Enabling Protein Degradation Drug Discovery

REDOXprofiler

Identify compounds with an undesired redox cycling 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, compounds that generate ROS, termed redox cycling compounds (RCCs), can cause inhibition of enzymes that utilise a catalytic cysteine.  These compounds will be identified as hits but are false positives.

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.  Within the DUB superfamily, 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 and eliminate false positives as early as possible in your drug discovery programme:

  • 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 – compound IC50 values against a particular DUB are reported 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, we would anticipate a reduction or elimination of inhibitory activity against the enzyme due to a reduction or elimination in ROS.  In contrast, the IC50 values of compounds with no RCC activity are unaffected by the absence or presence of Cat/SOD. Mixed mechanisms of action may also be detected.

Key features of Ubiquigent’s REDOXprofiler™ service:

  • False positive or genuine hit? REDOXprofiler™ is a critical triage assay –It’s important to understand whether the activity of your compound at a target enzyme may be due (at least in part) to RCC properties.
  • Cross-confirmation –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 screen any number of DUBs in 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 will 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 will 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 independently generate as many quotations as you like based on different combinations of service options.
  • Once you have finalised your study design, email us the completed submission form and send us your compounds.
  • Once we have received your compounds, we will immediately schedule them for profiling and keep you updated on project progress.
  • Once complete we will send you a fully annotated data report and address any questions by email or arrange a post project meeting.

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 services@ubiquigent.com or contact us here