Search PubMed⌕ Search

Biomedical subjects

S M Hecht

Publications and source records attributed to S M Hecht.

At least 19 recordsLinked to original sources
PubMed2001

Chemical and structural characterization of the interaction of bleomycin A2 with d(CGCGAATTCGCG)2. efficient, double-strand DNA cleavage accessible without structural reorganization.

A detailed description of the interaction between Fe(II).bleomycin A2 and the Dickerson-Drew dodecamer d(CGCGAATTCGCG)2 is presented. The reaction between bleomycin and this substrate leads to DNA cleavage at two major sites, adenosine5 and cytidine11, and two minor sites, cytidine3 and thymidine8. The pattern and relative intensities of cleavage at these sites was not entirely consistent with what would be predicted based on the preference of the drug for cleavage at the pyrimidines of 5'-GC-3' and 5'-GT-3' sites. Insight into the origins of the apparent alteration of selectivity was provided by examination of the structure of the duplex which had been determined by X-ray crystallography. This indicated that the C4' hydrogens of the two nucleotides located at the strongest cleavage sites, C11 on one strand and A5 on the other, were oriented toward each other in the minor groove. Two-dimensional NMR measurements and molecular dynamics modeling indicated that a metalloBLM could bind to the duplex in an orientation that positioned the metal center roughly equally close to each of these hydrogen atoms. On the basis of this observation, it was proposed that these two residues represented a double-stranded BLM cleavage site. This hypothesis was tested through the study of the BLM-mediated cleavage of the related decamer duplex, d(CGCGAATTCG).d(CGAATTCGCG), as well as the hairpin sequence d(CGCGAATTCGIIIITTTTCCCCCGAATTCGCG). By the use of the hairpin oligonucleotide 32P-labeled alternately at the 5' and 3'-ends, unequivocal evidence was obtained for BLM-mediated double-strand cleavage. Quantitative analysis of the proportion of damage involving double-strand cleavage was effected by the use of the hairpin substrate; for damage initiated at the predominant cleavage site (cytidine31, analogous to cytidine11 in the dodecanucleotide), it is estimated that 43% of all damage leads to double-stranded lesions. The exceptional efficiency of double-strand cleavage observed in this system must reflect the spatial proximity and orientation of the two sugar H's whose abstraction is required to produce double-stranded lesions.

Binding Sites↗
PubMed2001

Studies on the total synthesis of tallysomycin. Synthesis of the threonylbithiazole moiety containing a structurally unique glycosylcarbinolamide.

[structure: see text]. Tallysomycins are glycopeptide antibiotics that were first isolated from fermentation broths of Streptoalloteichus hindustanus. They are structurally related to the bleomycins but contain an additional talose sugar attached via a unique glycosylcarbinolamide linkage. Herein we report the synthesis of a key tallysomycin intermediate that incorporates the glycosylcarbinolamide moiety unique to the tallysomycins.

Anti-Bacterial Agents↗
PubMed2001

Analogues of bleomycin: synthesis of conformationally rigid methylvalerates.

[structure: see text]. Several conformationally rigid analogues of the methylvalerate subunit contained within the linker domain of the antitumor antibiotic bleomycin have been prepared. These compounds have been protected in a fashion suitable for the solid-phase synthesis of bleomycin. Bleomycin congeners containing these analogues should facilitate a more detailed understanding of the nature of the conformational bend that the methylvalerate moiety is thought to impart to the natural product.

Antibiotics, Antineoplastic↗
PubMed2001

Metallobleomycin-mediated cleavage of DNA not involving a threading-intercalation mechanism.

The DNA cleavage properties of metallobleomycins conjugated to three solid supports were investigated using plasmid DNA, relaxed covalently closed circular DNA, and linear duplex DNA as substrates. Cleavage of pBR322 and pSP64 plasmid DNAs by Fe(II).BLM A(5)-CPG-C(2) was observed with efficiencies not dissimilar to that obtained using free Fe(II).BLM A(5). Similar results were observed following Fe(II).BLM A(5)-CPG-C(2)-mediated cleavage of a relaxed plasmid, a substrate that lacks ends or negative supercoiling capable of facilitating strand separation. BLMs covalently tethered to solid supports, including Fe(II).BLM A(5)-Sepharose 4B, Fe(II).BLM A(5)-CPG-C(6), and Fe(II).BLM A(5)-CPG-C(2), cleaved a 5'-(32)P end labeled linear DNA duplex with a sequence selectivity identical to that of free Fe(II).BLM A(5); cleavage predominated at 5'-G(82)T(83)-3' and 5'-G(84)T(85)-3'. To verify that these results could also be obtained using other metallobleomycins, supercoiled plasmid DNA and a linear DNA duplex were employed as substrates for Co(III).BLM A(5)-CPG-C(2). Free green Co(III).BLM A(5) was only about 2-fold more efficient than green Co(III).BLM A(5)-CPG-C(2) in effecting DNA cleavage. A similar result was obtained using Cu(II).BLM A(5)-CPG-C(2) + dithiothreitol. In addition, the conjugated Co.BLM A(5) and Cu.BLM A(5) cleaved the linear duplex DNA with a sequence selectivity identical to that of the respective free metalloBLMs. Interestingly, when supercoiled plasmid DNA was used as a substrate, conjugated Fe.BLM A(5) and Co.BLM A(5) were both found to produce Form III DNA in addition to Form II DNA. The formation of Form III DNA by conjugated Fe.BLM A(5) was assessed quantitatively. When corrected for differences in the intrinsic efficiencies of DNA cleavage by conjugated vs free BLMs, conjugated Fe.BLM A(5) was found to produce Form III DNA to about the same extent as the respective free Fe.BLM A(5), arguing that this conjugated BLM can also effect double-strand cleavage of DNA. Although previous evidence supporting DNA intercalation by some metallobleomycins is convincing, the present evidence indicates that threading intercalation is not a requirement for DNA cleavage by Fe(II).BLM A(5), Co(III).BLM A(5), or Cu(I).BLM A(5).

Animals↗
PubMed2001

Synthesis and DNA cleavage activity of a novel bleomycin A(5) glycoconjugate.

[structure in text] To explore the possibility of modifying bleomycin in a fashion that could alter its physiological distribution in a therapeutic setting, a new analogue of bleomycin has been prepared. This analogue is intended to target the asialoglycoprotein receptor on liver cells. Critically, despite the large C-substituent, the bleomycin conjugate was found to degrade DNA in the same fashion as bleomycin A(5) itself, and with only modestly decreased efficiency.

Bleomycin↗
PubMed2001

Effect of 2'-5' phosphodiesters on DNA transesterification by vaccinia topoisomerase.

Vaccinia topoisomerase forms a covalent DNA-(3'-phosphotyrosyl)-enzyme intermediate at a pentapyrimidine target site 5'-CCCTTp downward arrow in duplex DNA. By introducing single 2'-5' phosphodiesters in lieu of a standard 3'-5' phosphodiester linkage, we illuminate the contributions of phosphodiester connectivity to DNA transesterification. We find that the DNA cleavage reaction was slowed by more than six orders of magnitude when a 2'-5' linkage was present at the scissile phosphodiester (CCCTT(2')p downward arrow(5')A). Thus, vaccinia topoisomerase is unable to form a DNA-(2'-phosphotyrosyl)-enzyme intermediate. We hypothesize that the altered geometry of the 2'-5' phosphodiester limits the ability of the tyrosine nucleophile to attain a requisite, presumably apical orientation with respect to the 5'-OH leaving group. A 2'-5' phosphodiester located to the 3' side of the cleavage site (CCCTTp downward arrowN(2')p(5')N) reduced the rate of transesterification by a factor of 500. In contrast, 2'-5' phosphodiesters at four other sites in the scissile strand (TpCGCCCTpT downward arrowATpTpC) and five positions in the nonscissile strand (3'-GGGpApApTpApA) had no effect on transesterification rate. The DNAs containing 2'-5' phosphodiesters were protected from digestion by exonuclease III. We found that exonuclease III was consistently arrested at positions 1 and 2 nucleotides prior to the encounter of its active site with the modified 2'-5' phosphodiester and that the 2'-5' linkage itself was poorly hydrolyzed by exonuclease III.

Base Sequence↗
PubMed2001

Studies toward the site specific incorporation of sugars into proteins: synthesis of glycosylated aminoacyl-tRNAs.

A series of glycosylated serine derivatives was synthesized from peracetylated sugars and Fmoc-protected serine; these were chemically esterified with the tris-(tetrabutylammonium) salt of pdCpA. The fully protected and deprotected glycosylated aminoacyl pdCpAs were ligated enzymatically to an abbreviated tRNA (tRNA-C(OH)) to provide the title compounds that are key intermediates in the elaboration of glycoproteins using readthrough of a nonsense codon.

Codon, Nonsense↗
PubMed2001

Photoactivated DNA cleavage by compounds structurally related to the bithiazole moiety of bleomycin.

The syntheses of several novel halogenated bithiazoles structurally related to the bithiazole moiety of bleomycin A(5) are described. Also described is the ability of these compounds to mediate photoactivated DNA cleavage. Chlorinated bithiazole analogues were shown to be much more active than an analogous brominated derivative. DNA strand scission activity was strictly light dependent and was accompanied by dechlorination of the bithiazole nucleus, apparently in a stoichiometric fashion. Inhibition of DNA cleavage in the presence of DMSO, as well as photoaddition to 1-octene by both brominated and chlorinated bithiazole derivatives, suggest strongly that the initial step in photoactivated DNA cleavage involves homolysis of the thiazole carbon-halogen bond. The chlorinated bithiazoles were found to mediate sequence selective cleavage of a (32)P-end labeled DNA, although the selectivity observed was not the same as that of bleomycin itself. The implications of this observation are discussed.

Alkenes↗
PubMed2000

Solid-phase synthesis of bleomycin group antibiotics. Elaboration of deglycobleomycin A(5).

The solid-phase syntheses of two deglycobleomycin A(5) analogues were achieved using a commercially available polystyrene resin containing triphenylmethyl-linked spermidine. The final products were deblocked and released from the resin, analyzed, and purified by C(18) reversed phase HPLC and characterized by high-field (1)H NMR spectroscopy and mass spectrometry. The purified products relaxed supercoiled plasmid DNA in a concentration-dependent fashion and to the same extent as authentic material derived from natural BLM A(5).

Bleomycin↗
PubMed2000

Probing the S1/S1' substrate binding pocket geometry of HIV-1 protease with modified aspartic acid analogues.

Aspartates 25 and 125, the active site residues of HIV-1 protease, participate functionally in proteolysis by what is believed to be a general acid-general base mechanism. However, the structural role that these residues may play in the formation and maintenance of the neighboring S1/S1' substrate binding pockets remains largely unstudied. Because the active site aspartic acids are essential for catalysis, alteration of these residues to any other naturally occurring amino acid by conventional site-directed mutagenesis renders the protease inactive, and hence impossible to characterize functionally. To investigate whether Asp-25 and Asp-125 may also play a structural role that influences substrate processing, a series of active site protease mutants has been produced in a cell-free protein synthesizing system via readthrough of mRNA nonsense (UAG) codons by chemically misacylated suppressor tRNAs. The suppressor tRNAs were activated with the unnatural aspartic acid analogues erythro-beta-methylaspartic acid, threo-beta-methylaspartic acid, or beta,beta-dimethylaspartic acid. On the basis of the specific activity measurements of the mutants that were produced, the introduction of the beta-methyl moiety was found to alter protease function to varying extents depending upon its orientation. While a beta-methyl group in the erythro orientation was the least deleterious to the specific activity of the protease, a beta-methyl group in the threo orientation, present in the modified proteins containing threo-beta-methylaspartate and beta,beta-dimethylaspartate, resulted in specific activities between 0 and 45% of that of the wild type depending upon the substrate and the substituted active site position. Titration studies of pH versus specific activity and inactivation studies, using an aspartyl protease specific suicide inhibitor, demonstrated that the mutant proteases maintained bell-shaped pH profiles, as well as suicide-inhibitor susceptibilities that are characteristic of aspartyl proteases. A molecular dynamics simulation of the beta-substituted aspartates in position 25 of HIV-1 protease indicated that the threo-beta-methyl moiety may partially obstruct the adjacent S1' binding pocket, and also cause reorganization within the pocket, especially with regard to residues Val-82 and Ile-84. This finding, in conjunction with the biochemical studies, suggests that the active site aspartate residues are in proximity to the S1/S1' binding pocket and may be spatially influenced by the residues presented in these pockets upon substrate binding. It thus seems possible that the catalytic residues cooperatively interact with the residues that constitute the S1/S1' binding pockets and can be repositioned during substrate binding to orient the active site carboxylates with respect to the scissile amide bond, a process that likely affects the facility of proteolysis.

Aspartic Acid↗
PubMed2000

Naturally occurring alkylresorcinols that mediate DNA damage and inhibit its repair.

A study of di- and trihydroxyalkylbenzenes and bis(dihydroxyalkylbenzenes) revealed that several compounds were capable of both mediating Cu(2+)-dependent DNA cleavage and strongly inhibiting DNA polymerase beta. The most potent DNA polymerase beta inhibitors were bis(dihydroxyalkylbenzenes) 5 and 6; compounds 3 and 4 were also reasonably potent. The length of the alkyl substituent was found to be a critical element for DNA polymerase beta inhibition, since compounds 1 and 2 had shorter substituents than 3 and were completely inactive. Lineweaver-Burk plots revealed that 3, 4, and 6 exhibited mixed inhibition of DNA polymerase beta with respect to both activated DNA and dTTP. Unsaturated bis(dihydroxyalkylbenzene) 5 was a pure noncompetitive inhibitor with respect to both substrates and associated avidly with the enzyme whether or not it was in complex with its substrate(s). Copper(II)-mediated DNA cleavage was the most pronounced for the trihydroxyalkylbenzene 3, consistent with an earlier report [Singh, U. S., Scannell, R. T., An, H., Carter, B. J., and Hecht, S. M. (1995) J. Am. Chem. Soc. 117, 12691-12699]. Unsaturated bis(dihydroxyalkylbenzene) 5 was the next most active DNA cleaving agent, followed by the dihydroxyalkylbenzene 4. The saturated bis(dihydroxyalkylbenzene) (6) did not cleave DNA well in a cell-free system under the conditions studied but nonetheless potentiated the effects of bleomycin to the greatest extent in cell culture studies. Interestingly, compound 5 produced a reduction in the numbers of viable cells when incubated in the presence of bleomycin and a further reduction in the numbers of viable cells in the presence of both bleomycin and Cu(2+). The same effect was noted to a lesser extent for compound 3 but not for 4 or 6.

Animals↗
PubMed2000

Pentacyclic triterpenoids from Freziera sp. that inhibit DNA polymerase beta.

In a survey of crude plant extracts for DNA polymerase 1 inhibitors, a methyl ethyl ketone extract prepared from Freziera sp. exhibited potent inhibition of DNA polymerase beta. Bioassay-guided fractionation of the extract, guided by an assay to detect DNA polymerase beta inhibition, resulted in the isolation of six active pentacyclic triterpenoids (1-6). These triterpenoids had IC50 values ranging from 7.5 to 16 microM in the presence of bovine serum albumin (BSA) and 2.6-5.8 microM in the absence of BSA, consistent with the possibility that these inhibitors may be of use in vivo.

Animals↗
PubMed2000

Use of COMPARE analysis to discover new natural product drugs: isolation of camptothecin and 9-methoxycamptothecin from a new source.

Analysis of cytotoxicity data of extracts from the National Cancer Institute's Active Repository by the COMPARE protocol was carried out using camptothecin as a reference point. Extracts identified by this process were further characterized by a selective yeast bioassay for inhibitors of topoisomerase I and by a biochemical assay for compounds that stabilize the topoisomerase I-DNA covalent binary complex. Five of the extracts were positive in the yeast bioassay, and eight extracts showed activity on the assay that monitors stabilization of the topoisomerase I-DNA complex. Four of the latter extracts were inactive in the yeast bioassay, and thus would not have been identified as hits without the COMPARE preselection process. One of the extracts, from Pyrenacantha klaineana, was selected for detailed investigation, and fractionation of this extract yielded camptothecin and 9-methoxycamptothecin as the bioactive constituents.

Algorithms↗
PubMed2000

Use of COMPARE analysis to discover functional analogues of bleomycin.

Bleomycin was used as the reference compound in a COMPARE analysis to identify extracts in the National Cancer Institute's Natural Products Repository exhibiting cytotoxicity profiles similar to this antitumor agent. One of the extracts so identified was a CH(2)Cl(2)-methanol extract prepared from Gymnosporia trigyna, which effected relaxation of supercoiled pSP64 DNA in the presence of Cu(2+). Bioassay-guided fractionation using DNA strand-scission activity as an end point resulted in the isolation of four active principles. These were identified as syringaldehyde (1), (-)-syringaresinol (2), (+)-catechin (3), and (+)-epicatechin (4). Compounds 1 and 2 represent a new type of DNA strand-scission agent.

Algorithms↗
PubMed2000

A new 7,8-euphadien-type triterpenoid from Brackenridgea nitida and Bleasdalea bleasdalei that inhibits DNA polymerase beta.

Bioassay-guided fractionation of extracts prepared from Brackenridgea nitida and Bleasdalea bleasdalei, using an assay to detect DNA polymerase beta inhibition, resulted in the isolation of the inhibitory principle, (24E)-3beta-hydroxy-7,24-euphadien-26-oic acid (1), a new euphane triterpenoid. The structure of 1 was established on the basis of HRMS and 1D and 2D NMR spectroscopic methods and was confirmed further by X-ray crystallographic analysis. Compound 1 inhibited rat DNA polymerase beta with an IC(50) value of 23 microM in the presence of bovine serum albumin (BSA) and 9.7 microM in the absence of BSA, consistent with the possibility that 1 may be of utility in vivo. This possibility was further supported by the finding that 1 potentiated the inhibitory action of the anticancer drug bleomycin in cultured P-388D(1) cells, reducing the number of viable cells by 48% when employed at a concentration of 25 microM in the presence of an otherwise nontoxic (75 nM) concentration of bleomycin. Compound 1 is the first euphane-type triterpenoid found to inhibit DNA polymerase beta.

Animals↗
PubMed2000

Isolation and biochemical characterization of a new topoisomerase I inhibitor from Ocotea leucoxylon.

In a continuation of our search for potential tumor inhibitors from plants, we found that a crude extract from Ocotea leucoxylon showed selective activity typical of inhibitors of the enzyme topoisomerase I in a yeast assay for DNA-damaging agents. Using a bioassay-directed fractionation approach, the major bioactive compound was isolated and identified as the known aporphine alkaloid dicentrinone (4); the inactive alkaloid dicentrine (3) was also isolated. Compound 4 showed selective bioactivity against the rad52 repair-deficient yeast strain RS322 (IC(12) 49 microg/mL) and was inactive against the rad52- and topo1-deficient strain RS321 (IC(12) > 2000 microg/mL) and against the repair-proficient strain RJ03 (IC(12) > 2000 microg/mL). Biochemical studies with recombinant human topoisomerase I indicated that dicentrinone (4) is an inhibitor of the human enzyme. Colony formation studies suggest that it is weakly cytotoxic, but that its mechanism of toxicity differs from that of camptothecin and its derivatives.

Aporphines↗
PubMed2000

Bleomycin: new perspectives on the mechanism of action.

The bleomycin group antitumor antibiotics have long been of interest as a consequence of their efficacy in the treatment of certain tumors, not to mention their unique structures and properties in mediating dioxygen activation and sequence selective degradation of DNA. At a chemical level, the structure originally assigned to bleomycin was subsequently reassigned and the new structure has been confirmed by total synthesis. Through the elaboration of structurally modified bleomycin congeners and fragments, synthetic efforts have also facilitated an understanding of the contribution of individual structural domains in bleomycin to sequence selective DNA binding and cleavage, and have also provided insights into the nature of the chemical processes by which DNA degradation takes place. Within the last several years, it has also become apparent that bleomycin can mediate the oxidative degradation of all major classes of cellular RNAs; it seems entirely plausible that RNA may also represent an important locus of action for this class of antitumor agent. In parallel with ongoing synthetic and mechanistic efforts using classical methods, the study of bleomycins attached to solid supports has been shown to provide important mechanistic insights, and the actual elaboration of modified bleomycins by solid phase synthesis constitutes a logical extension of such efforts.

Antibiotics, Antineoplastic↗