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Inhibition of Cdc25 phosphatases by indolyldihydroxyquinones
Jungsan Sohn1, Brendan Kiburz, Zhitao Li
1Department of Biochemistry, Duke University, and Duke University Medical Center, Durham, North Carolina 27710, USA.
Abstract:
Overexpression of the Cdc25A and Cdc25B dual-specificity phosphatases correlates with a wide variety of cancers, making the Cdc25s attractive drug targets for anticancer therapies. However, the search for good lead molecules has been hampered by the reactivity of the active site thiolate anion and the flat solvent-exposed active site region. We describe here the indolyldihydroxyquinones, a new class of inhibitors of Cdc25 that bind reversibly to the active site with submicromolar potency. Structure-activity relationships in the 50 derivatives of the lead molecule 2,5-dihydroxy-3-(1H-indol-3-yl)[1,4]benzoquinone show interesting and consistent trends identifying features required for inhibition of all three isoforms of Cdc25. The compounds do not show time-dependent inhibition, indicating that they form neither covalent adducts with nor oxidize the active site thiol. Our best compounds, 2,5-dihydroxy-3-(7-farnesyl-1H-indol-3-yl)[1,4]benzoquinone and 2,5-dihydroxy-3-(4,6-dichloro-7-farnesyl-1H-indol-3-yl)[1,4]benzoquinone, are competitive with substrate for the active site and yield K(i)s of 640 and 470 nM, respectively. Binding of the indolylhydroxyquinones is diminished by three, but not by six other, specific mutations in the active site region. Additionally, the flexible C-terminal tail required for binding of protein substrate is also required for binding derivatives with hydrophobic modifications at the 7-position. The indolyldihydroxyquinones compete effectively with the protein substrate for Cdc25 in vitro and lead to rapid cell death in vivo. Thus, the indolyldihydroxyquinones will serve as useful lead molecules for drug discovery and further cell-based studies on the role of Cdc25s in cell cycle control.
Insights
New indolyldihydroxyquinones inhibit Cdc25 phosphatases, crucial in cancer. These reversible inhibitors show submicromolar potency, offering promising lead molecules for anticancer drug discovery and cell cycle research.
Area of Science:
- Biochemistry
- Medicinal Chemistry
- Oncology
Background:
- Cdc25A and Cdc25B phosphatases are overexpressed in many cancers, making them key drug targets.
- Inhibitor development is challenging due to the Cdc25 active site's reactive thiolate anion and flat, solvent-exposed region.
Purpose of the Study:
- To identify and characterize a new class of reversible Cdc25 inhibitors.
- To explore structure-activity relationships for potent and selective Cdc25 inhibition.
Main Methods:
- Synthesis and testing of approximately 50 indolyldihydroxyquinone derivatives.
- Enzyme kinetics, including competitive inhibition assays and determination of inhibition constants (Ki).
- Site-directed mutagenesis to probe inhibitor-binding interactions.
Main Results:
- Indolyldihydroxyquinones exhibit reversible, non-time-dependent inhibition with submicromolar potency against Cdc25 isoforms.
- Structure-activity relationship studies identified key features for inhibition.
- The best compounds demonstrated competitive inhibition with substrate and induced rapid cell death in vivo.
- Mutagenesis studies revealed specific active site residues and the C-terminal tail are important for inhibitor binding.
Conclusions:
- Indolyldihydroxyquinones represent a novel class of reversible Cdc25 inhibitors with significant therapeutic potential.
- These compounds serve as valuable leads for developing anticancer drugs targeting Cdc25.
- Further studies can utilize these inhibitors to investigate Cdc25's role in cell cycle regulation.