Optimization and biological evaluation of celastrol derivatives as Hsp90–Cdc37 interaction disruptors with improved druglike properties

Heat shock protein 90 (Hsp90) as a molecular target for oncology therapeutics has attracted much attention in the last decade. The Hsp90 multichaperone complex has important roles in the growth and/or survival of cancer cells. Cdc37, as a cochaperone, associates kinase clients to Hsp90 and promotes the development of malignant tumors. Disrupting the Hsp90–Cdc37 interaction provides an alternative strategy to inhibit the function of Hsp90 for cancer therapy. Celastrol, as a natural product, can disrupt the Hsp90–Cdc37 interaction and induce degradation of kinase clients. The study conducted here attempted to elucidate the structure–activity relationship of celastrol derivatives as Hsp90–Cdc37 disruptors and to improve the druglike properties. 23 celastrol derivatives were designed, synthesized, and the biological activities and physicochemical properties were determined. The derivative CEL20 showed improved Hsp90–Cdc37 disruption activity, anti-proliferative activities as well as druglike properties. Additionally, CEL20 induced clients degradation, cell cycle arrest and apoptosis in Panc-1 cells. This study can provide reference for the discovery of novel Hsp90–Cdc37 disruptors.     

Chromosomal initiation in Bacillus subtilis may involve two closely linked origins

Summary When the dnaB37 initiation mutant of Bacillus subtilis is returned to a permissive temperature following a period at 45° C, a synchronous round of DNA replication immediately ensues. Using this system we have been able to analyse the first fragments to be replicated while avoiding the use of thymine starvation or inhibitors of DNA replication. Such treatments are necessary to achieve even modest synchrony in germinating spores. Our results showed that the first fragment to be replicated was a 4kb BamHI-SalI restriction fragment, BS6. In contrast, when the analysis was performed out in the presence of novobiocin, an inhibitor of DNA gyrase, replication from BS6 was inhibited and the first fragment to be replicated was BS5, a 5.6 kb fragment located 1.7 kb to the right of BS 6. Replication from both putative origins was suppressed by rifamycin and was dependent upon dnaB. The results are discussed in relation to previous attempts to identify the first replicating fragment in germinating spores. We also discuss the possibility that B. subtilis contains two origins and suggest that either can act as the primary origin under certain conditions, or alternatively that both origins may act in concert in normal bidirectional replication, each site being required for the leading strand in each direction.     

Comparative effectiveness of lymphotoxin anticarcinogenic and tumor cell growth-inhibitory activities

Prevention of ultraviolet radiation- or chemical carcinogen-induced morphologic transformation and inhibition of tumor-producing transformed cell growth by lymphotoxin and by normal spleen leukocytes were quantitatively compared to define the antineoplastic activity spectra of these natural immune mediators. When Syrian golden hamster embryo cells seeded for colony formation in culture dishes were treated simultaneously with carcinogen and lymphotoxin, the number of morphologically transformed cell colonies was irreversibly reduced by 50% in the presence of 6 units of lymphotoxin/ml. Lymphotoxin inhibition of tumor cell growth, however, was reversible and 50% reduction in tumor cell growth in three transformed lines required 124, 330, and 477 units/ml. Thus, the anticarcinogenic activity of lymphotoxin can be 20-fold or more greater than its tumor growth-inhibitory activity. Similarly spleen leukocytes also were more effective as an anticarcinogen than as an inhibitor of tumor cell growth, consistent with previous observations that naturally occurring spleen leukocyte antineoplastic activity may result from lymphotoxin secretion.     

Production of thermostable α-galactosidase from thermophilic fungusHumicola sp

The thermophilic fungus,Humicola sp isolated from soil, secreted extracellular 37/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-galactosidase in a medium cotaining wheat bran extract and yeast extract. Maximum enzyme production was found in a medium containing 5% wheat bran extract as a carbon source and 0.5% beef extract as a carbon and nitrogen source. Enzyme secretion was strongly inhibited by the presence of Cu²⁺, Ni²⁺ and Hg²⁺ (1mM) in the fermentation medium. Production of enzyme under stationary conditions resulted in 10-fold higher activity than under shaking conditions. The temperature range for production of the enzyme was 37° C to 55°C, with maximum activity (5.54 U ml^–1) at 45°C. Optimum pH and temperature for enzyme activity were 5.0 and 60° C respectively. One hundred per cent of the original activity was retained after heating the enzyme at 60°C for 1 h. At 5mM Hg²⁺ strongly inhibited enzyme activity. TheK _m andV _max forp-nitrophenyl-37/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-d-galactopyranoside were 6037/xxlarge956.gif" alt="mgr" align="MIDDLE" BORDER="0">M and 33.6 37/xxlarge956.gif" alt="mgr" align="MIDDLE" BORDER="0">mol min^–1 mg^–1, respectively, while for raffinose those values were 10.52 mM and 1.8 37/xxlarge956.gif" alt="mgr" align="MIDDLE" BORDER="0">mol min^–1 mg^–1, respectively.     

Hectogram-scale synthesis of [Aib8, Arg34]-GLP-1 (7–37) by liquid-phase fragment condensation

Based on small-scale synthesis (0.3 g), a 100-g scale-up synthesis of crude [Aib⁸, Arg³⁴]-glucagon-like peptide-1 (GLP-1) (7–37) was completed. The crude [Aib⁸, Arg³⁴]-GLP-1 (7–37) was purified using a dynamic axial compression column 200 (DAC-200). Approximately 61 g of [Aib⁸, Arg³⁴]-GLP-1 (7–37) with a purity of >99% was obtained through one-step reverse-phase chromatography. The purification yield was approximately 92%. The yield from the total reaction was approximately 60%. In summary, we developed an economical and environmentally friendly route to the synthesis and purification of crude [Aib⁸, Arg³⁴]-GLP-1 (7–37), laying a foundation for subsequent industrial production.     

Purification, properties, and immunological characterization of GalT-3 (UDP-galactose: GM2 ganglioside, β1-3 galactosyltransferase) from embryonic chicken brain

A 37n2347123313136/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">1-3 galactosyltransferase (GalT-3; UDP-Gal; GM2 37n2347123313136/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">1-3galactosyltransferase) was purified over 5100-fold from 19-day-old embryonic chicken brain homogenate employing detergent solubilization, 37n2347123313136/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-lactalbumin Sepharose, Q-Sepharose, UDP-hexanolamine Sepharose, and GalNAc37n2347123313136/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">1-4Gal37n2347123313136/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-Synsorb column chromatography. The purified enzyme was resolved into two bands on reducing gels with apparent molecular weights of 62 kDa and 65 kDa, respectively. GalT-3 activity was also localized in the same regions by activity gel analysis and sucrose-density gradient centrifugation of a detergent-solubilized extract of 19-day-old embryonic chicken brain. Purified GalT-3 exhibited apparentK _mS of 33 µm, 22 µm and 14.4mM with respect to the substrates GM2, UDP-galactose, and MnCl₂, respectively. Substrate specificity studies with the purified enzyme and a variety of glycosphingolipids, glycoproteins, and synthetic substrates revealed that the enzyme was highly specific only for the glycosphingolipid acceptors, GM2 and GgOse3Cer (asialo-GM2). Ovine-asialo-agalacto submaxillary mucin inhibited the transfer of galactose to GM2 but did not act as an acceptor in the range of concentrations tested. Polyclonal antibodies raised against purified GalT-3 inhibited GalT-3 activityin vitro and Western-immunoblot analysis of purified GalT-3 showed immunopositive bands at 62 and 65 kDa.Abbreviations CNS central nervous system - GM1 monosialotetraosylganglioside, Gal37n2347123313136/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">1-3GalNAc37n2347123313136/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">1-4(NeuAc37n2347123313136/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">2-3)Gal37n2347123313136/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">1-4Glc37n2347123313136/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">1-1Cer - GM2 monosialotriaosylganglioside, GalNAc37n2347123313136/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">1-4(NeuAc37n2347123313136/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">2-3)Gal37n2347123313136/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">1-4Glc37n2347123313136/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">1-1Cer - DSS detergent solubilized supernatant - ECB embryonic chicken brain - TBS Tris-buffered saline     

Biochemical and morphological characterization of a plasma membrane-enriched fraction from bovine parathyroid cells

A fraction of enriched plasma membranes from bovine parathyroid cells has been prepared by differential centrifugation. Biochemical characterization shows that this fraction has a specific activity enrichment of 7.2-fold in ouabain-sensitive Na+-K+ ATPase, and 3.5-fold in 5'-nucleotidase. Less than 4% of the total mitochondria and lysosomes are present within the plasma membranes, while microsomal contamination accounts for 14% of total specific activity. Parathyroid hormone radioimmunoassay also reveals the presence of some secretory granules within the plasma membrane fraction. The characteristic morphological aspect of the unusual surface membrane is shown by freeze-fracture electron microscopy. In the enriched pellets, vesicles identified as having a plasma membrane origin have variable sizes, and 50% show an inside-out conformation. Even though the plasma membrane fraction described herein is not absolutely free from contamination by other subcellular components, this protocol represents the first attempt to purify surface membrane from parathyroid tissue and provide the starting material for understanding, at a molecular level, the properties of extracellular Ca2+ regulation and its coupling with secretion of parathyroid hormone.     

Sensitivity of Escherichia coli acrA mutants to psoralen plus near-ultraviolet radiation

The sensitivity to psoralen plus near-ultraviolet radiation (PUVA) was compared in a pair of E. coli strains differing at the acrA locus. Survival was determined for both bacteria and phage lambda. AcrA mutant cells were 40 times more sensitive than wild type to the lethal effect of PUVA. Free lambda phage exposed to PUVA survived as well when plated on acrA mutants as on wild type. In contrast, prophage lambda CI857 ind carried in lysogenic acrA strains was hypersensitive to PUVA. The enhanced sensitivity of bacterial and lambda DNA, when inside acrA cells, was paralleled by an increased photobinding of radiolabelled psoralens in the mutant. Binding was increased specifically to DNA rather than to nucleic acids in general. The difference in psoralen-binding ability determined by the acrA gene persisted after permeabilizing treatment of the cells. The results suggest that the acrA mutation causes an alteration specifically in the environment of the cellular DNA so as to allow increased intercalation and photobinding of psoralens.     

Phospholamban: dissociation of the 22,000 molecular weight protein of cardiac sarcoplasmic reticulum into 11,000 and 5,500 molecular weight forms

Structural characterization of a 40 amino acid peptide with high intrinsic growth hormone releasing activity isolated from a human pancreatic tumor which had caused acromegaly was accomplished by gas phase sequence analyses of the intact peptide and its carboxy terminal cyanogen bromide digestion fragment. High pressure liquid chromatography of the native peptide and synthetic replicates showed that the molecule possessed a free acid rather than an amidated carboxy terminus. The structure of the peptide was established as: Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys- Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Met-Ser-Arg-Gln-Gln-Gly- Glu-Ser-Asn-Gln-Glu-Arg-Gly-Ala-OH using 1.8 nmoles of material. The structural identity of this material with a previously characterized fragment of a larger growth hormone releasing peptide isolated from a different human tumor is discussed.