Transformation of Adenine to 8-Hydroxyadenine by Strains of Oerskovia xanthineolytica

The 8-hydroxy derivative of adenine (6-amino-1,7-dihydro-8H-purin-8-one) is produced from adenine by two Oerskovia xanthineolytica strains. This transformation by a microorganism has not been reported previously. No novel products of dissimilation of xanthine (3,7-dihydro-1H-purine-2,6-dione) or hypoxanthine (1,7-dihydro-6H-purin-6-one) were found. Xanthine was oxidized to uric acid, but intermediates in the breakdown of hypoxanthine could not be demonstrated.     

Synthesis and antimicrobial activities of N6-hydroxyagelasine analogs and revision of the structure of ageloximes

(+)-N⁶-Hydroxyagelasine D, the enantiomer of the proposed structure of (?)-ageloxime D, as well as N⁶-hydroxyagelasine analogs were synthesized by selective N-7 alkylation of N⁶-[tert-butyl(dimethyl)silyloxy]-9-methyl-9H-purin-6-amine in order to install the terpenoid side chain, followed by fluoride mediated removal of the TBDMS-protecting group. N⁶-Hydroxyagelasine D and the analog carrying a geranylgeranyl side chain displayed profound antimicrobial activities against several pathogenic bacteria and protozoa and inhibited bacterial biofilm formation. However these compounds were also toxic towards mammalian fibroblast cells (MRC-5). The spectral data of N⁶-hydroxyagelasine D did not match those reported for ageloxime D before. Hence, a revised structure of ageloxime D was proposed. Basic hydrolysis of agelasine D gave (+)-N-[4-amino-6-(methylamino)pyrimidin-5-yl]-N-copalylformamide, a compound with spectral data in full agreement with those reported for (?)-ageloxime D.     

Discovery of selective EGFR modulator to inhibit L858R/T790M double mutants bearing a N-9-Diphenyl-9H-purin-2-amine scaffold

Based upon the modeling binding mode of marketed AZD9291 with T790M, a series of N-9-Diphenyl-9H-purin-2-amine derivatives were designed and synthesized with the purpose to overcome the drug resistance resulted from T790M/L858R double mutations. The most potent compound 23a showed excellent enzyme inhibitory activities and selectivity with nanomolar IC₅₀ values for both the single T790M and double T790M/L858R mutant EGFRs, and was more than 8-fold selective for wild type EGFR. Compound 23a displayed strong antiproliferative activity against the H1975 non-small cell lung cancer (NSCLC) cells bearing T790M/L858R. And it was less potent against A549 (WT EGFR and k-Ras mutation) and HT-29 (non-special gene type) cells, showing a high safety index.     

Triacontanol and Its Second Messenger 9-beta-l(+)-Adenosine as Plant Growth Substances

Triacontanol (TRIA), a common constituent of plant waxes, was first shown in 1977 to be an active growth substance which at nanomolar concentrations increased the growth and yield of crops. TRIA is used to increase crop yields on millions of hectares, particularly in Asia. Many investigators have shown that it affects several basic metabolic processes including photosynthesis, nutrient uptake, and enzyme activity. However, the initial site of action has not been elucidated. TRIA rapidly elicits a second messenger (TRIM) in rice (Oryza sativa L.), which at nanomolar concentrations causes plants to respond in a manner similar to TRIA. TRIM has been identified as 9-β-l(+)-adenosine (9H-purin-6-amine, 9-β-l-ribofuranosyl). During the process of isolating and identifying 9-β-l(+)-adenosine, it was shown that this enantiomer, which previously has not been reported as occurring in nature, made up about 1% of the total adenosine pool in roots from untreated rice seedlings.     

A class of novel conjugates of substituted purine and Gly-AA-OBzl: Synthesis and evaluation of orally analgesic activity

Aimed at the chemotherapy of chronic pain two kinds of analgesic pharmacophores, substituted purine and Gly-AA-OBzl, were coupled via a five-step-reaction procedure and 19 novel conjugates N-[2-chloro-9-(tetrahydropyran-2-yl)-9H-purin-6-yl]-N-cyclopropylglycylamino acid benzylesters were provided. On mouse-tail flick model their in vivo analgesic activities were assayed. The results indicate that introducing Gly-OC₂H₅ into the 6-position of the substituted purine leads to ambiguous increase of the analgesic activity, while introducing Gly-AA-OBzl into this position leads to significant increase of the analgesic activity.     

Growth stimulation ofEuphorbia lathyris L. by GA4+7 and BA

Container-grownEuphorbia lathyris plants were treated with foliar sprays of various combinations of BA and GA₄₊₇ or 0–3600 mg L^?1 Promalin (1∶1 BA + GA₄₊₇) in separate experiments. GA₄₊₇ and Promalin stimulated plants to grow taller. BA and Promalin promoted axillary shoot growth. Multiple applications of Promalin stimulated branching more than single treatments. Dry weight accumulation was stimulated only if the growth regulators were applied to 28–33-cm and not to 56-cm tall plants. Chemical names used: (1α, 2β, 4aα, 4bβ, 10β)-2,4a,7-trihydroxy-1-methyl-8-methylenegibb-3-ene-1,10-dicarboxylic acid 1,4a-lactone (GA₄₊₇),N-(phenylmethyl)-H-purin-6-amine (BA), and Promalin [1∶1 (wt/wt) GA₄₊₇ and BA].     

Discovery of 5-substituted 2-amino-4-chloro-8-((4-methoxy-3,5-dimethylpyridin-2-yl)methyl)-7,8-dihydropteridin-6(5H)-ones as potent and selective Hsp90 inhibitors

A new series of compounds, 5-substituted 2-amino-4-chloro-8-((4-methoxy-3,5-dimethylpyridin-2-yl)methyl)-7,8-dihydropteridin-6(5H)-ones, have been designed and identified as potent and selective inhibitors of Hsp90. These compounds demonstrated nanomolar potency toward both Hsp90-regulated Her2 degradation and the growth of a panel of human tumor cell lines in cell-based assays. High selectivity of these compounds toward Hsp90 was evident given that they did not inhibit a panel of 34 kinases at 10 μM. The structure–activity relationship (SAR) of this series is reported here.     

Novel anticancer Hsp90 inhibitor disubstituted pyrazolyl 2-aminopyrimidine compound 7t induces cell cycle arrest and apoptosis via mitochondrial pathway in MCF-7 cells

Compound 7t, 4-(4-bromophenyl)-6-(1-(4-chlorophenyl)-3-(4-nitrophenyl)-1H-pyrazol-4-yl) pyrimidin-2-amine, is a proven potent anticancer agent exhibiting Hsp90 inhibition in our previous studies. Herein, we explored the apoptotic potential of compound 7t by Annexin V assay. The mechanism underlining the apoptosis process is elucidated. As a potent Hsp90 inhibitor, compound 7t would induce the mitochondrial stress leading to increased permeability of its membrane, that would subsequently initiate the apoptosis in MCF-7 cells. This was proven by increased J-monomer formation using JC-1 stain. Moreover, due to the impaired mitochondrial function, compound 7t also exaggerated the apoptosis process by ROS generation as proved by DCFDA staining. The morphological and nuclear changes in MCF-7 cells following apoptosis were identified by AO/EB and DAPI staining techniques. It also induced subG1 phase cell cycle arrest. Thus, compound 7t could serve as potential drug in the treatment regimen of breast cancer.     

Synthesis of a hydrolytically stable,fluorescent-labeled ATP analog as a tool for probing adenylyl cyclases

(2R,3S,4R,5R)-5-(6-Amino-9H-purin-9-yl)-3-hydroxy-4-[2-(methylamino)benzamido]tetrahydrofuran-2-yl-methoxy[(hydroxy)phosphoryloxy][(hydroxy)phosphoryl]dichloromethylphosphonic acid was synthesized as a chemically and metabolically stable analog of ATP substituted with a fluorescent methylanthranoyl (MANT) residue. The compound is intended for studying the binding site and function of adenylyl cyclases (ACs), which was exemplified by studying its interaction with Bacillus anthracis edema factor (EF) AC exotoxin.     

The Non-canonical Hop Protein from Caenorhabditis elegans Exerts Essential Functions and Forms Binary Complexes with Either Hsc70 or Hsp90

Heat shock protein (Hsp) 70/Hsp90-organizing proteins (Hop/Sti1) are thought to function as adaptor proteins to link the two chaperone machineries Hsp70 and Hsp90 during the processing of substrate proteins in eukaryotes. Hop (Hsp70/Hsp90-organizing protein) is composed of three tetratricopeptide repeat (TPR) domains, of which the first (TPR1) binds to Hsp70, the second (TPR2A) binds to Hsp90, and the third (TPR2B) is of unknown function. Contrary to most other eukaryotes, the homologue closest to the Caenorhabditis elegans Hop homologue R09E12.3 (CeHop) lacks the TPR1 domain and the short linker region connecting it to TPR2A, questioning the reported function as an Hsp90/Hsp70 adaptor in vitro and in vivo. We observed high constitutive expression levels of CeHop and detected significant phenotypes upon knockdown, linking the protein to functions in gonad development. Interestingly, we observed physical interactions with both chaperones Hsp70 and Hsp90, albeit only the interaction with Hsp90 is strong and inhibition of the Hsp90 ATPase activity can be observed upon binding of CeHop. However, the formation of ternary complexes with both chaperone machineries is impaired, as Hsp70 and Hsp90 compete for CeHop interaction sites, in particular as Hsp90 binds to both TPR domains simultaneously and requires both TPR domains for ATPase regulation. These results imply that, at least in C. elegans, essential functions of Hop exist which apparently do not depend on the simultaneous binding of Hsp90 and Hsp70 to Hop.     

Production of cucumber fruits from the culture of ‘Marketmore-76’ plantlets

A procedure to produce fruits from cultured shoot tips of Cucumis sativus L. cultivar Marketmore-76 in vitro is described. Four-week-old shoot tips, derived from sterile germinated seedlings on a MS medium, were cultured in a 3.8-1 Mason jar using an automated plant culture system. Tips readily generated roots, leaves and flowers after another 4 to 8 weeks in culture. Administration of compressed air at a 300 ml/min flow rate for 30 min 10 or 15 times a day induced the development of parthenocarpic fruits from flowers. Fruits, up to 170 mm long by 35 mm diameter, were obtained within 30 to 45 d after flower opening.Abbreviations APCS automated plant culture system - BA N-(phenylmethyl)-1H-purin-6-amine - BM basal medium - cv cultivar - MS Murashige and Skoog - NAA 1-naphthaleneacetic acid     

Biological and Structural Basis for Aha1 Regulation of Hsp90 ATPase Activity in Maintaining Proteostasis in the Human Disease Cystic Fibrosis

The activator of Hsp90 ATPase 1, Aha1, has been shown to participate in the Hsp90 chaperone cycle by stimulating the low intrinsic ATPase activity of Hsp90. To elucidate the structural basis for ATPase stimulation of human Hsp90 by human Aha1, we have developed novel mass spectrometry approaches that demonstrate that the N- and C-terminal domains of Aha1 cooperatively bind across the dimer interface of Hsp90 to modulate the ATP hydrolysis cycle and client activity in vivo. Mutations in both the N- and C-terminal domains of Aha1 impair its ability to bind Hsp90 and stimulate its ATPase activity in vitro and impair in vivo the ability of the Hsp90 system to modulate the folding and trafficking of wild-type and variant (ΔF508) cystic fibrosis transmembrane conductance regulator (CFTR) responsible for the inherited disease cystic fibrosis (CF). We now propose a general model for the role of Aha1 in the Hsp90 ATPase cycle in proteostasis whereby Aha1 regulates the dwell time of Hsp90 with client. We suggest that Aha1 activity integrates chaperone function with client folding energetics by modulating ATPase sensitive N-terminal dimer structural transitions, thereby protecting transient folding intermediates in vivo that could contribute to protein misfolding systems disorders such as CF when destabilized.     

Identification of new Hsp90 inhibitors by structure-based virtual screening

Structure-based virtual screening identified pyrimidine-2,4,6-trione and 4H-1,2,4-triazole-3-thiol as novel scaffolds of Hsp90 ATPase inhibitors. Their binding modes in the ATP-binding pocket of Hsp90 were analyzed using AutoDoc program combined with molecular dynamics (MD) simulations.     

Functional characterization of orchardgrass endoplasmic reticulum-resident Hsp90 (DgHsp90) as a chaperone and an ATPase

Hsp90 proteins are essential molecular chaperones regulating multiple cellular processes in distinct subcellular organelles. In this study, we report the functional characterization of a cDNA encoding endoplasmic reticulum (ER)-resident Hsp90 from orchardgrass (DgHsp90). DgHsp90 is a 2742 bp cDNA with an open reading frame predicted to encode an 808 amino acid protein. DgHsp90 has a well conserved N-terminal ATPase domain and a C-terminal Hsp90 domain and ER-retention motif. Expression of DgHsp90 increased during heat stress at 35 °C or H₂O₂ treatment. DgHsp90 also functions as a chaperone protein by preventing thermal aggregation of malate dehydrogenase (EC 1.1.1.37) and citrate synthase (EC 2.3.3.1). The intrinsic ATPase activity of DgHsp90 was inhibited by geldanamycin, an Hsp90 inhibitor, and the inhibition reduced the chaperone activity of DgHsp90. Yeast cells overexpressing DgHsp90 exhibited enhanced thermotolerance.     

SBA1 Encodes a Yeast Hsp90 Cochaperone That Is Homologous to Vertebrate p23 Proteins

The Saccharomyces cerevisiae SBA1 gene was cloned by PCR amplification from yeast genomic DNA following its identification as encoding an ortholog of human p23, an Hsp90 cochaperone. The SBA1 gene product is constitutively expressed and nonessential, although a disruption mutant grew more slowly than the wild type at both 18 and 37°C. A double deletion of SBA1 and STI1, encoding an Hsp90 cochaperone, displayed synthetic growth defects. Affinity isolation of histidine-tagged Sba1p (Sba1^His6) after expression in yeast led to coisolation of Hsp90 and the cyclophilin homolog Cpr6. Using an in vitro assembly assay, purified Sba1^His6 bound to Hsp90 only in the presence of adenosine 5′-O-(3-thiotriphosphate) or adenyl-imidodiphosphate. Furthermore, interaction between purified Sba1^His6 and Hsp90 in yeast extracts was inhibited by the benzoquinoid ansamycins geldanamycin and macbecin. The in vitro assay was also used to identify residues in Hsp90 that are important for complex formation with Sba1^His6, and residues in both the N-terminal nucleotide binding domain and C-terminal half were characterized. In vivo analysis of known Hsp90 substrate proteins revealed that Sba1 loss of function had only a mild effect on the activity of the tyrosine kinase v-Src and steroid hormone receptors.     

Synthesis,cytotoxicity, antimicrobial and anti-biofilm activities of novel pyrazolo[3,4-b]pyridine and pyrimidine functionalized 1,2,3-triazole derivatives

A series of novel pyrazolo[3,4-b]pyridine and pyrimidine functionalized 1,2,3-triazole derivatives 8a–g and 9a–g were prepared starting from 6-trifluoromethylpyridine-2(1H)one 2 via selective O-alkylation, followed by cyclisation using hydrazine hydrate to obtain 6-(trifluoromethyl)-1H-pyrazolo[3,4-b]pyridin-3-amine 4. Compound 4 was diazotized followed by reaction with sodium azide, resulted in 3-azido-6-(trifluoromethyl)-1H-pyrazolo[3,4-b]pyridine 5. Compound 5 was further cyclized with N-/O-propargylated pyrimidine derivatives under Sharpless conditions and obtained compounds 6 and 7, respectively. Each set of compounds 6 and 7 were alkylated with different alkyl halides and obtained respective products 8 and 9. All the products were screened for cytotoxicity against four human cancer cell lines such as A549-Lung (CCL-185), MCF7-Breast (HTB-22), DU145-Prostate (HTB-81) and HeLa-Cervical (CCL-2), compounds 9d, 9e and 9f which showed promising activity have been identified. The products were also screened for antimicrobial, anti bio-film and MBC activities. Promising compounds in each case have been identified.     

Synthesis,single crystal and antitumor activities of benzimidazole–quinazoline hybrids

A series of novel regioisomeric hybrids of quinazoline/benzimidazole viz. (3-allyl-2-methyl-3H-benzimidazol-5-yl)-(2-substituted-quinazolin-4-yl)-amine and (1-allyl-2-methyl-1H-benzimidazol-5-yl)-(2-substituted-quinazolin-4-yl)-amine of biological interest were synthesized. All the synthesized compounds were well characterized by ¹H and ¹³C NMR as well as mass spectroscopy. The newly synthesized compounds were screened for in vitro antitumor activities against 60 tumor cell lines panel assay. A significant inhibition for cancer cells were observed with compound 9 and also more active against known drug 5-fluorouracil (5-FU) in some tumor cell lines. Compound 9 displayed appreciable anticancer activity against leukemia, colon, melanoma, renal and breast cancer cell lines.     

Hsp90 selectively modulates phenotype in vertebrate development

Compromised heat shock protein 90 (Hsp90) function reveals cryptic phenotypes in flies and plants. These observations were interpreted to suggest that this molecular stress-response chaperone has a capacity to buffer underlying genetic variation. Conversely, the protective role of Hsp90 could account for the variable penetrance or severity of some heritable developmental malformations in vertebrates. Using zebrafish as a model, we defined Hsp90 inhibitor levels that did not induce a heat shock response or perturb phenotype in wild-type strains. Under these conditions the severity of the recessive eye phenotype in sunrise, caused by a pax6b mutation, was increased, while in dreumes, caused by a sufu mutation, it was decreased. In another strain, a previously unobserved spectrum of severe structural eye malformations, reminiscent of anophthalmia, microphthalmia, and nanophthalmia complex in humans, was uncovered by this limited inhibition of Hsp90 function. Inbreeding of offspring from selected unaffected carrier parents led to significantly elevated malformation frequencies and revealed the oligogenic nature of this phenotype. Unlike in Drosophila, Hsp90 inhibition can decrease developmental stability in zebrafish, as indicated by increased asymmetric presentation of anophthalmia, microphthalmia, and nanophthalmia and sunrise phenotypes. Analysis of the sunrise pax6b mutation suggests a molecular mechanism for the buffering of mutations by Hsp90. The zebrafish studies imply that mild perturbation of Hsp90 function at critical developmental stages may underpin the variable penetrance and expressivity of many developmental anomalies where the interaction between genotype and environment plays a major role.     

Heat Shock Protein 90 as a Drug Target against Protozoan Infections: BIOCHEMICAL CHARACTERIZATION OF HSP90 FROM PLASMODIUM FALCIPARUM AND TRYPANOSOMA EVANSI AND EVALUATION OF ITS INHIBITOR AS A CANDIDATE DRUG*

Using a pharmacological inhibitor of Hsp90 in cultured malarial parasite, we have previously implicated Plasmodium falciparum Hsp90 (PfHsp90) as a drug target against malaria. In this study, we have biochemically characterized PfHsp90 in terms of its ATPase activity and interaction with its inhibitor geldanamycin (GA) and evaluated its potential as a drug target in a preclinical mouse model of malaria. In addition, we have explored the potential of Hsp90 inhibitors as drugs for the treatment of Trypanosoma infection in animals. Our studies with full-length PfHsp90 showed it to have the highest ATPase activity of all known Hsp90s; its ATPase activity was 6 times higher than that of human Hsp90. Also, GA brought about more robust inhibition of PfHsp90 ATPase activity as compared with human Hsp90. Mass spectrometric analysis of PfHsp90 expressed in P. falciparum identified a site of acetylation that overlapped with Aha1 and p23 binding domain, suggesting its role in modulating Hsp90 multichaperone complex assembly. Indeed, treatment of P. falciparum cultures with a histone deacetylase inhibitor resulted in a partial dissociation of PfHsp90 complex. Furthermore, we found a well known, semisynthetic Hsp90 inhibitor, namely 17-(allylamino)-17-demethoxygeldanamycin, to be effective in attenuating parasite growth and prolonging survival in a mouse model of malaria. We also characterized GA binding to Hsp90 from another protozoan parasite, namely Trypanosoma evansi. We found 17-(allylamino)-17-demethoxygeldanamycin to potently inhibit T. evansi growth in a mouse model of trypanosomiasis. In all, our biochemical characterization, drug interaction, and animal studies supported Hsp90 as a drug target and its inhibitor as a potential drug against protozoan diseases.