This review presents recommended nomenclature for the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), a rapidly growing class of natural products. The current knowledge regarding the biosynthesis of the >20 distinct compound classes is also reviewed, and commonalities are discussed.

The efficient total synthesis of the recently described natural substance largazole (1) and it’s active metabolite largazole thiol (2) is described. The synthesis required eight linear steps and proceeded in 37% overall yield. It is demonstrated that largazole is a pro-drug, that is activated by removal of the octanoyl residue from the 3-hydroxy-7-mercaptohept-4-enoic acid moiety to generate the active metabolite largazole thiol (2) which is an extraordinarily potent Class I histone deacetylase inhibitor. Synthetic largazole and the largazole thiol (2) have been evaluated side-by-side with FK228 and SAHA for inhibition of HDACs 1, 2, 3, and 6. Largazole and largazole thiol were further assayed for cytotoxic activity against a panel of chemoresistant melanoma cell lines and it was found that largazole is substantially more cytotoxic than largazole thiol; this difference being attributed to differences in cell permeability of the two substances, respectively.

Sactipeptides are ribosomally synthesized peptides that contain a characteristic thioether bridge (sactionine bond) that is installed posttranslationally and is absolutely required for their antibiotic activity. Sactipeptide biosynthesis requires a unique family of radical SAM enzymes, which contain multiple [4Fe-4S] clusters, to form the requisite thioether bridge between a cysteine and the α-carbon of an opposing amino acid through radical-based chemistry. Here we present the structure of the sactionine bond-forming enzyme CteB, from Clostridium thermocellum ATCC 27405, with both SAM and an N-terminal fragment of its peptidyl-substrate at 2.04 Å resolution. CteB has the (β/α)-TIM barrel fold that is characteristic of radical SAM enzymes, as well as a C-terminal SPASM domain that contains two auxiliary [4Fe-4S] clusters. Importantly, one [4Fe-4S] cluster in the SPASM domain exhibits an open coordination site in absence of peptide substrate, which is coordinated by a peptidyl-cysteine residue in the bound state. The crystal structure of CteB also reveals an accessory N-terminal domain that has high structural similarity to a recently discovered motif present in several enzymes that act on ribosomally synthesized and post-translationally modified peptides (RiPPs), known as a RiPP precursor peptide recognition element (RRE). This crystal structure is the first of a sactionine bond forming enzyme and sheds light on structures and mechanisms of other members of this class such as AlbA or ThnB.

Fourteen analogs of the marine natural product largazole have been prepared and assayed against HDACs 1,2, 3, and 6. Olefin cross-metathesis was used to efficiently access six variants of the side-chain zinc-binding domain, while adaptation of our previously reported modular synthesis allowed probing of the macrocyclic cap group

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Thiolutin is a disulfide-containing antibiotic and anti-angiogenic compound produced by Streptomyces. Its biological targets are not known. We show that reduced thiolutin is a zinc-chelator that inhibits the JAB1/MPN/Mov34 (JAMM) domain-containing metalloprotease Rpn11, a de-ubiquinating enzyme of the 19S proteasome. Thiolutin also inhibits the JAMM metalloproteases Csn5, the deneddylase of the COP9 signalosome, Associated-molecule-with-the-SH3-Domain-of-STAM (AMSH), which regulates ubiquitin-dependent sorting of cell-surface receptors, and Brcc36, a K63-specific deubiquitnase of BRCC36-containing isopeptidase complex (BRISC) and BRCA1-BRCA2-containing complex (BRCC). We provide evidence that other dithiolopyrrolones also function as inhibitors of JAMM metalloproteases.

Bacillus cereus ATCC 14579 converts the C-terminal 14 residues of a 52-mer prepeptide into a related set of eight variants of the thiocillin subclass of thiazolyl peptide antibiotics by a cascade of posttranslational modifications that alter 13 of those 14 residues. We have introduced prepeptide gene variants into a knockout strain to conduct an alanine scan of all 14 progenitor residues, as well as a serine scan of the six cysteine residues that are converted to thiazoles in the mature natural product. No mature scaffolds were detected for the S1A and S10A mutants, consistent with their roles as the source of the pyridine core. In both the alanine and serine scans, only one substitution mutant failed to produce a mature scaffold: cysteine 11. Cysteine to serine mutants gave mixture of dehydrations, aromatizations, and unaltered alcohol side chains depending on position. Overall, substitutions that altered the trithiazolylpyridine core or reduced the conformational rigidity of the 26-membered macrocyclic loop led to loss of antibiotic activity. In total, 21 peptide mutants were cultured, from which production of 107 compounds was observed and 94 compounds, representing 17 structural mutants were assayed for antibiotic activity. High-resolution NMR solution structures were determined for one mutant and one wild type compound. These structures demonstrate that the tight conformational rigidity of the natural product is severely disrupted by loss of even a single heterocycle, perhaps accounting for the attendant loss of activity in such mutants.

The peptide isosteres (10 and 11) of the naturally occurring and potent histone deacetylase (HDAC) inhibitors FK228 and largazole have been synthesized and evaluated side-by-side with FK228, largazole and SAHA for inhibition of the class I HDACs 1, 2, 3, and 6.Histone deacetylase (HDAC) inhibitors are under intense investigation as targeted therapies for the treatment of cancer. 1 The zinc-dependent, class I hydrolases (HDAC1,2,3,8) are implicated in numerous events associated with malignant transformation, notably epigenetic maintenance of the malignant phenotype. Numerous small-molecule HDAC inhibitors have been identified in nature or developed with medicinal chemistry. Several naturally occurring and synthetic HDAC inhibitors are shown in Figure 1 and include SAHA (1), 2 trichostatin A (2), 2 apicidin (3), 4 trapoxin B (4), 3,4 azumamide A (5), 5-7 FK228 (6), [8][9][10] and FR901375 (7), [8][9][10] spiruchostatins (8), 11 and largazole (9).12 -14 A common pharmacophore model has been used to describe this set of seemingly diverse chemical probes: (1) a metal binding domain, which interacts with the active-site Zn 2+ , (2) a predominantly hydrophobic linking group, Correspondence to: James E. Bradner. james_bradner@dfci.harvard.edu . Supporting Information Available. Spectroscopic data and experimental details for the preparation of all new compounds as well as procedures for the biochemical HDAC assay used in these experiments are provided. This material is available free of charge via the internet at http://pubs.acs.org. Pymol sessions of the conformations and docked enzyme-ligand complexes are available from the authors upon request. NIH Public AccessAuthor Manuscript J Am Chem Soc. Author manuscript; available in PMC 2010 June 4. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript which occupies the channel of the enzyme and mimics the histone protein N-acetyl-lysine side chain, and (3) a surface recognition domain. 15 The opportunity to study structure-activity relationships among HDAC inhibitors with more granularity has recently caught our attention. We specifically have become interested in the analogous depsipeptide natural products, FK228 (6) and largazole (9). The unique, remarkable HDAC inhibition exhibited by these ligands suggests that the complex, macrocyclic capping region likely confers high potency surface interactions. We sought to explore this hypothesis in comparing the biochemical activity and predicted conformational space of these two natural products with their corresponding peptide isosteres.FK228 (6) was originally isolated from the fermentation broth of Chromobacterium violaceum (No. 968) by Fujisawa Pharmaceutical Co., Ltd. in 1991 along with the closely related depsipeptide, FR901375 (7). The unique structural features of FK228 include a 16-membered cyclic depsipeptide bridged by a fifteen-membered macrocyclic disulfide ring. The D-cysteine residue of the peptide portion enables FK228 to act as a pro-drug, in which the disulfide bond is reduced in v...