The oxidative mechanism of action of ortho-quinone inhibitors of protein-tyrosine phosphatase α is mediated by hydrogen peroxide

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

“…[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][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 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 sesquiterpene quinone, dysidine, from the sponge Dysidea villosa, activates the insulin pathway through inhibition of PTPases

“…Covalent addition [41,42] and irreversible oxidation [43] of cysteine in the active site are the main mechanisms. Since p-benzoquinone was contained in the structure of the three sesquiterpene quinones, the inhibition of dysidine against PTP1B was measured in the presence of either DTT (5 mmol/L) or catalase (1000 U/mL) to investigate whether these quinone-dependent mechanisms were involved.…”

“…For example, a number of ortho-quinone-containing compounds inhibited PTPα in a catalase-sensitive manner [43] , suggesting that these compounds inhibited PTPα via generation of ROS (including H 2 O 2 ) and subsequent oxidation of cysteine in the active site. Moreover, menadione (vitamin K3) [41] and other vitamin K analogues [42] containing a benzoquinone in their structure were reported to inhibit the CDC25 phosphatase by covalent interaction between the compound and the enzyme.…”