Linking Ubiquitin Research to Drug Discovery


Identify compounds with a REDOX mechanism of action

Certain compounds may generate Reactive Oxygen Species (ROS) such as hydrogen peroxide (H2O2) or superoxide (O2), particularly in the presence of reducing agents including dithiothreitol (DTT). In screening campaigns such ROS generating or redox cycling compounds (RCCs) can cause false positive inhibition of enzymes that utilise a catalytic cysteine. This occurs through the oxidation of that cysteine to a reversible sulphenic acid (SOH) or an irreversible sulphinic (SO2H) or sulphonic (SO3H) acid.

The identification of RCCs is important when screening for inhibitors of enzymes including deubiquitylases (DUBs), cysteine proteases such as cathepsins and caspases as well as protein tyrosine phosphatases. In the case of the DUBs the ubiquitin-specific proteases (USPs), ubiquitin C-terminal hydrolases (UCHs), Machado-Josephin domain proteases (MJDs or Josephins), ovarian tumour proteases (OTUs) and the motif interacting with Ub-containing novel DUB family (MINDY) subfamilies may all be susceptible to RCCs.

Ubiquigent has developed two complementary assays that may be used to identify RCCs:

  • REDOXprofiler™-Resazurin: A surrogate assay for measuring the ability of compounds to oxidise susceptible targets. In the presence of an RCC the resazurin substrate is converted to resorufin thus dequenching its fluorescence.
  • REDOXprofiler™-Cat/SOD: In this – a modified DUBprofiler assay – a compound’s IC50 at a DUB is reported both in the presence and absence of catalase and superoxide dismutase (SOD). Catalase catalyzes the disproportionation of H2O2 to O2 and H2O while SOD disproportionates O2 to O2 and H2O2. Thus if the compound has an RCC mechanism of action then in the presence of catalase and SOD an elimination or a reduction in the target inhibitory activity of the compound may be seen if there is an elimination or a sufficient fall in ROS respectively; whereas if the compound does not have RCC activity then the IC50 will remain the same in the presence or absence of catalase and SOD. Mixed mechanisms of action may also be detected.

Key features of Ubiquigent’s REDOXprofiler™ service:

  • A critical triage assay – It’s important to understand whether your compound’s activity at a target enzyme may be due (at least in part) to RCC properties.
  • Cross confirmatory methods – Two complementary RCC detection assays REDOXprofiler™-Resazurin and REDOXprofiler™-Cat/SOD.
  • Context specific – Ability to determine RCC effects in the context of specific DUBs.
  • Enzyme target agnostic – The REDOXprofiler™-Resazurin assay is applicable to detecting RCCs independent of target class (DUBs, cathepsins, caspases and other cysteine proteases). REDOXprofiler™-Cat/SOD is designed for DUB analysis but may be adapted for addressing non-DUB targets (please enquire here).
  • Full curve analysis – REDOXprofiler™-Resazurin: One EC50 curve reported, REDOXprofiler™-Cat/SOD: Two DUB IC50 curves reported (plus and minus Cat/SOD).
  • Free control data – The standard RCC beta-lapachone is analysed in each REDOXprofiler™-Resazurin and REDOXprofiler™-Cat/SOD project and the data provided free of charge.
  • Flexible – screen any combination of compound(s) in either REDOX assay and vs. any DUBs (in the context of REDOXprofiler™-Cat/SOD)
  • Scalable – Any scale and complexity of screening available.
  • Efficient – Simple service procedure using one electronic submission form (similar to that used in our DUBprofiler service and supplied on request) to order either or both assays.
  • Fast – Rapid screening cycles.

The process

  • Contact us and we’ll send you a single electronic submission form that lets you order the REDOXprofiler™-Resazurin and/or REDOXprofiler™-Cat/SOD profiling service(s) that you require to characterise your compounds. We’ll also send you a set of barcoded tubes for the submission of your compounds, a guide to take you through the ordering process and a Master Services Agreement if you are not already one of our service clients.
  • Our submission forms allow you to generate as many quotations as you like on your desktop based on different combinations of service options.
  • Once you’ve finalised your study design, email us the completed submission form and send us your compounds.
  • Once we’ve received your compounds we’ll immediately schedule them for profiling and keep you updated on project progress.
  • Once complete we’ll send you a fully annotated data report and arrange a post project meeting to answer any questions that you might have.

Learn more

Please download our REDOXprofiler™ brochure which provides more detail and example data.

To learn more about our REDOXprofiler™ screening services and to discuss your specific requirements, email us at or contact us here