Lizzy S. Kappen

Neocarzinostatin induction of DNA repair synthesis in HeLa cells and isolated nuclei.

The antitumor antibiotic neocarzinostatin that causes DNA strand breaks in vivo and in vitro is shown to induce DNA repair synthesis in HeLa S3 cells. In the repair assay, the parental DNA was prelabeled with 32P and a density label (bromodeoxyuridine) was introduced into the new synthesized DNA. Quantitation of the repair synthesis as measured by the incorporation of [3H]thymidine into the light parental DNA at varying doses of the drug indicate that there is a significant repair response at low levels of the drug (0.2--0.5 microgram/ml) which cause DNA strand breakage and inhibition of DNA synthesis. In isolated HeLa nuclei neocarzinostatin stimulates the incorporation of dTMP many-fold. This enhancement of dTMP incorporation, which requires the presence of a sulfhydryl agent, is a consequence of the drug-induced DNA strand breakage and is in the parental DNA. These results suggest that an intact cell membrane is not required for DNA strand breakage and its subsequent repair.

Inhibition of globin chain initiation in reticulocyte lysates by pactamycin: accumulation of methionyl-valine.

Abstract In a globin synthesizing system containing reticulocyte lysate and [ 35 S] met-tRNA f , low levels of pactamycin cause an accumulation of radioactivity on the monosomes and small oligosomes concomitant with the breakdown of polysomes. About 50% of the ribosome-bound radioactivity corresponds to methionyl-valine, the initial dipeptide of globin chains, with negligible amounts of tri- or other oligopeptides. This suggests that the site of its action is after the formation of the first peptide bond. The ribosome-bound radioactivity in the presence of sparsomycin, an inhibitor of chain elongation, is in di-, tri- and oligopeptides. Sparsomycin levels (10 −5 M) that cause almost complete inhibition of polypeptide synthesis have only a small inhibitory effect on the pactamycin-induced accumulation of methionyl-valine. Fusidic acid and chlortetracycline do not cause accumulation of any significant amounts of methionyl-valine.

The nature and mechanism of the damage induced in DNA by a protein antibiotic.

NCS, an antitumor protein, that causes DNA strand scissions both in vitro and in vivo lowers the template activity of DNA for DNA polymerase I in vitro. A correlation exists between the extent of strand scission and the degree of inhibition of the polymerase. NCS-induced strand breakage generates non-functional binding sites for the polymerase. By the use of bacterial alkaline phosphatase and exonuclease III it is shown that NCS generates gaps in the DNA that bear phosphomonoester groups at both the 3′- and 5′-ends. In the intact cell possessing the complete system for DNA repair, these lesions in the DNA are repaired to a considerable degree as indicated by the marked stimulation of dTMP incorporation into DNA induced by NCS. Experiments on the stoichiometry of the DNA cutting reaction reveal a zero order reaction with regard to DNA. Possible mechanisms for inducing the lesion described and accounting for the characteristics of the NCS-DNA interaction are discussed.

Molecular basis of action of cytotoxic antibiotics.

Abstract We have described ongoing studies designed to elucidate the mechanisms of action of the cytotoxic antibiotics PM and NCS. PM selectively blocks polypeptide chain initiation in eukaryotes by two effects, which may be related: (1) the joining of the 60S ribosomal subunit to the smaller initiation complex is interfered with. This action can be overcome by adding excess of a factor, obtained by DEAE-cellulose chromatography of a crude initiation factor preparation, that is normally required for joining activity; (2) under conditions where joining is not prevented, there is still a block in the formation of peptides beyond the initial dipeptide. These data can be explained by a model of PM action in which PM does not interfere with the ribosomal joining reaction per se , but prevents the release and reuse of the joining factor, and in so doing, blocks a step in elongation after formation of the initial dipeptide and its translocation to the P-site on the ribosome. It is possible that the block in tripeptide formation is at the level of the use of the ribosomal A-site. The selectivity of PM for polypeptide initiation may be determined by the availability of its binding site only at any early stage in initiation. NCS has been found to inhibit thymidine incorporation into DNA in vivo and to place single stranded nicks in DNA both in vivo and in vitro . While it is premature to relate the cutting of DNA by NCS in vitro to these and other effects of the drug in vivo , the test tube reaction is interesting in its own right and has revealed information on the molecular requirements for the cutting reaction. Mercaptoethanol or some other sulfhydryl compound and a pH of 7.5 are required for maximal cutting. DNA strand scission by NCS produces 5′-phosphoryl end groups on all four deoxyribonucleotides, although in preliminary experiments dGMP and TMP appear to be favored. From protection experiments, it appears that thymidylic acid in DNA is required for interaction between DNA and NCS. How these effects of NCS are related, if at all, to the block at G2 of the cell cycle (45) remains to be elucidated.

Congeners of the Enediyne Neocarzinostatin Chromophore: Designed Agents for bulged Nucleic Acid Targets

Of the commonly recognized structural elements within nucleic acids, bulges are among the least developed as targets for small molecules. Bulges in DNA and RNA have been linked to biomolecular processes involved in numerous diseases, thus probes with affinity for these targets would be of considerable utility to chemical biologists and medicinal chemists. Despite such opportunity, there is a dearth of small molecules available with affinity for bulges, which has hampered exploitation of these key targets. We have used guided chemical synthesis to prepare small molecules capable of binding to DNA and RNA bulges. Our design is based on a template which mimics a metabolite of the enediyne neocarzinostatin. The key spirocylic building block was formed through an intramolecular aldol process and the parent template shows pronounced affinity for 2 base bulges. Functionalization with specific aminosugar moieties confers nanomolar binding affinity for selected bulged DNA targets, and installation of reactive functional groups allows covalent modification of bulges. These rationally designed agents can now be used to study the stereochemistry and architecture of bulge-drug complexes and investigate the molecular biology of bulge induced processes. Members of this class have been shown to induce slipped synthesis of DNA, suggesting the agents, in addition to recognizing and binding to pre-formed bulges, can also induce bulge formation on demand.

Designed DNA probes from the neocarzinostatin family: Impact of glycosyl linkage stereochemistry on bulge base binding

Bulged sites in DNA and RNA have become targets for rational drug design due to their suspected involvement in a number of key biomolecular processes. A lead compound, derived from the enediyne natural product NCS-chrom has been used to inform chemical synthesis of a family of designed probes of DNA bulges, one of which shows 80 nM affinity for a two base bulged target. Key contributors to binding of these spirocyclic compounds have been studied in order to correlate affinity and specificity with structural features. Herein, we demonstrate that the glycosyl linkage stereochemistry of the pendant aminofucosyl group plays a pivotal role in binding, and coupled with insight obtained with various bulged targets, will allow rational design of second generation ligands.

Roles of oxygen and oxygen substitutes in DNA sugar damage by antitumor antibiotics.

Oxidative damage to the deoxyribose backbone of DNA produced by ionizing radiation and antitumor antibiotics (e.g., bleomycin) generally involves oxygen at two stages in the damage process: 1) “reactive oxygen” (OH radical or its equivalent) as the hydrogen atom abstracting species, generating a carbon-centered radical on the deoxyribose, and 2) addition of dioxygen to the latter to “fix” the lesion in a form not readily repaired. By contrast, neocarzinostatin (NCS) is a member of a class of antitumor antibiotics that binds specifically to DNA and is itself converted to a radical species that directly attacks the DNA sugar. Dioxygen or its substitute is involved only after generation of the DNA damage intermediate. This review will focus on the roles of dioxygen and oxygen substitutes, the nitroaromatic radiation sensitizers, in the formation of novel types of oxidative DNA sugar damage by NCS.

DNA as a Target for a Protein Antibiotic: Molecular Basis of Action

The antitumor antibiotic neocarzinostatin (NSC), isolated from the culture filtrates of Streptomyces carzinostaticus variant F-41 (Ishida et al. 1965), is an acidic single-chain polypeptide with a molecular weight of 10,700 (Meienhofer et al. 1972a). The protein has been purified to homogeneity and its amino acid sequence (Fig. 1) (Meienhofer et al. 1972a, b; Maeda et al. 1974; Samy et al. 1977) and physical properties (Maeda et al. 1973; Samy and Meienhofer 1974) have been determined. There are high degrees of homology of some regions of NCS and the protein antibiotics actinoxanthin (Khokhlov et al. 1969, 1976) and macromomycin (Sawyer et al. 1979). NCS exists in a tight, proteolysis-resistant conformation with an antiparallel β-pleated sheet structure (Samy et al. 1974). It possesses two reduction-resistant disulfide bridges and lacks methionine and histidine. The positions of the disulfides have not yet been unambiguously assigned. NCS contains two tryptophan residues in positions 46 (buried) and 79 and one buried tyrosine residue at position 32. Oxidation of tryptophan 79 does not result in loss of biological activity (Samy et al. 1974). Similarly, acylation of the amino groups (alanine 1 and lysine 20) does not affect the activity of NCS (Maeda 1974; Samy 1977). On the other hand, modification of the carboxyl groups results in loss of activity (Samy 1977). Further, spontaneous deamidation of asparagine 83 at a weakly acidic pH generates “preneocarzinostatin” which lacks biological activity (Maeda and Kuromizu 1977). The chemically deamidated compound is thought to be the same as the material isolated from culture filtrates that antagonizes NCS activity (Kikuchi et al. 1974).