Novobiocin and related coumarins and depletion of heat shock protein 90-dependent signaling proteins.
BACKGROUND
Heat shock protein 90 (Hsp90) interacts with and stabilizes several oncogenic protein kinases (e.g., p185(erbB2), p60(v-src), and Raf-1) and is required for the stability and dominant-negative function of mutated p53 protein. Two unrelated antibiotics, geldanamycin and radicicol, bind specifically to an atypical nucleotide-binding pocket of Hsp90, a site that shares homology with the adenosine triphosphate (ATP)-binding domain of bacterial DNA gyrase B. This interaction leads to destabilization of proteins that interact with Hsp90. Since the nucleotide-binding site of gyrase B is targeted 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 either endogenous Hsp90 from cell lysates or Hsp90 deletion fragments translated in vitro. Effects of the coumarin antibiotics novobiocin, chlorobiocin, and coumermycin A1 on several proteins interacting with Hsp90 were assessed in 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 immobilized geldanamycin, while a geldanamycin-binding amino-terminal fragment did not bind novobiocin. All three coumarins markedly reduced cellular levels of p185(erbB2), p60(v-src), Raf-1, and mutated p53. Furthermore, novobiocin reduced Raf-1 levels in the spleens of mice treated with the drug.


CONCLUSIONS
These coumarin antibiotics, particularly novobiocin, represent a first-generation alternative to other Hsp90-targeting drugs that are not as well tolerated. Novobiocin's unique interaction with Hsp90 identifies an additional site on this protein amenable to pharmacologic interference with small molecules.
Marcu
M G
MG
Department of Cell and Cancer Biology, Medicine Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20850, USA.
Schulte
T W
TW
Neckers
L
L
D049933:Aminocoumarins; D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000737:chemistry / Q000494:pharmacology*; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000188:drug therapy; D002277:Carcinoma / Q000188:drug therapy; D002352:Carrier Proteins / Q000187:drug effects / Q000378:metabolism; D003374:Coumarins / Q000494:pharmacology*; D004250:DNA Topoisomerases, Type II / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D005347:Fibroblasts; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D009675:Novobiocin / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology*; D015688:Oncogene Protein pp60(v-src) / Q000187:drug effects / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D019908:Proto-Oncogene Proteins c-raf / Q000187:drug effects / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013154:Spleen / Q000166:cytology / Q000378:metabolism; D059005:Topoisomerase II Inhibitors; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000187:drug effects / Q000378:metabolism; D025521:Tumor Suppressor Proteins
D049933:Aminocoumarins; D000903:Antibiotics, Antineoplastic; D002352:Carrier Proteins; D003374:Coumarins; D004791:Enzyme Inhibitors; D018841:HSP90 Heat-Shock Proteins; D047908:Intracellular Signaling Peptides and Proteins; C098669:TOB1 protein, human; C490903:Tob1 protein, mouse; D059005:Topoisomerase II Inhibitors; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; D009675:Novobiocin; C006260:clorobiocin; D015688:Oncogene Protein pp60(v-src); D019908:Proto-Oncogene Proteins c-raf; D004250:DNA Topoisomerases, Type II; C004628:coumermycin