Potent Trypsin Inhibitor Research Articles

Combined Antitumor Effect of the Serine Protease Urokinase Inhibitor Upamostat and the Sphingosine Kinase 2 Inhibitor Opaganib on Cholangiocarcinoma Patient-Derived Xenografts.

Upamostat is an orally available small-molecule serine protease inhibitor that is a highly potent inhibitor of trypsin 1, trypsin 2, trypsin 3 (PRSS1/2/3), and the urokinase-type plasminogen activator (uPA). These enzymes are expressed in many cancers, especially during tissue remodeling and subsequent tumor cell invasion. Opaganib (ABC294640), a novel, orally available small molecule is a selective inhibitor of the phosphorylation of sphingosine to sphingosine-1-phosphate (S-1-P) by sphingosine kinase 2 (SPHK2). Both sphingosine kinase 1 (SPHK1) and SPHK2 are known to regulate the proliferation-inducing compound S-1-P. However, SPHK2 is more critical in cancer pathogenesis. The goal of this project was to investigate the potential antitumor effects of upamostat and opaganib, individually and in combination, on cholangiocarcinoma (CCA) xenografts in nude mice. PAX165, a patient-derived xenograft (PDX) from a surgically resected CCA, expresses substantial levels of SPHK2, PRSS1, PRSS2, and PRSS3. Four groups of 18 mice each were treated with upamostat, opaganib, both, or vehicle. Mouse weights and PAX165 tumor volumes were measured. Tumor volumes in the upamostat, opaganib, and upamostat plus opaganib groups were significantly decreased compared to the control group.

Preclinical testing of dabigatran in trypsin-dependent pancreatitis.

Pancreatitis, the inflammatory disorder of the pancreas, has no specific therapy. Genetic, biochemical, and animal model studies revealed that trypsin plays a central role in the onset and progression of pancreatitis. Here, we performed biochemical and preclinical mouse experiments to offer proof of concept that orally administered dabigatran etexilate can inhibit pancreatic trypsins and shows therapeutic efficacy in trypsin-dependent pancreatitis. We found that dabigatran competitively inhibited all human and mouse trypsin isoforms (Ki range 10-79 nM) and dabigatran plasma concentrations in mice given oral dabigatran etexilate well exceeded the Ki of trypsin inhibition. In the T7K24R trypsinogen mutant mouse model, a single oral gavage of dabigatran etexilate was effective against cerulein-induced progressive pancreatitis, with a high degree of histological normalization. In contrast, spontaneous pancreatitis in T7D23A mice, which carry a more aggressive trypsinogen mutation, was not ameliorated by dabigatran etexilate, given either as daily gavages or by mixing it with solid chow. Taken together, our observations showed that benzamidine derivatives such as dabigatran are potent trypsin inhibitors and show therapeutic activity against trypsin-dependent pancreatitis in T7K24R mice. Lack of efficacy in T7D23A mice is probably related to the more severe pathology and insufficient drug concentrations in the pancreas.

Evaluation of interaction between Ponceau 4R (P4R) and trypsin using kinetic, spectroscopic, and molecular dynamics simulation methods

Azo-dyes, a diverse group of synthetic dyes, are commonly used for coloring food products. However, these dyes are persistent in nature with the potential to cause harmful effects on the human body. Hence, it seems that studies are required about the effects of azo dyes on the structure and function of biomacromolecules. Ponceau 4R (P4R) is an azo-dye found in foods, beverages, and pharmaceuticals, but its potential risk on gastrointestinal enzymes such as trypsin is poorly characterized. Thus, this study focused on analyzing the binding behavior between trypsin and P4R by spectroscopic, kinetic, and theoretical methods. Intrinsic fluorescence revealed that there was an interaction between P4R and trypsin, and P4R could quench the trypsin fluorescence by a static model. The resonance light scattering (RLS) method was used to confirm the formation of the P4R-trypsin complex. The thermodynamic data indicated that the predominant interactions between P4R and trypsin were Van der Waals and hydrogen bonds, consistent with docking results. The distance between P4R and trypsin after the interaction was calculated based on fluorescence resonance energy transfer (FRET) theory and found to be 3.36 nm. Synchronous fluorescence, three-dimensional fluorescence, UV–Visible spectrophotometry, and circular dichroism (CD) analyses indicated that the conformation of trypsin was changed upon binding with P4R, which could affect the trypsin function. Kinetic studies revealed that P4R inhibited trypsin activity in a non-competitive model, and docking analysis confirmed that P4R was not bound to the active site of the trypsin. The molecular dynamics (MD) simulation studies showed that the root mean square deviation (RMSD) in the trypsin-P4R complex (0.270 ± 0.02 nm) was larger than that of the free trypsin system (0.221 ± 0.01 nm), which agreed with CD and thermal stability experiments. Thus, P4R was shown to be a potent inhibitor and quencher of trypsin.

Identification and Target-Modification of SL-BBI: A Novel Bowman-Birk Type Trypsin Inhibitor from Sylvirana latouchii.

The peptides from the ranacyclin family share similar active disulphide loop with plant-derived Bowman–Birk type inhibitors, some of which have the dual activities of trypsin inhibition and antimicrobial. Herein, a novel Bowman–Birk type trypsin inhibitor of the ranacyclin family was identified from the skin secretion of broad-folded frog (Sylvirana latouchii) by molecular cloning method and named as SL-BBI. After chemical synthesis, it was proved to be a potent inhibitor of trypsin with a Ki value of 230.5 nM and showed weak antimicrobial activity against tested microorganisms. Modified analogue K-SL maintains the original inhibitory activity with a Ki value of 77.27 nM while enhancing the antimicrobial activity. After the substitution of active P1 site to phenylalanine and P2′ site to isoleucine, F-SL regenerated its inhibitory activity on chymotrypsin with a Ki value of 309.3 nM and exhibited antiproliferative effects on PC-3, MCF-7 and a series of non-small cell lung cancer cell lines without cell membrane damage. The affinity of F-SL for the β subunits in the yeast 20S proteasome showed by molecular docking simulations enriched the understanding of the possible action mode of Bowman–Birk type inhibitors. Further mechanistic studies have shown that F-SL can activate caspase 3/7 in H157 cells and induce apoptosis, which means it has the potential to become an anticancer agent.

Abstract 3078: Effects of upamostat and opaganib on cholangiocarcinoma patient derived xenografts

Abstract Background: Upamostat is an orally available small molecule serine protease inhibitor that is a highly potent inhibitor of trypsin 1, trypsin 2 and trypsin 3 (PRSS1/2/3) as well as urokinase-type plasminogen activator (uPA) which are expressed in many cancers and mediate cell migration, invasion and tissue remodeling. Opaganib (ABC294640), a novel, orally available small molecule is a specific inhibitor of sphingosine kinase 2 (SPHK2), which phosphorylates sphingosine to sphingosine-1-phosphate (S-1-P). While proliferation induced by S-1-P is regulated by both sphingosine kinase 1 (SPHK1) and SPHK2, SPHK2 appears to be more involved in cancer. We aimed to investigate the potential antitumor effect of upamostat and opaganib, individually and in combination, on cholangiocarcinoma (CCA) patient derived xenografts (PDX) in nude mice. Methods: PAX165, a PDX from a surgically resected CCA, expresses substantial levels of SPHK2, PRSS1, PRSS2 and PRSS3. 4 groups of 18 mice were treated with either drug or both. Mouse weights and tumor volumes were measured. In addition, experiments were conducted using the chorioallantoic membrane (CAM) of chicken embryos. Results: Table 1 shows the average tumor size for the control, upamostat, opaganib, and upamostat+opaganib groups at the study end point (Day 42). Tumor volumes in the upamostat, opaganib, and upamostat+opagnib groups were significantly decreased compared to the control group. The CAM experiments are ongoing and will be presented at the AACR Annual Meeting. Change in tumor volumes (mean) of CCA PDX after opaganib, upamostat or combination treatmentControlOpaganibUpamostatOpaganib+UpamostatPre-treatment129.9128.7118.8126.8Day 42198.6102.093.3186.09Percent change Day 0-42+53%-21%-21%-32%P value vs. control0.00020.00100.0008 Conclusion: This preclinical study demonstrated that upamostat and opaganib resulted in tumor regression in mice. Body weights of the mice showed no significant inter- or intra- group differences. The combination of upamostat and opaganib treatment showed greater regression compared to either upamostat or opaganib alone. Studies are underway to identify the molecular mechanisms of their interaction. Citation Format: Faizal Z. Asumda, Mohamed A. Hassan, Yo Han Kim, Nellie A. Campbell, Xin Luo, Daniel R. O'Brien, Sarah A. Buhrow, Joel M. Reid, Michael J. Moore, Vered Katz Ben-Yair, Reza Fathi, Mark L. Levitt, Fabrice Lucien-Matteoni, Jennifer L. Leiting, Mark J. Truty, Lewis R. Roberts. Effects of upamostat and opaganib on cholangiocarcinoma patient derived xenografts [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3078.

Head-to-Tail Cyclization after Interaction with Trypsin: A Scorpion Venom Peptide that Resembles Plant Cyclotides.

Peptidase inhibitors (PIs) have been broadly studied due to their wide therapeutic potential for human diseases. A potent trypsin inhibitor from Tityus obscurus scorpion venom was characterized and named ToPI1, with 33 amino acid residues and three disulfide bonds. The X-ray structure of the ToPI1:trypsin complex, in association with the mass spectrometry data, indicate a sequential set of events: the complex formation with the inhibitor Lys32 in the trypsin S1 pocket, the inhibitor C-terminal residue Ser33 cleavage, and the cyclization of ToPI1 via a peptide bond between residues Ile1 and Lys32. Kinetic and thermodynamic characterization of the complex was obtained. ToPI1 shares no sequence similarity with other PIs characterized to date and is the first PI with CS-α/β motif described from animal venoms. In its cyclic form, it shares structural similarities with plant cyclotides that also inhibit trypsin. These results bring new insights for studies with venom compounds, PIs, and drug design.

In Silico Investigation of the Binding of MCoTI-II Plant Defense Knottin to the γ-NGF Serine Protease of the 7S Nerve Growth Factor Complex and Biological Activity of Its NGF Mimetic Properties.

Nerve growth factor (NGF) is an endogenously produced polypeptide that promotes the differentiation, survival, and repair of neurons in the central and peripheral nervous systems. While trophic proteins hold promise for the treatment of neuronal injury and disease, use of NGF is limited by its large molecular weight, lack of permeability through the blood-brain barrier, and peripheral side effects. Previously, we found that an extract of the Momordica cochinchinensis seed stimulated PC-12 neurite outgrowth. Bioactivity-guided fractioning of the seed extract suggested that the NGF mimetic agent was one of few defined proteins from this plant: one group being the defense Knottins and the other group of the lowest mass is the potent trypsin inhibitor MCoTI-II. Here, the NGF mimetic potential of this latter protein was investigated using two concurrent but different approaches. A biological study used recombinant purified MCoTI-II, which when tested in rat PC-12 cells grown on collagen, failed to initiate outgrowth relative to the positive control 7S NGF. In a separate computational study, the possibility was investigated such that MCoTI-II could exert an effect through binding to the serine protease γ-NGF subunit of the 7S NGF complex, analogous to its binding to its native receptor trypsin. Molecular dynamics simulations showed that MCoTI-II can bind stably to γ-NGF for >350 ns. Modeling indicated that this interaction could sterically inhibit 7S NGF complex formation, potentially altering the equilibrium between inactive complexed and free active NFG protein. In conclusion, the biological study now excludes the MCoTI-II protein as the NGF mimetic factor in the Momordica extract, an important and required step to identify the active component in this seed. On the other hand, the theoretical study has revealed a novel observation that may be of use in the development of strategies to affect NGF activity.

Abstract 4841: Rapid screening of cyclotide-based libraries for the selection of potent E3 ligase antagonists

Abstract We report novel methods for the biosynthesis of natively-folded MCoTI-based cyclotides inside live E. coli cells using a split protein splicing unit. The cyclotide MCoTI-cylotides are potent trypsin inhibitors recently isolated from the seeds of Momordica cochinchinensis, a plant member of cucurbitaceae family. Biosynthesis of genetically encoded cyclotide-based libraries opens the possibility of using single cells as microfactories where the biosynthesis and screening of a particular inhibitor can take place in a single process within the same cellular cytoplasm. The cyclotide scaffold has a tremendous potential for the development of therapeutic leads based on their extraordinary stability and potential for grafting applications. We will show an example, where a large cyclotide-based genetically-encoded library was used to screen for low nanomolar antagonists for the Hdm2-HdmX RING-mediated E3 ligase activity. We will also present different strategies to improve the cellular uptake and pharmacokinetic profiles of bioactive cyclotides. Citation Format: Julio A. Camarero. Rapid screening of cyclotide-based libraries for the selection of potent E3 ligase antagonists [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4841.

Engineering potent mesotrypsin inhibitors based on the plant-derived cyclic peptide, sunflower trypsin inhibitor-1

Plants produce a diverse range of peptides and proteins that inhibit the activity of different serine proteases. The value of these inhibitors not only stems from their native role(s) in planta, but they are also regarded as promising templates for inhibitor engineering. Interest in this field has grown rapidly in recent years, particularly for therapeutic applications. The serine protease mesotrypsin has been implicated in several cancers, but is a challenging target for inhibitor engineering as a number of serine protease inhibitors that typically display broad-range activity show limited activity against mesotrypsin. In this study, we use a cyclic peptide isolated from sunflower seeds, sunflower trypsin inhibitor-1 (SFTI-1), as a scaffold for engineering potent mesotrypsin inhibitors. SFTI-1 comprises 14-amino acids and is a potent inhibitor of human cationic trypsin (Ki = 30 ± 0.8 pM) but shows 165,000-fold weaker activity against mesotrypsin (Ki = 4.96 ± 0.2 μM). Using an inhibitor library based on SFTI-1, we show that the inhibitor's P2′ residue (Ile) is a key contributor to SFTI-1's limited activity against mesotrypsin. Substituting P2′ Ile with chemically diverse amino acids, including non-canonical aromatic residues, produced new inhibitor variants that maintained a similar structure to SFTI-1 and showed marked improvements in activity (exceeding 100-fold). An assessment of the activity of the new inhibitors against closely-related trypsin paralogs revealed that the improved activity against mesotrypsin was accompanied by a loss in activity against off-target proteases, such that several engineered variants showed comparable activity against mesotrypsin and human cationic trypsin. Together, these findings identify potent mesotrypsin inhibitors that are suitable for further optimisation studies and demonstrate the potential gains in activity and selectivity that can be achieved by optimising the P2′ residue, particularly for engineered SFTI-based inhibitors.

Divergent Protein Synthesis of Bowman–Birk Protease Inhibitors, their Hydrodynamic Behavior and Co-crystallization with α-Chymotrypsin

A divergent protein synthesis strategy was executed to effectively synthesize Bowman–Birk protease inhibitor (BBI) analogues using native chemical ligation of peptide hydrazides. Grafting selected residues from a potent trypsin inhibitor, sunflower trypsin inhibitor-1, onto the α-chymotrypsin-binding loop of BBI, resulted in a fourfold improvement of α-chymotrypsin inhibition. The crystal structure of a synthetic BBI analogue co-crystallized with α-chymotrypsin confirmed the correct protein fold and showed a similar overall structure to unmodified BBI in complex with α-chymotrypsin. Dynamic light scattering showed that C-terminal truncation of BBI led to increased self-association.

A case of neofunctionalization of a Putranjiva roxburghii PNP protein to trypsin inhibitor by disruption of PNP-UDP domain through an insert containing inhibitory site

The attainment of new function by a protein is achieved through convergent/divergent evolution. In present work, the sequence analysis of a 34kDa protein from Putranjiva roxburghii, earlier reported as a potent trypsin inhibitor, showed resemblance to some of the wound inducible and vegetative storage proteins. A detailed sequence analysis revealed that these proteins belong to PNP-UDP family. In case of P. roxburghii protein, an approximately 46 residue insert disrupts the PNP domain. Similar disruption of PNP domain is observed in related plant proteins. The characterization of recombinant full length and truncated (without 46 residue insert) forms of P. roxburghii PNP family protein (PRpnp) unraveled that trypsin inhibitory active site is located within the insert. The truncated form containing uninterrupted PNP domain showed strong PNP enzymatic activity where it hydrolyzed the N-glycosidic bond of inosine and guanosine. The full length protein, however, showed weak PNP enzyme activity which may be due to presence of the insert. These results indicate towards the neofunctionalization of PRpnp to a potent trypsin inhibitor through an insert containing inhibitory residue to cater to the needs of plant defense. The similar wound inducible and vegetative storage proteins may have also evolved due to evolutionary needs.

Natural structural diversity within a conserved cyclic peptide scaffold.

We recently isolated and described the evolutionary origin of a diverse class of small single-disulfide bonded peptides derived from Preproalbumin with SFTI-1 (PawS1) proteins in the seeds of flowering plants (Asteraceae). The founding member of the PawS derived peptide (PDP) family is the potent trypsin inhibitor SFTI-1 (sunflower trypsin inhibitor-1) from Helianthus annuus, the common sunflower. Here we provide additional structures and describe the structural diversity of this new class of small peptides, derived from solution NMR studies, in detail. We show that although most have a similar backbone framework with a single disulfide bond and in many cases a head-to-tail cyclized backbone, they all have their own characteristics in terms of projections of side-chains, flexibility and physiochemical properties, attributed to the variety of their sequences. Small cyclic and constrained peptides are popular as drug scaffolds in the pharmaceutical industry and our data highlight how amino acid side-chains can fine-tune conformations in these promising peptides.

Trypsin inhibitors for the treatment of pancreatitis

Proline-based trypsin inhibitors occupying the S1–S2–S1′ region were identified by an HTS screening campaign. It was discovered that truncation of the P1′ moiety and appropriate extension into the S4 region led to highly potent trypsin inhibitors with excellent selectivity against related serine proteases and a favorable hERG profile.

Kunitzins: Prototypes of a new class of protease inhibitor from the skin secretions of European and Asian frogs

Amphibian skin secretions contain biologically-active compounds, such as anti-microbial peptides and trypsin inhibitors, which are used by biomedical researchers as a source of potential novel drug leads or pharmacological agents. Here, we report the application of a recently developed technique within our laboratory to “shotgun” clone the cDNAs encoding two novel but structurally-related peptides from the lyophilised skin secretions of one species of European frog, Rana esculenta and one species of Chinese frog, Odorrana schmackeri. Bioanalysis of the peptides established the structure of a 17-mer with an N-terminal Ala (A) residue and a C-terminal Cys (C) residue with a single disulphide bridge between Cys 12 and 17, which is a canonical Kunitz-type protease inhibitor motif (-CKAAFC-). Due to the presence of this structural attribute, these peptides were named kunitzin-RE (AAKIILNPKFRCKAAFC) and kunitzin-OS (AVNIPFKVHLRCKAAFC). Synthetic replicates of these two novel peptides were found to display a potent inhibitory activity against Escherichia coli but were ineffective at inhibiting the growth of Staphylococcus aureus and Candida albicans at concentrations up to 160 μM, and both showed little haemolytic activity at concentrations up to 120 μM. Subsequently, kunitzin-RE and kunitzin-OS were found to be a potent inhibitor of trypsin with a Ki of 5.56 μM and 7.56 μM that represent prototypes of a novel class of highly-attenuated amphibian skin protease inhibitor. Substitution of Lys-13, the predicted residue occupying the P1 position within the inhibitory loop, with Phe (F) resulted in decrease in trypsin inhibitor effectiveness and antimicrobial activity against Esherichia coli, but exhibits a potential inhibition activity against chymotrypsin.

Computational analysis of the MCoTI-II plant defence knottin reveals a novel intermediate conformation that facilitates trypsin binding.

MCoTI-I and II are plant defence proteins, potent trypsin inhibitors from the bitter gourd Momordica cochinchinensis. They are members of the Knottin Family, which display exceptional stability due to unique topology comprising three interlocked disulfide bridges. Knottins show promise as scaffolds for new drug development. A crystal structure of trypsin-bound MCoTI-II suggested that loop 1, which engages the trypsin active site, would show decreased dynamics in the bound state, an inference at odds with an NMR analysis of MCoTI-I, which revealed increased dynamics of loop 1 in the presence of trypsin. To investigate this question, we performed unrestrained MD simulations of trypsin-bound and free MCoTI-II. This analysis found that loop 1 of MCoTI-II is not more dynamic in the trypsin-bound state than in the free state. However, it revealed an intermediate conformation, transitional between the free and bound MCoTI-II states. The data suggest that MCoTI-II binding involves a process in which initial interaction with trypsin induces transitions between the free and intermediate conformations, and fluctuations between these states account for the increase in dynamics of loop 1 observed for trypsin-bound MCoTI-I. The MD analysis thus revealed new aspects of the inhibitors’ dynamics that may be of utility in drug design.

Partial purification and characterization of a trypsin inhibitor isolated from Adenanthera pavonina L. seeds

Trypsin inhibitors are important tools used by plants against parasites and once isolated may be directed to biotechnological applications. In this work, a trypsin inhibitor was extracted and isolated from the seeds of Adenanthera pavonina L. (Fabaceae). After, the biomolecule was purified and characterized in terms of its thermal stability and molecular weight. In addition its toxic effect against Artemia salina was investigated. The purification process was based in cetonic precipitation with acetone 70% (v/v) and anion-exchange chromatography on DEAE-Sephadex G-50. SDS–PAGE analysis, under reducing condition, showed that protein consists of a single polypeptide chain with molecular mass of approximately 14.1kDa. The biomolecule is a competitive inhibitor with an inhibition stoichiometry of 1:2 for bovine trypsin. The inhibitor purified presented thermal stability in wide range of temperature (25–100°C), and showed still effective inhibition (61%) until 100°C during 30min. Furthermore a volume of 0.16mg/mL of the inhibitor was enough to present toxicity against the larvae of Artermia salina (100% of death rate after 72h). The species A. pavonina is therefore source of trypsin inhibitors with high activity and could be used for obtaining the biomolecule in accord to its demand mostly in pharmaceutical and environment industry, medicine and others. As a likely source of potent trypsin inhibitors, the extract obtained from seeds of A. pavonina is discussed to be useful directly in the agro-industry, in the production of value-added bioproducts or in general biotechnological applications.

Cloning and Characterization of Two Potent Kunitz Type Protease Inhibitors from Echinococcus granulosus.

The tapeworm Echinococcus granulosus is responsible for cystic echinococcosis (CE), a cosmopolitan disease which imposes a significant burden on the health and economy of affected communities. Little is known about the molecular mechanisms whereby E. granulosus is able to survive in the hostile mammalian host environment, avoiding attack by host enzymes and evading immune responses, but protease inhibitors released by the parasite are likely implicated. We identified two nucleotide sequences corresponding to secreted single domain Kunitz type protease inhibitors (EgKIs) in the E. granulosus genome, and their cDNAs were cloned, bacterially expressed and purified. EgKI-1 is highly expressed in the oncosphere (egg) stage and is a potent chymotrypsin and neutrophil elastase inhibitor that binds calcium and reduced neutrophil infiltration in a local inflammation model. EgKI-2 is highly expressed in adult worms and is a potent inhibitor of trypsin. As powerful inhibitors of mammalian intestinal proteases, the EgKIs may play a pivotal protective role in preventing proteolytic enzyme attack thereby ensuring survival of E. granulosus within its mammalian hosts. EgKI-1 may also be involved in the oncosphere in host immune evasion by inhibiting neutrophil elastase and cathepsin G once this stage is exposed to the mammalian blood system. In light of their key roles in protecting E. granulosus from host enzymatic attack, the EgKI proteins represent potential intervention targets to control CE. This is important as new public health measures against CE are required, given the inefficiencies of available drugs and the current difficulties in its treatment and control. In addition, being a small sized highly potent serine protease inhibitor, and an inhibitor of neutrophil chemotaxis, EgKI-1 may have clinical potential as a novel anti-inflammatory therapeutic.

Pseudoaeruginosins, nonribosomal peptides in Nodularia spumigena.

Nodularia spumigena is a filamentous cyanobacterium that forms toxic blooms in brackish waters around the world through the production of the pentapeptide toxin nodularin. This cyanobacterium also produces large amounts of protease inhibitors belonging to the aeruginosin and spumigin families. Here we report the discovery of previously unknown protease inhibitors, pseudoaeruginosins NS1 (1) and NS2 (2), from 33 strains of N. spumigena isolated from the Baltic Sea. Pseudoaeruginosin NS1 (1) and NS2 (2) contain hexanoic acid, tyrosine, 4-methylproline, and argininal/argininol. The chemical structure of the two pseudoaeruginosins was verified by thorough comparison of the liquid chromatography-mass spectrometry (LC-MS) analyses of the extracts from the N. spumigena strains with synthetic peptides. The structures of the synthetic pseudoaeruginosins were confirmed using nuclear magnetic resonance spectroscopy. Surprisingly, the structure of pseudoaeruginosin NS1 (1) and NS2 (2) combines features of both aeruginosins and spumigins, suggesting that they have been produced through the joint action of both the spumigin and aeruginosin biosynthesis pathways. We screened with polymerase chain reaction and LC-MS 68 N. spumigena strains from the Baltic Sea and Australia. Pseudoaeruginosins were present in half of the Baltic Sea strains but were not found from the Australian strains. The production of pseudoaeruginosin seems to be coupled to the production of aeruginosins and 4-methylproline-containing spumigins. Pseudoaeruginosin NS1 was found to be as potent trypsin inhibitor as the most potent aeruginosins and spumigins with an IC50 of 0.19 ± 0.04 μM. This finding suggests that cooperation between the spumigin and aeruginosin biosynthetic pathways results in hybrid pseudoaeruginosin peptides.

The evolution of Momordica cyclic peptides.

Cyclic proteins have evolved for millions of years across all kingdoms of life to confer structural stability over their acyclic counterparts while maintaining intrinsic functional properties. Here, we show that cyclic miniproteins (or peptides) from Momordica (Cucurbitaceae) seeds evolved in species that diverged from an African ancestor around 19 Ma. The ability to achieve head-to-tail cyclization of Momordica cyclic peptides appears to have been acquired through a series of mutations in their acyclic precursor coding sequences following recent and independent gene expansion event(s). Evolutionary analysis of Momordica cyclic peptides reveals sites that are under selection, highlighting residues that are presumably constrained for maintaining their function as potent trypsin inhibitors. Molecular dynamics of Momordica cyclic peptides in complex with trypsin reveals site-specific residues involved in target binding. In a broader context, this study provides a basis for selecting Momordica species to further investigate the biosynthesis of the cyclic peptides and for constructing libraries that may be screened against evolutionarily related serine proteases implicated in human diseases.

Enhancement of Quantum Dot Förster Resonance Energy Transfer within Paper Matrices and Application to Proteolytic Assays

Brightly luminescent semiconductor quantum dots (QDs) continue to play an increasing role in biophotonic research and applications such as bioassays. Here, we present methods for the immobilization of QDs on the cellulose fibers of paper substrates for Förster resonance energy transfer (FRET)-based assays of proteolytic activity. Steady-state and time-resolved fluorescence characterization of FRET between immobilized QDs and self-assembled dye-labeled peptides within the paper matrix revealed a substantial enhancement in energy transfer efficiency. Compared to bulk solution, the rate of energy transfer increased approximately fourfold resulting in a concomitant sevenfold increase in the ratio of FRET-sensitized acceptor dye emission and quenched QD emission. Spots of immobilized QDs with different amounts of dye-labeled peptide had bright luminescence under UV/violet illumination and the net QD and Alexa Fluor 555 (A555) dye emission was visible by eye as different colors. Tryptic digestion of the peptides linking the QD donor and the acceptor dyes resulted in loss of FRET. Changes in the dye/QD photoluminescence (PL) ratio permitted the tracking of proteolytic activity, including the effect of increasing amounts of aprotinin, a potent inhibitor of trypsin. The combination of QDs, a paper substrate, and enhanced FRET has strong potential for developing bioassays.