""""""""HSP90 and GRP94 are homologous cellular chaperones found in cytosol and endoplasmic reticulum, respectively. Several years ago, we discovered that members of the benzoquinone and ansamycin class of antibiotic, including herbimycin A and geldanamycin (GA) bound to HSP90 and GRP94 and disrupted certain multi-molecular complexes of which these proteins were a part. We have utilized pharmacologic disruption of HSP90 and GRP94 activity to study the function of these chaperones in cellular signal transduction. Multiple signal transduction proteins interact with these charperones, including the kinasesz src, erbB2 and c-raf-1, and mutated (but not wild type) p53. A general consequence of pharmacologic disruption of the chaperone/signal protein complex is the resultant marked instability and incorrect subcellular localization of the signalling protein. The instability is due to stimulation of targeted degradation of the signalling protein by the 26S proteasome proteolytic complex following chaperone dissociation. With respect to c-raf-1, no other members of the MAP kinase signalling pathway appear to be affected by GA. With respect to erbB2, it appears to be the most sensitive member of the EGF receptor family of tyrosine kinases to this drug. In the case of mutated p53, attainment of the mutated (but not wild type) conformation of the protein requires transient interaction with HSP90 and an accessory protein termed p23. GA treatment destroys the conformation and the function of mutated p53, providing evidence that pharmacologic manipulation of protein comformation is feasible. In collaboration with Pfizer Central Research, we have tested more than 35 benzoquinone ansamycin derivatives for their ability to bind HSP90/GRP94 and to affect the stability and function of mutant p53, c-raf-1 and erbB2. Benzoquinone ansamycins are currently the only agents capable of specifically interfering in HSP90/GRP94 function and these drugs will be useful agents in further studying the role of these chaperones proteins in regulating the stability and function of oncogenes and other signalling proteins. Most recently, we have identified the ansamycin binding site on hsp90 and localized this site to the amino terminal portion of the molecule. In addition, we have shown that this site is identical to the recently characterized ATP binding site on hsp90. We further demonstrated that ansamycins block ATP from binding to hsp90, thereby altering the chaperone's conformation and restricting its association with other co-chaperones. It is throught this mechanism that the ansamycins appear to exert their anti-oncogene effects in cells. Further, we have shown that 17-AAG, a derivative of geldanamcyin with less in vivo toxicity, has similar biochemical propeties as geldanamycin on hsp90 function. In the last year, we have identified radicicol as a novel natural product which also binds to hsp90 and interferes with its function in a manner very similar to the ansamycins. This is the case even though radicicol is structurally dissimilar from the ansamycins. Radicicol thus represents a second type of natural structure capable of interfering with hsp90 function by binding to and blocking the ATP binding site on the chaperone. Radicicol also binds to the amino terminal nucleotide binding pocket conserved in all hsp90 family proteins from bacteria to man. Using immobilized radicicol, we have been able to determine approximate affinities of all hsp90 family members for the drug. Most recently, we have identified a third class of natural product, novobiocin, which also binds to hsp90, although at a lower affinity than either benzoquinone ansamycins or radicicol. Nonetheless, novobiocin appears to cause the same biologic effects as ansamycins and radicicol by preventing ATP from binding to hsp90. Ongoing studies are further characterizing this interaction. Preliminary animal testing has revealed no toxicity after twice daily administration of novobiocin for more than one month. This regimen demonstrates significant anti-tumor activity in a transgenic murine model of erbB2-driven breast cancer.""""""""
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