Biomedical subjects

Sidney M Hecht

Publications and source records attributed to Sidney M Hecht.

At least 37 records · Page 2Linked to original sources

PubMed2005

Analogues of vaccinia virus DNA topoisomerase I modified at the active site tyrosine.

The mechanism of type IB topoisomerase-mediated DNA relaxation was studied by modification of vaccinia topoisomerase I at the active site tyrosine (position 274) with several tyrosine analogues. These analogues had varied steric, electronic, and stereochemical features to permit assessment of those structural elements required to support topoisomerase function. Eleven tyrosine analogues were successfully incorporated into the active site of vaccinia topoisomerase I. It was found that only tyrosine analogues having the phenolic -OH group in the normal position relative to the protein backbone were active. Modifications that replaced the nucleophilic tyrosine OH (pKa approximately 10.0) group with NH2 (pKa 4.6), SH (pKa approximately 7.0), or I groups or that changed the orientation of the nucleophilic OH group essentially eliminated topoisomerase I function. For the active analogues, the electronic effects and H-bonding characteristics of substituents in the meta-position of the aromatic ring may be important in modulating topoisomerase I function. The pH profile for the functional analogues revealed a small shift toward lower pH when compared with wild-type topoisomerase I.

Amino Acid Sequence↗

PubMed2005

Synthesis of 14-azacamptothecin, a water-soluble topoisomerase I poison.

14-Azacamptothecin, a potent, water-soluble analogue of the antitumor agent camptothecin, has been prepared by a convergent synthesis. The key condensation of the AB and DE rings with concomitant formation of ring C of 14-aza CPT was carried out in two stages, the latter of which involved a radical cyclization strategy. [structure: see text]

Antineoplastic Agents↗

PubMed2005

On the role of E-ring oxygen atoms in the binding of camptothecin to the topoisomerase I-DNA covalent binary complex.

A recent X-ray crystallographic analysis of the binding of a water soluble camptothecin analogue to the human topoisomerase I-DNA covalent binary complex has suggested the existence of some novel features in the way that camptothecin is bound to the binary complex. Four additional models based on chemical and biochemical data have also been proposed. Presently we describe S-containing analogues of camptothecin prepared on the basis of these models, and report their ability to form stable ternary complexes with human topoisomerase I, and to mediate cytotoxicity at the locus of topoisomerase I. The results indicate that replacement of the 20-OH group of CPT with a SH functionality results in diminution of the potency of CPT as a topoisomerase I poison, while replacement of the O atoms at positions 20 and 21 with S atoms results in essentially complete loss of topoisomerase I inhibitory activity.

Autoradiography↗

PubMed2005

Polybrominated diphenyl ethers from a sponge of the Dysidea genus that inhibit Tie2 kinase.

Tie2 kinase, an enzyme that supports angiogenesis essential for tumor growth and survival, was selected as a target in a search for naturally occurring inhibitors of potential utility for antitumor therapy. Two polybrominated diphenyl ethers, 3,5-dibromo-2-(2',4'-dibromophenoxy)phenol (1) and 4,6-dibromo-2-(2',4'-dibromophenoxy)phenol (2) were isolated from an extract prepared from Dysidea sp. after bioassay-guided fractionation.

Animals↗

PubMed2005

DNA topoisomerase I inhibitors from Rinorea anguifera.

An organic extract prepared from Rinorea anguifera was investigated in order to identify the natural principle(s) responsible for stabilization of a topoisomerase I-DNA covalent binary complex. Bioassay-guided fractionation resulted in the isolation of mauritianin and (+)-syringaresinol as new topoisomerase I inhibitors, and also of the known inhibitor camptothecin.

Camptothecin↗

PubMed2005

Identification of the first specific inhibitor of p90 ribosomal S6 kinase (RSK) reveals an unexpected role for RSK in cancer cell proliferation.

p90 ribosomal S6 kinase (RSK) is an important downstream effector of mitogen-activated protein kinase, but its biological functions are not well understood. We have now identified the first small-molecule, RSK-specific inhibitor, which we isolated from the tropical plant Forsteronia refracta. We have named this novel inhibitor SL0101. SL0101 shows remarkable specificity for RSK. The major determinant of SL0101-binding specificity is the unique ATP-interacting sequence in the amino-terminal kinase domain of RSK. SL0101 inhibits proliferation of the human breast cancer cell line MCF-7, producing a cell cycle block in G(1) phase with an efficacy paralleling its ability to inhibit RSK in intact cells. RNA interference of RSK expression confirmed that RSK regulates MCF-7 proliferation. Interestingly, SL0101 does not alter proliferation of a normal human breast cell line MCF-10A, although SL0101 inhibits RSK in these cells. We show that RSK is overexpressed in approximately 50% of human breast cancer tissue samples, suggesting that regulation of RSK has been compromised. Thus, we show that RSK has an unexpected role in proliferation of transformed cells and may be a useful new target for chemotherapeutic agents. SL0101 will provide a powerful new tool to dissect the molecular functions of RSK in cancer cells.

Amino Acid Sequence↗

PubMed2005

14-azacamptothecin: a potent water-soluble topoisomerase I poison.

On the basis of an analysis of luotonin A and its D-ring deaza analogue as topoisomerase I poisons and topoisomerase I-dependent cytotoxic agents, a novel analogue of the structurally related antitumor antibiotic camptothecin (CPT) was prepared. 14-Azacamptothecin was found to have much greater aqueous solubility than CPT, to inhibit topoisomerase I-mediated DNA relaxation more efficiently than CPT, and to stabilize the covalent binary complex to almost the same extent. 14-Aza CPT was found to be slightly less active than CPT in mediating cytotoxicity toward yeast expressing human topoisomerase I, possibly as a consequence of its greater off-rate from the CPT-topoisomerase I-DNA ternary complex.

Camptothecin↗

PubMed2005

Mechanistic plasticity of DNA topoisomerase IB: phosphate electrostatics dictate the need for a catalytic arginine.

Four conserved amino acids of type IB topoisomerases (Arg130, Lys167, Arg223, and His265 in vaccinia topoisomerase) catalyze the attack by tyrosine on the scissile phosphodiester to form a DNA-(3'-phosphotyrosyl)-enzyme intermediate. The mechanism entails general acid catalysis (by Lys167 and Arg130) and transition-state stabilization (via contact of His265 with the pro-Sp oxygen). Here we query the function of Arg223, which accelerates transesterification by a factor of 10(5). The requirement for Arg223 is alleviated by a neutral Sp methylphosphonate (MeP) linkage at the cleavage site. Arg223 is not required for the 30,000-fold activation of the latent endonuclease activity of topoisomerase by the Sp MeP. The rate of autohydrolysis by the DNA-(3'-MeP)-topoisomerase intermediate approaches 10% of the rate of religation to a 5'-OH DNA strand. These findings underscore the importance of transition-state electrostatics in determining the composition of the active site and dictating the balance between strand transferase and hydrolase functions.

Arginine↗

PubMed2005

The N-pentenoyl protecting group for aminoacyl-tRNAs.

The elaboration of misacylated transfer RNAs by T4 RNA ligase-mediated condensation of an aminoacylated pdCpA derivative and a tRNA (transcript) missing the two 3'-terminal nucleotides requires that the aminoacyl moiety of the dinucleotide be stabilized during the ligation reaction. This can be done conveniently by the use of a simple 4-pentenoyl group attached to N(alpha) of the amino acid. The pentenoyl amide can be deblocked readily with aqueous iodine, presumably via an iodolactone intermediate. This protecting group can be used in conjunction with side chain protecting group for amino acids having side chain functionality, thus permitting the elaboration of proteins bearing side chain protecting groups that can be removed in a subsequent step (e.g., caged proteins). In addition, an aminated analogue of the pentenoyl protecting group, the unnatural amino acid allylglycine, can be employed as part of the peptide backbone to afford a protein cleavable by iodine.

Nucleic Acid Conformation↗

PubMed2005

A dihydroflavonol glucoside from Commiphora africana that mediates DNA strand scission.

A crude CH(2)Cl(2)-MeOH extract prepared from Commiphora africana was found to mediate Cu(2+)-dependent relaxation of supercoiled plasmid DNA. Bioassay-guided fractionation of this extract was carried out and was monitored by the use of an in vitro DNA strand scission assay. The dihydroflavonol glucoside phellamurin (1) was identified as the active principle responsible for the DNA cleavage activity of the crude extract.

Commiphora↗

PubMed2005

Umbelactonyl cinnamate derivatives from Crypteronia paniculata that mediate DNA strand scission.

As part of an initiative to discover functional natural product analogues of bleomycin guided by the use of the COMPARE algorithm, a CH(2)Cl(2)-MeOH extract prepared from Crypteronia paniculata was found to exhibit relaxation of supercoiled pSP64 DNA in the presence of Cu(2+). Bioassay-guided fractionation employing a DNA strand scission assay resulted in the isolation of three novel DNA cleavage agents. Their structures were elucidated as umbelactonyl cinnamate derivatives 1-3 through their NMR and MS spectral data analyses. This is the first example of the isolation and structural characterization of naturally occurring umbelactonyl cinnamate derivatives. Compound 1 exhibited strong Cu(2+)-dependent relaxation of supercoiled pSP64 DNA, while compounds 2 and 3 had only weak DNA cleavage activity.

Algorithms↗

PubMed2005

Alkaloids from Alangium javanicum and Alangium grisolleoides that mediate Cu2+-dependent DNA strand scission.

Crude CH2Cl2-MeOH extracts prepared from Alangium javanicum and A. grisolleoides were found to induce DNA strand breakage in the presence of Cu2+ and were subjected to bioassay-guided fractionation to permit identification of the active principle(s). Javaniside (1), a novel alkaloid possessing an unusual monoterpenoid oxindole skeleton, was identified as an active principle contributing to the DNA cleavage activity observed for the crude extract of A. javanicum. Alangiside (2), a tetrahydroisoquinoline monoterpene glucoside widely distributed in the genus Alangium, was also isolated from A. grisolleoides as a new type of Cu2+-dependent DNA cleavage agent. The relative configuration of the asymmetric centers in javaniside was established by analysis of 1H-1H coupling constants and NOESY correlations. Semisynthesis of javaniside from secologanin (3) established the absolute stereochemistry of javaniside.

Alangiaceae↗

PubMed2005

(+)-Myristinins A and D from Knema elegans, which inhibit DNA polymerase beta and cleave DNA.

A survey of crude plant extracts for DNA polymerase beta inhibitors resulted in the identification of a methyl ethyl ketone extract prepared from Knema elegans that strongly inhibited the enzyme. Subsequent bioassay-guided fractionation of the extract, using an assay to monitor the activity of DNA polymerase beta, led to the isolation of two potent inhibitors, (+)-myristinins A (1) and D (2), which are known flavans having unusual structures. (+)-Myristinins A and D exhibited IC50 values of 12 and 4.3 microM, respectively, as inhibitors of DNA polymerase beta in the presence of bovine serum albumin (BSA), and 2.7 and 1.2 microM in the absence of BSA. As such, they are the most potent DNA polymerase beta inhibitors reported to date. Compounds 1 and 2 potentiated the cytotoxicity of bleomycin toward cultured P388D1 cells, reducing the number of viable cells by at least 30% when employed at 9 microM concentration for 6 h in the presence of an otherwise nontoxic concentration of bleomycin (75 nM). Principles 1 and 2 also induced strong Cu2+-dependent DNA strand scission in a DNA cleavage assay. Accordingly, 1 and 2 exhibit two activities, namely, DNA polymerase beta inhibition and DNA damage.

Animals↗

PubMed2005

Camptothecin: roles of the D and E rings in binding to the topoisomerase I-DNA covalent binary complex.

The alkaloid camptothecin is the prototypical DNA topoisomerase I poison. This core structure has formed the basis for two marketed antitumor agents and numerous clinical candidates, and has been the focus of many synthetic and medicinal chemistry studies. Recent reports have furthered our understanding of the roles played by the D and E rings of camptothecin in stabilization of the enzyme-DNA-camptothecin ternary complex. Important parameters for further study and optimization include the facility of E-ring lactone hydrolysis and the prospects for replacing the E ring with more stable structures, the role of the 14-CH group in binary complex binding, and the effect of ternary complex dynamics on the expression of cytotoxicity by the camptothecins.

Animals↗

PubMed2004

New lupane triterpenoids from Solidago canadensis that inhibit the lyase activity of DNA polymerase beta.

Bioassay-directed fractionation of a methyl ethyl ketone extract of Solidago canadensis L. (Asteraceae), using an assay to detect the lyase activity of DNA polymerase beta, resulted in the isolation of the four new lupane triterpenoids 1-4 and the seven known compounds lupeol, lupeyl acetate, ursolic acid, cycloartenol, cycloartenyl palmitate, alpha-amyrin acetate, and stigmasterol. The structures of the new compounds were established as 3beta-(3R-acetoxyhexadecanoyloxy)-lup-20(29)-ene (1), 3beta-(3-ketohexadecanoyloxy)-lup-20(29)-ene (2), 3beta-(3R-acetoxyhexadecanoyloxy)-29-nor-lupan-20-one (3), and 3beta-(3-hetohexadecanoyloxy)-29-nor-lupan-20-one (4), respectively, on the basis of extensive 1D and 2D NMR spectroscopic interpretation and chemical modification studies. All 11 compounds were inhibitory to the lyase activity of DNA polymerase beta.

DNA Polymerase beta↗

PubMed2004

Synthesis and topoisomerase I inhibitory properties of luotonin A analogues.

Luotonin A, a naturally occurring pyrroloquinazolinoquinoline alkaloid, has been previously demonstrated to be a topoisomerase I poison. A number of luotonin A derivatives have now been prepared through the condensation of anthranilic acid derivatives and 1,2-dihydropyrrolo[3,4-b]quinoline-3-one in the presence of phosphorus oxychloride. When dichloromethane was used as solvent the reaction proceeded to a single product. In contrast when the reaction was carried out in tetrahydrofuran or in phosphorus oxychloride, an additional isomeric product was obtained. The luotonin A analogues were evaluated for their ability to effect stabilization of the covalent binary complex formed between human topoisomerase I and DNA, and for cytotoxicity toward a yeast strain expressing the human topoisomerase I.

DNA↗

PubMed2004

Plant sterols as selective DNA polymerase beta lyase inhibitors and potentiators of bleomycin cytotoxicity.

In a survey of crude plant extracts for DNA polymerase beta lyase inhibitors, the hexanes extracts of Cladogynus orientalis, Hymenache donacifolia, and Heteropsis integerrima, and the methyl ethyl ketone extract of Acacia pilispina were found to exhibit good inhibition of the dRP lyase activity of DNA polymerase beta. Bioassay-guided fractionation of these extracts led to the isolation of three DNA polymerase beta lyase inhibitory phytosterols, namely stigmasterol (1) and beta-sitosterol (2), isolated from the hexanes extracts, and beta-sitosterol-beta-d-glucoside (3), isolated from the methyl ethyl ketone extract. Compounds 1-3 inhibited the DNA polymerase beta lyase activity with IC(50) values of 43.6, 43.3, and 72.4 microM, respectively. Compounds 1 and 2 were found capable of potentiating the action of bleomycin in cultured human tumor cells, consistent with the possibility that lyase inhibitors may find utility in vivo.

Base Sequence↗

PubMed2004

Phenolic acid amides: a new type of DNA strand scission agent from Piper caninum.

In a survey of the active components of crude plant extracts for their ability to cleave DNA, a crude extract prepared from Piper caninum was found to induce the relaxation of supercoiled pBR322 plasmid DNA in the presence of Cu(2+). Bioassay-guided fractionation was carried out on this extract, guided by an in vitro DNA strand scission assay. Three active principles were isolated and identified as N-cis-feruloyl tyramine (1),N-trans-feruloyl tyramine (2), and 1-cinnamoylpyrrolidine (3). Compounds 1-3 represent a structurally new type of DNA strand scission agent.

Amides↗