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.

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 stemand-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.calicheamicin ͉ esperamicin ͉ assay ͉ molecular beacon ͉ bleomycin

Two new naphthoquinone macrolides, hygrocins A (1) and B (2), were isolated from the fermentation broth of Streptomyces hygroscopicus. Hygrocin A is not stable due to the presence of an active methylene group (C-22), which undergoes intramolecular aldol condensation with the quinone ring to yield a gamma-lactam derivative, 6. Its structural elucidation was achieved by chemical conversion to 3, an unusual diazomethane derivative, and confirmed by its alkaline hydrolysis product 4, hydrogenation derivative 5, and "degradation" product 6. The structure of hygrocin B was determined by combined chemical and spectroscopic methods.

Deregulated phosphatidylinositol 3-kinase (PI3K) signaling pathway is widely implicated in tumor growth and resistance to chemotherapy. While a strong rationale exists for pharmacological targeting of PI3K, only a few proof-of-principle in vivo efficacy studies are currently available. PWT-458, pegylated-17-hydroxywortmannin, is a novel and highly potent inhibitor of PI3K in animal models. Upon in vivo cleavage of its poly(ethyleneglycol) (PEG), PWT-458 releases its active moiety 17-hydroxywortmannin (17-HWT), the most potent inhibitor in its class. Here we show that a single intravenous injection of PWT-458 rapidly inhibited PI3K signaling, as measured by a complete loss of AKT (Ser-473) phosphorylation in xenograft tumors grown in nude mice. Following a daily X5 dosing regimen, PWT-458 demonstrated single-agent antitumor activity in nude mouse xenograft models of U87MG glioma, nonsmall cell lung cancer (NSCLC) A549, and renal cell carcinoma (RCC) A498. Efficacious doses ranged from 0.5 mg/kg to 10 mg/kg, achieving a superior therapeutic index over 17-HWT. PWT-458 augmented anticancer efficacy of a suboptimal dose of paclitaxel against A549 and U87MG tumors. Combination treatment of PWT-458 and an mTOR inhibitor, Pegylated-Rapamycin (Peg-Rapa), resulted in an enhanced antitumor efficacy in U87MG. Finally, PWT-458 in combination with interferon-alpha (Intron-A) caused a dramatic regression of RCC A498, which was not achieved by either agent alone. These studies identify PWT-458 as an effective anticancer agent and provide strong proof-of-principle for targeting the PI3K pathway as novel anticancer therapy.

Hyperactivation of the PI3K/AKT/mTOR signaling pathway is common in cancer, and PI3K and mTOR act synergistically in promoting tumor growth, survival, and resistance to chemotherapy. Thus, combined targeting of PI3K and mTOR presents an opportunity for robust and synergistic anticancer efficacy. 17-Hydroxywortmannin (2a) analogues conjugated to rapamycin (3a) analogues via a prodrug linker are uniquely positioned for this approach. Our efforts led to the discovery of diester-linked conjugates that, upon in vivo hydrolysis, released two highly potent inhibitors. Conjugate 7c provided enhanced solubility relative to 3a and to an equivalent mixture of 3a and 9a and demonstrated profound activity in U87MG mouse xenografts, achieving an MED of 1.5 mg/kg, following weekly intravenous dosing. At 15 mg/kg, 7c completely inhibited the growth of HT29 tumors, whereas an equivalent mixture of the inhibitors was poorly tolerated. In the A498 renal tumor model, 7c exhibited superior efficacy over 3a or 9a when administered as a single agent or in combination with bevacizumab. Thus, we have uncovered a novel approach to target both PI3K and mTOR via hybrid inhibitors, leading to a broader and more robust anticancer efficacy.