indicates the potential utility of this assay method for HTS applications. In addition, the HEK293 autophagy reporter cells can be induced to form 3D cell spheroids, thus allowing investigation of assay performance in this more complex model. Autophagy reporter levels increased with increasing spheroid size (up to 650 μm diameter tested) in a manner proportional to a surrogate measure of viable cell number. Importantly, both induction and inhibition of autophagic activity was easily detected following PP242 and bafilomycin A1 treatment, respectively. Using this novel plate-based assay system for the determination of autophagic flux, it is possible to screen test agents and quantitatively determine both the potency and efficacy of autophagy modulation. Citation Format: Dan F. Lazar, Amani A. Gillette, Braeden L. Butler, Christopher T. Eggers, Brock F. Binkowski, Gediminas Vidugiris, Michael R. Slater, Dongping Ma, James J. Cali. A novel plate-based assay for screening autophagic activity in 2D and 3D cell culture models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3312. doi:10.1158/1538-7445.AM2017-3312