Identification and localization of the gene cluster encoding biosynthesis of the antitumor macrolactam leinamycin in Streptomyces atroolivaceus S-140

Leinamycin (LNM), produced by Streptomyces atroolivaceus, is a thiazole-containing hybrid peptide-polyketide natural product structurally characterized with an unprecedented 1,3-dioxo-1,2-dithiolane moiety that is spiro-fused to a 18-member macrolactam ring. LNM exhibits a broad spectrum of antimicrobial and antitumor activities, most significantly against tumors that are resistant to clinically important anticancer drugs, resulting from its DNA cleavage activity in the presence of a reducing agent. Using a PCR approach to clone a thiazole-forming nonribosomal peptide synthetase (NRPS) as a probe, we localized a 172-kb DNA region from S. atroolivaceus S-140 that harbors the lnm biosynthetic gene cluster. Sequence analysis of 11-kb DNA revealed three genes, lnmG, lnmH, and lnmI, and the deduced product of lnmI is characterized by domains characteristic to both NRPS and polyketide synthase (PKS). The involvement of the cloned gene cluster in LNM biosynthesis was confirmed by disrupting the lnmI gene to generate non-LNM-producing mutants and by characterizing LnmI as a hybrid NRPS-PKS megasynthetase, the NRPS module of which specifies for L-Cys and catalyzes thiazole formation. These results have now set the stage for full investigations of LNM biosynthesis and for generation of novel LNM analogs by combinatorial biosynthesis.     

Aminyl and iminyl radicals from arylhydrazones in the photo-induced DNA cleavage

Photolytic cleavage of the nitrogen-nitrogen single bond in benzaldehyde phenylhydrazones produced aminyl (R2N*) and iminyl (R2C=N*) radicals. This photochemical property was utilized in the development of hydrazones as photo-induced DNA-cleaving agents. Irradiation with 350 nm UV light of arylhydrazones bearing substituents of various types in a phosphate buffer solution containing the supercoiled circular phiX174 RFI DNA at pH 6.0 resulted in single-strand cleavage of DNA. Attachment of the electron-donating OMe group to arylhydrazones increased their DNA-cleaving activity. Results from systematic studies indicate that both the aminyl and the iminyl radicals possessed DNA-cleaving ability.     

Covalent modification and single-strand scission of DNA by a new antitumor antibiotic kapurimycin A3

Kapurimycin A3 is a new antitumor antibiotic isolated from a Streptomyces. It contains the anthrapyrone skeleton and a beta,gamma-unsaturated delta-keto carboxylic acid moiety in the structure. In vitro, kapurimycin causes single-strand cleavage of supercoiled pBR322 DNA. The diminished cytotoxicity and DNA cleaving activity for 13-decarboxykapurimycin A3 indicates that the beta, gamma-unsaturated delta-keto carboxylic acid moiety is important for the activity of kapurimycin. Kapurimycin A3 binds to calf thymus DNA at 4 degrees C, and the thermal treatment of this adduct results in release of a guanine covalently attached to C-16 of kapurimycin via one of its nitrogen atoms. Thus, the epoxide is the alkylating functional group of kapurimycin, and this is consistent with the lack of DNA cleaving and cytotoxic activities for 14,16-deoxy-14,16-dihydroxykapurimycin. These findings have revealed that DNA strand scission by kapurimycin is due to the alkylation of guanine by ring opening of the epoxide group of kapurimycin, depurination of modified guanine, and presumably subsequent hydrolysis of the phosphate ester backbone at the resultant apurinic sites.     

Synthesis and Biological Activity of 1,2-Dithiolanes and 1,2-Dithianes Bearing a Nitrogen-containing Substituent

A series of 1,2-dithiolanes, 1,2-dithianes and related compounds bearing a nitrogen-containing substituent were synthesized and their pesticidal activity was tested. A new general synthetic route to 1,2-dithiolanes was established from 1,3-diols. A variation in the position and character of the nitrogen atom is shown to be allowable to some extent for promoting insecticidal activity, unlike the case of sulfur atoms. Most compounds showed acaricidal activity, the strongest being displayed by cis-3,5-bis(dimethylaminomethyl)-1,2-dithiolane.     

DNA alkylation by leinamycin can be triggered by cyanide and phosphines.

Previous work has shown that alkylation of DNA by the antitumor agent leinamycin (1) is potentiated by reaction of the antibiotic with thiols. Here, it is shown that other soft nucleophiles such as cyanide and phosphines can also trigger DNA alkylation by leinamycin. Overall, the results suggest that reactions of cyanide and phosphines with leinamycin produce the oxathiolanone intermediate (2), which is known to undergo rearrangement to the DNA-alkylating episulfonium ion 4.     

Thiol-dependent DNA cleavage by 3H-1,2-benzodithiol-3-one 1,1-dioxide

Hydrodisulfides (RSSH) have previously been implicated as key intermediates in thiol-triggered oxidative DNA damage by the antitumor agent leinamycin. In an effort to better understand DNA damage by RSSH and to expand on the number and type of chemical systems that produce this reactive intermediate, the ability of 3H-1,2-benzodithiol-3-one 1,1-dioxide (11) to serve as a thiol-dependent DNA cleaving agent has been investigated. The findings reported here indicate that reaction of 11 with thiols results in release of RSSH and subsequent oxidative DNA strand cleavage.     

Escherichia coli RecA promotes strand invasion with cisplatin-damaged DNA

The antitumor drug cisplatin causes intrastrand cross-linking of adjacent guanine residues that severely distorts the DNA backbone. These DNA adducts impede the progress of the replisome and may result in replication fork arrest. In Escherichia coli, the response to cisplatin involves the action of the prototypic recombinase RecA. Here we show that RecA can utilize, albeit at reduced levels, oligonucleotides that bear site-specific cisplatin-induced 1,2 d(GpG) intrastrand cross-links in strand invasion reactions. Binding of RecA to cisplatin-damaged oligonucleotides was not affected, indicating that the impediment was in the pairing step. The cognate E. coli single-strand DNA-binding protein specifically stimulated strand invasion particularly with cisplatin-damaged DNA. These results indicate that RecA is capable of processing the major cisplatin-induced lesion via a recombination mechanism.     

Design, synthesis, and biological evaluation of new mitonafide derivatives as potential antitumor drugs

A series of potential DNA-binding antitumor agents, 2-[omega-(alkylamino)alkyl]-6-{[omega-(alkylamino)alkyl]amino}-1H-benzo[de]isoquinolin-1,3(2H)-diones and 1,7-bis{6-[(omega-(dimethylamino)alkyl)amino]-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl}-4-methyl-4-azaheptanes, have been prepared as mitonafide derivatives. Their DNA-binding ability and cytotoxic activity have been evaluated. Some of the target compounds have shown high DNA affinity as well as relevant cytotoxic properties.     

DNA unwinding protein from meiotic cells of Lilium.

An ATP-dependent DNA unwinding protein is present at a high level of activity in meiotic cells of lilies. The protein also acts as a DNA-dependent ATPase, the single strand form being the preferred cofactor. It binds in the absence of ATP to single-strand DNA and to ends or nicks in duplex DNA. A 3'-OH terminus is required for binding at duplex ends; such binding is highly stable. Unwinding occurs in the presence of ATP, and it is limited to about 50 base pairs per end or 400-500 base pairs per nick. The ATP hydrolyzed during unwinding is distinguishable from ATP hydrolysis in the presence of single-strand DNA.     

Synthesis and antitumor activity of novel C-8 ester derivatives of leinamycin

A novel series of C-8 ester derivatives of leinamycin are described. Condensation of N-substituted amino acids or carboxylic acids containing polyether moiety with leinamycin resulted in the C-8 ester derivatives with good antitumor activity in several experimental models. Among these derivatives, compound 4e, which has five ethylene glycol ether units in the C-8 acyl group, showed potent antitumor activity against human tumor xenograft. Combination with the modification of the dithiolanone moiety was applied to these C-8 ester derivatives and some of them also showed good antitumor activity.     

Synthesis and characterization of a small analogue of the anticancer natural product leinamycin

Leinamycin (1) is a Streptomyces-derived natural product that displays nanomolar IC₅₀ values against human cancer cell lines. In the work described here, we report the synthesis and characterization of a small leinamycin analogue 19 that closely resembles the ‘upper-right quadrant’ of the natural product, consisting of an alicyclic 1,2-dithiolan-3-one 1-oxide heterocycle connected to an alkene by a two-carbon linker. The results indicate that this small analogue contains the core set of functional groups required to enable thiol-triggered generation of both redox active polysulfides and an episulfonium ion intermediate via the complex reaction cascade first seen in the natural product leinamycin. The small leinamycin analogue 19 caused thiol-triggered oxidative DNA strand cleavage in a manner similar to the natural product, but did not alkyate duplex DNA effectively. This highlights the central role of the 18-membered macrocycle of leinamycin in driving efficient DNA alkylation by the natural product.     

Raman spectroscopy of DNA modified by intrastrand cross-links of antitumor cisplatin

Raman spectroscopy was employed to characterize the perturbations to DNA conformation induced in DNA by two different intrastrand adducts of antitumor cis-diamminedichloroplatinum(II) (cisplatin), namely by its 1,2-GG or 1,3-GTG intrastrand cross-links. We examined short deoxyribooligonucleotide duplexes containing single, site-specific cross-link by Raman spectroscopy and assigned the spectral alterations to conformational changes induced in DNA by 1,2-GG or 1,3-GTG intrastrand CLs determined earlier by other biochemical and biophysical methods. The results confirmed significant perturbations to the B-form DNA backbone due to the intrastrand lesions and that several nucleotides changed their conformation from C2'-endo to C3'-endo. Evidence for a partial transition from B- to A-form was found in several regions of the Raman spectra as well. The spectra also confirmed the different and more extensive distortion induced in B-DNA by 1,3-GTG in comparison with 1,2-GG intrastrand CLs, consistent with their already known high resolution structures. The results of the present work demonstrate that Raman spectroscopy represents a suitable tool to provide insights into structural factors involved in the mechanisms underlying antitumor effects of platinum drugs.     

Evaluation of Solid Substrates for the Biosynthesis of Cellulase by Trichoderma viride

A series of 4-alkylthio-1,2-dithiolanes were synthesized from S,S′-(2-alkylthiotrimethylene) di(benzenethiosulfonates), and their biological activities were tested on Culex pipience molestus, Laodelphax striatellus and Tetranychus urticae. These compounds showed potent activity against both insect species, the strongest being displayed by 4-ethylthio-1,2-dithiolane and 4-isopropylthio-1,2-dithiolane.     

Cleavage of supercoiled circular double-stranded DNA induced by a eukaryotic cambialistic superoxide dismutase from Cinnamomum camphora

Superoxide dismutase (SOD, EC 1.15.1.1) that protectsorganisms from O2?– toxicity is a family of transitionmetal-containing enzyme existing in all oxygen-consu-ming living beings [1,2]. SOD catalyzes the dismutationof the toxic superoxide anion O2?– int…     

The macrocycle of leinamycin imparts hydrolytic stability to the thiol-sensing 1,2-dithiolan-3-one 1-oxide unit of the natural product

Reaction of cellular thiols with the 1,2-dithiolan-3-one 1-oxide moiety of leinamycin triggers the generation of DNA-damaging reactive intermediates. Studies with small, synthetic analogues of leinamycin reveal that the macrocyclic portion of the natural product imparts remarkable hydrolytic stability to the 1,2-dithiolan-3-one 1-oxide heterocycle without substantially compromising its thiol-sensing property.     

A CT promoter element binding protein: definition of a double-strand and a novel single-strand DNA binding motif.

Numerous genes contain promoter elements that are nuclease hypersensitive. These elements frequently possess polypurine/polypyrimidine stretches and are usually associated with altered chromatin structure. We have previously isolated a clone that binds a class of CT-rich promoter elements. We have further characterized this clone, termed the nuclease-sensitive element protein-1, or NSEP-1. NSEP-1 binds both duplex CT elements and the CT-rich strand of these elements in a 'generic' sequence specific manner and has overlapping but distinct single-and double-strand DNA binding domains. The minimal peptide region sufficient for both duplex and single-strand DNA binding includes two regions rich in basic amino acids flanking an RNP-CS-1 like octapeptide motif. Deletion analysis shows that the single-strand DNA binding activity is mediated by the RNP-CS-1 like octapeptide motif and is the key peptide region necessary for single-strand binding. NSEP-1's affinity for CT rich promoter elements with strand asymmetry in addition to its double- and single-strand DNA binding properties suggests that it may be a member of a class of DNA binding proteins that modulate gene expression by their ability to recognize DNA with unusual secondary structure.     

In vitro lipofection with novel series of symmetric 1,3-dialkoylamidopropane-based cationic surfactants containing single primary and tertiary amine polar head groups

A novel series of symmetric double-chained primary and tertiary 1,3-dialkoylamido monovalent cationic lipids were synthesized and evaluated for their transfection activities. In the absence of the helper lipid DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), only the primary and tertiary dioleoyl derivatives 1,3lmp5 and 1,3lmt5, respectively elicited transfection activity. This is a striking difference between symmetrical 1,2-diacyl glycerol-based monovalent cationic lipids that always found both dioleoyl and dimyristoyl analogues being efficient transfection reagents. In the presence of helper lipid, all cationic derivatives induced marker gene expression, except the dilauroyl analogues 1,3lmp1 and 1,3lmt1 that elicited no transfection activity. Combining electrophoretic mobility data of the lipoplexes at different charge ratios with transfection activity suggested two requirements for high transfection activity with monovalent double-chained cationic lipids, that is, binding/association of the lipid to the plasmid DNA and membrane fusion properties of the lipid layers surrounding the DNA.     

Selective recognition of a cisplatin-DNA adduct by human mismatch repair proteins.

The antitumor agent cis-diamminedichloroplatinum(II) (cisplatin) introduces cytotoxic DNA damage predominantly in the form of intrastrand crosslinks between adjacent purines. Binding assays using a series of duplex oligonucleotides containing a single 1,2 diguanyl intrastrand crosslink indicate that human cell extracts contain factors that preferentially recognise this type of damage when the complementary strand contains T opposite the 3', and C opposite the 5'guanine in the crosslink. Under the conditions of the band-shift assay used, little binding is observed if the positions of the T and C are reversed in the complementary strand. Similarly, duplexes containing CC or TT opposite the crosslink are recognised relatively poorly. The binding activity is absent from extracts of the colorectal carcinoma cell lines LoVo and DLD-1 in which the hMutSalpha mismatch recognition complex is inactivated by mutation. Extensively purified human hMutSalpha exhibits the same substrate preference and binds to the mismatched platinated DNA at least as well as to an identical unplatinated duplex containing a single G.T mismatch. It is likely, therefore, that human mismatch repair may be triggered by 1,2 diguanyl intrastrand crosslinks that have undergone replicative bypass.     

Solution structure of the single-strand break repair protein XRCC1 N-terminal domain.

XRCC1 functions in the repair of single-strand DNA breaks in mammalian cells and forms a repair complex with beta-Pol, ligase III and PARP. Here we describe the NMR solution structure of the XRCC1 N-terminal domain (XRCC1 NTD). The structural core is a beta-sandwich with beta-strands connected by loops, three helices and two short two-stranded beta-sheets at each connection side. We show, for the first time, that the XRCC1 NTD specifically binds single-strand break DNA (gapped and nicked). We also show that the XRCC1 NTD binds a gapped DNA-beta-Pol complex. The DNA binding and beta-Pol binding surfaces were mapped by NMR and found to be well suited for interaction with single-strand gap DNA containing a 90 degrees bend, and for simultaneously making contacts with the palm-thumb of beta-Pol in a ternary complex. The findings suggest a mechanism for preferential binding of the XRCC1 NTD to flexible single-strand break DNA.