Therapies from Thiopeptides.

The first part of this contribution describes solutions that were developed to achieve progressively more efficient syntheses of the thiopeptide natural products, micrococcins P1 and P2 (MP1-MP2), with an eye toward exploring their potential as a source of new antibiotics. Such efforts enabled investigations on the medicinal chemistry of those antibiotics, and inspired the development of the kinase inhibitor, Masitinib®, two candidate oncology drugs, and new antibacterial agents. The studies that produced such therapeutic resources are detailed in the second part. True to the theme of this issue, "Organic Synthesis and Medicinal Chemistry: Two Inseparable Partners", an important message is that the above advances would have never materialized without the support of curiosity-driven, academic synthetic organic chemistry: a beleaguered science that nonetheless has been-and continues to be-instrumental to progress in the biomedical field.

Enrichment of Cysteine-Containing Peptide by On-Resin Capturing and Fixed Charge Tag Derivatization for Sensitive ESI-MS Detection.

High complexity of cell and tissue proteomes limits the investigation of proteomic biomarkers. Therefore, the methods of enrichment of some chemical groups of peptides including thiopeptides are important tools that may facilitate the proteomic analysis by reducing sample complexity and increasing proteome coverage. Here, we present a new method of cysteine-containing tryptic peptide enrichment using commercially available TentaGel R RAM resin modified by the linker containing the maleimide group, allowing thiol conjugation. The captured tryptic peptides containing lysine residue were then tagged by 2,4,6-triphenylpyrylium salt to form 2,4,6-triphenylpyridinium derivatives, which increases the ionization efficiency during mass spectrometry analysis. This makes it possible to conduct an ultrasensitive analysis of the trace amount of compounds. The proposed strategy was successfully applied in the enrichment of model tryptic podocin peptide and podocin tryptic digest.

Selective Modification of Ribosomally Synthesized and Post-Translationally Modified Peptides (RiPPs) through Diels-Alder Cycloadditions on Dehydroalanine Residues.

We report the late-stage chemical modification of ribosomally synthesized and post-translationally modified peptides (RIPPs) by Diels-Alder cycloadditions to naturally occurring dehydroalanines. The tail region of the thiopeptide thiostrepton could be modified selectively and efficiently under microwave heating and transition-metal-free conditions. The Diels-Alder adducts were isolated and the different site- and endo/exo isomers were identified by 1D/2D 1 H NMR. Via efficient modification of the thiopeptide nosiheptide and the lanthipeptide nisin Z the generality of the method was established. Minimum inhibitory concentration (MIC) assays of the purified thiostrepton Diels-Alder products against thiostrepton-susceptible strains displayed high activities comparable to that of native thiostrepton. These Diels-Alder products were also subjected successfully to inverse-electron-demand Diels-Alder reactions with a variety of functionalized tetrazines, demonstrating the utility of this method for labeling of RiPPs.

Recombinant thiopeptides containing noncanonical amino acids.

Thiopeptides are a subclass of ribosomally synthesized and posttranslationally modified peptides (RiPPs) with complex molecular architectures and an array of biological activities, including potent antimicrobial activity. Here we report the generation of thiopeptides containing noncanonical amino acids (ncAAs) by introducing orthogonal amber suppressor aminoacyl-tRNA synthetase/tRNA pairs into a thiocillin producer strain of Bacillus cereus .We demonstrate that thiopeptide variants containing ncAAs with bioorthogonal chemical reactivity can be further postbiosynthetically modified with biophysical probes, including fluorophores and photo-cross-linkers. This work allows the site-specific incorporation of ncAAs into thiopeptides to increase their structural diversity and probe their biological activity; similar approaches can likely be applied to other classes of RiPPs.

Ynamide-Mediated Thiopeptide Synthesis.

Exploration of the full potential of thioamide substitution as a tool in the chemical biology of peptides and proteins has been hampered by insufficient synthetic strategies for the site-specific introduction of a thioamide bond into a peptide backbone. A novel ynamide-mediated two-step strategy for thiopeptide bond formation with readily available monothiocarboxylic acids as thioacyl donors is described. The α-thioacyloxyenamide intermediates formed from the ynamides and monothiocarboxylic acids can be purified, characterized, and stored. The balance between their activity and stability enables them to act as effective thioacylating reagents to afford thiopeptide bonds under mild reaction conditions. Amino acid functional groups such as OH, CONH2 , and indole NH groups need not be protected during thiopeptide synthesis. The modular nature of this strategy enables the site-specific incorporation of a thioamide bond into peptide backbones in both solution and the solid phase.

Thiopeptide antibiotics stimulate biofilm formation in Bacillus subtilis.

Bacteria have evolved the ability to produce a wide range of structurally complex natural products historically called "secondary" metabolites. Although some of these compounds have been identified as bacterial communication cues, more frequently natural products are scrutinized for antibiotic activities that are relevant to human health. However, there has been little regard for how these compounds might otherwise impact the physiology of neighboring microbes present in complex communities. Bacillus cereus secretes molecules that activate expression of biofilm genes in Bacillus subtilis. Here, we use imaging mass spectrometry to identify the thiocillins, a group of thiazolyl peptide antibiotics, as biofilm matrix-inducing compounds produced by B. cereus. We found that thiocillin increased the population of matrix-producing B. subtilis cells and that this activity could be abolished by multiple structural alterations. Importantly, a mutation that eliminated thiocillin's antibiotic activity did not affect its ability to induce biofilm gene expression in B. subtilis. We go on to show that biofilm induction appears to be a general phenomenon of multiple structurally diverse thiazolyl peptides and use this activity to confirm the presence of thiazolyl peptide gene clusters in other bacterial species. Our results indicate that the roles of secondary metabolites initially identified as antibiotics may have more complex effects--acting not only as killing agents, but also as specific modulators of microbial cellular phenotypes.

Structural basis and dynamics of multidrug recognition in a minimal bacterial multidrug resistance system.

TipA is a transcriptional regulator found in diverse bacteria. It constitutes a minimal autoregulated multidrug resistance system against numerous thiopeptide antibiotics. Here we report the structures of its drug-binding domain TipAS in complexes with promothiocin A and nosiheptide, and a model of the thiostrepton complex. Drug binding induces a large transition from a partially unfolded to a globin-like structure. The structures rationalize the mechanism of promiscuous, yet specific, drug recognition: (i) a four-ring motif present in all known TipA-inducing antibiotics is recognized specifically by conserved TipAS amino acids; and (ii) the variable part of the antibiotic is accommodated within a flexible cleft that rigidifies upon drug binding. Remarkably, the identified four-ring motif is also the major interacting part of the antibiotic with the ribosome. Hence the TipA multidrug resistance mechanism is directed against the same chemical motif that inhibits protein synthesis. The observed identity of chemical motifs responsible for antibiotic function and resistance may be a general principle and could help to better define new leads for antibiotics.

The Catalytic Mechanism of the Class C Radical S-Adenosylmethionine Methyltransferase NosN.

S-Adenosylmethionine (SAM) is one of the most common co-substrates in enzyme-catalyzed methylation reactions. Most SAM-dependent reactions proceed through an SN 2 mechanism, whereas a subset of them involves radical intermediates for methylating non-nucleophilic substrates. Herein, we report the characterization and mechanistic investigation of NosN, a class C radical SAM methyltransferase involved in the biosynthesis of the thiopeptide antibiotic nosiheptide. We show that, in contrast to all known SAM-dependent methyltransferases, NosN does not produce S-adenosylhomocysteine (SAH) as a co-product. Instead, NosN converts SAM into 5'-methylthioadenosine as a direct methyl donor, employing a radical-based mechanism for methylation and releasing 5'-thioadenosine as a co-product. A series of biochemical and computational studies allowed us to propose a comprehensive mechanism for NosN catalysis, which represents a new paradigm for enzyme-catalyzed methylation reactions.

Identification and classification of known and putative antimicrobial compounds produced by a wide variety of Bacillales species.

BACKGROUND: Gram-positive bacteria of the Bacillales are important producers of antimicrobial compounds that might be utilized for medical, food or agricultural applications. Thanks to the wide availability of whole genome sequence data and the development of specific genome mining tools, novel antimicrobial compounds, either ribosomally- or non-ribosomally produced, of various Bacillales species can be predicted and classified. Here, we provide a classification scheme of known and putative antimicrobial compounds in the specific context of Bacillales species. RESULTS: We identify and describe known and putative bacteriocins, non-ribosomally synthesized peptides (NRPs), polyketides (PKs) and other antimicrobials from 328 whole-genome sequenced strains of 57 species of Bacillales by using web based genome-mining prediction tools. We provide a classification scheme for these bacteriocins, update the findings of NRPs and PKs and investigate their characteristics and suitability for biocontrol by describing per class their genetic organization and structure. Moreover, we highlight the potential of several known and novel antimicrobials from various species of Bacillales. CONCLUSIONS: Our extended classification of antimicrobial compounds demonstrates that Bacillales provide a rich source of novel antimicrobials that can now readily be tapped experimentally, since many new gene clusters are identified.

The many roles of glutamate in metabolism.

The amino acid glutamate is a major metabolic hub in many organisms and as such is involved in diverse processes in addition to its role in protein synthesis. Nitrogen assimilation, nucleotide, amino acid, and cofactor biosynthesis, as well as secondary natural product formation all utilize glutamate in some manner. Glutamate also plays a role in the catabolism of certain amines. Understanding glutamate's role in these various processes can aid in genome mining for novel metabolic pathways or the engineering of pathways for bioremediation or chemical production of valuable compounds.

Mechanistic Insights into the Radical S-adenosyl-l-methionine Enzyme NosL From a Substrate Analogue and the Shunt Products.

The radical S-adenosyl-l-methionine (SAM) enzyme NosL catalyzes the transformation of l-tryptophan into 3-methyl-2-indolic acid (MIA), which is a key intermediate in the biosynthesis of a clinically interesting antibiotic nosiheptide. NosL catalysis was investigated by using the substrate analogue 2-methyl-3-(indol-3-yl)propanoic acid (MIPA), which can be converted into MIA by NosL. Biochemical assays with different MIPA isotopomers in D2 O and H2 O unambiguously indicated that the 5'-deoxyadenosyl (dAdo)-radical-mediated hydrogen abstraction is from the amino group of l-tryptophan and not a protein residue. Surprisingly, the dAdo-radical-mediated hydrogen abstraction occurs at two different sites of MIPA, thereby partitioning the substrate into different reaction pathways. Together with identification of an α,ß-unsaturated ketone shunt product, our study provides valuable mechanistic insight into NosL catalysis and highlights the remarkable catalytic flexibility of radical SAM enzymes.

Expanding Radical SAM Chemistry by Using Radical Addition Reactions and SAM Analogues.

Radical S-adenosyl-l-methionine (SAM) enzymes utilize a [4Fe-4S] cluster to bind SAM and reductively cleave its carbon-sulfur bond to produce a highly reactive 5'-deoxyadenosyl (dAdo) radical. In almost all cases, the dAdo radical abstracts a hydrogen atom from the substrates or from enzymes, thereby initiating a highly diverse array of reactions. Herein, we report a change of the dAdo radical-based chemistry from hydrogen abstraction to radical addition in the reaction of the radical SAM enzyme NosL. This change was achieved by using a substrate analogue containing an olefin moiety. We also showed that two SAM analogues containing different nucleoside functionalities initiate the radical-based reactions with high efficiencies. The radical adduct with the olefin produced in the reaction was found to undergo two divergent reactions, and the mechanistic insights into this process were investigated in detail. Our study demonstrates a promising strategy in expanding radical SAM chemistry, providing an effective way to access nucleoside-containing compounds by using radical SAM-dependent reactions.

Substrate-Tuned Catalysis of the Radical S-Adenosyl-L-Methionine Enzyme NosL Involved in Nosiheptide Biosynthesis.

NosL is a radical S-adenosyl-L-methionine (SAM) enzyme that converts L-Trp to 3-methyl-2-indolic acid, a key intermediate in the biosynthesis of a thiopeptide antibiotic nosiheptide. In this work we investigated NosL catalysis by using a series of Trp analogues as the molecular probes. Using a benzofuran substrate 2-amino-3-(benzofuran-3-yl)propanoic acid (ABPA), we clearly demonstrated that the 5'-deoxyadenosyl (dAdo) radical-mediated hydrogen abstraction in NosL catalysis is not from the indole nitrogen but likely from the amino group of L-Trp. Unexpectedly, the major product of ABPA is a decarboxylated compound, indicating that NosL was transformed to a novel decarboxylase by an unnatural substrate. Furthermore, we showed that, for the first time to our knowledge, the dAdo radical-mediated hydrogen abstraction can occur from an alcohol hydroxy group. Our study demonstrates the intriguing promiscuity of NosL catalysis and highlights the potential of engineering radical SAM enzymes for novel activities.

Thiopeptide engineering: a multidisciplinary effort towards future drugs.

The recent development of thiopeptide analogues of antibiotics has allowed some of the limitations inherent to these naturally occurring substances to be overcome. Chemical synthesis, semisynthetic derivatization, and engineering of the biosynthetic pathway have independently led to complementary modifications of various thiopeptides. Some of the new substances have displayed improved profiles, not only as antibiotics, but also as antiplasmodial and anticancer drugs. The design of novel molecules based on the thiopeptide scaffold appears to be the only strategy to exploit the high potential they have shown in vitro. Herein we present the most relevant achievements in the production of thiopeptide analogues and also discuss the way the different approaches might be combined in a multidisciplinary strategy to produce more sophisticated structures.

Total synthesis and stereochemical assignment of baringolin.

The thiopeptide antibiotic baringolin has been synthesized, and its structure and stereochemistry have been confirmed. The use of a strategy based on palladium-catalyzed cross-couplings permitted a modular construction of this natural product.

Synthetic studies towards bottromycin.

Thio-Ugi reactions are described as an excellent synthetic tool for the synthesis of sterically highly hindered endothiopeptides. S-Methylation and subsequent amidine formation can be carried out in an inter- as well as in an intramolecular fashion. The intramolecular approach allows the synthesis of the bottromycin ring system in a straightforward manner.

Partial thioamide scan on the lipopeptaibiotic trichogin GA IV. Effects on folding and bioactivity.

Backbone modification is a common chemical tool to control the conformation of linear peptides and to explore potentially useful effects on their biochemical and biophysical properties. The thioamide, ψ[CS-NH], group is a nearly isosteric structural mimic of the amide (peptide) functionality. In this paper, we describe the solution synthesis, chemical characterization, preferred conformation, and membrane and biological activities of three, carefully selected, peptide analogues of the lipopeptaibiotic [Leu(11)-OMe] trichogin GA IV. In each analogue, a single thioamide replacement was incorporated. Sequence positions near the N-terminus, at the center, and near the C-terminus were investigated. Our results indicate that (i) a thioamide linkage is well tolerated in the overall helical conformation of the [Leu(11)-OMe] lipopeptide analogue and (ii) this backbone modification is compatible with the preservation of its typical membrane leakage and antibiotic properties, although somewhat attenuated.

Whole genome sequencing of Streptomyces actuosus ISP-5337, Streptomyces sioyaensis B-5408, and Actinospica acidiphila B-2296 reveals secondary metabolomes with antibiotic potential.

Streptomycetes are bacteria of biotechnological importance since they are avid producers of secondary metabolites, including antibiotics. Progress in genome mining has recently shown that Streptomyces species encode for many biosynthetic gene clusters which are mostly unexplored. Here, we selected three Actinomycetes species for whole genome sequencing that are known to produce potent thiopeptide antibiotics. Streptomyces actuosus biosynthesizes nosiheptide, Streptomyces sioyaensis produces siomycin, and Actinospica acidiphila is a member of the Actinomycete subfamily. Bioinformatic analyses demonstrated diverse secondary metabolomes with multiple antibiotic-encoding gene clusters. Detailed mass spectrometry analysis of metabolite extracts verified the active expression of nosiheptide and siomycin from S. actuosus and S. sioyaensis while fractionation of the bacterial extracts and subsequent challenge against Staphylococcus aureus demonstrated potent antibiotic activity of fractions containing these compounds. Whole genome sequencing of these species facilitates future bioengineering efforts for thiopeptides and characterization of relevant secondary metabolites.

Introduction to Thiopeptides: Biological Activity, Biosynthesis, and Strategies for Functional Reprogramming.

Thiopeptides (also known as thiazolyl peptides) are structurally complex natural products with rich biological activities. Known for over 70 years for potent killing of Gram-positive bacteria, thiopeptides are experiencing a resurgence of interest in the last decade, primarily brought about by the genomic revolution of the 21st century. Every area of thiopeptide research-from elucidating their biological function and biosynthesis to expanding their structural diversity through genome mining-has made great strides in recent years. These advances lay the foundation for and inspire novel strategies for thiopeptide engineering. Accordingly, a number of diverse approaches are being actively pursued in the hope of developing the next generation of natural-product-inspired therapeutics. Here, we review the contemporary understanding of thiopeptide biological activities, biosynthetic pathways, and approaches to structural and functional reprogramming, with a special focus on the latter.

Large-Scale Bioinformatics Analysis of Bacillus Genomes Uncovers Conserved Roles of Natural Products in Bacterial Physiology.

Bacteria possess an amazing capacity to synthesize a diverse range of structurally complex, bioactive natural products known as specialized (or secondary) metabolites. Many of these specialized metabolites are used as clinical therapeutics, while others have important ecological roles in microbial communities. The biosynthetic gene clusters (BGCs) that generate these metabolites can be identified in bacterial genome sequences using their highly conserved genetic features. We analyzed an unprecedented 1,566 bacterial genomes from Bacillus species and identified nearly 20,000 BGCs. By comparing these BGCs to one another as well as a curated set of known specialized metabolite BGCs, we discovered that the majority of Bacillus natural products are comprised of a small set of highly conserved, well-distributed, known natural product compounds. Most of these metabolites have important roles influencing the physiology and development of Bacillus species. We identified, in addition to these characterized compounds, many unique, weakly conserved BGCs scattered across the genus that are predicted to encode unknown natural products. Many of these "singleton" BGCs appear to have been acquired via horizontal gene transfer. Based on this large-scale characterization of metabolite production in the Bacilli, we go on to connect the alkylpyrones, natural products that are highly conserved but previously biologically uncharacterized, to a role in Bacillus physiology: inhibiting spore development. IMPORTANCEBacilli are capable of producing a diverse array of specialized metabolites, many of which have gained attention for their roles as signals that affect bacterial physiology and development. Up to this point, however, the Bacillus genus's metabolic capacity has been underexplored. We undertook a deep genomic analysis of 1,566 Bacillus genomes to understand the full spectrum of metabolites that this bacterial group can make. We discovered that the majority of the specialized metabolites produced by Bacillus species are highly conserved, known compounds with important signaling roles in the physiology and development of this bacterium. Additionally, there is significant unique biosynthetic machinery distributed across the genus that might lead to new, unknown metabolites with diverse biological functions. Inspired by the findings of our genomic analysis, we speculate that the highly conserved alkylpyrones might have an important biological activity within this genus. We go on to validate this prediction by demonstrating that these natural products are developmental signals in Bacillus and act by inhibiting sporulation.