Selective inhibition of MAPKK Wis1 in the stress-activated MAPK cascade of Schizosaccharomyces pombe by novel berberine derivatives

Intracellular molecular targets of novel berberine derivatives, HWY 289 and HWY 336, were identified by a screen of a variety of mutants in fission yeast Schizosaccharomyces pombe. HWY 289 and HWY 336 completely inhibited the proliferation of wild type as well as various mutant fission yeast cells (minimal inhibitory concentrations were 29.52 muM for HWY 289 and 11.83 muM for HWY 336), but did not affect the proliferation of Wis1 mitogen-activated protein kinase kinase (MAPKK) deletion mutants. In addition, HWY 289 with an IC50 value of 7.3 muM or HWY 336 with IC50 of 5.7 muM specifically inhibited in vitro kinase activities of purified Wis1, whereas either compound did not affect the activities of other kinases in the mitogen-activated protein kinase (MAPK) cascades of fission yeast. These genetic and biochemical results demonstrate the high degree of specificity of HWY 289 and HWY 336 to MAPKK Wis1 and suggest that the cytotoxicity of these compounds is not simply due to the inhibition of Wis1 kinase activity. High salt wash experiments have shown that strong noncovalent binding occurs between Wis1 and either HWY 289 or HWY 336. The preincubation of Wis1 kinase with ATP did not affect the inhibition of Wis1 by HWY 289 and HWY 336, but when Wis1 was preincubated with MBP, a protein substrate, Wis1 kinase activity was no longer inhibited. These observations demonstrate that HWY 289/HWY 336 do inhibit Wis1 kinase, not by binding to the ATP-binding site but by disturbing the binding of substrate to the kinase. Target validation of the complex of HWY 289/HWY 336 and Wis1 kinase will provide important clues for the mechanism of specific cytotoxicity of these compounds in S. pombe. On a broader aspect, it would create an initiative to further modify and develop compounds that selectively inhibit kinases and cause cytotoxicity in various MAPK cascades including those of mammals.