DMSO-tolerant ornithine decarboxylase (ODC) tandem assay optimised for high-throughput screening

Abstract

Ornithine decarboxylase (ODC), the first rate-limiting enzyme in polyamine synthesis, has emerged as a therapeutic target for cancer and Alzheimer's disease (AD). To inhibit ODC, α-difluoromethylornithine (DFMO), an irreversible ODC inhibitor, has been widely used. However, due to its poor pharmacokinetics, the need for discovery of better ODC inhibitors is inevitable. For high-throughput screening (HTS) of ODC inhibitors, an ODC enzyme assay using supramolecular tandem assay has been introduced. Nevertheless, there has been no study utilising the ODC tandem assay for HTS, possibly due to its intolerability to dimethyl sulfoxide (DMSO), a common amphipathic solvent used for drug libraries. Here we report a DMSO-tolerant ODC tandem assay in which DMSO-dependent fluorescence quenching becomes negligible by separating enzyme reaction and putrescine detection. Furthermore, we optimised human cell-line-based mass production of ODC for HTS. Our newly developed assay can be a crucial first step in discovering more effective ODC modulators than DFMO.

Keywords: Dimethyl sulfoxide; Ornithine decarboxylase; cucurbit[6]uril; high-throughput screening assay; trans-4-(4-(dimethylamino)-styryl)-1-methylpyridinium iodide.

Figure 1.

CB6/DSMI complex is sensitive to DMSO at high concentrations. A. Working principle of the ODC tandem assay. B. A linear relationship between the CB6 concentration and the fluorescence of CB6/DSMI complex. C. Standard calibrations with putrescine and ornithine at varying concentrations to determine the optimal CB6 concentration for 4 µM DSMI. D. Time-course plots of the fluorescence of DSMI with DMSO at different concentrations. E. Time-course plots of the fluorescence of CB6/DSMI with DMSO at different concentrations. F. A dose-response curve of the fluorescence quenching of CB6/DSMI by DMSO [%]. G. A dose-response curve of the fluorescence quenching of CB6/DSMI by DMSO [mM].

Figure 2.

Human cell-line-based mass production of recombinant human ODC homodimers. A. Chromatograms of standard solution (top) and sample solution purified via His-tag system (middle) and ODC enzyme activity of each fraction from size-exclusion chromatography (bottom). B. Workflow for Expi293F^TM-based overexpression of Twin-Strep-tagged human ODC followed by Strep-Tacin chromatography and size-exclusion chromatography. C. Coomasie blue staining (top) and western blot analysis (bottom) of ODC-immunoreactive protein eluates from Strep-Tactin chromatography. D. Coomasie blue staining (top) and western blot analysis (bottom) of ODC-immunoreactive protein fractions (11th to 22nd) from size-exclusion chromatography. E. Chromatograms of standard solution (top) and sample solution purified via Strep-tag system (middle) and ODC enzyme activity of each fraction from size-exclusion chromatography (bottom). F. Time-course plots of the fluorescence of CB6/DSMI with ODC enzyme reaction (in red) and without ODC enzyme reaction (in orange, non-ODC blank). G. A time-course plot of ODC enzyme reaction corrected for background and positively sloped.

Figure 3.

Optimised ODC tandem assay minimises the DMSO-dependent fluorescence quenching of CB6/DSMI and further expands the dynamic range. A. Workflows for traditional ODC tandem assay (top) and optimised ODC tandem assay (bottom). B. Time-course plots of ODC enzyme reaction with DFMO dissolved in DW at varying concentrations (traditional assay). C. A dose-response curve of human ODC inhibition by DFMO dissolved in DW (traditional assay). D. Time-course plots of ODC enzyme reaction with DFMO dissolved in DMSO at varying concentrations (traditional assay). E. A dose-response curve of human ODC inhibition by DFMO dissolved in DMSO (traditional assay). F. Time-course plots of ODC enzyme reaction with DFMO dissolved in DW at varying concentrations (optimised assay). G. A dose-response curve of human ODC inhibition by DFMO dissolved in DW (optimised assay). H. Time-course plots of ODC enzyme reaction with DFMO dissolved in DMSO at varying concentrations (optimised assay). I. A dose-response curve of human ODC inhibition by DFMO dissolved in DMSO (optimised assay).

Figure 4.

Validation of traditional ODC tandem assay and optimised ODC tandem assay by calculating Z-factor. (A–D) The scatter plots showing the positive control (100 µM DFMO, ornage circle) and negative control (0 µM DFMO, red circle) data for the Z-factor calculation in a 96-well microplate format. 42 samples were used for calculating the means and standard deviations. (E–F) The scatter plots showing the positive control (100 µM DFMO, ornage circle) and negative control (0 µM DFMO, red circle) data for the Z-factor calculation in a 384-well microplate format. 42 samples were used for calculating the means and standard deviations.

Figure 5.

The dynamic range of ODC tandem assay is enhanced by the use of 10 mM Tris buffer over 50 mM Tris buffer. Dose-response curves of human ODC inhibition by DFMO dissolved in DW or DMSO. (A–B) Traditional assay with 10 mM Tris buffer. (C–D) Traditional assay with 50 mM Tris buffer. (E–F) Optimised assay with 10 mM Tris buffer. (G–H) Optimised assay with 50 mM Tris buffer.