Redox cycling compounds generate H2O2 in HTS buffers containing strong reducing reagents--real hits or promiscuous artifacts?

Abstract

Redox cycling compounds (RCCs) generate μM concentrations of hydrogen peroxide (H(2)O(2)) in the presence of strong reducing agents, common buffer components used to maintain the catalytic activity and/or folding of target proteins for high throughput screening (HTS) assays. H(2)O(2) generated by RCCs can indirectly inhibit the catalytic activity of proteins by oxidizing accessible cysteine, tryptophan, methionine, histidine, or selenocysteine residues, and indeed several important classes of protein targets are susceptible to H(2)O(2)-mediated inactivation; protein tyrosine phosphatases, cysteine proteases, and metalloenzymes. The main sources of H(2)O(2) in cells are the Nox enzyme/SOD systems, peroxisome metabolism, and the autoxidation of reactive chemicals by enzyme mediated redox cycling at both the microsomal and mitochondrial sites of electron transport. Given the role of H(2)O(2) as a second messenger involved in the regulation of many signaling pathways it is hardly surprising that compounds that can generate intracellular H(2)O(2) by enzyme mediated redox cycling would have pleiotropic effects. RCCs can therefore have serious negative consequences for the probe and/or lead generation process: primary HTS assay hit rates may be inflated by RCC false positives; crucial resources will be diverted to develop and implement follow up assays to distinguish RCCs from real hits; and screening databases will become annotated with the promiscuous activity of RCCs. In an attempt to mitigate the serious impact of RCCs on probe and lead generation, two groups have independently developed assays to indentify RCCs.

Figure 1. Redox Cycling Compounds Generate H₂O₂ in Reducing Environments and Exhibit Promiscuous Bioactivity Profiles in HTS Databases

A) Aqueous solutions containing dithiothreitol (DTT) and oxygen at neutral to acidic pH generate H₂O₂ via a chain reaction, and if compounds capable of redox cycling are added to this environment, micromolar (µM) concentrations of H₂O₂ can be produced. H₂O₂ generated by placing RCCs like 1-ethyl-6-methyl-3-phenyl-1H-pyrimido[5,4-e][1,2,4]triazine-5,7-dione, PubChem substance identifier (SID) 845167, in HTS assay buffers containing DTT can indirectly inhibit the catalytic activity of proteins by oxidizing accessible cysteine, tryptophan, methionine, histidine or selenocysteine residues. H₂O₂ generated by RCCs may also inhibit assays by having adverse effects on cofactor binding, or by disrupting disulfide bonds that contribute to the dimerization and/or folding of proteins. B) The PubChem database (http://pubchem.ncbi.nlm.nih.gov/) was queried using the PubChem SID 845167 for the RCC 1-ethyl-6-methyl-3-phenyl-1H-pyrimido [5,4-e][1,2,4]triazine-5,7-dione. The bioassay activity information is captured in two PubChem data fields, the activity outcome and the activity score. A substance may be flagged active in a bioassay data uploaded to PubChem if it meets the active criterion for the assay that has been designated appropriate by the depositor. A substance may be flagged a “confirmed” active in bioassay data uploaded to PubChem if it exhibits a concentration response that has been designated appropriate by the depositor. RCCs represent nuisance/pan assay interference compounds with the potential to annotate screening databases with promiscuous bioactivity profiles. The RCC SID 845167 has been tested in 541 bioassays and was designated active in 173 (32%) of them. This RCC was confirmed active in 77 concentration response assays including a number of protein targets that are susceptible to H₂O₂-mediated inactivation; protein tyrosine phosphatases (PTPs), and cysteine proteases (cathepsins and caspases).

Figure 2. Redox Cycling Reaction Scheme for a Quinone RCC in DTT

A reaction scheme for the generation of H₂O₂ by redox cycling between a quinone RCC and DTT in the presence of oxygen is presented. (1) DTT reacts with the quinone RCC to form dihydroxydithiane (ox-DTT) and a hydroquinone. (2) When the hydroquinone and quinone RCC are present together they undergo a synproportionation to form a transient semiquinone radical anion species (RCC^*-) and 2H⁺. (3) The semiquinone radical anion reacts with O₂ to form superoxide anion (O₂^*-) and regenerate the quinone RCC. (4) In neutral to acidic pH DTT may also react with O₂ in aqueous buffers to generate O2^.- and ox-DTT. (5) O2^.- produced in reactions 3 and 4 can oxidize a hydroquinone resulting in the production of H₂O₂ and the regeneration of the RCC^.- semiquinone radical anion. In aqueous buffers containing a strong reducing agent and molecular oxygen, quinone RCCs cycle between the quinone, hydroquinone and semiquinone radical anion species to generate the reactive oxygen species O2^.- and H₂O₂.

Figure 3. HTS Assays to Identify Compounds that Redox Cycle in Reducing Environments

A) Surrogate assay using the conversion of resazurin to resorufin to detect small molecule redox activity [9]. 100 nL of compounds are added to the wells of black low volume plates to which is added 10 µL of a mixture containing 5 µM resazurin and 50 µM DTT in 50 mM HEPES, 50 mM NaCl at pH 7.5, and after a 60 minute incubation at ambient temperature the fluorescent intensity of resorufin (Ex 560 nm. Em 590 nm) is captured. B) Colorimetric assay to measure H₂O₂ generated by RCCs incubated with strong reducing agents based on the H₂O₂-dependent horseradish peroxidase (HRP) mediated oxidation of phenol red (PR) that produces a change in its absorbance at 610 nm in alkaline pH [8,10]. The assay is performed in 384-well flat bottomed clear polystyrene microtiter plates and involves three liquid transfer steps of 20 µL each of compounds/controls, DTT, and the HRP-PR detection reagent to give a final assay volume of 60 µL. Compounds (0.01 to 50 µM, final), plate controls (100 µM H₂O₂ or 1 % DMSO), DTT (0.5 to 1.0 mM, final), and the HRP-PR detection reagent (100 µg/mL phenol red and 60 µg/ml HRP, final) are all prepared in Hank’s balanced salt solution (HBSS). Compounds and DTT are incubated together at ambient temperature for a minimum of 15 min prior to the addition of the HRP-PR detection reagent and after an additional incubation period at ambient temperature, minimally 5 min, the assay is terminated by the addition of 10 µL of 1N NaOH to all wells and the absorbance of the phenol red is measured at 610 nm on a plate reader.

Figure 4. Representative Structural Classes of Redox Cycling Compounds

Representative RCCs reported in the literature [–,,,–18] were subjected to structure based clustering and classification techniques based on recursive partitioning using the Leadscope Enterprise 2.4.6-1 software as described previously [8,10,15,16].