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Updated: May 24, 2025
Functionalized Spirocyclic Heterocycle Synthesis and Cytotoxicity Assay
Published on: February 9, 2021
Synthesis and Biological Evaluation of Novel Triazine Analogs as Rad6 Inhibitors
Qian Lin1, Ambikai Gajan2, Ignatius Nguyen1
1Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, Detroit, MI, 48201, USA.
Abstract:
Rad6 is an E2 ubiquitin-conjugating enzyme that plays critical roles in genome maintenance and proteostasis. Rad6 is frequently overexpressed in many cancers and promotes cancer development, progression, and chemotherapy resistance.
Purpose:
Given its role in cancer development and progression, Rad6 is an underexplored therapeutic target. Previous research identified compound SMI#9 as a small molecule inhibitor of Rad6. Despite its potency, SMI#9 has limited efficacy in vivo due to its limiting water solubility and the presence of a labile ester group.
Methods:
To address these limitations, we prepared a series of SMI#9 analogs in which the ester group was replaced with a secondary amine, and their effects on Rad6B-mediated ubiquitination of histone H2A were evaluated. In vivo interaction with Rad6 was assessed using cellular thermal shift assays. SMI#9 analog effects on cell survival and migration of triple negative and endocrine-resistant breast cancer, and melanoma cells were measured using MTT and Boyden chamber assays. Autophagy, mitochondrial function, and β-catenin localization were measured using CytoID, Mitotracker, and immunostaining, respectively. Cellular uptakes of analogs were determined by mass spectroscopy.
Results:
Analogs #4 and #6 inhibited H2A ubiquitination, induced autophagy and mitochondrial dysfunction, downregulated intracellular β-catenin, and inhibited proliferation. #6 targets Rad6 in vivo. #4 and #6 are chemically related, and #4 undergoes in vivo conversion to #6.
Conclusions:
#6 retains all the properties of SMI#9 but with lesser potency. However, its improved water solubility and metabolic stability allows for in vivo studies that were previously precluded due to the poor physicochemical properties of SMI#9.
Insights
New Rad6 inhibitors show promise for cancer therapy. Analogs of SMI#9 improve solubility and stability, enabling in vivo studies for cancer treatment development.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Rad6, an E2 ubiquitin-conjugating enzyme, is crucial for genome maintenance and proteostasis.
- Overexpression of Rad6 is linked to cancer development, progression, and chemotherapy resistance, making it a potential therapeutic target.
- Previous inhibitor SMI#9 showed potency but suffered from poor water solubility and metabolic instability.
Purpose of the Study:
- To develop novel Rad6 inhibitors with improved physicochemical properties for in vivo cancer research.
- To synthesize and evaluate SMI#9 analogs with modified structures to enhance efficacy and drug-likeness.
Main Methods:
- Synthesized SMI#9 analogs by replacing the ester group with a secondary amine.
- Assessed inhibition of Rad6B-mediated histone H2A ubiquitination.
- Evaluated in vivo Rad6 interaction using cellular thermal shift assays.
- Measured effects on cancer cell survival, migration, autophagy, mitochondrial function, and β-catenin localization.
Main Results:
- Analogs #4 and #6 effectively inhibited H2A ubiquitination and demonstrated anti-cancer properties.
- These analogs induced autophagy, mitochondrial dysfunction, and downregulated β-catenin.
- #6 was confirmed to target Rad6 in vivo, with analog #4 converting to #6 in vivo.
Conclusions:
- Analog #6, despite slightly lower potency than SMI#9, offers superior water solubility and metabolic stability.
- These improved properties facilitate in vivo studies, overcoming the limitations of the original compound.
- The developed analogs represent promising candidates for further investigation as anti-cancer therapeutics targeting Rad6.
