We have extended the search for selective inhibitors of the kinases of MAPK cascades by screening a derivative library of one of the isoquinoline rings of the protoberberine backbone. HWY 5069 inhibited the proliferation of wild-type and all mutants of Schizosaccharomyces pombe examined, except spc1Delta, at a minimal inhibitory concentration (MIC) of 3.76 microM. HWY 5069 also completely inhibited Spc1 kinase activity in vitro with an IC(50) of 16.4 microM as a competitive inhibitor of substrate binding. It was highly selective for Spc1 and did not affect the activity of other kinases in the MAPK cascades of fission yeast and mammals, including functional homologs of Spc1.