Redox Regulation of Cdc25B by Cell-Active Quinolinediones

Abstract

Intracellular reduction and oxidation pathways regulate protein functionality through both reversible and irreversible mechanisms. The Cdc25 phosphatases, which control cell cycle progression, are potential subjects of oxidative regulation. Many of the more potent Cdc25 phosphatase inhibitors reported to date are quinones, which are capable of redox cycling. Therefore, we used the previously characterized quinolinedione Cdc25 inhibitor DA3003-1 [NSC 663284 or 6-chloro-7-(2-morpholin-4-yl-ethylamino)-quinoline-5,8-dione] and a newly synthesized congener JUN1111 [7-(2-morpholin-4-yl-ethylamino)-quinoline-5,8-dione] to test the hypothesis that quinone inhibitors of Cdc25 regulate phosphatase activity through redox mechanisms. Like DA3003-1, JUN1111 selectively inhibited Cdc25 phosphatases in vitro in an irreversible, time-dependent manner and arrested cells in the G1 and G2/M phases of the cell cycle. It is noteworthy that both DA3003-1 and JUN1111 directly inhibited Cdc25B activity in cells. Depletion of glutathione increased cellular sensitivity to DA3003-1 and JUN1111, and in vitro Cdc25B inhibition by these compounds was sensitive to pH, catalase, and reductants (dithiothreitol and glutathione), consistent with oxidative inactivation. In addition, both DA3003-1 and JUN1111 rapidly generated intracellular reactive oxygen species. Analysis of Cdc25B by mass spectrometry revealed sulfonic acid formation on the catalytic cysteine of Cdc25B after in vitro treatment with DA3003-1. These results indicate that irreversible oxidation of the catalytic cysteine of Cdc25B is indeed a mechanism by which these quinolinediones inactivate this protein phosphatase.

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.…”

“…[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. 7): (1) DTT reacts with the RCC to form dihydroxydithiane (ox-DTT) and a hydro-RCC; (2) when the hydro-RCC and RCC are present together they undergo a synproportionation to form a transient radical anion species (RCC˙-) and 2H + ; (3) the RCC˙-radical anion reacts with O 2 to form superoxide (O 2˙-) and regenerates the RCC; (4) DTT may also react with O 2 in the buffer to generate O 2˙-and ox-DTT; (5) O 2˙-can oxidize a hydro-RCC resulting in the production of a RCC˙-radical anion and H 2 O 2 .…”

“…[1][2][3][4]6,7 Several important classes of protein targets are susceptible to H 2 O 2 -mediated inactivation, including protein tyrosine phosphatases (PTPs), cysteine proteases (cathepsins and caspases), and metalloenzymes. [1][2][3][4][6][7][8][9][10][11][12][13][14] Redox cycling compounds (RCCs) generate H 2 O 2 in the presence of strong reducing agents such as DTT and tris(2-carboxyethyl)phosphine (TCEP), but not in the presence of weaker reducing agents like β-mercaptoethanol (BME), glutathione (GSH), or cysteine (Cys). 1,3,7 However, DTT and TCEP are commonly utilized in high-throughput screening (HTS) buffers to preserve the reduced state of critical amino acids and to maintain the catalytic activity or the folding of target proteins.…”

“…1-3-,6,7,15,16 Compounds capable of redox cycling in buffers containing DTT or TCEP generate reactive oxygen species (ROS) including H 2 O 2 that may indirectly inhibit the target activity of proteins that are susceptible to oxidation. [1][2][3][4]6,7 A small molecule library of between 200,000 and 300,000 compounds has been assembled under the auspices of the National Institutes of Health (NIH) Roadmap Initiative, for distribution to the academic screening laboratories involved in the pilot and probe production phases of the Molecular Library Screening Centers Network (MLSCN and MLPCN). 15,[17][18][19][20][21] The NIH Small Molecule Repository (SMR) library contains chemically diverse substances that are being acquired from multiple sources: (1) a specialty set of FDA approved drugs, known bioactives, toxins, and metabolites; (2) natural products and derivatives; (3) targeted libraries of modulators for specifi c target protein classes including proteases, kinases, G-protein coupled receptors, ion channels, and nuclear hormone receptor families; and (4) compounds with chemically diverse scaffolds that include assorted small molecule libraries from commercial and academic sources.…”

“…[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.…”