What question did this study set out to answer?

To identify and analyze molecular targets of dehydrodiisoeugenol (DHE) and its mechanisms in bladder cancer.

April 24, 2026Pharmaceuticals0 citationsOpen Access

Identification and Functional Analysis of Targets of Dehydrodiisoeugenol in Bladder Cancer Based on Chemoproteomics-Based Profiling

Key Points

To identify and analyze molecular targets of dehydrodiisoeugenol (DHE) and its mechanisms in bladder cancer.
Integrated phenotypic screening with 2D cell lines and 3D organoids
Used chemoproteomics-based activity-based protein profiling (ABPP)
Conducted cellular thermal shift assays (CETSA) and molecular dynamics simulations.
DHE showed dose-dependent cytotoxicity with IC50 values of 39.23μM and 34.58μM in T24 and 5637 cells, respectively.
DHE significantly reduced viability in 3D organoids (p < 0.0001).
Identified 65 high-confidence candidate targets, with PTPN1 prioritized as a key functional interactor.

Abstract

Background/Objectives: The clinical management of bladder cancer is severely impeded by high recurrence rates and the rapid emergence of chemoresistance, necessitating the discovery of novel therapeutic agents with distinct mechanisms of action. Dehydrodiisoeugenol (DHE), a bioactive neolignan, exhibits potent anti-tumor efficacy, yet its direct molecular targets and mode of action remain elusive. Methods: To deconvolute the mechanism of DHE, we integrated a phenotypic screening approach using 2D cell lines and 3D patient-derived organoids with a chemoproteomics-based activity-based protein profiling (ABPP) strategy. We synthesized a functionalized photoaffinity probe to capture the specific interactome of DHE under physiological conditions and validated targets via cellular thermal shift assays (CETSA), quantitative mass spectrometry, and 100 ns molecular dynamics (MD) simulations. Results: DHE exhibited potent dose-dependent cytotoxicity in bladder cancer cells, with IC50 values of 39.23μM in T24 and 34.58μM in 5637 cells. In 3D patient-derived organoids, DHE significantly reduced viability (p < 0.0001). Using a dual-filtering ABPP strategy, we identified 65 high-confidence candidate targets, prioritizing PTPN1 (PTP1B) as the primary functional interactor. Comparative molecular docking and 100 ns MD analyses showed that multiple stereoisomers of DHE could adopt plausible PTPN1-binding modes. Mechanistically, organoid proteomics indicated that DHE engagement with PTPN1 disrupts ER membrane homeostasis, thereby modulating the PI3K-Akt signaling axes. Conclusions: These findings establish PTPN1 as a critical druggable vulnerability in bladder cancer and define the molecular basis for the therapeutic potential of DHE. This study highlights the power of combining chemoproteomics with physiological 3D models to accelerate the translation of natural products into precision cancer therapies.

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