A Simple Assay for Detection of Small-Molecule Redox Activity

Abstract

In addition to selecting molecules of pharmacological interest, high-throughput screening campaigns often generate hits of undesirable mechanism, which cannot be exploited for drug discovery as they lead to obvious problems of specificity and developability. Examples of undesirable mechanisms are target alkylation/acylation and compound aggregation. Both types of "promiscuous" mechanisms have been described in the literature, as have methods for their detection. In addition to these mechanisms, compounds can also inhibit by oxidizing susceptible enzyme targets, such as metalloenzymes and cysteine-using enzymes. However, this redox phenomenon has been documented infrequently, and an easy method for detecting this behavior is missing. In this article, the authors describe direct proof of small-molecule oxidation of a cysteine protease by liquid chromatography/tandem mass spectrometry, develop a simple assay to predict this oxidizing behavior by compounds, and show the utility of this assay by demonstrating its ability to distinguish nuisance redox compounds from well-behaved inhibitors in 3 historical GlaxoSmithKline drug discovery efforts. (Journal of Biomolecular Screening 2007:881-890)

Profiling the NIH Small Molecule Repository for Compounds That Generate H2O2by Redox Cycling in Reducing Environments

“…[1][2][3][4]6,7 Typically, the follow-up process to identify and eliminate RCCs from HTS hit lists requires the development and implementation of several secondary assays that signifi cantly lengthen the timelines for hit characterization and lead selection while consuming critical resources: (i) conducting a detailed enzyme kinetic analysis to verify the time-and concentration-dependent inhibition of target activity by redox active compounds in the presence of DTT, (ii) testing whether catalase abolishes the target inhibition by compounds in DTT, (iii) examining compound inhibition in the presence of weaker reducing agents such as GSH or Cys, (iv) measuring the UV/Vis spectra of the compound in the presence and absence of the reducing agent to determine if it is reduced in a time-dependent manner, and (v) liquid chromatography and mass spectrometry analysis to confi rm the oxidation of active site cysteines. [1][2][3]6,7 Recently, we have reported the development and optimization of a rapid, economical, 384-well colorimetric HTS compatible assay to measure H 2 O 2 generated by the redox cycling of compounds incubated with reducing agents. 3 The assay readily detects H 2 O 2 in the 1-100 μM range and is based on the H 2 O 2 -dependent horseradish peroxidase (HRP)-mediated oxidation of phenol red (PR) that produces a change in its absorbance at 610 nm in alkaline pH.…”

A Homogeneous HTRF Assay for the Identification of Inhibitors of the TWEAK-Fn14 Protein Interaction

“…Therefore, an additional counterassay was applied that measured the oxidation potential of compounds in the presence of resazurin and DTT. 34 Four hit compounds were not active in the redox assay (data not shown), but we found that the original batch of compound B displayed a clear concentration-dependent activity, whereas the resynthesized batch was inactive (Fig. 5B).…”

“…34 To increase sensitivity, a concentration of 1 mM dithiothreitol (DTT) was applied in the assay instead of 50 µM. A solution of DTT and 5 µM resazurin in 50 mM NaCl, 50 mM HEPES (pH 7.5) was mixed with increasing concentrations of the compound, incubated for 60 min at RT, and analyzed on a Victor 2V plate reader.…”

Discovery of Potent and Selective Inhibitors of Trypanosoma brucei Ornithine Decarboxylase

“…R7017, Sigma) to resorufin as described previously (44). Briefly, 25 l of assay solution (5 M resazurin, 50 mM HEPES, 50 mM NaCl, pH 7.5, 50 M DTT, prepared immediately prior to usage) was added to a black 384-well polystyrene plate (catalog no.…”

“…Inhibition of TbODC was determined to be ratelimiting in all cases. The compounds were also screened for redox activity to eliminate nonspecific radical based inhibitors (44). A representative compound from each scaffold series was also tested using the radiolabeled ornithine assay to confirm inhibition of TbODC in an orthogonal assay system.…”