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 Macrolactamization of mRNA Display Libraries for Inhibitor Selection.

mRNA display is a powerful, high-throughput technology for discovering novel, peptide ligands for protein targets. A number of methods have been used to expand the chemical diversity of mRNA display libraries beyond the 20 canonical amino acids, including genetic code reprogramming and biorthogonal chemistries. To date, however, there have been few reports using enzymes as biocompatible reagents for diversifying mRNA display libraries. Here, we report the evaluation and implementation of the common industrial enzyme, microbial transglutaminase (mTG), as a versatile biocatalyst for cyclization of mRNA display peptide libraries via lysine-to-glutamine isopeptide bonds. We establish two separate display-based assays to validate the compatibility of mTG with mRNA-linked peptide substrates. These assays indicate that mTG has a high degree of substrate tolerance and low single round bias. To demonstrate the potential benefits of mTG-mediated cyclization in ligand discovery, high diversity mTG-modified libraries were employed in two separate affinity selections: (1) one against the calcium and integrin binding protein, CIB1, and (2) the second against the immune checkpoint protein and emerging therapeutic target, B7-H3. Both selections resulted in the identification of potent, cyclic, low nanomolar binders, and subsequent structure-activity studies demonstrate the importance of the cyclization to the observed activity. Notably, cyclization in the CIB1 binder stabilizes an α-helical conformation, while the B7-H3 inhibitor employs two bridges, one mTG-derived lactam and a second disulfide to achieve its potency. Together, these results demonstrate potential benefits of enzyme-based biocatalysts in mRNA display ligand selections and establish a framework for employing mTG in mRNA display.

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.

mRNA Display Reaches for the Clinic with New PCSK9 Inhibitor.

Merck & Co. recently reported one of the first mRNA display-derived clinical candidates in a bioavailable inhibitor of proprotein convertase subtilisin/kexin type 9 (PCSK9). Herein, we discuss the chemical and pharmacological challenges surmounted in bringing this compound to trials and the current outlook for mRNA display-based therapeutic development.

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.

Expanding the Chemical Diversity of Genetically Encoded Libraries.

The power of ribosomes has increasingly been harnessed for the synthesis and selection of molecular libraries. Technologies, such as phage display, yeast display, and mRNA display, effectively couple genotype to phenotype for the molecular evolution of high affinity epitopes for many therapeutic targets. Genetic code expansion is central to the success of these technologies, allowing researchers to surpass the intrinsic capabilities of the ribosome and access new, genetically encoded materials for these selections. Here, we review techniques for the chemical expansion of genetically encoded libraries, their abilities and limits, and opportunities for further development. Importantly, we also discuss methods and metrics used to assess the efficiency of modification and library diversity with these new techniques.

Interception of the Bycroft-Gowland Intermediate in the Enzymatic Macrocyclization of Thiopeptides.

Thiopeptides are a broad class of macrocyclic, heavily modified peptide natural products that are unified by the presence of a substituted, nitrogen-containing heterocycle core. Early work indicated that this core might be fashioned from two dehydroalanines by an enzyme-catalyzed aza-[4 + 2] cycloaddition to give a cyclic-hemiaminal intermediate. This common intermediate could then follow a reductive path toward a dehydropiperidine, as in the thiopeptide thiostrepton, or an aromatization path to yield the pyridine groups observed in many other thiopeptides. Although several of the enzymes proposed to perform this cycloaddition have been reconstituted, only pyridine products have been isolated and any hemiaminal intermediates have yet to be observed. Here, we identify the conditions and substrates that decouple the cycloaddition from subsequent steps and allow interception and characterization of this long hypothesized intermediate. Transition state modeling indicates that the key amide-iminol tautomerization is the major hurdle in an otherwise energetically favorable cycloaddition. An anionic model suggests that deprotonation and polarization of this amide bond by TbtD removes this barrier and provides a sufficient driving force for facile (stepwise) cycloaddition. This work provides evidence for a mechanistic link between disparate cyclases in thiopeptide biosynthesis.

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.

Exploring the Post-translational Enzymology of PaaA by mRNA Display.

PaaA is a RiPP enzyme that catalyzes the transformation of two glutamic acid residues within a substrate peptide into the bicyclic core of Pantocin A. Here, for the first time, we use mRNA display techniques to understand RiPP enzyme-substrate interactions to illuminate PaaA substrate recognition. Additionally, our data revealed insights into the enzymatic timing of glutamic acid modification. The technique developed is quite sensitive and a significant advancement over current RiPP studies and opens the door to enzyme modified mRNA display libraries for natural product-like inhibitor pans.

Dehydroamino acids: chemical multi-tools for late-stage diversification.

α,ß-Dehydroamino acids (dhAAs) are noncanonical amino acids that are found in a wide array of natural products and can be easily installed into peptides and proteins. dhAAs exhibit remarkable synthetic flexibility, readily undergoing a number of reactions, such as polar and single-electron additions, transition metal catalyzed cross-couplings, and cycloadditions. Because of the relatively mild conditions required for many of these reactions, dhAAs are increasingly being used as orthogonal chemical handles for late-stage modification of biomolecules. Still, only a fraction of the chemical reactivity of dhAAs has been exploited in such biorthogonal applications. Herein, we provide an overview of the broad spectrum of chemical reactivity of dhAAs, with special emphasis on recent efforts to adapt such transformations for biomolecules such as natural products, peptides, and proteins. We also discuss examples of enzymes from natural product biosynthetic pathways that have been found to catalyze many similar reactions; these enzymes provide mild, regio- and stereoselective, biocatalytic alternatives for future development. We anticipate that the continued investigation of the innate reactivity of dhAAs will furnish a diverse portfolio dhAA-based chemistries for use in chemical biology and drug discovery.

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.

Flexizyme-Enabled Benchtop Biosynthesis of Thiopeptides.

Thiopeptides are natural antibiotics that are fashioned from short peptides by multiple layers of post-translational modification. Their biosynthesis, in particular the pyridine synthases that form the macrocyclic antibiotic core, has attracted intensive research but is complicated by the challenges of reconstituting multiple-pathway enzymes. By combining select RiPP enzymes with cell free expression and flexizyme-based codon reprogramming, we have developed a benchtop biosynthesis of thiopeptide scaffolds. This strategy side-steps several challenges related to the investigation of thiopeptide enzymes and allows access to analytical quantities of new thiopeptide analogs. We further demonstrate that this strategy can be used to validate the activity of new pyridine synthases without the need to reconstitute the cognate prior pathway enzymes.

Mapping the sites of the lipoprotein lipase (LPL)-angiopoietin-like protein 4 (ANGPTL4) interaction provides mechanistic insight into LPL inhibition.

Cardiovascular disease has been the leading cause of death throughout the world for nearly 2 decades. Hypertriglyceridemia affects more than one-third of the population in the United States and is an independent risk factor for cardiovascular disease. Despite the frequency of hypertriglyceridemia, treatment options are primarily limited to diet and exercise. Lipoprotein lipase (LPL) is an enzyme responsible for clearing triglycerides from circulation, and its activity alone can directly control plasma triglyceride concentrations. Therefore, LPL is a good target for triglyceride-lowering therapeutics. One approach for treating hypertriglyceridemia may be to increase the amount of enzymatically active LPL by preventing its inhibition by angiopoietin-like protein 4 (ANGPTL4). However, little is known about how these two proteins interact. Therefore, we used hydrogen-deuterium exchange MS to identify potential binding sites between LPL and ANGPTL4. We validated sites predicted to be located at the protein-protein interface by using chimeric variants of LPL and an LPL peptide mimetic. We found that ANGPTL4 binds LPL near the active site at the lid domain and a nearby α-helix. Lipase lid domains cover the active site to control both enzyme activation and substrate specificity. Our findings suggest that ANGPTL4 specifically inhibits LPL by binding the lid domain, which could prevent substrate catalysis at the active site. The structural details of the LPL-ANGPTL4 interaction uncovered here may inform the development of therapeutics targeted to disrupt this interaction for the management of hypertriglyceridemia.