Novobiocin and related coumarins and depletion of heat shock protein 90-dependent signaling proteins

Background: Heat shock protein 90 (Hsp90) interacts with and stabilizes sev eral oncogenic protein kinases (e.g., p185(erbB2), p60(v-src), and Raf-1) a nd is required for the stability and dominant-negative function of mutated p53 protein. Two unrelated antibiotics, geldanamycin and radicicol, bind sp ecifically to an atypical nucleotide-binding pocket of Hsp90, a site that s hares homology with the adenosine triphosphate (ATP)-binding domain of bact erial DNA gyrase B, This interaction leads to destabilization of proteins t hat interact with Hsp90, Since the nucleotide-binding site of gyrase B is t argeted by coumarin antibiotics (e.g., novobiocin), we investigated whether these drugs can also interact with Hsp90 and affect its activity. Methods: We used immobilized novobiocin, geldanamycin, or radicicol to isolate eith er endogenous Hsp90 from cell lysates or Hsp90 deletion fragments translate d in vitro. Effects of the coumarin antibiotics novobiocin, chlorobiocin, a nd coumermycin A1 on several proteins interacting with Hsp90 were assessed ill vitro and in vivo. Results: Hsp90 binding to immobilized novobiocin was competed by soluble coumarins and ATP but not by geldanamycin or radicicol , A carboxy-terminal Hsp90 fragment bound immobilized novobiocin but not im mobilized geldanamycin, while a geldanamycin-binding amino-terminal fragmen t did not bind novobiocin. All three coumarins markedly reduced cellular le vels of p185(erbB2), p60(v-src), Raf-l, and mutated p53, Furthermore, novob iocin reduced Raf-l levels in the spleens of mice treated with the drug. Co nclusions: These coumarin antibiotics, particularly novobiocin, represent a first-generation alternative to other Hsp90-targeting drugs that are not a s well tolerated. Novobiocin's unique interaction with Hsp90 identifies an additional site on this protein amenable to pharmacologic interference with small molecules.