F. Vinyl Sulfones and Other Michael Acceptors 4683 G. Azodicarboxamides 4695 IV. Acylating Agents 4695 A. Aza-peptides 4695 B. Carbamates 4699 C. Peptidyl Acyl Hydroxamates 4700 D. â-Lactams and Related Inhibitors 4704 E. Heterocyclic Inhibitors 4714 1. Isocoumarins 4715 2. Benzoxazinones 4722 3. Saccharins 4725 4. Miscellaneous Heterocyclic Inhibitors 4728 V. Phosphonylation Agents 4728 A. Peptide Phosphonates 4728 B. Phosphonyl Fluorides 4734 VI. Sulfonylating Agents 4735 A. Sulfonyl Fluorides 4735 VII. Miscellaneous Inhibitors 4736 VIII. Summary and Perspectives 4737 IX. Acknowledgments 4740 X. Note Added in Proof 4740 XI. References 4740

Irreversible inhibitors of serine, cysteine, and threonine proteases.

The proteasome regulates cellular processes as diverse as cell cycle progression and NF-kappaB activation. In this study, we show that the potent antitumor natural product epoxomicin specifically targets the proteasome. Utilizing biotinylated-epoxomicin as a molecular probe, we demonstrate that epoxomicin covalently binds to the LMP7, X, MECL1, and Z catalytic subunits of the proteasome. Enzymatic analyses with purified bovine erythrocyte proteasome reveal that epoxomicin potently inhibits primarily the chymotrypsin-like activity. The trypsin-like and peptidyl-glutamyl peptide hydrolyzing catalytic activities also are inhibited at 100- and 1,000-fold slower rates, respectively. In contrast to peptide aldehyde proteasome inhibitors, epoxomicin does not inhibit nonproteasomal proteases such trypsin, chymotrypsin, papain, calpain, and cathepsin B at concentrations of up to 50 microM. In addition, epoxomicin is a more potent inhibitor of the chymotrypsin-like activity than lactacystin and the peptide vinyl sulfone NLVS. Epoxomicin also effectively inhibits NF-kappaB activation in vitro and potently blocks in vivo inflammation in the murine ear edema assay. These results thus define epoxomicin as a novel proteasome inhibitor that likely will prove useful in exploring the role of the proteasome in various in vivo and in vitro systems.

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Epoxomicin, a potent and selective proteasome inhibitor, exhibits in vivo antiinflammatory activity

Although the protein synthesis inhibitor cycloheximide (CHX) has been known for decades, its precise mechanism of action remains incompletely understood. The glutarimide portion of CHX is seen in a family of structurally related natural products including migrastatin, isomigrastatin and lactimidomycin (LTM). We found that LTM, isomigrastatin and analogs have a potent antiproliferative effect on tumor cell lines and selectively inhibit translation. A systematic comparative study of the effects of CHX and LTM on protein synthesis revealed both similarities and differences between the two inhibitors. Both LTM and CHX were found to block the translocation step in elongation. Footprinting experiments revealed protection of a single cytidine nucleotide (C3993) in the E-site of the 60S ribosomal subunit, thus defining a common binding pocket for the two inhibitors in the ribosome. These results shed new light on the molecular mechanism of inhibition of translation elongation by both CHX and LTM.

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Inhibition of eukaryotic translation elongation by cycloheximide and lactimidomycin

Understanding translational control in gene expression relies on precise and comprehensive determination of translation initiation sites (TIS) across the entire transcriptome. The recently developed ribosome-profiling technique enables global translation analysis, providing a wealth of information about both the position and the density of ribosomes on mRNAs. Here we present an approach, global translation initiation sequencing, applying in parallel the ribosome E-site translation inhibitors lactimidomycin and cycloheximide to achieve simultaneous detection of both initiation and elongation events on a genome-wide scale. This approach provides a view of alternative translation initiation in mammalian cells with single-nucleotide resolution. Systemic analysis of TIS positions supports the ribosome linear-scanning mechanism in TIS selection. The alternative TIS positions and the associated ORFs identified by global translation initiation sequencing are conserved between human and mouse cells, implying physiological significance of alternative translation. Our study establishes a practical platform for uncovering the hidden coding potential of the transcriptome and offers a greater understanding of the complexity of translation initiation.

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Global mapping of translation initiation sites in mammalian cells at single-nucleotide resolution.

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.

Bleomycin: new perspectives on the mechanism of action.

Streptomyces hygroscopicus No. P247-71 (ATCC 53709) produced a novel antibiotic eponemycin which exhibited specific in vivo antitumor effect against B16 melanoma. Structural studies assigned (4S)-1, 2-epoxy-2-hydroxymethyl-4-(N-isooctanoyl-L-serylamino)-6-methylhept-6-ene-3-one to eponemycin which is unrelated to the known antitumor antibiotics.

Eponemycin, a new antibiotic active against b16 melanoma i. production, isolation, structure and biological activity

Streptomyces amphibiosporus R310-104 (ATCC 53964) produced a novel antibiotic lactimidomycin which showed inhibitory activity against fungi and prolonged the survival time of mice transplanted with experimental tumors. Structural studies clarified that lactimidomycin is a new glutarimide antibiotic having a unique unsaturated 12-membered lactone ring.

Lactimidomycin, a new glutarimide group antibiotic. Production, isolation, structure and biological activity.

The unusual actinomycetes strain No. E465-94 produced a complex of new glycopeptide antibiotics tallysomycin, which was separated by CM-Sephadex chromatography into two major components, A (C68H107N21O27S2) and B (C62H95N19O26S2). They were isolated first in a copperchelated form and showed physico-chemical properties similar to those of the bleomycingroup of antibiotics. Tallysomycin exhibited broad antibacterial and antifungal activity, and was highly active in vivo against bacterial infections in mice. Tallysomycins A and B demonstrated potent activity in the prophage induction of lysogenic bacteria.

Tallysomycin, a new antitumor antibiotic complex related to bleomycin

Melanostatin, a new antibiotic with melanin synthesis inhibitor activity, was isolated from the fermentation broth of Streptomyces clavifer No. N924-2. Its structure was determined by spectral analysis and degradation experiments. Melanostatin strongly inhibited melanin formation in Streptomyces bikiniensis NRRL B-1049 and B16 melanoma cells.

Melanostatin, a new melanin synthesis inhibitor. Production, isolation, chemical properties, structure and biological activity.

Pradimicin FA-1 was produced via directed biosynthesis with substitution of D-serine for D-alanine in the 15-position of pradimicin A. This substitution was achieved by the addition of D-serine to the culture medium of Actinomadura hibisca P157-2. Likewise, pradimicin FA-2 was co-produced along with pradimicin FA-1 when the pradimicins A and C producing strain, A. hibisca A2493 was grown in D-serine-supplemented medium. The new pradimicin analogs share a common core structure of 5,6-dihydrobenzo[a]naphthacenequinone substituted by D-serine at C-15, but differ in the disaccharide moiety at C-5. Pradimicin FA-1 has an N-methylamino sugar and D-xylose. Pradimicin FA-2 is the des-N-methyl analog of pradimicin FA-1. The in vitro and in vivo antifungal activity of the analogs was comparable to that of pradimicin A.

Masami Hatori

Papers

Eponemycin, a new antibiotic active against b16 melanoma i. production, isolation, structure and biological activity

Lactimidomycin, a new glutarimide group antibiotic. Production, isolation, structure and biological activity.

Tallysomycin, a new antitumor antibiotic complex related to bleomycin

Melanostatin, a new melanin synthesis inhibitor. Production, isolation, chemical properties, structure and biological activity.

New antifungal antibiotics pradimicins FA-1 and FA-2: D-serine analogs of pradimicins A and C.