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Published on: September 30, 2016
Physical and Functional Analysis of the Putative Rpn13 Inhibitor RA190
Paige Dickson1, Daniel Abegg1, Ekaterina Vinogradova2
1Department of Chemistry, The Scripps Research Institute, 130 Scripps Way, Jupiter, FL 33458, USA.
Abstract:
Rpn13 is one of several ubiquitin receptors in the 26S proteasome. Cys88 of Rpn13 has been proposed to be the principal target of RA190, an electrophilic small molecule with interesting anti-cancer activities. Here, we examine the claim that RA190 mediates its cytotoxic effects through engagement with Rpn13. We find no evidence that this is the case. In vitro, RA190 is has no measurable effect on any of the known interactions of Rpn13. In cellulo, we see no physical engagement of Rpn13 by RA190, either on C88 or any other residue. However, chemical proteomics experiments in two different cell lines reveal that dozens of other proteins are heavily engaged by RA190. Finally, increasing or reducing the level of Rpn13 in HeLa and melanoma cells had no effect on the sensitivity of HeLa or melanoma cells to RA190. We conclude that Rpn13 is not the physiologically relevant target of RA190.
Insights
This study found that RA190 does not target Rpn13, a protein in the 26S proteasome, despite previous suggestions. Experiments showed no interaction between RA190 and Rpn13, indicating Rpn13 is not the drug's relevant target.
Area of Science:
- Biochemistry
- Molecular Biology
- Proteasome Biology
Background:
- Rpn13 is a ubiquitin receptor within the 26S proteasome.
- RA190, an electrophilic small molecule, exhibits anti-cancer properties.
- Cysteine 88 (Cys88) of Rpn13 was hypothesized as the primary target of RA190.
Purpose of the Study:
- To investigate whether RA190 exerts its cytotoxic effects by interacting with Rpn13.
- To validate or refute the proposed mechanism of RA190 action via Rpn13 engagement.
Main Methods:
- In vitro assays to assess RA190's effect on Rpn13 interactions.
- In cellulo studies to detect physical engagement of Rpn13 by RA190.
- Chemical proteomics to identify proteins targeted by RA190.
- Genetic manipulation of Rpn13 levels in cancer cell lines to assess drug sensitivity.
Main Results:
- RA190 showed no measurable effect on known Rpn13 interactions in vitro.
- No physical engagement of Rpn13 by RA190 was observed in cells.
- Chemical proteomics identified numerous other proteins as targets of RA190.
- Altering Rpn13 levels did not affect cellular sensitivity to RA190.
Conclusions:
- Rpn13 is not the physiologically relevant target of RA190.
- The anti-cancer activity of RA190 likely results from interactions with other cellular proteins.
