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Deazaflavin Inhibitors of TDP2 with Cellular Activity Can Affect Etoposide Influx and/or Efflux
Emilia Komulainen1, Lewis Pennicott2, Darren Le Grand2
1Genome Damage and Stability Centre, School of Life Sciences , University of Sussex , Falmer, Brighton , BN1 9RQ , United Kingdom.
Abstract:
Tyrosyl DNA phosphodiesterase 2 (TDP2) facilitates the repair of topoisomerase II (TOP2)-linked DNA double-strand breaks and, as a consequence, is required for cellular resistance to TOP2 "poisons". Recently, a deazaflavin series of compounds were identified as potent inhibitors of TDP2, in vitro. Here, however, we show that while some deazaflavins can induce cellular sensitivity to the TOP2 poison etoposide, they do so independently of TDP2 status. Consistent with this, both the cellular level of etoposide-induced TOP2 cleavage complexes and the intracellular concentration of etoposide was increased by incubation with deazaflavin, suggesting an impact of these compounds on etoposide uptake/efflux. In addition, deazaflavin failed to increase the level of TOP2 cleavage complexes or sensitivity induced by m-AMSA, which is a different class of TOP2 poison to which TDP2-defective cells are also sensitive. In conclusion, while deazaflavins are potent inhibitors of TDP2 in vitro, their limited cell permeability and likely interference with etoposide influx/efflux limits their utility in cells.
Insights
Deazaflavin compounds inhibit Tyrosyl DNA phosphodiesterase 2 (TDP2) in vitro. However, they increase sensitivity to etoposide independently of TDP2, suggesting effects on drug uptake rather than direct TDP2 inhibition in cells.
Area of Science:
- Biochemistry
- Molecular Biology
- Genetics
Background:
- Tyrosyl DNA phosphodiesterase 2 (TDP2) repairs topoisomerase II (TOP2)-linked DNA breaks.
- TDP2 is crucial for cellular resistance to TOP2 poisons like etoposide.
- Deazaflavins were recently identified as potent in vitro inhibitors of TDP2.
Purpose of the Study:
- To investigate the cellular effects of deazaflavins in relation to TDP2 inhibition.
- To determine if deazaflavins enhance sensitivity to TOP2 poisons through TDP2.
- To assess the utility of deazaflavins as therapeutic agents targeting TDP2.
Main Methods:
- Cellular sensitivity assays with etoposide and m-AMSA.
- Measurement of TOP2-DNA cleavage complexes.
- Analysis of intracellular etoposide concentration.
- Assessment of deazaflavin effects independent of TDP2 status.
Main Results:
- Deazaflavins increased cellular sensitivity to etoposide, but not dependent on TDP2.
- Deazaflavins altered etoposide intracellular concentration, indicating effects on uptake/efflux.
- Deazaflavins did not enhance sensitivity to m-AMSA in TDP2-defective cells.
- In vitro TDP2 inhibition did not translate to cellular efficacy.
Conclusions:
- Deazaflavins' potent in vitro TDP2 inhibition is limited by poor cell permeability.
- Interference with etoposide transport affects cellular response to deazaflavins.
- Deazaflavins have limited utility in cells for targeting TDP2 due to these factors.
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