11-mer Peptide Research Articles

Development of a mesothelin-based prophylactic vaccine against ovarian cancer

Abstract Mesothelin is a tumor-associated antigen overexpressed in ovarian cancer and normally expressed by mesothelial linings. While mesothelin function remains to be fully elucidated, the lack of phenotype of mesothelin-knockout mice makes it plausible that mesothelin can safely serve as a vaccine target. We designed a new vaccination strategy based on mesothelin targeting with activation of type I IFN signaling via cyclic dinucleotides (CDN). Vaccines first combined human mesothelin protein with alum- vs. CDN-based adjuvants. C57Bl/6 mice received a priming immunization followed by 2 boosts; two weeks later, immunized mice were injected orthotopically with luciferase-transduced mouse ovarian cancer cells (Luc-ID8). More than 2/3 of the mesothelin/CDN-immunized mice were protected against invasive cancer and presented increased cytotoxic T cells (CTL) and plasmacytoid dendritic cells in spleens. Effector memory T cells were also increased and MDSC were profoundly decreased in peritoneal lavages after immunization with mesothelin +/− CDN compared with all the other groups. IFNγ ELISPOT assays performed with protected animal splenocytes stimulated with mesothelin peptides identified 2 immunogenic regions. We next tested a peptide-based subunit vaccine derived from mouse mesothelin sequence. Immunization and 2 boosts of 25-mer peptides with CDN were sufficient to mount an immune response against 2 overlapping mouse mesothelin peptides. Immunizations with peptide pools mapping in the immunogenic regions are currently assessed for protection against Luc-ID8 growth and progression. Peptide-based prophylactic vaccination against ovarian cancer may be possible in combination with adjuvants that trigger cellular immunity.

Small tumor necrosis factor receptor biologics inhibit the tumor necrosis factor-p38 signalling axis and inflammation

Despite anti-TNF therapy advancements for inflammatory diseases such as rheumatoid arthritis, the burden of diseases remains high. An 11-mer TNF peptide, TNF70–80, is known to stimulate selective functional responses compared to the parent TNF molecule. Here, we show that TNF70–80 binds to the TNF receptor, activating p38 MAP kinase through TNF receptor-associated factor 2. Using truncated TNFR mutants, we identify the sequence in TNFRI which enables p38 activation by TNF70–80. Peptides with this TNFRI sequence, such as TNFRI206–211 bind to TNF and inhibit TNF-induced p38 activation, respiratory burst, cytokine production and adhesion receptor expression but not F-Met-Leu-Phe-induced respiratory burst in neutrophils. TNFRI206–211 does not prevent TNF binding to TNFRI or TNF-induced stimulation of ERK, JNK and NF-κB. TNFRI206–211 inhibits bacterial lipopolysaccharide-induced peritonitis, carrageenan-induced and antigen-induced paw inflammation, and respiratory syncytial virus-induced lung inflammation in mice. Our findings suggest a way of targeting TNF-p38 pathway to treat chronic inflammatory disorders.

A novel synthetic peptide inspired on Lys49 phospholipase A2 from Crotalus oreganus abyssus snake venom active against multidrug-resistant clinical isolates

Currently, the evolving and complex mechanisms of bacterial resistance to conventional antibiotics are increasing, while alternative medicines are drying up, which urges the need to discover novel agents able to kill antibiotic-resistant bacteria. Lys49 phospholipase A2s (PLA2s) from snake venoms are multifunctional toxins able to induce a huge variety of therapeutic effects and consequently serve as templates for new drug leads. Hence, the present study was aimed at the synthesis of oligopeptides mimicking regions of the antibacterial Lys49 PLA2 toxin (CoaTx-II), recently isolated from Crotalus oreganus abyssus snake venom, to identify small peptides able to reproduce the therapeutic action of the toxin. Five peptides, representing major regions of interest within CoaTx-II, were synthesized and screened for their antibacterial properties. The 13-mer peptide pC-CoaTxII, corresponding to residues 115–129 of CoaTx-II, was able to reproduce the promising bactericidal effect of the toxin against multi-resistant clinical isolates. Peptide pC-CoaTxII is mainly composed by positively charged and hydrophobic amino acids, a typical trait in most antimicrobial peptides, and presented no defined secondary structure in aqueous environment. The physicochemical properties of pC-CoaTxII are favorable towards a strong interaction with anionic lipid membranes as those in bacteria. Additional in silico studies suggest formation of a water channel across the membrane upon peptide insertion, eventually leading to bacterial cell disruption and death. Overall, our findings confirm the valuable potential of snake venom toxins towards design and synthesis of novel antimicrobials, thus representing key insights towards development of alternative efficient antimicrobials to fight bacterial resistance to current antibiotics.

Design and NMR Structural Studies of New Antimicrobial Peptides with Higher Activity

The availability of antibiotics has allowed for the successful treatment of many bacterial infections as well as the ability to perform invasive medical procedures including surgery and chemotherapy. However, their wide use has led to pathogens' increased drug resistance and the need to find novel classes of antimicrobial peptides as alternatives to antibiotics. Lactophoricin (LPcin), a cationic amphipathic peptide consists of 23-mer peptide, corresponds to the carboxy terminal 113-135 region of component-3 of proteose-peptone and is a good candidate as a peptide antibiotic because it has an antibacterial activity but no hemolytic activity. Three different analogs of LPcin, LPcin-yk2 which has mutant amino acids, LPcin-yk1 and LPcin-yk3 that has shorter mutant amino acids are recently developed by using peptide engineering techniques in our laboratory and show better antibioitic activities than wild-type LPcin and no toxicity at all. Recently, we designed and selected more antimicrobial peptides LPcin-YK5, LPcub-YK8 and LPcin-YK11 based on LPcin-YK3. Designed all analogues were expressed and purified using several biophysical techniques and characterized using antimicrobial activity tests and various spectroscopic methods like MALDI-TOF MS and CD spectrometry, as well as 1D/2D solution NMR and solid-state NMR techniques in membrane environments. In here, we will present the optimizing processes with high-yield expression and purification of new LPcin analogs and solid- state NMR structural studies to figure out antibacterial killing mechanisms.

Targeted Imaging of Brain Tumors with a Framework Nucleic Acid Probe.

Development of agents for delivering drugs and imaging probes across the blood-brain barrier (BBB) remains a major challenge. In this study, we designed a biocompatible framework nucleic acid (FNA)-based imaging probe for brain tumor-targeting. We employed a typical type of FNAs, tetrahedral DNA nanostructures (TDNs), as the building block, which were modified with angiopep-2 (ANG), a 19-mer peptide derived from human Kunitz domain of aprotinin. This probe exhibited high binding efficiency with low-density lipoprotein receptor-related protein-1 (LRP-1) of BBB and glioma. We found that ANG-functionalized TDNs (ANG-TDNs) stayed intact for at least 12 h in serum, and that ANG modification effectively enhanced cellular uptake of TDNs in brain capillary endothelial cells and Uppsala 87 malignant glioma (U87MG) cells. Remarkably, studies in both in vitro and in vivo models revealed that ANG-TDNs could cross the BBB. Especially, in vivo imaging showed strong fluorescent signals in U87MG human glioblastoma xenograft in nude mice. This study establishes that the FNA-based platform provides a new theranostic tool for the study and therapy of brain tumors.

Mutation Based Structural Modelling and Dynamics Study of Alpha Fetoprotein: An Insight to Inhibitory Mechanism in Breast Cancer

The incidence of breast cancer is amassed in the current era due to the advent in urbanization, increase in sophisticated vicissitude living, and espousal of western lifestyles. Alpha- fetoprotein, a serum glycoprotein produced during embryonic development tends to act as the curative mediator and has anti-estrotrophic properties to inhibit the growth of estrogen- dependent tumor’s in breast cancer and metastasis. This oncofetal protein exhibits pharmaceutical activity during en route cancer metastasis and pathways lead to tumor cell progression and proliferation. In this work, the maximal inhibitory action of the peptide derived from the active site segment, which was previously suggested in experimental works against mice xenografts (8-mer Peptide), was derived from the structural model generated by homology modelling that retains the inhibitory activity exhibited by the derived AFPep P489- P496 (EMTPVNPG). A comparable mutation study has been undertaken in the derived peptide region to maximize the inhibitory action of the above-said activities. Comparative molecular dynamics study of each mutation has been carried out to know the stability of the octapeptide 489-496 to ensure the curative perspective that is indulged in inhibiting the progression and proliferation of oncofetal proteins in breast cancer. Another modification to the derived peptide was done by addition of hydroxyproline group to the region selected that was previously suggested with the combined effect of tamoxifen and hydroxyproline associated peptide. Molecular docking studies have also been carried out for the octapeptide against Hsp70 which might help in stabilising the anti tumour associated peptide AFPep for better binding efficacy for maximal inhibitory action and treatment of breast cancer.

In vivo therapeutic evaluation of polymeric nanomedicines: effect of different targeting peptides on therapeutic efficacy against breast cancer.

Targeted nanomedicines offer many advantages over macromolecular therapeutics that rely only on passive accumulation within the tumour environment. The aim of this work was to investigate the in vivo anticancer efficiency of polymeric nanomedicines that were conjugated with peptide aptamers that show high affinity for receptors on many cancer cells. In order to assess the ability for the nanomedicine to treat cancer and investigate how structure affected the behavior of the nanomedicine, three imaging modalities were utilized, including in vivo optical imaging, multispectral optoacoustic tomography (MSOT) and ex vivo confocal microscopy. An 8-mer (A8) or 13-mer (A13) peptide aptamer that have been shown to exhibit high affinity for heat shock protein 70 (HSP70) was covalently-bound to hyperbranched polymer (HBP) nanoparticles with the purpose of both cellular targeting, as well as the potential to impart some level of chemo-sensitization to the cells. Furthermore, doxorubicin was bound to the polymeric carrier as the anticancer drug, and Cyanine-5.5 (Cy5.5) was incorporated into the polymer as a monomeric fluorophore to aid in monitoring the behavior of the nanomedicine. Enhanced tumour regression was observed in nude mice bearing MDA-MB-468 xenografts when the nanocarriers were targeted using the peptide ligands, compared to control groups treated with free DOX or HBP without aptamer. The accumulated DOX level in solid tumours was 5.5 times higher in mice treated with the targeted therapeutic, than mice treated with free DOX, and 2.6 times higher than the untargeted nanomedicine that relied only on passive accumulation. The results suggest that aptamer-targeted therapeutics have great potential for improving accumulation of nanomedicines in tumours for therapy.

A novel peptidic inhibitor derived from Streptococcus cristatus ArcA attenuates virulence potential of Porphyromonas gingivalis

Periodontitis is a global health problem and the 6th most common infectious disease worldwide. Porphyromonas gingivalis is considered a keystone pathogen in the disease and is capable of elevating the virulence potential of the periodontal microbial community. Strategies that interfere with P. gingivalis colonization and expression of virulence factor are therefore attractive approaches for preventing and treating periodontitis. We have previously reported that an 11-mer peptide (SAPP) derived from Streptococcus cristatus arginine deiminase (ArcA) was able to repress the expression and production of several well-known P. gingivalis virulence factors including fimbrial proteins and gingipains. Herein we expand and develop these studies to ascertain the impact of this peptide on phenotypic properties of P. gingivalis related to virulence potential. We found that growth rate was not altered by exposure of P. gingivalis to SAPP, while monospecies and heterotypic biofilm formation, and invasion of oral epithelial cells were inhibited. Additionally, SAPP was able to impinge the ability of P. gingivalis to dysregulate innate immunity by repressing gingipain-associated degradation of interleukin-8 (IL8). Hence, SAPP has characteristics that could be exploited for the manipulation of P. gingivalis levels in oral communities and preventing realization of virulence potential.

Binding of calcium and target peptide to calmodulin-like protein CML19, the centrin 2 of Arabidopsis thaliana

Calmodulin-like protein 19 (CML19) is an Arabidopsis centrin that modulates nucleotide excision repair (NER) by binding to RAD4 protein, the Arabidopsis homolog of human Xeroderma pigmentosum complementation group C protein. Although the necessity of CML19 as a part of the RAD4 plant recognition complex for functional NER is known at a cellular level, little is known at a molecular level. Herein, we used a combination of biophysical and biochemical approaches to investigate the structural and ion and target-peptide binding properties of CML19. We found that CML19 possesses four Ca2+-specific binding sites, two of high affinity in the N-terminal domain and two of low affinity in the C-terminal domain. Binding of Ca2+ to CML19 increases its alpha-helix content, stabilizes the tertiary structure, and triggers a conformational change, resulting in the exposure of a hydrophobic patch instrumental for target protein recognition. Using bioinformatics tools we identified a CML19-binding site at the C-terminus of RAD4, and through in vitro binding experiments we analyzed the interaction between a 17-mer peptide representing this site and CML19. We found that the peptide shows a high affinity for CML19 in the presence of Ca2+ (stoichiometry 1:1) and the interaction primarily involves the C-terminal half of CML19.

Identification of a peptide-peptide binding motif in the coating of nab-paclitaxel nanoparticles with clinical antibodies: bevacizumab, rituximab, and trastuzumab

Antibody directed chemotherapy (ADC) takes advantage of the selectivity of the monoclonal antibody to increase the efficacy of the chemotherapeutic agent, while reducing toxicity. Previously we described three nab-paclitaxel (Abraxane) nanoparticles coated with commercial monoclonal antibodies. Identifying the binding sites responsible for these particles could allow reverse engineering of nab-paclitaxel binding antibodies, creating a modular platform for antibody directed chemotherapeutic nanoparticles. Herein, Biacore surface plasmon resonance is used to identify an antibody binding site, HSA Peptide 40, on human serum albumin with nanomolar affinity for all three monoclonal antibodies. This 18-mer peptide, which lies in Subdomain IIIA of human serum albumin, blocks binding of all three antibodies to nab-paclitaxel when added in excess. We furthermore show the complementary binding region on all three monoclonal antibodies to be the CDR H3 loop of the Fab region, and show that they all have nano to micromolar affinity for HSA Peptide 40 and nab-paclitaxel nanoparticles. The presented data identify the nature of the critical protein-protein interaction that enables antibody coating of nab-paclitaxel.

A Linear 19-Mer Plant Defensin-Derived Peptide Acts Synergistically with Caspofungin against Candida albicans Biofilms.

Public health problems are associated with device-associated biofilm infections, with Candida albicans being the major fungal pathogen. We previously identified potent antibiofilm combination treatment in which the antifungal plant defensin HsAFP1 is co-administered with caspofungin, the preferred antimycotic to treat such infections. In this study, we identified the smallest linear HsAFP1-derived peptide that acts synergistically with caspofungin or anidulafungin against C. albicans as HsLin06_18, a 19-mer peptide derived from the C-terminal part of HsAFP1. The [caspofungin + HsLin06_18] combination significantly reduced in vitro biofilm formation of Candida glabrata and C. albicans on catheters, as well as biofilm formation of a caspofungin-resistant C. albicans strain. The [caspofungin + HsLin06_18] combination was not cytotoxic and reduced biofilm formation of C. albicans in vivo using a subcutaneous rat catheter model, as compared to control treatment. Mode of action research on the [caspofungin + HsLin06_18] combination pointed to caspofungin-facilitated HsLin06_18 internalization and immediate membrane permeabilization. All these findings point to broad-spectrum antibiofilm activity of a combination of HsLin06_18 and caspofungin.

Bifunctional Hydrogels Containing the Laminin Motif IKVAV PromoteNeurogenesis.

SummaryEngineering of biomaterials with specific biological properties has gained momentum as a means to control stem cell behavior. Here, we address the effect of bifunctionalized hydrogels comprising polylysine (PL) and a 19-mer peptide containing the laminin motif IKVAV (IKVAV) on embryonic and adult neuronal progenitor cells under different stiffness regimes. Neuronal differentiation of embryonic and adult neural progenitors was accelerated by adjusting the gel stiffness to 2 kPa and 20 kPa, respectively. While gels containing IKVAV or PL alone failed to support long-term cell adhesion, in bifunctional gels, IKVAV synergized with PL to promote differentiation and formation of focal adhesions containing β1-integrin in embryonic cortical neurons. Furthermore, in adult neural stem cell culture, bifunctionalized gels promoted neurogenesis via the expansion of neurogenic clones. These data highlight the potential of synthetic matrices to steer stem and progenitor cell behavior via defined mechano-adhesive properties.

Using Peptide Aptamer Targeted Polymers as a Model Nanomedicine for Investigating Drug Distribution in Cancer Nanotheranostics.

Theranostics is a strategy that combines multiple functions such as targeting, stimulus-responsive drug release, and diagnostic imaging into a single platform, often with the aim of developing personalized medicine.1,2 Based on this concept, several well-established hyperbranched polymeric theranostic nanoparticles were synthesized and characterized as model nanomedicines to investigate how their properties affect the distribution of loaded drugs at both the cell and whole animal levels. An 8-mer peptide aptamer was covalently bound to the periphery of the nanoparticles to achieve both targeting and potential chemosensitization functionality against heat shock protein 70 (Hsp70). Doxorubicin was also bound to the polymeric carrier as a model chemotherapeutic drug through a degradable hydrazone bond, enabling pH-controlled release under the mildly acid conditions that are found in the intracellular compartments of tumor cells. In order to track the nanoparticles, cyanine-5 (Cy5) was incorporated into the polymer as an optical imaging agent. In vitro cellular uptake was assessed for the hyperbranched polymer containing both doxorubicin (DOX) and Hsp70 targeted peptide aptamer in live MDA-MB-468 cells, and was found to be greater than that of either the untargeted, DOX-loaded polymer or polymer alone due to the specific affinity of the peptide aptamer for the breast cancer cells. This was also validated in vivo with the targeted polymers showing much higher accumulation within the tumor 48 h postinjection than the untargeted analogue. More detailed assessment of the nanomedicine distribution was achieved by directly following the polymeric carrier and the doxorubicin at both the in vitro cellular level via compartmental analysis of confocal images of live cells and in whole tumors ex vivo using confocal imaging to visualize the distribution of the drug in tumor tissue as a function of distance from blood vessels. Our results indicate that this polymeric carrier shows promise as a cancer theranostic, demonstrating active targeting to tumor cells with the capability for simultaneous drug release.

Structural Study of a New HIV-1 Entry Inhibitor and Interaction with the HIV-1 Fusion Peptide in Dodecylphosphocholine Micelles.

Previous studies support the hypothesis that the envelope GB virus C (GBV-C) E1 protein interferes the HIV-1 entry and that a peptide, derived from the region 139-156 of this protein, has been defined as a novel HIV-1 entry inhibitor. In this work, we firstly focus on the characterization of the structural features of this peptide, which are determinant for its anti-HIV-1 activity and secondly, on the study of its interaction with the proposed viral target (i.e., the HIV-1 fusion peptide). We report the structure of the peptide determined by NMR spectroscopy in dodecylphosphocholine (DPC) micelles solved by using restrained molecular dynamics calculations. The acquisition of different NMR experiments in DPC micelles (i.e., peptide-peptide titration, diffusion NMR spectroscopy, and addition of paramagnetic relaxation agents) allows a proposal of an inhibition mechanism. We conclude that a 18-mer peptide from the non-pathogenic E1 GBV-C protein, with a helix-turn-helix structure inhibits HIV-1 by binding to the HIV-1 fusion peptide at the membrane level, thereby interfering with those domains in the HIV-1, which are critical for stabilizing the six-helix bundle formation in a membranous environment.

Abstract LB-203: VXL100, a MUC1 signal peptide based immunotherapy, acts through a neoantigen-like mechanism, and shows promising effects in hematological cancers

Abstract VXL100 (ImMucin®), a 21mer peptide, pan-HLA vaccine derived from the MUC1 signal peptide (SP) domain, has been shown to be safe and to elicit a robust and specific T and B-cell responses in stable and progressive multiple myeloma patients in a phase I/II clinical trial. It is now becoming recognized that the advantages of using the signal peptide domain for immunization stem from its unique B and T cells epitope restriction density and TAP independent presentation. Additionally, the low avidity of signal peptides as antigens, is believed to result in escaping negative selection in the thymus during development, and preservation of active clones in the peripheral blood. This may allow signal peptides to be presented as true neoantigens during tumor development and generate a robust immune response. To prove the neoantigenic properties of MUC1 SP, we have recently tested endogenous levels of specific anti-VXL100 T-cells by staining for VXL100-specific or control multimers. We also performed proliferation assays for patient TIL and normal donors PBMCs using cells stimulated with VXL100 or other MUC1 antigenic control peptides. VXL100 specific T-cell were found in 100% of patients (1.3-6%), VS. 0% in normal donors. We have found a direct correlation between the MUC1 signal peptide expression levels on tumor cells and levels of endogenous VXL100 specific T-cells. Positive signals were observed for multimers of other MUC1 peptides as well, but these were also seen in normal donors, suggesting a non-specific binding. Moreover, we were able to show that VXL100 specific exhausted CD8 cells exist at a non-significant levels as compared to the total exhausted CD8. When stimulated in-vitro with VXL100, PBMCs from 70% of naive normal donors and 94% of cancer patients showed a strong proliferative response (average of 8.9% and 16.5% proliferating cells, respectively). When stimulated with control peptides 0.5% - 5.2% of patients and 0 - 13% of donors showed positive proliferation response. Conclusions: VXL100, a novel immunotherapy agent shows the capacity to elicit a specific and robust T-cell response in healthy individuals and hematological cancer patients. These features suggest that MUC1 SP is a native sequence functioning as a cancer neoantigen and preserved through normal and tumor development. It presents an attractive opportunity for the development of an effective cancer immunotherapy. Citation Format: Limor Chen, Riva Kovjazin, Michael Shapira. VXL100, a MUC1 signal peptide based immunotherapy, acts through a neoantigen-like mechanism, and shows promising effects in hematological cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-203. doi:10.1158/1538-7445.AM2017-LB-203

Identification and molecular cloning of novel antimicrobial peptides from skin secretions of the Chinese bamboo leaf odorous frog (Odorrana versabilis) and the North American pickerel frog (Rana palustris)

ObjectiveAmphibian skin secretions are an abundant source of bioactive peptides, some of which could be developed as candidate drugs. Among these natural peptides, cytolytic peptides have attracted the most attention given that they might replace conventional antibiotics and help deal with the problem of microbial resistance. This study discovered two bioactive peptides, Brevinin-1-PLr and Nigrocin-2-OV, from two species frogs, the Chinese bamboo leaf odorous frog (Odorrana versabilis) and the North American pickerel frog (Rana palustris), respectively. Their antimicrobial, anticancer and hemolytic activities were also investigated. MethodscDNA sequences encoding peptides were cloned from cDNA libraries constructed from the lyophilized secretions of the Chinese bamboo leaf odorous frog and the North American pickerel frog. By reversed-phase HPLC and MS/MS fragmentation sequencing, the encoded novel peptides, named Nigrocin-2-OV and Brevinin-1-PLr, were identified in skin secretions and their structures were confirmed. Replicates of both peptides were produced through solid phase peptide synthesis. Their antimicrobial and anticancer activity was studied against three types of microorganisms (Staphylococcus aureus, Candida albicans, and Escherichia coli) and five cancer cell lines (NCI-H157, PC-3, MDA-MB-435s, MCF-7, and U251MG). Their hemolytic activity was investigated using whole horse blood. ResultsIn this research, cDNA sequences encoding two novel 24-mer peptides were cloned from cDNA libraries constructed from the lyophilized skin secretions of the Chinese bamboo leaf odorous frog and the North American pickerel frog. Both of the peptides had the strongest inhibitory effect against C. albicans, and IC50 values against five cancer cell lines were all under 6 μM. ConclusionsNigrocin-2-OV and Brevinin-1-PLr had the strong ability to inhibit the proliferation of studied microorganisms and tumor cell lines, with slight hemolytic activity. Compared with Brevinin-1-PLr, Nigrocin-2-OV exhibited higher antimicrobial and anticancer activity but slightly higher hemolytic activity.

Free Energy Calculations of Microcin J25 Variants Binding to the FhuA Receptor.

Computer simulations were performed to study the antimicrobial peptide microcin J25 (MJ25), a 21-mer peptide with an unusual lasso structure and high activity against Gram-negative bacteria. MJ25 has intracellular targets. The initial step of MJ25 acquisition in bacterial cells is binding to the outer-membrane receptor FhuA. Molecular dynamics simulations were implemented to study the binding mechanism of MJ25 to FhuA and to search for important binding residues. The absolute binding free energy calculated from combined free energy perturbation and thermodynamic integration methods agrees well with experimental data. In addition, computational mutation analysis revealed that His5 is the key residue responsible for MJ25 and FhuA association. We found that the number of hydrogen bonds is essential for binding of MJ25 to FhuA. This atomistic, quantitative insight sheds light on the mechanism of action of MJ25 and may pave a path for designing active MJ25 analogues.

The human digestive tract has proteases capable of gluten hydrolysis

ObjectiveTo identify, purify, and characterize the proteins responsible for glutenase activity in the feces of healthy subjects and patients with celiac disease (CD). MethodsSixteen subjects were included in this study; 8 were healthy with no known food intolerances, and 8 were treated CD patients on a gluten-free diet. Fecal samples were homogenized, and precipitated proteins were purified by chromatography. Glutenase activity was evaluated by bioassays, zymography, and high-performance liquid chromatography with immunogenic 33-mer, 19-mer, and 13-mer gliadin peptides. ResultsThe gastrointestinal elastase 3B (CEL3B), elastase 2A (CEL2A), and carboxypeptidase A1 (CBPA1) enzymes degraded human gluten. These proteins fully hydrolyzed 13-mer and 19-mer gliadin peptides that trigger immune-mediated enteropathy in individuals genetically predisposed to CD and partially digested a 33-mer. Feces from patients with CD showed more glutenase activity than feces from individuals without CD (171–466% higher). Peptidase activity against the gliadin peptides also increased in patients with CD. ConclusionThe digestive tracts of patients with CD and healthy subjects have enzymatic machinery needed for gluten degradation. Patients with CD showed more gluten hydrolysis than did healthy individuals, although, in both cases, a fraction of 33-mer peptide remained intact. Gliadin peptides derived from gastrointestinal digestion, especially the 33-mer, can potentially be used by commensal microbiota from both CD-positive and CD-negative individuals, and differences in bacterial hydrolysis can modify its immunogenic capacity.

A potent and selective natriuretic peptide receptor-3 blocker 11-mer peptide created by hybridization of musclin and atrial natriuretic peptide

The natriuretic peptide (NP) system is a critical endocrine, autocrine, and paracrine system and has been investigated for potential use against cardiovascular and metabolic diseases. The clearance of NPs is regulated by the proteolysis of neutral endopeptidase (NEP) and by endocytosis via natriuretic peptide receptor-3 (NPR3). A linear NPR3-selective peptide, [Cha8]-ANP(7-16)-NH2 (1), showed potent binding affinity for NPR3 but poor predicted chemical stability due to its free thiol group. A 12-mer peptide (9) without a thiol group was designed by the hybridization of two NPR3-binding peptides: a linear ANP fragment peptide analog and musclin, a murine member of the bHLH family of transcription factors, possessed high binding affinity and strict selectivity for NPR3. To increase the proteolytic resistance of 9, amino acid substitutions at the cleavage sites led to hydroxyacetyl-[d-Phe5,d-Hyp7,Cha8,d-Ser9,Hyp11,Arg(Me)14]-ANP(5-15)-NHCH3 (23), showing high and selective binding affinity for NPR3 over NPR1 and excellent stability in mouse serum. Compound 23 increased intracellular cGMP concentrations in primary cultured adipocytes, and continuous administration induced substantial plasma cGMP elevation in mice, suggesting its potential to clarify the physiological role of NPR3 and its therapeutic application.

Inside Back Cover: Folding Topology of a Short Coiled‐Coil Peptide Structure Templated by an Oligonucleotide Triplex (Chem. Eur. J. 39/2017)

Peptide-oligonucleotide conjugates (POC) were used for the self-assembly of miniature coiled-coil α-helical peptides guided by oligonucleotide duplex and triplex formation. The POC consisted of three oligonucleotides each with a 23-mer peptide attached. The oligonucleotide domains gave a parallel triplex that templated the peptides to form a small coiled-coil motif with high degree of α-helicity. Cooperativity was observed between the trimeric peptide and the oligonucleotide triplex domains. More information can be found in the Full Paper by J. Wengel et al. on page 9297.