Linker Length in Biotinylated Probes is Key to Identifying Target Proteins for Anticancer Natural Product OSW-1

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January 10, 2025

?Graduate students Myat Nyein Khine, Naho Isogai (former student), and Tomoya Takeshita (former student), led by Professor Kaori Sakurai from the Division of Biotechnology and Life Science,? Institute of Engineering at Tokyo University of Agriculture and Technology, have developed an optimized biotinylated probe to study OSW-1, a potent anticancer natural product. In this study, they demonstrated that the length of the linker in the biotinylated probe of OSW-1 is crucial for effectively capturing the target proteins (Figure 1).
?This study was supported by the JSPS ACBI program, KAKENHI (18K05331 and 17H06110), Kobayashi Foundation and Global Innovation Research (GIR) of TUAT. M.N.K. was additionally funded by the Japanese Government (MEXT) Scholarship, the Monbukagakusho Honors Scholarship for Privately-Financed International Students, the Doctoral Program for World-Leading Innovative & Smart Education (WISE Program) of TUAT, granted by the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), Japan, and the Kaneko Foundation for International Cultural Communication.

- Background
?OSW-1 is a natural product found in the constituents of an African lily, which is recognized for its strong and selective anticancer activity. In addition to its potency, OSW-1 is considered to have a novel mechanism of action, suggesting it may interact with previously unexplored biological pathways. While OSW-1 has been shown to target two proteins, OSBP and ORP4, its precise anticancer mechanism remains unclear.
?Many natural products with therapeutic potential are known to act on multiple targets, and in many cases, these targets remain unidentified or poorly understood. Similarly, fully identifying all the proteins OSW-1 interacts with, or its "drug targets," is essential for decoding its mechanism of action. This knowledge could not only advance our understanding of OSW-1¡¯s anticancer properties but also reveal new targets for drug development and cancer research.

- Using Chemical Probes for Target Identification
?In recent years, chemical biology, an interdisciplinary field that interfaces chemistry and biology, has been attracting considerable attention. In this field, chemical methods and tools are used to elucidate the complex mechanisms and functions of living organisms at the molecular level. Chemical biology provides powerful tools for studying drug targets, with biotinylated chemical probes being particularly effective. These probes enable the isolation of binding proteins through a method called affinity pulldown. A biotinylated probe, consisting of a "bait" compound and a biotin tag is first attached to avidin-coated beads. Then, the bait compound can capture target proteins in a cell lysate onto the beads, which can then be separated and analyzed. This method helps identify potential drug targets and understand how compounds work biologically.
?The design of biotinylated probes plays a key role in their success. Two critical factors are:
1. Position of functionalization ¨C Where the biotin tag is attached to the compound.
2. Linker structure ¨C The length and flexibility of the linker connecting the biotin tag to the compound.
?If the linker is too short or rigid, it may hinder the probe¡¯s ability to bind both the target protein and the avidin-coated beads, reducing capture efficiency (Figure 2). Conversely, an overly long or flexible linker may cause nonspecific binding or dilute the compound¡¯s effective concentration on the beads. Optimizing linker length and flexibility is thus essential, though its impact on affinity pulldown experiments has not been straightforward to predict.

Figure 1. Biotinylated OSW-1 probes with different linker lengths for affinity pull-down of target proteins. The biotin probe is shown in molecular formula. The biotin moiety (blue) binds to avidin (cyan), while the bait compound (red) interacts with the target protein (pink) via the linker (black). This structure enables the capture of target proteins using biotinylated probes.

- Research Highlights
?To tackle the challenge of designing effective chemical probes, we developed three biotinylated OSW-1 probes with different lengths of polyethylene glycol (PEG) linkers. PEG is a commonly used material known for its compatibility with biological systems and ability to improve solubility. These probes were tested to evaluate their ability to suppress cancer cell growth and isolate target proteins by affinity pulldown.
?The results showed that all three probes maintained the anticancer activity of the original OSW-1 compound, indicating that adding a biotin tag and varying the linker length did not significantly affect its biological function.
?Among the probes, the one with a medium-length PEG linker (PEG5) was the most effective at isolating two known OSW-1 binding proteins, OSBP and ORP4. Interestingly, while the probe with the longest linker had the highest anticancer activity, it was less effective at isolating target proteins than the PEG 5 probe. This finding highlights that probes designed for biological effectiveness may not always be the best suited for identifying protein targets. Taken together, we expect that biotinylated probes with PEG5 linkers will lead to efficient identification of OSW-1 target proteins.

- Future Directions
?This study demonstrated how chemical probes can be carefully designed to improve affinity pulldown experiments, enabling more efficient identification of drug targets.
In the next phase, researchers will use the optimized OSW-1 probe to elucidate new target proteins. By uncovering new pathways related to OSW-1's anticancer effects, this approach may lead to the discovery of novel therapeutic targets and contribute to advancing drug development research.

- Glossary of Terms
1)Biotinylated Probe
A compound with a biotin tag attached, which is used to efficiently capture target proteins. Biotin tags bind avidin proteins with extremely high affinity.?
2)Affinity Pulldown
A biochemical method to capture only strongly bound proteins by centrifugation after the compound-immobilized beads were incubated with intracellular proteins.
3)Linker
Molecular structure linking the biotin tag to the compound.
4)Target Protein
A protein that plays an essential role in the binding of drugs and compounds to exert their pharmacological effects.

Figure 2. Impact of linker length on target capture efficiency of biotin probes.?

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¡ôContact Information¡ô
For inquiries about this research:
Sakurai Kaori
Professor,
Division of Biotechnology and Life Science,
Institute of Engineering, Tokyo University of Agriculture and Technology
TEL/FAX: 042-388-7374
E-mail: sakuraik (please insert @ here) cc.jskrtf.com