New,Non‐quinone Fluorogeldanamycin Derivatives Strongly Inhibit Hsp90

Streptomyces hygroscopicus is a natural producer of geldanamycin. Mutasynthetic supplementation of an AHBA‐blocked mutant with all possible monofluoro 3‐aminobenzoic acids provided new fluorogeldanamycins. These showed strong antiproliferative activity and inhibitory effects on human heat shock protein Hsp90. Binding to Hsp90 in the low nanomolar range was determined from molecular modelling, AFM analysis and by calorimetric studies.     

Rational Biosynthetic Engineering for Optimization of Geldanamycin Analogues

Tailor made : We report the rational biosynthesis of C15 hydroxylated non‐quinone geldanamycin analogues by site‐directed mutagenesis of the geldanamycin polyketide synthase (PKS), together with a combination of post‐PKS tailoring genes. Rational biosynthetic engineering allowed the generation of geldanamycin derivatives, such as DHQ3 illustrated in the figure, which had superior pharmacological properties in comparison to the parent compound.

     

Determination of the cryptic stereochemistry of the first PKS chain-extension step in ansamitocin biosynthesis by Actinosynnema pretiosum

The biosynthesis of the antitumor antibiotic, ansamitocin, involves the assembly of a linear octaketide on the ansamitocin (asm) polyketide synthase (PKS), which is then cyclized to proansamitocin and further modified to the final product. In the first chain-extension step on the asm PKS, a stereocenter is generated which is then obliterated in a subsequent double-bond migration. The cryptic configuration at this stereocenter was determined by first synthesizing the two enantiomers of the intermediate diketide as their N-acetylcysteamine (SNAC) thioesters. These were then used to demonstrate that only the R enantiomer complements a 3-amino-5-hydroxybenzoic acid (AHBA) deficient mutant of Actinosynnema pretiosum to restore ansamitocin formation. The low efficiency of complementation by the diketide, compared to AHBA, is due to inefficient loading onto the PKS and not the inhibition of the enzyme. A presumed next chain-extension intermediate-the triketide with an unrearranged double bond-was also synthesized as its SNAC ester, but did not complement the AHBA(-) mutant.     

Effects of Geldanamycin on Hatching and Juvenile Motility in <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis> and <Emphasis Type="Italic">Heterodera glycines</Emphasis>

Several Streptomyces species are known to produce metabolites that inhibit plant pathogens. One such compound is geldanamycin (GA), a benzoquinone ansamycin originally isolated from Streptomyces hygroscopicus. We examined the effect of geldanamycin on egg hatch and juvenile motility in Caenorhabditis elegans and in two populations of the plant-parasitic nematode Heterodera glycines. When C. elegans eggs were exposed to geldanamycin, both hatch and motility were reduced by GA doses between 2 and 50 μg/ml. The H. glycines inbred populations TN17 and TN18 exhibited low dose stimulation of hatch and motility, whereas levels occurring at higher GA doses were at or below control levels. These experiments represent the first demonstration of geldanamycin effects in C. elegans and H. glycines and suggest that the heat shock chaperone Hsp90, the known molecular target of geldanamycin, may be involved in nematode egg hatch and motility. This study also indicates that geldanamycin-producing strains of Streptomyces may be useful as biocontrol agents for nematodes.     

Involvement of Calcium-Mediated Reactive Oxygen Species in Inductive GRP78 Expression by Geldanamycin in 9L Rat Brain Tumor Cells

Treatment with geldanamycin (GA) leads to an increase in [Ca²⁺]_c and the production of reactive oxygen species (ROS) in rat brain tumor 9L RBT cells. GA-exerted calcium signaling was blocked by BAPTA/AM and EGTA. The effect of GA on [Ca²⁺]_c was significantly reduced in the presence of thapsigargin (TG) and ruthenium red (RR). GA-induced GRP78 expression is significantly decreased in the presence of BAPTA/AM, EGTA and RR, suggesting that the calcium influx from the extracellular space and intracellular calcium store oscillations are contributed to by the calcium mobilization and GRP78 expression induced by GA. The induced GRP78 expression is sensitive to added {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 and Ro-31-8425, pinpointing the involvement of phospholipase C (PLC) and protein kinase C (PKC) in GA-induced endoplasmic reticulum (ER) stress. The antioxidants N-acetylcysteine (NAC), BAPTA/AM, EGTA and H7 also have significant inhibitory effects on ROS generation. Finally, neither H7 nor NAC was able to affect the calcium response elicited by GA. Our results suggest that the causal signaling cascade during GA-inducted GRP78 expression occurs via a pathway that connects PLC to cytoplasmic calcium increase, PKC activation and, then, finally, ROS generation. Our data provides new insights into the influence of GA on ER stress response in 9L RBT cells.     

格尔德霉素对SMYD3核转位的影响

采用Western blot检测法研究了不同浓度的格尔德霉素对人乳腺癌细胞MDA-MB-231中SET和MYND结构域蛋白3(SMYD3)核转位的影响,采用MTT比色法检测了格尔德霉素对人乳腺癌细胞MDA-MB-231增殖的影响。结果表明:格尔德霉素可以抑制SMYD3的核转位,并且这种抑制作用是浓度依赖性的;与对照组相比,人乳腺癌细胞MDA-MB-231的增殖速率被显著地抑制了。说明格尔德霉素可以通过抑制SMYD3的核转位显著抑制人乳腺癌细胞MDA-MB-231的增殖。     

Heat Shock Proteins Revisited: Using a Mutasynthetically Generated Reblastatin Library to Compare the Inhibition of Human and Leishmania Hsp90s

Thirteen new reblastatin derivatives, with alkynyl, amino and fluoro substituents on the aromatic ring, were prepared by a chemo‐biosynthetic approach using an AHBA(?) mutant strain of Streptomyces hygroscopicus, the geldanamycin producer. The inhibitory potencies of these mutaproducts and of an extended library of natural products and derivatives were probed with purified heat shock proteins (Hsps), obtained from Leishmania braziliensis (LbHsp90) as well as from human sources (HsHsp90). We determined the activities of potential inhibitors by means of a displacement assay in which fluorescence‐labelled ATP competes for the ATP binding sites of Hsps in the presence of the inhibitor in question. The results were compared with those of cell‐based assays and, in selected cases, of isothermal titration calorimetry (ITC) measurements. In essence, reblastatin derivatives are also able to bind effectively to the ATP‐binding site of LbHsp90, and for selected derivatives, moderate differences in binding to LbHsp90 and HsHsp90 were encountered. This work demonstrates that parasitic heat shock proteins can be developed as potential pharmaceutical targets.