Paradoxical activation of Raf by a novel Raf inhibitor

Background: Raf is a proto-oncogene that is activated in response to growth factors or phorbol eaters, and is thought to activate MAP kinase kinase-1 (MKK1) and hence the classical MAP kinase (MAPK) cascade, Results: The compound ZM 336372 is identified as a potent and specific inhi bitor of Raf isoforms in vitro, Paradoxically, exposure of cells to ZM 3363 72 induces >100-fold activation of c-Raf (measured in the absence of compou nd), but without triggering any activation of MKK1 or p42 MAPK/ERK2, The ZM 336372-induced activation of c-Raf occurs without any increase in the GTP- loading of Pas and is not prevented by inhibition of the MAPK cascade, prot ein kinase C or phosphatidylinositide 3-kinase, ZM 336372 does not prevent growth factor or phorbol ester induced activation of MKK1 or P42 MAPK/ERK2, or reverse the phenotype of Ras- or Raf-transformed cell lines. The only o ther protein kinase inhibited by ZM 336372 out of 20 tested was SAPK2/p38, Although ZM 336372 is structurally unrelated to SB 203580; a potent inhibit or of SAPK2/p38, the mutation of Thr106-->Met made SAPK2/p38 insensitive to ZM 336372 as well as to SE 203580. Conclusions: Raf appears to suppress its own activation by a novel feedback loop, such that inhibition is always counterbalanced by reactivation. Thes e observations imply that some agonists reported to trigger the cellular ac tivation of c-Rai might actually be inhibitors of this enzyme, and that com pounds which inhibit the kinase activity of Raf might not be useful as anti cancer drugs. The binding sites for ZM 336372 and SE 203580 on Raf and SAPK 2/p38 are likely to overlap.