Ansamycin

Abstract: Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric transcription factor composed of alpha and beta subunits. HIF-1 is critically involved in cellular responses to hypoxia, glycolysis, and angiogenesis. Here, we show that treatment of prostate cancer PC-3 and LNCaP cells with the benzoquinone ansamycin geldanamycin, an Hsp90-specific inhibitor, induced degradation of HIF-1alpha protein in a dose- and time-dependent manner under both normoxia and hypoxia. This inhibition was also shown in other common cancer types tested. Rapid degradation of nuclear HIF-1alpha protein levels was accompanied by respective inhibition in HIF-1alpha functional transcription activity of VEGF. No difference between HIF-1alpha mRNA levels before or after geldanamycin treatment was found. Moreover, [35S]methionine pulse-chase analysis revealed that HIF-1alpha protein half-life was markedly decreased in the presence of geldanamycin compared with that in control. The geldanamycin-induced degradation of HIF-1alpha was reversed by proteosome inhibitors lactacystin and MG-132. We conclude that geldanamycin induces reduction of HIF-1alpha levels and its downstream transcriptional activity by accelerating protein degradation independent of O2 tension. Thus, benzoquinone ansamycin drugs and their derivatives, such as 17-allyl-aminogeldanamycin, are excellent candidates as small molecule drug inhibitors of HIF-1 overexpression in cancer cells.

Abstract: Three benzenoid ansamycin antibiotics (herbimycin, macbecin, and geldanamycin) were found to reduce the intracellular phosphorylation of p60src at a permissive temperature (33 degrees C) in a rat kidney cell line infected with a temperature-sensitive mutant of Rous sarcoma virus. This effect was accompanied by morphological changes from the transformed to the normal phenotype. The filamentous staining pattern of actin fibers was observed in the cells treated with these antibiotics at 33 degrees C. Removal of the antibiotics allowed the cells to revert to the transformed morphology. Ansamitocin, another benzenoid ansamycin, and naphthalenoid ansamycins such as streptovaricin and rifamycins did not show this effect. Pulse-labeling of the antibiotic-treated cultures with 32Pi showed a marked reduction of 32P radioactivity incorporated into p60src. A parallel experiment with [35S]methionine showed that synthesis of p60src was slightly inhibited. The immune complex prepared by mixing the herbimycin-treated cell extracts with antibody against p60src was inactive in vitro in phosphorylating the complex itself. On the contrary, the immune complex derived from untreated cells was active in vitro even in the presence of the antibiotics. These results suggest that benzoquinonoid ansamycins have no direct effect on src kinase but destroy its intracellular environment, resulting in an irreversible alteration of p60src and loss of catalytic activity.

Abstract: Ansamycins are a very specific class of macrocyclic antibiotics of which the rifamycins are among the better known members. Rifamycins bind to and inhibit DNA polymerase. Rifamycin B (the most easily obtained ansamycin) is negatively charged and is shown to associate with and enantioselectively resolve several chiral amino alcohols including terbutaline, isoproterenol, bamethan, metaproterenol, synephrine, metanephrine, salbutamol, epinephrine, norphenylephrine, ephedrine, psi-ephedrine, octopamine, norepinephrine, normetanephrine, metoprolol, alprenolol, atenolol, and oxprenolol. A description of the structure and properties of rifamycins, in general, and rifamycin B, in particular, is given. The complexation and chiral recognition of the aforementioned racemic compounds by rifamycin B is afforded by multiple interactions of which charge-charge, hydrogen-bonding, and hydrophobic inclusion interactions most likely dominate in hydroorganic solvents. The effect of various experimental factors on enantiomeric resolution is discussed in terms of optimizing the CE separations. Since most chiral antibiotic macrocycles are ionizable, somewhat flexible, and contain hydrophobic and hydrophilic moieties, they tend to be significantly affected by variations in the solution environment.

Abstract: The ansamycin antibiotic, geldanamycin, targets the hsp 90 protein chaperone and promotes ubiquitin-dependent proteasomal degradation of its numerous client proteins. Bortezomib is a specific and potent proteasome inhibitor. Both bortezomib and the geldanamycin analogue, 17-N-allylamino-17-demethoxy geldanamycin, are in separate clinical trials as new anticancer drugs. We hypothesized that destabilization of hsp 90 client proteins with geldanamycin, while blocking their degradation with bortezomib, would promote the accumulation of aggregated, ubiquitinated, and potentially cytotoxic proteins. Indeed, geldanamycin plus bortezomib inhibited MCF-7 tumor cell proliferation significantly more than either drug alone. Importantly, while control cells were unaffected, human papillomavirus E6 and E7 transformed fibroblasts were selectively sensitive to geldanamycin plus bortezomib. Geldanamycin alone slightly increased protein ubiquitination, but when geldanamycin was combined with bortezomib, protein ubiquitination was massively increased, beyond the amount stabilized by bortezomib alone. In geldanamycin plus bortezomib-treated cells, ubiquitinated proteins were mostly detergent insoluble, indicating that they were aggregated. Individually, both geldanamycin and bortezomib induced hsp 90, hsp 70, and GRP78 stress proteins, but the drug combination superinduced these chaperones and caused them to become detergent insoluble. Geldanamycin plus bortezomib also induced the formation of abundant, perinuclear vacuoles, which were neither lysosomes nor autophagosomes and did not contain engulfed cytosolic ubiquitin or hsp 70. Fluorescence marker experiments indicated that these vacuoles were endoplasmic reticulum derived and that their formation was prevented by cycloheximide, suggesting a role for protein synthesis in their genesis. These observations support a mechanism whereby the geldanamycin plus bortezomib combination simultaneously disrupts hsp 90 and proteasome function, promotes the accumulation of aggregated, ubiquitinated proteins, and results in enhanced antitumor activity.