Cheminformatics-Guided Cell-Free Exploration of Peptide Natural Products.

There have been significant advances in the flexibility and power of in vitro cell-free translation systems. The increasing ability to incorporate noncanonical amino acids and complement translation with recombinant enzymes has enabled cell-free production of peptide-based natural products (NPs) and NP-like molecules. We anticipate that many more such compounds and analogs might be accessed in this way. To assess the peptide NP space that is directly accessible to current cell-free technologies, we developed a peptide parsing algorithm that breaks down peptide NPs into building blocks based on ribosomal translation logic. Using the resultant data set, we broadly analyze the biophysical properties of these privileged compounds and perform a retrobiosynthetic analysis to predict which peptide NPs could be directly synthesized in augmented cell-free translation reactions. We then tested these predictions by preparing a library of highly modified peptide NPs. Two macrocyclases, PatG and PCY1, were used to effect the head-to-tail macrocyclization of candidate NPs. This retrobiosynthetic analysis identified a collection of high-priority building blocks that are enriched throughout peptide NPs, yet they had not previously been tested in cell-free translation. To expand the cell-free toolbox into this space, we established, optimized, and characterized the flexizyme-enabled ribosomal incorporation of piperazic acids. Overall, these results demonstrate the feasibility of cell-free translation for peptide NP total synthesis while expanding the limits of the technology. This work provides a novel computational tool for exploration of peptide NP chemical space, that could be expanded in the future to allow design of ribosomal biosynthetic pathways for NPs and NP-like molecules.

Identification of Covalent Cyclic Peptide Inhibitors in mRNA Display.

Peptides have historically been underutilized for covalent inhibitor discovery, despite their unique abilities to interact with protein surfaces and interfaces. This is in part due to a lack of methods for screening and identifying covalent peptide ligands. Here, we report a method to identify covalent cyclic peptide inhibitors in mRNA display. We combine co- and post-translational library diversification strategies to create cyclic libraries with reactive dehydroalanines (Dhas), which we employ in selections against two model targets. The most potent hits exhibit low nanomolar inhibitory activities and disrupt known protein-protein interactions with their selected targets. Overall, we establish Dhas as electrophiles for covalent inhibition and showcase how separate library diversification methods can work synergistically to dispose mRNA display to novel applications like covalent inhibitor discovery.

Tyrosinase-Catalyzed Peptide Macrocyclization for mRNA Display.

mRNA display of macrocyclic peptides has proven itself to be a powerful technique to discover high-affinity ligands for a protein target. However, only a limited number of cyclization chemistries are known to be compatible with mRNA display. Tyrosinase is a copper-dependent oxidase that oxidizes tyrosine phenol to an electrophilic o-quinone, which is readily attacked by cysteine thiol. Here we show that peptides containing tyrosine and cysteine are rapidly cyclized upon tyrosinase treatment. Characterization of the cyclization reveals it to be widely applicable to multiple macrocycle sizes and scaffolds. We combine tyrosinase-mediated cyclization with mRNA display to discover new macrocyclic ligands targeting melanoma-associated antigen A4 (MAGE-A4). These macrocycles potently inhibit the MAGE-A4 binding axis with nanomolar IC50 values. Importantly, macrocyclic ligands show clear advantage over noncyclized analogues with ∼40-fold or greater decrease in IC50 values.

Enabling Genetic Code Expansion and Peptide Macrocyclization in mRNA Display via a Promiscuous Orthogonal Aminoacyl-tRNA Synthetase.

mRNA display is revolutionizing peptide drug discovery through its ability to quickly identify potent peptide binders of therapeutic protein targets. Methods to expand the chemical diversity of display libraries are continually needed to increase the likelihood of identifying clinically relevant peptide ligands. Orthogonal aminoacyl-tRNA synthetases (ORSs) have proven utility in cellular genetic code expansion, but are relatively underexplored for in vitro translation (IVT) and mRNA display. Herein, we demonstrate that the promiscuous ORS p-CNF-RS can incorporate noncanonical amino acids at amber codons in IVT, including the novel substrate p-cyanopyridylalanine (p-CNpyrA), to enable a pyridine-thiazoline (pyr-thn) macrocyclization in mRNA display. Pyr-thn-based selections against the deubiquitinase USP15 yielded a potent macrocyclic binder that exhibits good selectivity for USP15 and close homologues over other ubiquitin-specific proteases (USPs). Overall, this work exemplifies how promiscuous ORSs can both expand side chain diversity and provide structural novelty in mRNA display libraries through a heterocycle forming macrocyclization.

Designer installation of a substrate recruitment domain to tailor enzyme specificity.

Promiscuous enzymes that modify peptides and proteins are powerful tools for labeling biomolecules; however, directing these modifications to desired substrates can be challenging. Here, we use computational interface design to install a substrate recognition domain adjacent to the active site of a promiscuous enzyme, catechol O-methyltransferase. This design approach effectively decouples substrate recognition from the site of catalysis and promotes modification of peptides recognized by the recruitment domain. We determined the crystal structure of this novel multidomain enzyme, SH3-588, which shows that it closely matches our design. SH3-588 methylates directed peptides with catalytic efficiencies exceeding the wild-type enzyme by over 1,000-fold, whereas peptides lacking the directing recognition sequence do not display enhanced efficiencies. In competition experiments, the designer enzyme preferentially modifies directed substrates over undirected substrates, suggesting that we can use designed recruitment domains to direct post-translational modifications to specific sequence motifs on target proteins in complex multisubstrate environments.

Enzymatic Pyridine Aromatization during Thiopeptide Biosynthesis.

Thiazole-containing pyritides (thiopeptides) are ribosomally synthesized and post-translationally modified peptides (RiPPs) that have attracted interest owing to their potent biological activities and structural complexity. The class-defining feature of a thiopeptide is a six-membered, nitrogenous heterocycle formed by an enzymatic [4 + 2]-cycloaddition. In rare cases, piperidine or dehydropiperidine (DHP) is present; however, the aromatized pyridine is considerably more common. Despite significant effort, the mechanism by which the central pyridine is formed remains poorly understood. Building on our recent observation of the Bycroft-Gowland intermediate (i.e., the direct product of the [4 + 2]-cycloaddition), we interrogated thiopeptide pyridine synthases using a combination of targeted mutagenesis, kinetic assays, substrate analogs, enzyme-substrate cross-linking, and chemical rescue experiments. Collectively, our data delineate roles for several conserved residues in thiopeptide pyridine synthases. A critical tyrosine facilitates the final aromatization step of pyridine formation. This work provides a foundation for further exploration of the [4 + 2]-cycloaddition reaction and future customization of pyridine-containing macrocyclic peptides.

Discovery and Structural Basis of the Selectivity of Potent Cyclic Peptide Inhibitors of MAGE-A4.

MAGE proteins are cancer testis antigens (CTAs) that are characterized by highly conserved MAGE homology domains (MHDs) and are increasingly being found to play pivotal roles in promoting aggressive cancer types. MAGE-A4, in particular, increases DNA damage tolerance and chemoresistance in a variety of cancers by stabilizing the E3-ligase RAD18 and promoting trans-lesion synthesis (TLS). Inhibition of the MAGE-A4:RAD18 axis could sensitize cancer cells to chemotherapeutics like platinating agents. We use an mRNA display of thioether cyclized peptides to identify a series of potent and highly selective macrocyclic inhibitors of the MAGE-A4:RAD18 interaction. Co-crystal structure indicates that these inhibitors bind in a pocket that is conserved across MHDs but take advantage of A4-specific residues to achieve high isoform selectivity. Cumulatively, our data represent the first reported inhibitor of the MAGE-A4:RAD18 interaction and establish biochemical tools and structural insights for the future development of MAGE-A4-targeted cellular probes.

Discovery and Development of Cyclic Peptide Inhibitors of CIB1.

Calcium and integrin binding protein 1 (CIB1) is a small, intracellular protein recently implicated in survival and proliferation of triple-negative breast cancer (TNBC). Considering its interactions with PAK1 and downstream signaling, CIB1 has been suggested as a potential therapeutic target in TNBC. As such, CIB1 has been the focus of inhibitor discovery efforts. To overcome issues of potency and stability in previously reported CIB1 inhibitors, we deploy mRNA display to discover new cyclic peptide inhibitors with improved biophysical properties and cellular activity. We advance UNC10245131, a cyclic peptide with low nanomolar affinity and good selectivity for CIB1 over other EF-hand domain proteins and improved permeability and stability over previously identified linear peptide inhibitor UNC10245092. Unlike UNC10245092, UNC10245131 lacks cytotoxicity and does not affect downstream signaling. Despite this, UNC10245131 is a potent ligand that could aid in clarifying roles of CIB1 in TNBC survival and proliferation and other CIB1-associated biological phenotypes.

Thiopeptides Induce Proteasome-Independent Activation of Cellular Mitophagy.

Thiopeptide antibiotics are emerging clinical candidates that exhibit potent antibacterial activity against a variety of intracellular pathogens, including Mycobacterium tuberculosis (Mtb). Many thiopeptides directly inhibit bacterial growth by disrupting protein synthesis. However, recent work has shown that one thiopeptide, thiostrepton (TSR), can also induce autophagy in infected macrophages, which has the potential to be exploited for host-directed therapies against intracellular pathogens, such as Mtb. To better define the therapeutic potential of this class of antibiotics, we studied the host-directed effects of a suite of natural thiopeptides that spans five structurally diverse thiopeptide classes, as well as several analogs. We discovered that thiopeptides as a class induce selective autophagic removal of mitochondria, known as mitophagy. This activity is independent of other biological activities, such as proteasome inhibition or antibiotic activity. We also find that many thiopeptides exhibit potent activity against intracellular Mtb in macrophage infection models. However, the thiopeptide-induced mitophagy occurs outside of pathogen-containing autophagosomes and does not appear to contribute to thiopeptide control of intracellular Mtb. These results expand basic understanding of thiopeptide biology and provide key guidance for the development of new thiopeptide antibiotics and host-directed therapeutics.

Discovery and Characterization of Peptide Inhibitors for Calcium and Integrin Binding Protein 1.

Calcium and integrin binding protein 1 (CIB1) is an EF-hand-containing, small intracellular protein that has recently been implicated in cancer cell survival and proliferation. In particular, CIB1 depletion significantly impairs tumor growth in triple-negative breast cancer (TNBC). Thus, CIB1 is a potentially attractive target for cancer chemotherapy that has yet to be validated by a chemical probe. To produce a probe molecule to the CIB1 helix 10 (H10) pocket and demonstrate that it is a viable target for molecular intervention, we employed random peptide phage display to screen and select CIB1-binding peptides. The top peptide sequence selected, UNC10245092, was produced synthetically, and binding to CIB1 was confirmed by isothermal titration calorimetry (ITC) and a time-resolved fluorescence resonance energy transfer (TR-FRET) assay. Both assays showed that the peptide bound to CIB1 with low nanomolar affinity. CIB1 was cocrystallized with UNC10245092, and the 2.1 Å resolution structure revealed that the peptide binds as an α-helix in the H10 pocket, displacing the CIB1 C-terminal H10 helix and causing conformational changes in H7 and H8. UNC10245092 was further derivatized with a C-terminal Tat-derived cell penetrating peptide (CPP) to demonstrate its effects on TNBC cells in culture, which are consistent with results of CIB1 depletion. These studies provide a first-in-class chemical tool for CIB1 inhibition in cell culture and validate the CIB1 H10 pocket for future probe and drug discovery efforts.

Inter-Modular Linkers play a crucial role in governing the biosynthesis of non-ribosomal peptides.

MOTIVATION: Non-ribosomal peptide synthetases (NRPSs) are modular enzymatic machines that catalyze the ribosome-independent production of structurally complex small peptides, many of which have important clinical applications as antibiotics, antifungals and anti-cancer agents. Several groups have tried to expand natural product diversity by intermixing different NRPS modules to create synthetic peptides. This approach has not been as successful as anticipated, suggesting that these modules are not fully interchangeable. RESULTS: We explored whether Inter-Modular Linkers (IMLs) impact the ability of NRPS modules to communicate during the synthesis of NRPs. We developed a parser to extract 39 804 IMLs from both well annotated and putative NRPS biosynthetic gene clusters from 39 232 bacterial genomes and established the first IMLs database. We analyzed these IMLs and identified a striking relationship between IMLs and the amino acid substrates of their adjacent modules. More than 92% of the identified IMLs connect modules that activate a particular pair of substrates, suggesting that significant specificity is embedded within these sequences. We therefore propose that incorporating the correct IML is critical when attempting combinatorial biosynthesis of novel NRPS. AVAILABILITY AND IMPLEMENTATION: The IMLs database as well as the NRPS-Parser have been made available on the web at https://nrps-linker.unc.edu. The entire source code of the project is hosted in GitHub repository (https://github.com/SWFarag/nrps-linker). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Thiopeptide Pyridine Synthase TbtD Catalyzes an Intermolecular Formal Aza-Diels-Alder Reaction.

Thiopeptide pyridine synthases catalyze a multistep reaction involving a unique and nonspontaneous intramolecular aza-[4 + 2] cycloaddition between two dehydroalanines to forge a trisubstituted pyridine core. We discovered that the in vitro activity of pyridine synthases from the thiocillin and thiomuracin pathways are significantly enhanced by general base catalysis and that this broadly expands the enzymes substrate tolerance. Remarkably, TbtD is competent to perform an intermolecular cyclization in addition to its cognate intramolecular reaction, underscoring its versatility as a biocatalyst. These data provide evidence that pyridine synthases use a two-site substrate recognition model to engage and process their substrates.

Structural Insights into Thioether Bond Formation in the Biosynthesis of Sactipeptides.

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 (ß/α)6-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.

P450-Mediated Non-natural Cyclopropanation of Dehydroalanine-Containing Thiopeptides.

Thiopeptides are a growing class of ribosomally synthesized and post-translationally modified peptide (RiPP) natural products. Many biosynthetic enzymes for RiPPs, especially thiopeptides, are promiscuous and can accept a wide range of peptide substrates with different amino acid sequences; thus, these enzymes have been used as tools to generate new natural product derivatives. Here, we explore an alternative route to molecular complexity by engineering thiopeptide tailoring enzymes to do new or non-native chemistry. We explore cytochrome P450 enzymes as biocatalysts for cyclopropanation of dehydroalanines, chemical motifs found widely in thiopeptides and other RiPP-based natural products. We find that P450TbtJ1 and P450TbtJ2 selectively cyclopropanate dehydroalanines in a number of complex thiopeptide-based substrates and convert them into 1-amino-2-cyclopropane carboxylic acids (ACCAs), which are important pharmacophores. This chemistry takes advantage of the innate affinity of these biosynthetic enzymes for their substrates and enables incorporation of new pharmacophores into thiopeptide architectures. This work also presents a strategy for diversification of natural products through rationally repurposing biosynthetic enzymes as non-natural biocatalysts.

Thiolutin is a zinc chelator that inhibits the Rpn11 and other JAMM metalloproteases.

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 deubiquitinating enzyme of the 19S proteasome. Thiolutin also inhibits the JAMM metalloproteases Csn5, the deneddylase of the COP9 signalosome; AMSH, which regulates ubiquitin-dependent sorting of cell-surface receptors; and BRCC36, a K63-specific deubiquitinase of the BRCC36-containing isopeptidase complex and the BRCA1-BRCA2-containing complex. We provide evidence that other dithiolopyrrolones also function as inhibitors of JAMM metalloproteases.

Production of Sactipeptides in Escherichia coli: Probing the Substrate Promiscuity of Subtilosin A Biosynthesis.

Sactipeptides are peptide-derived natural products that are processed by remarkable, radical-mediated cysteine sulfur to α-carbon coupling reactions. The resulting sactionine thioether linkages give rise to the unique defined structures and concomitant biological activities of sactipeptides. An E. coli heterologous expression system, based on the biosynthesis of one such sactipeptide, subtilosin A, is described and this expression system is exploited to probe the promiscuity of the subtilosin A sactionine bond-forming enzyme, AlbA. These efforts allowed the facile expression and isolation of a small library of mutant sactipeptides based on the subtilosin A precursor peptide, demonstrating broad substrate promiscuity where none was previously known. Importantly, we show that the positions of the sactionine linkages can be moved, giving rise to new, unnatural sactipeptide structures. E. coli heterologous expression also allowed incorporation of unnatural amino acids into sactipeptides by means of amber-suppression technology, potentially opening up new chemistry and new applications for unnatural sactipeptides.

Chemoenzymatic synthesis of thiazolyl peptide natural products featuring an enzyme-catalyzed formal [4 + 2] cycloaddition.

Thiocillins from Bacillus cereus ATCC 14579 are members of the well-known thiazolyl peptide class of natural product antibiotics, the biosynthesis of which has recently been shown to proceed via post-translational modification of ribosomally encoded precursor peptides. It has long been hypothesized that the final step of thiazolyl peptide biosynthesis involves a formal [4 + 2] cycloaddition between two dehydroalanines, a unique transformation that had eluded enzymatic characterization. Here we demonstrate that TclM, a single enzyme from the thiocillin biosynthetic pathway, catalyzes this transformation. To facilitate characterization of this new class of enzyme, we have developed a combined chemical and biological route to the complex peptide substrate, relying on chemical synthesis of a modified C-terminal fragment and coupling to a 38-residue leader peptide by means of native chemical ligation (NCL). This strategy, combined with active enzyme, provides a new chemoenzymatic route to this promising class of antibiotics.