Cdc25B Dual-Specificity Phosphatase Inhibitors Identified in a High-Throughput Screen of the NIH Compound Library

Abstract

ABSTRACT

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

“…[1][2][3][4]6,7,26,27 For example, the active site cysteines of PTPs have a low pK a of 4.7-5.4 and exist as thiolate anions at neutral pH, which enhances their catalytic activity and also their susceptibility to inactivation by H 2 O 2 generated by RCCs in the presence of DTT. [1][2][3][4] A reaction scheme for the generation of H 2 O 2 by redox cycling between a RCC and DTT in the presence of oxygen has been proposed 1,3,5 ( Fig.…”

“…There are several hallmark characteristics that have been used to distinguish RCCs from hit compounds that modulate target protein activities directly. [1][2][3][4]7,27 Caspase 8 inhibition by quinone RCCs is time-dependent, abolished by catalase, and occurs in strong reducing agents like DTT or TCEP, but not in GSH or Cys. 7 Inhibition of PTPα by orthoquinone RCCs is also time-dependent, is sensitive to superoxide dismutase (SOD) and catalase, and depends on the concentrations been screened in a comparable number of bioassays (Fig.…”

“…2,26 Rather than expend critical resources and time to identify RCCs in a complex follow-up testing paradigm, 1,2,6,7 we have developed an assay that accurately measures the H 2 O 2 generating capability of RCCs in DTT containing buffers with respect to time and concentration dependence, catalase sensitivity, and the requirement for strong reducing agents. [1][2][3][4]7,27 Recently a surrogate assay to detect small molecule redox activity has been used to identify undesirable promiscuous nuisance compounds in the hits from historical HTS campaigns conducted at GlaxoSmithKline (GSK). 6 Pyrimidotriazinedione hits identifi ed in a Cathepsin L (CatL) cysteine protease inhibitor screen were subsequently found to be promiscuous hits in many GSK HTS campaigns, predominantly those against cysteine proteases, metalloenzymes, or other drug targets with an active site cysteine.…”

“…6 There are, however, inconsistencies between the performance of the resazurin surrogate assay 6 and the behavior of RCCs. [1][2][3][4]7,26,27 The pyrimidotriazinedione control compound (5 μM) generated O 2˙-in the presence of DTT (10 μM) that was readily detected in a cytochrome c assay, but was prevented by the inclusion of SOD. 6 However, the inclusion of SOD and/or catalase in the resazurin assay reaction did not prevent the generation of resorufi n by compounds in DTT, indicating that the reaction is not mediated by either the O 2˙-or H 2 O 2 ROS.…”

“…1,6,7,15,27 Pyrimidotriazinedione and ortho-quinone RCCs were among the promiscuous hits identifi ed in HTS campaigns against cysteine proteases, metalloenzymes, and other target proteins with active site cysteines. 6,7 Seven confi rmed pyrimidotriazinedione hits, identical to the RCCs presented here (Fig.…”