Giang K. T. Nguyen

Orally Active Peptidic Bradykinin B1 Receptor Antagonists Engineered from a Cyclotide Scaffold for Inflammatory Pain Treatment

Chronic pain is a universal health issue associated with numerous medical conditions, for example after severe burns or following major surgery. Compelling evidence suggests that bradykinin (BK) antagonists could be useful in treating chronic pain and inflammatory pain. Bradykinin and its homolog kallidin (lysyl-BK or KD), which are collectively known as kinins, participate in many pathophysiological insults. They are short-lived peptide mediators and the most potent endogenous pain inducers. Kinins are released during tissue injury or noxious stimulation and modulate pain through the activation of both the B1 and the B2 receptor, which are two G-protein-coupled receptors; the carboxypeptidase metabolites of kinins, des-Arg-BK and des-Arg-KD, activate the B1 receptor. [2b, 4] The B1 receptor stimulates the chronic phase of the inflammatory pain response, while the B2 receptor stimulates the acute phase owing to their differences in ligand dissociation, receptor desensitization, downregulation as well as internalization. Emerging evidence also suggests that the B1 receptor mediates various chronic pain responses through the activation of phospholipase C, thereby leading to the production of diacylglycerol and inositol triphosphate, which further activate protein kinase C and Ca mobilization. Numerous BK-antagonist peptides have been discovered from natural sources and structure–activity studies. Kinestatin isolated from frog skin and helokinestatin from lizard venom are examples of natural BK-antagonist peptides. Structure–activity studies have shown that removing the Cterminal Arg residue and concurrently replacing the penultimate residue Phe to Leu of bradykinin to des-Arg-[Leu]bradykinin or kallidin to des-Arg-[Leu]-kallidin (DALK) changes a bradykinin B2 receptor agonist to a B1 receptor antagonist (Figure 1). To increase potency and in vivo stability, several laboratories also developed BK antagonists

Butelase 1 is an Asx-specific ligase enabling peptide macrocyclization and synthesis

Proteases are ubiquitous in nature, whereas naturally occurring peptide ligases, enzymes catalyzing the reverse reactions of proteases, are rare occurrences. Here we describe the discovery of butelase 1, to our knowledge the first asparagine/aspartate (Asx) peptide ligase to be reported. This highly efficient enzyme was isolated from Clitoria ternatea, a cyclic peptide-producing medicinal plant. Butelase 1 shares 71% sequence identity and the same catalytic triad with legumain proteases but does not hydrolyze the protease substrate of legumain. Instead, butelase 1 cyclizes various peptides of plant and animal origin with yields greater than 95%. With Kcat values of up to 17 s(-1) and catalytic efficiencies as high as 542,000 M(-1) s(-1), butelase 1 is the fastest peptide ligase known. Notably, butelase 1 also displays broad specificity for the N-terminal amino acids of the peptide substrate, thus providing a new tool for C terminus-specific intermolecular peptide ligations.

Discovery and Characterization of Novel Cyclotides Originated from Chimeric Precursors Consisting of Albumin-1 Chain a and Cyclotide Domains in the Fabaceae Family

The tropical plant Clitoria ternatea is a member of the Fabaceae family well known for its medicinal values. Heat extraction of C. ternatea revealed that the bioactive fractions contained heat-stable cysteine-rich peptides (CRPs). The CRP family of A1b (Albumin-1 chain b/leginsulins), which is a linear cystine knot CRP, has been shown to present abundantly in the Fabaceae. In contrast, the cyclotide family, which also belongs to the cystine knot CRPs but with a cyclic structure, is commonly found in the Rubiaceae, Violaceae, and Cucurbitaceae families. In this study, we report the discovery of a panel of 15 heat-stable CRPs, of which 12 sequences (cliotide T1–T12) are novel. We show unambiguously that the cliotides are cyclotides and not A1bs, as determined by their sequence homology, disulfide connectivity, and membrane active properties indicated by their antimicrobial activities against Escherichia coli and cytotoxicities to HeLa cells. We also show that cliotides are prevalent in C. ternatea and are found in every plant tissue examined, including flowers, seeds, and nodules. In addition, we demonstrate that their precursors are chimeras, half from cyclotide and the other half from Albumin-1, with the cyclotide domain displacing the A1b domain in the precursor. Their chimeric structures likely originate from either horizontal gene transfer or convergent evolution in plant nuclear genomes, which are exceedingly rare events. Such atypical genetic arrangement also implies a different mechanism of biosynthetic processing of cyclotides in the Fabaceae and provides new understanding of their evolution in plants.

Discovery of linear cyclotides in monocot plant Panicum laxum of Poaceae family provides new insights into evolution and distribution of cyclotides in plants.

Background: Cyclotides are biologically active, plant-derived macrocyclic peptides. All cyclotides and their acyclic variants have been isolated from dicots. Results: We characterized nine novel linear cyclotides from monocot plant Panicum laxum. Conclusion: Our study provides the first evidence of linear cyclotides at the protein level in the Poaceae. Significance: Ancient linear cyclotide analogs may have existed before the divergence of dicots and monocots. Cyclotides are disulfide-rich macrocyclic peptides that display a wide range of bioactivities and represent an important group of plant defense peptide biologics. A few linear variants of cyclotides have recently been identified. They share a high sequence homology with cyclotides but are biosynthetically unable to cyclize from their precursors. All hitherto reported cyclotides and their acyclic variants were isolated from dicot plants of the Rubiaceae, Violaceae, Cucurbitaceae, and recently the Fabaceae and Solanaceae families. Although several cyclotide-like genes in the Poaceae family were known from the data mining of the National Center for Biotechnology Information (NCBI) nucleotide database, their expression at the protein level has yet to be proven. Here, we report the discovery and characterization of nine novel linear cyclotides, designated as panitides L1–9, from the Panicum laxum of the Poaceae family and provide the first evidence of linear cyclotides at the protein level in a monocot plant. Disulfide mapping of panitide L3 showed that it possesses a cystine knot arrangement similar to cyclotides. Several panitides were shown to be active against Escherichia coli and cytotoxic to HeLa cells. They also displayed a high stability against heat and proteolytic degradation. Oxidative folding of the disulfide-reduced panitide L1 showed that it can fold efficiently into its native form. The presence of linear cyclotides in both dicots and monocots suggests their ancient origin and existence before the divergence of these two groups of flowering plants. Moreover, the Poaceae family contains many important food crops, and our discovery may open up new avenues of research using cyclotides and their acyclic variants in crop protection.

Novel Cyclotides and Uncyclotides with Highly Shortened Precursors from Chassalia chartacea and Effects of Methionine Oxidation on Bioactivities

Background: Cyclotides are biologically active, plant-derived macrocyclic peptides. Only three naturally occurring linear variants have been discovered. Results: We discovered 14 novel cyclotides and four uncyclotides from Chassalia chartacea. Their precursors are the shortest of all known cyclotides. Conclusion: Uncyclotides have comparable activities to cyclotides. Met oxidation disrupts the hydrophobic patch causing loss of activity. Significance: Our study broadens knowledge of cyclotides and uncyclotides. Cyclotides are a new class of plant biologics that display a diverse range of bioactivities with therapeutic potentials. They possess an unusual end-to-end cyclic backbone combined with a cystine knot arrangement, making them exceptionally stable to heat, chemical and enzymatic degradation. Currently, >200 cyclotides have been discovered but only three naturally occurring linear variants (also known as uncyclotides) have been isolated. In this study, we report the discovery of 18 novel peptides, chassatides C1 to C18, composed of 14 new cyclotides and four uncyclotides from Chassalia chartacea (Rubiaceae family). Thus far, this is the largest number of uncyclotides being reported in a single species. Activity testing showed that the uncyclotides not only retain the effectiveness but also are the most potent chassatides in the assays for antimicrobial, cytotoxic, and hemolytic activities. Genetic characterization of novel chassatides revealed that they have the shortest precursors of all known cyclotides hitherto isolated, which represents a new class of cyclotide precursors. This is the first report of cyclotide genes in a second genus, the Chassalia, other than the Hedyotis (Oldenlandia) of the Rubiaceae family. In addition, we also report the characterization of two Met-oxidized derivatives of chassatides C2 and C11. The oxidation of Met residue causes loss of bioactivities, strengthening the importance of the hydrophobic patch for membrane interaction.

Discovery of a Linear Cyclotide from the Bracelet Subfamily and Its Disulfide Mapping by Top-down Mass Spectrometry

Background: Cyclotides are plant-derived cyclic peptides that are divided into Möbius and bracelet subfamilies. Currently, only four linear variants of the Möbius subfamily have been isolated. Results: We discovered hedyotide B2 as the first linear representative of the bracelet subfamily. Conclusion: Hedyotide B2 shares the same connectivity as conventional cyclotides. Its linear structure is genetically predetermined. Significance: Our study broadens our knowledge of linear cyclotides. Cyclotides are heat-stable macrocyclic peptides from plants that display a wide range of biological activities. They can be divided into two subfamilies: Möbius or bracelet, based on the presence or absence of a cis-proline residue in loop 5, respectively. Currently, over 150 cyclotides have been discovered, but only four linear variants of the Möbius subfamily have been hitherto isolated. In this study, we report the discovery of two novel cyclotides, hedyotide B1 and hedyotide B2, from the aerial parts of Hedyotis biflora. Hedyotide B1 has a cyclic cystine knot structure typical of cyclotides. Interestingly, hedyotide B2 possesses a linear backbone and is the first linear representative of the bracelet subfamily. Disulfide mapping of hedyotide B2 by a top-down MS/MS approach showed that it shares the same knotted disulfide arrangement as conventional cyclotides. Its unfolding pathway also showed that the penetrating disulfide bond Cys III–VI is the most stable disulfide linkage. Cloning of the gene encoding hedyotide B2 revealed a nonsense mutation that introduces a premature stop codon at the conserved Asn residue position, which is essential for an end-to-end backbone ligation. Biophysical characterization showed that hedyotide B2 was more susceptible to exopeptidase degradation as compared with hedyotide B1. Hedyotide B2 was also inactive against all four tested bacterial strains, whereas hedyotide B1 was bactericidal to Escherichia coli and Streptococcus salivarius at low micromolar concentration. Our results provide a deeper understanding of the structures, functions, and biosynthetic processing of cyclotides and uncyclotides in plants.

Butelase 1: A Versatile Ligase for Peptide and Protein Macrocyclization.

Macrocyclization is a valuable tool for drug design and protein engineering. Although various methods have been developed to prepare macrocycles, a general and efficient strategy is needed. Here we report a highly efficient method using butelase 1 to macrocyclize peptides and proteins ranging in sizes from 26 to >200 residues. We achieved cyclizations that are 20,000 times faster than sortase A, the most widely used ligase for protein cyclization. The reactions completed within minutes with up to 95% yields.

Total Synthesis of Circular Bacteriocins by Butelase 1.

Circular bacteriocins, ranging from 35 to 70 amino acids, are the largest cyclic peptides produced by lactic acid bacteria to suppress growth of other bacteria. Their end-to-end cyclized backbone that enhances molecular stability is an advantage to survive in pasteurization and cooking processes in food preservation, but becomes a disadvantage and challenge in chemical synthesis. They also contain unusually long and highly hydrophobic segments which pose an additional synthetic challenge. Here we report the total synthesis of the three largest circular bacteriocins, AS-48, uberolysin, and garvicin ML, by an efficient chemoenzymatic strategy. A key feature of our synthetic scheme is the use of an Asn-specific butelase-mediated cyclization of their linear precursors, prepared by microwave stepwise synthesis. Antimicrobial assays showed that the AS-48 linear precursor is inactive at concentrations up to 100 μM, whereas the macrocyclic AS-48 is potently active against pathogenic and drug-resistant bacteria, with minimal inhibitory concentrations in a sub-micromolar range.

Butelase-mediated cyclization and ligation of peptides and proteins

Enzymes that catalyze efficient macrocyclization or site-specific ligation of peptides and proteins can enable tools for drug design and protein engineering. Here we describe a protocol to use butelase 1, a recently discovered peptide ligase, for high-efficiency cyclization and ligation of peptides and proteins ranging in size from 10 to >200 residues. Butelase 1 is the fastest known ligase and is found in pods of the common medicinal plant Clitoria ternatea (also known as butterfly pea). It has a very simple C-terminal-specific recognition motif that requires Asn/Asp (Asx) at the P1 position and a dipeptide His–Val at the P1′ and P2′ positions. Substrates for butelase-mediated ligation can be prepared by standard Fmoc (9-fluorenylmethyloxycarbonyl) chemistry or recombinant expression with the minimal addition of this tripeptide Asn–His–Val motif at the C terminus. Butelase 1 achieves cyclizations that are 20,000 times faster than those of sortase A, a commonly used enzyme for backbone cyclization. Unlike sortase A, butelase is traceless, and it can be used for the total synthesis of naturally occurring peptides and proteins. Furthermore, butelase 1 is also useful for intermolecular ligations and synthesis of peptide or protein thioesters, which are versatile activated intermediates necessary for and compatible with many chemical ligation methods. The protocol describes steps for isolation and purification of butelase 1 from plant extract using a four-step chromatography procedure, which takes ∼3 d. We then describe steps for intramolecular cyclization, intermolecular ligation and butelase-mediated synthesis of protein thioesters. Butelase reactions are generally completed within minutes and often achieve excellent yields.

Engineering a Catalytically Efficient Recombinant Protein Ligase

Breaking and forming peptidyl bonds are fundamental biochemical reactions in protein chemistry. Unlike proteases that are abundantly available, fast-acting ligases are rare. OaAEP1 is an enzyme isolated from the cyclotide-producing plant oldenlandia affinis that displayed weak peptide cyclase activity, despite having a similar structural fold with other asparaginyl endopeptidases (AEP). Here we report the first atomic structure of OaAEP1, at a resolution of 2.56 Å, in its preactivation form. Our structure and biochemical analysis of this enzyme reveals its activation mechanism as well as structural features important for its ligation activity. Importantly, through structure-based mutagenesis of OaAEP1, we obtained an ultrafast variant having hundreds of times faster catalytic kinetics, capable of ligating well-folded protein substrates using only a submicromolar concentration of enzyme. In contrast, the protein-protein ligation activity in the original wild-type OaAEP1 enzyme described previously is extremely weak. Thus, the structure-based engineering of OaAEP1 described here provides a unique and novel recombinant tool that can now be used to conduct various protein labeling and modifications that were extremely challenging before.