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DMSO-tolerant ornithine decarboxylase (ODC) tandem assay optimised for high-throughput screening
Mingu Gordon Park1,2, Suyeon Yellena Kim2, C Justin Lee1,2
1KU-KIST Graduate School of Converging Science and Technology, Korea University, Seoul, South Korea.
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.
Insights
Researchers developed a new assay to screen for better Ornithine Decarboxylase (ODC) inhibitors, crucial for treating cancer and Alzheimer's disease (AD). This assay overcomes limitations of previous methods, enabling efficient drug discovery.
Area of Science:
- Biochemistry
- Enzymology
- Pharmacology
Background:
- Ornithine decarboxylase (ODC) is a key enzyme in polyamine synthesis and a therapeutic target for cancer and Alzheimer's disease (AD).
- Current ODC inhibitor, α-difluoromethylornithine (DFMO), has limitations due to poor pharmacokinetics, necessitating the development of novel inhibitors.
- High-throughput screening (HTS) is essential for identifying new ODC inhibitors, but existing assays have limitations.
Purpose of the Study:
- To develop a novel, DMSO-tolerant Ornithine Decarboxylase (ODC) tandem assay for high-throughput screening (HTS).
- To optimize the production of ODC from human cell lines for HTS applications.
- To facilitate the discovery of more effective ODC modulators compared to existing drugs like DFMO.
Main Methods:
- Development of a DMSO-tolerant ODC tandem assay by separating enzyme reaction and putrescine detection to minimize fluorescence quenching.
- Optimization of mass production of ODC using human cell lines for HTS.
- Implementation of the assay for screening potential ODC inhibitors.
Main Results:
- A novel ODC tandem assay was successfully developed, demonstrating tolerance to dimethyl sulfoxide (DMSO).
- The assay effectively minimizes DMSO-dependent fluorescence quenching by separating enzymatic reaction and detection steps.
- Optimized protocols for human cell-line-based ODC production were established for HTS.
Conclusions:
- The newly developed DMSO-tolerant ODC tandem assay is a significant advancement for HTS of ODC inhibitors.
- This assay overcomes previous limitations, enabling efficient screening of drug libraries.
- It represents a critical step towards discovering superior ODC modulators for treating diseases like cancer and AD.
