Understanding and exploiting nature's chemical arsenal: the past, present and future of calicheamicin research.

The enediyne antitumor antibiotics are appreciated for their novel molecular architecture, their remarkable biological activity and their fascinating mode of action and many have spawned considerable interest as anticancer agents in the pharmaceutical industry. Of equal importance to these astonishing properties, the enediynes also offer a distinct opportunity to study the unparalleled biosyntheses of their unique molecular scaffolds and what promises to be unprecedented modes of self-resistance to highly reactive natural products. Elucidation of these aspects should unveil novel mechanistic enzymology, and may provide access to the rational biosynthetic modification of enediyne structure for new drug leads, the construction of enediyne overproducing strains and eventually lead to an enediyne combinatorial biosynthesis program. This article strives to compile and present the critical research discoveries relevant to the clinically most promising enediyne, calicheamicin, from a historical perspective. Recent progress, particularly in the areas of biosynthesis, self-resistance, bio-engineering analogs and clinical studies are also highlighted.

A continuous assay for DNA cleavage: the application of "break lights" to enediynes, iron-dependent agents, and nucleases.

Although extensive effort has been applied toward understanding the mechanism by which enediynes cleave DNA, a continuous assay for this phenomenon is still lacking. In fact, with the exception of assays for DNase, continuous assays for most DNA cleavage events are unavailable. This article describes the application of "molecular break lights" (a single-stranded oligonucleotide that adopts a stem-and-loop structure and carries a 5'-fluorescent moiety, a 3'-nonfluorescent quenching moiety, and an appropriate cleavage site within the stem) to develop the first continuous assay for cleavage of DNA by enediynes. Furthermore, the generality of this approach is demonstrated by using the described assay to directly compare the DNA cleavage by naturally occurring enediynes [calicheamicin and esperamicin), non-enediyne small molecule agents (bleomycin, methidiumpropyl-EDTA-Fe(II), and EDTA-Fe(II]), as well as the restriction endonuclease BamHI. Given the simplicity, speed, and sensitivity of this approach, the described methodology could easily be extended to a high throughput format and become a new method of choice in modern drug discovery to screen for novel protein-based or small molecule-derived DNA cleavage agents.

Control of intracellular serine protease expression in Bacillus subtilis.

Expression of the major intracellular serine protease (ISP-1) gene of Bacillus subtilis was studied by using a translational fusion plasmid in which the isp promoter region was fused to the lacZ gene. beta-Galactosidase activity, used to measure transcription from the isp promoter, was produced immediately after the end of exponential growth, whereas intracellular protease activity was not detected until 4 h later. These results are consistent with a previous suggestion that ISP-1 initially accumulates in the cell in an enzymatically inactive form. ISP-1 activity was detected in all of the sporulation-deficient strains examined, and the amount of protease activity always corresponded to the amount of beta-galactosidase activity. These results indicate that the activation of ISP-1 is not dependent on a sporulation-specific gene product. Expression of ISP-1 is regulated by a number of mutations known to affect the expression of extracellular enzymes. In sacU(h) and sacQ(h) mutants, the expression of ISP-1 was 10-fold higher than in the wild-type strain. In catA, hpr, and scoC strains, expression of ISP was stimulated two- to threefold, whereas in sacU mutants the expression of ISP-1 was reduced to less than 10% of the wild-type level. The temporal expression and activation of ISP-1 was not affected by any of these mutations. This is the first evidence that the expression of a native intracellular protein is affected by these hyperproduction mutations.

Construction and properties of an intracellular serine protease mutant of Bacillus subtilis.

An intracellular serine protease (ISP-1) mutant of Bacillus subtilis was created by introducing a frameshift into the coding region of the cloned gene. Intracellular protease activity in the mutant was very low, yet sporulation in both nutrient broth and minimal medium was normal. The rate of bulk protein turnover in the mutant was slightly slower than that in the wild-type strain. These results suggest that the gene for ISP-1 is not essential and that ISP-1 is not the major enzyme involved in protein turnover during sporulation.

Protein turnover in Azotobacter vinelandii during encystment and germination.

Protein turnover occurs during differentiation of Azotobacter vinelandii 12837 to the extent of 50% during encystment and 7% during germination. The addition of rifampin at the initiation of encystment prevents encystment and inhibits turnover. In germinating cysts, protein turnover is essential owing to an apparent lack of certain amino acid biosynthetic enzymes. The capacity to synthesize sulfur-containing amino acids from inorganic precursors is regained about halfway through the germination process.

Involvement of the stringent response in degradation of glutamine phosphoribosylpyrophosphate amidotransferase in Bacillus subtilis.

Glutamine phosphoribosylpyrophosphate amidotransferase, the first enzyme of purine biosynthesis, has previously been shown to be rapidly inactivated and degraded in Bacillus subtilis cells at the end of growth. The loss of enzyme activity appears to involve the oxidation of an iron-sulfur cluster in the enzyme. The degradation of the inactive enzyme involves some elements of the stringent response because it is inhibited in relA and relC mutants. Intracellular pools of guanosine tetra- and pentaphosphate were measured by an improved extraction procedure in cells that had been manipulated in various ways to induce or inhibit amidotransferase degradation. The results are consistent with the hypothesis that one or both of these nucleotides stimulates the synthesis of a protein involved in degradation. An elevated level of these nucleotides was not required for the continued degradation of amidotransferase once it had begun.

Degradation of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase in vivo.

Glutamine phosphoribosylpyrophosphate amidotransferase, the first enzyme of purine nucleotide biosynthesis, is inactivated and degraded in Bacillus subtilis during carbon, nitrogen, or amino acid starvation. Amidotransferase is stable in exponentially growing cells, and synthesis of the enzyme ceases prior to its inactivation at the end of exponential growth. Inactivation has been previously shown to result from reaction of an essential [4Fe-4S] center with oxygen. In this work, monospecific antibodies against amidotransferase have been used to demonstrate that inactivation is followed by proteolytic degradation in vivo and that the metabolic requirements for degradation differ from those for inactivation. Unlike inactivation, degradation is inhibited by addition of 10 mM KCN or antibiotic inhibitors of RNA and protein synthesis to glucose-starved cells. The cross-reactive material that accumulates when degradation is inhibited by chloramphenicol initially has native subunit molecular weight, but lower molecular weight polypeptides slowly accumulate. Degradation, but not inactivation, of amidotransferase is strongly inhibited during amino acid starvation of a relA strain. Degradation of amidotransferase is inhibited by pseudomonic acid, an antibiotic that blocks protein synthesis but permits a normal stringent response. This result indicates that both protein synthesis and normal relA gene function are required for degradation.