Thioamide Analogues of MHC I Antigen Peptides.

Short, synthetic peptides that are displayed by major histocompatibility complex I (MHC I) can stimulate CD8 T cells in vivo to destroy virus-infected or cancer cells. The development of such peptides as vaccines that provide protective immunity, however, is limited by rapid proteolytic degradation. Introduction of unnatural amino acid residues can suppress MHC I antigen proteolysis, but the modified peptides typically display lower affinity for MHC I and/or diminished ability to activate CD8 T cells relative to native antigen. Here, we report a new strategy for modifying MHC I antigens to enhance resistance to proteolysis while preserving MHC I affinity and T cell activation properties. This approach, replacing backbone amide groups with thioamides, was evaluated in two well-characterized antigens presented by HLA-A2, a common human MHC I. For each antigen, singly modified thioamide analogues retained affinity for HLA-A2 and activated T cells specific for the native antigen, as measured via interferon-γ secretion. In each system, we identified a highly potent triply substituted thioamide antigen ("thio-antigen") that displayed substantial resistance to proteolytic cleavage. Collectively, our results suggest that thio-antigens may represent a general and readily accessible source of potent vaccine candidates that resist degradation.

Functional elucidation of TfuA in peptide backbone thioamidation.

YcaO enzymes catalyze several post-translational modifications on peptide substrates, including thioamidation, which substitutes an amide oxygen with sulfur. Most predicted thioamide-forming YcaO enzymes are encoded adjacent to TfuA, which when present, is required for thioamidation. While activation of the peptide amide backbone is well established for YcaO enzymes, the function of TfuA has remained enigmatic. Here we characterize the TfuA protein involved in methyl-coenzyme M reductase thioamidation and demonstrate that TfuA catalyzes the hydrolysis of thiocarboxylated ThiS (ThiS-COSH), a proteinaceous sulfur donor, and enhances the affinity of YcaO toward the thioamidation substrate. We also report a crystal structure of a TfuA, which displays a new protein fold. Our structural and mutational analyses of TfuA have uncovered conserved binding interfaces with YcaO and ThiS in addition to revealing a hydrolase-like active site featuring a Ser-Lys catalytic pair.

Formation of an aminovinyl-cysteine residue in thioviridamides occurs through a path independent of known lanthionine synthetase activity.

2-Aminovinyl-cysteine (AviCys) is a thioether amino acid shared by a variety of ribosomally synthesized and posttranslationally modified peptides (RiPPs). Based on investigations into the biosynthesis of thioviridamide RiPPs in Streptomyces sp. NRRL S-87, we here report a path for the formation of this unusual thioether residue. This path relies on four dedicated proteins: phosphotransferase TvaCS-87, Lyase TvaDS-87, kinase homolog TvaES-87, and LanD-like flavoprotein TvaFS-87. TvaES-87 plays a critical role in effective AviCys formation. During the posttranslational modifications of the precursor peptide, it works with TvaFS-87 to form a minimum AviCys synthetase complex, which follows the combined activity of TvaCDS-87 for Thr dehydration and catalyzes Cys oxidative decarboxylation and subsequent Michael addition of the resulting enethiol nucleophile onto the newly formed dehydroamino acid residue for cyclization. With TvaES-87, TvaFS-87 activity for Cys processing can be coordinated with TvaCDS-87 activity for minimizing competitive or unexpected spontaneous reactions and forming AviCys effectively.

An Unexpected Split-Merge Pathway in the Assembly of the Symmetric Nonribosomal Peptide Antibiotic Closthioamide.

Closthioamide (CTA) is a symmetric nonribosomal peptide (NRP) comprised of two diaminopropane-linked polythioamidated monomers. CTA is biosynthesized by Ruminiclostridium cellulolyticum via an atypical NRP synthetase (NRPS)-independent biosynthetic pathway. Although the logic for monomer assembly was recently elucidated, the strategy for the biosynthesis and incorporation of the diamine linker remained a mystery. By means of genome editing, synthesis, and in vitro biochemical assays, we demonstrate that the final steps in CTA maturation proceed through a surprising split-merge pathway involving the dual use of a thiotemplated intermediate. This pathway includes the first examples of an aldo-keto reductase catalyzing the reductive release of a thiotemplated product, and of a transthioamidating transglutaminase. In addition to clarifying the remaining steps in CTA assembly, our data shed light on largely unexplored pathways for NRPS-independent peptide biosynthesis.

Non-Heme Monooxygenase ThoJ Catalyzes Thioholgamide ß-Hydroxylation.

Thioviridamide-like compounds, including thioholgamides, are ribosomally synthesized and post-translationally modified peptide natural products with potent anticancer cell activity and an unprecedented structure. Very little is known about their biosynthesis, and we were intrigued by the ß-hydroxy-N1, N3-dimethylhistidinium moiety found in these compounds. Here we report the construction of a heterologous host capable of producing thioholgamide with a 15-fold increased yield compared to the wild-type strain. A knockout of thoJ, encoding a predicted nonheme monooxygenase, shows that ThoJ is essential for thioholgamide ß-hydroxylation. The crystal structure of ThoJ exhibits a typical mono/dioxygenase fold with conserved key active-site residues. Yet, ThoJ possesses a very large substrate binding pocket that appears suitable to receive a cyclic thioholgamide intermediate for hydroxylation. The improved production of the heterologous host will enable the dissection of the individual biosynthetic steps involved in biosynthesis of this exciting RiPP family.

Silver complexes with heterocyclic thioamide and tertiary arylphosphane ligands: Synthesis, crystal structures, in vitro and in silico antibacterial and cytotoxic activity, and interaction with DNA.

Herein we report on the synthesis and molecular structures of six silver(I) mixed-ligand complexes containing a heterocyclic thioamide [4-phenyl-imidazole-2-thione (phimtH) or 2,2,5,5-tetramethyl-imidazolidine-4-thione (tmimdtH)] and a tertiary arylphosphane [triphenylphosphine (PPh3), tri-o-tolylphosphane (totp)] or diphosphane [(1,2-bis(diphenylphosphano)ethane (dppe), bis(2-diphenylphosphano-phenyl)ether (DPEphos) or 4,5-bis(diphenylphosphano)-9,9-dimethylxanthene) (xantphos)]. The interaction of the compounds with calf-thymus DNA (CT DNA), as monitored directly via UV-vis spectroscopy and DNA-viscosity measurements and indirectly via its competition with ethidium bromide for DNA-intercalation sites, is suggested to take place via an intercalative mode. The new complexes show selective significant in vitro antibacterial activity against four bacterial strains. The antiproliferative effects and cytostatic efficacies of the complexes against four human cancer cell lines were evaluated. The best cytostatic and cytotoxic activity was appeared for the complexes bearing the phimtH moiety. In order to explain the described in vitro activity of the complexes, and to approach a possible mechanism of action, molecular docking studies were adopted on the crystal structure of CT DNA, DNA-gyrase, human estrogen receptor alpha and a cell-cycle specific target protein, human cyclin-dependent kinase 6.

Synthesis of morpholine derivatives using the Castagnoli-Cushman reaction as BACE1 inhibitors: Unexpected binding activity of cyclic thioamides.

The Castagnoli-Cushman reaction between diglycolic anhydride and imines was applied for the synthesis of morpholine derivatives containing a thioamide or an amidino group. Enzyme inhibition assays towards BACE1 revealed an unexpected role of the cyclic thioamide group in providing inhibition in the micromolar range. Molecular docking calculations showed the thioamido group interacting with catalytic aspartic acid, and calculated BBB permeability indicated this molecular scaffold as a promising hit for further optimization.

Reconstitution of polythioamide antibiotic backbone formation reveals unusual thiotemplated assembly strategy.

Closthioamide (CTA) is a rare example of a thioamide-containing nonribosomal peptide and is one of only a handful of secondary metabolites described from obligately anaerobic bacteria. Although the biosynthetic gene cluster responsible for CTA production and the thioamide synthetase that catalyzes sulfur incorporation were recently discovered, the logic for peptide backbone assembly has remained a mystery. Here, through the use of in vitro biochemical assays, we demonstrate that the amide backbone of CTA is assembled in an unusual thiotemplated pathway involving the cooperation of a transacylating member of the papain-like cysteine protease family and an iteratively acting ATP-grasp protein. Using the ATP-grasp protein as a bioinformatic handle, we identified hundreds of such thiotemplated yet nonribosomal peptide synthetase (NRPS)-independent biosynthetic gene clusters across diverse bacterial phyla. The data presented herein not only clarify the pathway for the biosynthesis of CTA, but also provide a foundation for the discovery of additional secondary metabolites produced by noncanonical biosynthetic pathways.

Oxyanion transport across lipid bilayers: direct measurements in large and giant unilamellar vesicles.

A simple di(thioamido)carbazole 1 serves as a potent multispecific transporter for various biologically relevant oxyanions, such as drugs, metabolites and model organic phosphate. The transport kinetics of a wide range of oxyanions can be easily quantified by a modified lucigenin assay in both large and giant unilamellar vesicles.

Ribosomal Formation of Thioamide Bonds in Polypeptide Synthesis.

It has been well established that the ribosome can accept various nucleophiles on the Xacyl-tRNA in the A site during elongation, where X can be amino, N-alkyl-amino, hydroxy, and thiol groups. However, it remains elusive that the ribosome is able to accept an electrophile in the P site other than the carboxyl group during elongation. Here we report ribosomal formation of a thioamide bond in the mRNA-dependent polypeptide synthesis. In this study, amino(carbothio)acyl-tRNA was prepared by flexizyme and used for the expression of peptides containing a thioamide bond in the nascent peptide chain. We give strong evidence that the thioamide-peptide was formed but accompanied by the amide counterpart due to rapid carbo(S-to-O) exchange during the preparation of amino(carbothio)acyl-tRNA. We also demonstrate the ribosomal formation of thioamide and N-methyl-thioamide bonds in linear as well as macrocyclic peptide scaffolds in the mRNA-dependent manner, showing its potential for applications in peptide-based drug discovery and studying peptide/protein structure and function.

One-Atom Substitution Enables Direct and Continuous Monitoring of Histone Deacylase Activity.

We developed a one-step direct assay for the determination of histone deacylase (HDAC) activity by substituting the carbonyl oxygen of the acyl moiety with sulfur, resulting in thioacylated lysine side chains. This modification is recognized by class I HDACs with different efficiencies ranging from not accepted for HDAC1 to kinetic constants similar to that of the parent oxo substrate for HDAC8. Class II HDACs can hydrolyze thioacylated substrates with approximately 5-10-fold reduced kcat values, which resembles the effect of thioamide substitution in metallo-protease substrates. Class IV HDAC11 accepts thiomyristoyl modification less efficiently with an ∼5-fold reduced specificity constant. On the basis of the unique spectroscopic properties of thioamide bonds (strong absorption in spectral range of 260-280 nm and efficient fluorescence quenching), HDAC-mediated cleavage of thioamides could be followed by ultraviolet-visible and fluorescence spectroscopy in a continuous manner. The HDAC activity assay is compatible with microtiter plate-based screening formats up to 1536-well plates with Z' factors of >0.75 and signal-to-noise ratios of >50. Using thioacylated lysine residues in p53-derived peptides, we optimized substrates for HDAC8 with a catalytic efficiency of >250000 M-1 s-1, which are more than 100-fold more effective than most of the known substrates. We determined inhibition constants of several inhibitors for human HDACs using thioacylated peptidic substrates and found good correlation with the values from the literature. On the other hand, we could introduce N-methylated, N-acylated lysine residues as inhibitors for HDACs with an IC50 value of 1 µM for an N-methylated, N-myristoylated peptide derivative and human HDAC11.

Reconstitution of Iterative Thioamidation in Closthioamide Biosynthesis Reveals Tailoring Strategy for Nonribosomal Peptide Backbones.

Thioamide-containing nonribosomal peptides (NRPs) are exceedingly rare. Recently the biosynthetic gene cluster for the thioamidated NRP antibiotic closthioamide (CTA) was reported, however, the enzyme responsible for and the timing of thioamide formation remained enigmatic. Here, genome editing, biochemical assays, and mutational studies are used to demonstrate that an Fe-S cluster containing member of the adenine nucleotide α-hydrolase protein superfamily (CtaC) is responsible for sulfur incorporation during CTA biosynthesis. However, unlike all previously characterized members, CtaC functions in a thiotemplated manner. In addition to prompting a revision of the CTA biosynthetic pathway, the reconstitution of CtaC provides the first example of a NRP thioamide synthetase. Finally, CtaC is used as a bioinformatic handle to demonstrate that thioamidated NRP biosynthetic gene clusters are more widespread than previously appreciated.

Comprehensive Derivatization of Thioviridamides by Heterologous Expression.

New technology for the derivatization of peptide natural products is required for drug development. Despite the recent advances in the genome sequencing technique enabling us to search for the biosynthetic genes for wide variety of natural products, the technical methods to get access to them are limited. A class of RiPPs, a recently emerged natural product family such as thioviridamide, is one of those possessing such unexplored chemical space. In this paper, we report a streamlined method to generate new thioviridamide derivatives and to assess their biological activities. Heterologous expression of 42 constructs in an engineered Streptomyces avermitilis host gave 35 designed thioviridamide derivatives, along with several unprecedented analogues. Moreover, cytotoxicity assay revealed that several derivatives showed more potent activities than those of prethioviridamide. These results indicate that this strategy can become one of the potential ways to produce supreme unnatural products.

Insights into the thioamidation of thiopeptins to enhance the understanding of the biosynthetic logic of thioamide-containing thiopeptides.

Thiopeptins are a complex of thiopeptide antibiotics similar in structure to thiostrepton and harboring a thioamide, a rare moiety among natural products. Here, we illustrate through a series of in vivo experiments that the thioamide moiety of thiopeptins is generated posttranslationally by a TfuA-YcaO pair, encoded in the thiopeptin biosynthetic gene cluster, before the maturation of the thiopeptide bicyclic scaffold, enhancing the understanding of the biosynthetic logic of thioamide-containing thiopeptides.

Biosynthesis and Chemical Applications of Thioamides.

Thioamidation as a posttranslational modification is exceptionally rare, with only a few reported natural products and exactly one known protein example (methyl-coenzyme M reductase from methane-metabolizing archaea). Recently, there has been significant progress in elucidating the biosynthesis and function of several thioamide-containing natural compounds. Separate developments in the chemical installation of thioamides into peptides and proteins have enabled cell biology and biophysical studies to advance the current understanding of natural thioamides. This review highlights the various strategies used by Nature to install thioamides in peptidic scaffolds and the potential functions of this rare but important modification. We also discuss synthetic methods used for the site-selective incorporation of thioamides into polypeptides with a brief discussion of the physicochemical implications. This account will serve as a foundation for the further study of thioamides in natural products and their various applications.

Thiodipeptides targeting the intestinal oligopeptide transporter as a general approach to improving oral drug delivery.

The broad substrate capacity of the intestinal oligopeptide transporter, PepT1, has made it a key target of research into drug delivery. Whilst the substrate capacity of this transporter is broad, studies have largely been limited to small peptides and peptide-like drugs. Here, we demonstrate for the first time that a diverse range of drugs can be targeted towards transport by PepT1 using a hydrolysis resistant carrier. Eleven prodrugs were synthesized by conjugating modified dipeptides containing a thioamide bond to the approved drugs ibuprofen, gabapentin, propofol, aspirin, acyclovir, nabumetone, atenolol, zanamivir, baclofen and mycophenolate. Except for the aspirin and acyclovir prodrugs, which were unstable in the assay conditions and were not further studied, the prodrugs were tested for affinity and transport by PepT1 expressed in Xenopus laevis oocytes: binding affinities ranged from approximately 0.1 to 2 mM. Compounds which showed robust transport in an oocyte trans-stimulation assay were then tested for transcellular transport in Caco-2 cell monolayers: all five tested prodrugs showed significant PepT1-mediated transcellular uptake. Finally, the ibuprofen and propofol prodrugs were tested for absorption in rats: following oral dosing the intact prodrugs and free ibuprofen were measured in the plasma. This provides proof-of-concept for the idea of targeting poorly bioavailable drugs towards PepT1 transport as a general means of improving oral permeability.

Enzymatic Thioamide Formation in a Bacterial Antimetabolite Pathway.

6-Thioguanine (6TG) is a DNA-targeting therapeutic used in the treatment of various cancers. While 6TG was rationally designed as a proof of concept for antimetabolite therapy, it is also a rare thioamide-bearing bacterial natural product and critical virulence factor of Erwinia amylovorans, plant pathogens that cause fire blight. Through gene expression, biochemical assays, and mutational analyses, we identified a specialized bipartite enzyme system, consisting of an ATP-dependent sulfur transferase (YcfA) and a sulfur-mobilizing enzyme (YcfC), that is responsible for the peculiar oxygen-by-sulfur substitution found in the biosynthesis of 6TG. Mechanistic and phylogenetic studies revealed that YcfA-mediated 6TG biosynthesis evolved from ancient tRNA modifications that support translational fidelity. The successful in vitro reconstitution of 6TG thioamidation showed that YcfA employs a specialized sulfur shuttle that markedly differs from universal RNA-related systems. This study sheds light on underexplored enzymatic C-S bond formation in natural product biosynthesis.

Glucosinolate turnover in Brassicales species to an oxazolidin-2-one, formed via the 2-thione and without formation of thioamide.

Glucosinolates are found in plants of the order Brassicales and hydrolyzed to different breakdown products, particularly after tissue damage. In Barbarea vulgaris R.Br. (Brassicaceae), the dominant glucosinolate in the investigated "G-type" is glucobarbarin, (S)-2-hydroxy-2-phenylethylglucosinolate. Formation of the nitrile from glucobarbarin was observed in vitro, while a previously suggested thioamide (synonym thionamide) was not confirmed. Resedine (5-phenyl-1,3-oxazolidin-2-one) was detected after glucobarbarin hydrolysis in crushed B. vulgaris leaves and siliques, but not in intact parts. The abundance increased for several hours after completion of hydrolysis. The corresponding 1,3-oxazolidine-2-thione (OAT), with the common name barbarin, was also formed, and appeared to be the precursor of resedine. Addition of each of two non-endogenous OATs, (S)-5-ethyl-5-methylOAT and (R)-5-vinylOAT (R-goitrin), to a leaf homogenate resulted in formation of the corresponding 1,3-oxazolidin-2-ones (OAOs), confirming the metabolic connection of OAT to OAO. Formation of OAOs was inhibited by prior brief heating of the homogenate, suggesting enzyme involvement. We suggest the conversion of OATs to OAOs to be catalyzed by an enzyme ("oxazolidinethionase") responsible for turnover of OAT formed in intact plants. Resedine had been reported as an alkaloid from another species - Reseda luteola L. (Resedaceae) - naturally containing the glucosinolate glucobarbarin. However, resedine was not detected in intact R. luteola plants, but formed after tissue damage. The formation of resedine in two families suggests a broad distribution of putative OATases in the Brassicales; potentially involved in glucosinolate turnover that needs myrosinase activity as the committed step. In agreement with the proposed function of OATase, several candidate genes for myrosinases in glucosinolate turnover in intact plants were discovered in the B. vulgaris genome. We also suggest that biotechnological conversion of OATs to OAOs might improve the nutritional value of Brassicales protein. HPLC-MS/MS methods for detection of these glucobarbarin products are described.

Enzymatic reconstitution of ribosomal peptide backbone thioamidation.

Methyl-coenzyme M reductase (MCR) is an essential enzyme found strictly in methanogenic and methanotrophic archaea. MCR catalyzes a reversible reaction involved in the production and consumption of the potent greenhouse gas methane. The α-subunit of this enzyme (McrA) contains several unusual posttranslational modifications, including the only known naturally occurring example of protein thioamidation. We have recently demonstrated by genetic deletion and mass spectrometry that the tfuA and ycaO genes of Methanosarcina acetivorans are involved in thioamidation of Gly465 in the MCR active site. Modification to thioGly has been postulated to stabilize the active site structure of MCR. Herein, we report the in vitro reconstitution of ribosomal peptide thioamidation using heterologously expressed and purified YcaO and TfuA proteins from M. acetivorans Like other reported YcaO proteins, this reaction is ATP-dependent but requires an external sulfide source. We also reconstitute the thioamidation activity of two TfuA-independent YcaOs from the hyperthermophilic methanogenic archaea Methanopyrus kandleri and Methanocaldococcus jannaschii Using these proteins, we demonstrate the basis for substrate recognition and regioselectivity of thioamide formation based on extensive mutagenesis, biochemical, and binding studies. Finally, we report nucleotide-free and nucleotide-bound crystal structures for the YcaO proteins from M. kandleri Sequence and structure-guided mutagenesis with subsequent biochemical evaluation have allowed us to assign roles for residues involved in thioamidation and confirm that the reaction proceeds via backbone O-phosphorylation. These data assign a new biochemical reaction to the YcaO superfamily and paves the way for further characterization of additional peptide backbone posttranslational modifications.

Neothioviridamide, a Polythioamide Compound Produced by Heterologous Expression of a Streptomyces sp. Cryptic RiPP Biosynthetic Gene Cluster.

During genome mining for thioviridamide-like biosynthetic gene clusters that could produce polythioamide RiPP (ribosomally synthesized and post-translationally modified peptides), we discovered a novel cryptic biosynthetic gene cluster. During efforts to express this biosynthetic gene using heterologous expression of this biosynthetic gene cluster, a novel compound designated as neothioviridamide was produced. We report herein the cloning and heterologous expression of the neothioviridamide biosynthetic gene cluster and the isolation, structure determination, and cytotoxic activity of neothioviridamide.