12-mer Peptide Research Articles

Guanidinium-Functionalized Flexible Azaproline Transporter for Efficient Intracellular Delivery of Proapoptotic Peptide and PDL1 Antisense Morpholino Oligo in Human Carcinoma Cells In Vitro.

Cell-penetrating peptides (CPPs) are structurally diverse sophisticated tools endowed with high arginine content, amphipathicity, and well-adopted suitable secondary structures. Despite its capability of breaching the lipid barriers, CPP has major limitations such as in vivo metabolic instability, poor bioavailability, and reduced endosomal escape tendency, which are yet to be improved. In this context, we first have introduced a new class of cellular transporter having a guanidinium-functionalized δ-azaproline (δ-azp)-containing peptide where the δ-azp structurally resembles the "proline" amino acid having an additional "N" at the δ-position. This non-natural peptidic backbone was found to impart proteolytic stability, as reported earlier by our group. Herein, we report the synthesis of a flexible azaproline-tetraguanidinium transporter named FAT along with a revised scalable methodology for δ-azp compared to our previously reported procedure. FAT shows a random-coil-like structure as determined by CD spectroscopy, and is hence structurally different from the polyproline PPII helix. Direct translocation is predicted to be the possible mode of the cellular entrance of FAT into CHO cells when the "Bodipy" fluorophore is covalently attached as the cargo. Simultaneously, two other macromolecular therapeutics, e.g., proapoptotic domain peptide (PAD, a 14-mer peptide) and programmed death ligand 1 (PDL1) morpholino (a 25-mer antisense oligo), were successfully conjugated with FAT and delivered into human carcinoma cells, and their efficacy was analyzed by MTT assay and western blot technique, respectively. Having obtained promising results in internalizing different types of cargos, FAT could be envisaged as a potential drug delivery agent as an alternative to natural CPPs for future application.

Identification of functionally important domains of human cytomegalovirus gO that act after trimer binding to receptors.

Human cytomegalovirus (HCMV) entry involves trimer (gH/gL/gO) that interacts with PDGFRα in fibroblasts. Entry into epithelial and endothelial cells requires trimer, which binds unidentified receptors, and pentamer (gH/gL/UL128-131), which binds neuropilin-2. To identify functionally important domains in trimer, we screened an overlapping 20-mer gO peptide library and identified two sets of peptides: 19/20 (a.a. 235–267) and 32/33 (a.a. 404–436) that could block virus entry. Soluble trimer containing wild type gO blocked HCMV entry, whereas soluble trimers with the 19/20 or 32/33 sequences mutated did not block entry. Interestingly, the mutant trimers retained the capacity to bind to cellular receptors including PDGFRα. Peptide 19/20 and 32/33 sequences formed a lobe extending from the surface of gO and an adjacent concave structure, respectively. Neither of these sets of sequences contacted PDGFRα. Instead, our data support a model in which the 19/20 and 32/33 trimer sequences function downstream of receptor binding, e.g. trafficking of HCMV into endosomes or binding to gB for entry fusion. We also screened for peptides that bound antibodies (Abs) in human sera, observing that peptides 20 and 26 bound Abs. These peptides engendered neutralizing Abs (NAbs) after immunization of rabbits and could pull out NAbs from human sera. Peptides 20 and 26 sequences represent the first NAb epitopes identified in trimer. These studies describe two important surfaces on gO defined by: i) peptides 19/20 and 32/33, which apparently act downstream of receptor binding and ii) peptide 26 that interacts with PDGFRα. Both these surfaces are targets of NAbs.

Cutting Edge: Unconventional CD8+ T Cell Recognition of a Naturally Occurring HLA-A*02:01-Restricted 20mer Epitope.

Unconventional HLA class I-restricted CD8+ T cell epitopes, longer than 10 aa, have been implicated to play a role in human immunity against viruses and cancer. T cell recognition of long peptides, centrally bulging from the HLA cleft, has been described previously. Alternatively, long peptides can contain a linear HLA-bound core peptide, with a N- or C-terminal peptide "tail" extending from the HLA peptide binding groove. The role of such a peptide "tail" in CD8+ T cell recognition remains unclear. In this study, we identified a 20mer peptide (FLPTPEELGLLGPPRPQVLA [FLP]) derived from the IL-27R subunit α gene restricted to HLA-A*02:01, for which we solved the crystal structure and demonstrated a long C-terminal "tail" extension. FLP-specific T cell clones demonstrated various recognition modes, some T cells recognized the FLP core peptide, while for other T cells the peptide tail was essential for recognition. These results demonstrate a crucial role for a C-terminal peptide tail in immunogenicity.

A novel navigated doxorubicin delivery formulation to breast cancer therapy

Breast cancer (BC) is the most frequently diagnosed cancer and leading cause of the death in women. Current chemotherapy usually results in poor prognosis with low treatment efficacy and high side effects because of weak targeting specificity and fast elicitation of multidrug resistance (MDR). It is emergently needed to develop a novel BC targeting drug delivery formulation for the chemotherapy of BC and drug resistant BC. In our study, we screened out BC targeting 12-mer peptide (BSP1) from a phage-displayed peptide library, then a novel doxorubicin (DOX) delivery formulation (BSP1-Lipo-DOX-miR101) targeting BC automatically was synthesized and used in a series of experiments to test its efficacy of chemotherapy of BC and DOX resistant BC in vitro. The consequently results indicated that BSP1 bound to MCF-7 cells and BC tissues specifically and sensitively with high affinity, the BC automatically targeted drug vehicles exhibited its high specificity for BC targeting, marked suppression for the development of cell motility relevant microstructures, such as the polymerization of the pseudopodia and filopodia in MCF-7 and MCF-7/ADR cells. The novel drug formulation presented the satisfied therapeutic efficacy for BC therapy, particularly for DOX resistant BC in vitro. The results of western blot explored the potential mechanism by that BSP1-Lipo-DOX-miR101 inhibits the expression of EMT (epithelial/endothelial mesenchymal transition) and MDR relevant genes. Our current results provide a novel chemotherapeutic formulation with precise, satisfying therapeutic efficacy to improve the current poor BC clinical prognosis including drug resistant BC.

Mitigation of sulfate reducing Desulfovibrio ferrophilus microbiologically influenced corrosion of X80 using THPS biocide enhanced by Peptide A

• Peptide A is a non-biocidal biofilm dispersal agent for biocide enhancement. • 20 ppm (w/w) THPS is insufficient to mitigate D. ferrophilus MIC of X80 steel. • 20 ppm THPS is enhanced by 100 ppb Peptide A, achieving 83% lower weight loss. • It also achieves 4-log extra sessile cell reduction compared with 20 ppm THPS. • 20 ppm THPS + 100 ppb Peptide A weight loss is similar to that for 50 ppm THPS. Tetrakis hydroxymethyl phosphonium sulfate (THPS) was enhanced by a 14-mer Peptide A, with its core 12-mer sequence mimicking part of Equinatoxin II protein, in the mitigation of sulfate reducing Desulfovibrio ferrophilus MIC (microbiologically influenced corrosion) of X80 carbon steel. Results proved that 50 ppm (w/w) THPS was sufficient to mitigate the D. ferrophilus biofilm, and its very agressive MIC (19.7 mg/cm 2 in 7 days or 1.31 mm/a), but not 20 ppm THPS. To achieve effective mitigation at a low dosage of THPS, biofilm-dispersing Peptide A was added to 20 ppm THPS in the culture medium. Sessile cell counts were reduced by 2-log and 4-log after enhancement by 10 ppb and 100 ppb Peptide A, respectively. Enhancement efficiency (further reduction in corrosion rate) reached 69% for 10 ppb Peptide A and 83% for 100 ppb Peptide A compared with 20 ppm THPS alone treatment, indicating that Peptide A was a good biocide enhancer for THPS.

A short peptide derived from pigment epithelial-derived factor exhibits an angioinhibitory effect

BackgroundPigment epithelial-derived factor (PEDF), a 50 kDa secreted glycoprotein, exhibits distinct effects on a range of cell types. PEDF has been shown to inhibit vascular endothelial growth factor (VEGF)-mediated angiogenesis and widely accepted as a promising agent for treatment eye diseases related to neovascularization. A pool of short peptide fragments derived from PEDF reportedly manifests angioinhibitory activity. This study aims to determine the minimal PEDF fragment which can exert the anti-VEGF effect.MethodsA series of shorter synthetic peptides, derived from the 34-mer (PEDF amino acid positions Asp44-Asn77), were synthesized. An MTT assay was used to evaluate the ability of the 34-mer-derived peptides to inhibit VEGF-induced proliferation of multiple myeloma RPMI8226 cells. Cell apoptosis was monitored by annexin V-FITC staining. Western blot analysis was used to detect phosphorylated kinases, including c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), and the expression of apoptosis-associated proteins, including p53, bax and caspase-3. VEGF-mediated angiogenesis of human umbilical vein endothelial cells (HUVECs), rat aortic ring and mouse cornea were used to detect the angioinhibitory activity of the PEDF-derived peptides.ResultsThe MTT assay showed that the anti-VEGF effect of a 7-mer (Asp64-Ser70) was 1.5-fold greater than the 34-mer. In addition, massive apoptosis (37%) was induced by 7-mer treatment. The 7-mer induced JNK phosphorylation in RPMI8226 cells. Cell apoptosis and apoptosis-associated proteins induced by the 7-mer were blocked by pharmacological inhibition of JNK, but not p38 MAPK. Moreover, the 7-mer prevented VEGF-mediated angiogenesis of endothelial cells (ECs), including tube formation, aortic EC spreading and corneal neovascularization in mice.ConclusionsThis is the first study to show that the PEDF 7-mer peptide manifests anti-VEGF activity, further establishing its potential as an anti-angiogenic agent.

Utilizing the autoantibody immune response to tumor antigens for kidney cancer early detection.

369 Background: Kidney cancer (renal cell carcinoma, RCC), the 8th most common U.S. cancer, is in need for better cure rates through early detection (5-year relative survival for stage I RCC: ̃95%; for stage IV RCC ̃19%). Autoantibodies are common in cancer and result from the altered expression, localization, or post-translational modification of endogenous proteins in tumor cells (autoantigens) and from the expression of mutated genes that give rise to new proteins (neoantigens). In contrast to cellular immune responses in cancer, autoantibodies are less well characterized, yet hold promise to enable cancer early detection by immune amplification of the ‘cancer signal’ while retaining specificity to cancer types including RCC. Autoantibodies may therefore be useful for kidney cancer early detection and diagnosis. Our goal was to profile the autoantibody repertoire in blood from patients with clear cell RCC (ccRCC), the most common form of RCC, in order to: 1) determine if autoantibodies can be detected in patients with early-stage and late-stage ccRCC; 2) identify common epitopes amongst ccRCC patients that could suggest common RCC antigens; and 3) determine specificity and sensitivity of potential autoantibody biomarkers for ccRCC vs. other non-cancer conditions. Methods: We use the SERA platform (https://serimmune.com/publications/) to compare putative autoantibody signal in blood from 177 patients with ccRCC, 23 with benign kidney lesions, and ̃800 healthy controls. SERA utilizes a random bacterial display 12mer peptide library of 1010 diversity in conjunction with next-generation sequencing to ascertain epitope enrichment across the entire human proteome. Results: We find significant differences in epitope repertoires in ccRCC compared to the healthy human cohort. Patients with ccRCC exhibit a rich repertoire of rare, enriched epitopes which may comprise putative autoantibody signal. This epitope signal is present with high abundance in all ccRCC stages, including stage I ccRCC. In contrast, healthy controls and patients with benign kidney lesions demonstrate more restricted repertoires. However, we do not find evidence of common ccRCC antigens: epitopes are not conserved across large subsets of ccRCC patients. Conclusions: Our initial results suggest that each patient may develop an individualized tumor-associated antibody response. Whether assessing a select epitope panel in a patient’s blood could be useful for ccRCC early detection, or even epitope diversity without needing to identify specific epitopes, warrants further study.

Selection of new diagnostic markers for Dirofilaria repens infections with the use of phage display technology

Dirofilaria repens is a parasitic nematode causing vector-borne disease (dirofilariasis), considered an emerging problem in veterinary and human medicine. Although main hosts are carnivores, particularly dogs, D. repens shows high zoonotic potential. The disease spreads uncontrollably, affecting new areas. Since there is no vaccine against dirofilariasis, the only way to limit disease transmission is an early diagnosis. Currently, diagnosis depends on the detection of microfilariae in the host bloodstream using modified Knott's test or multiplex PCR. However, the efficacy of tests relying on microfilariae detection is limited by microfilariae periodic occurrence. Therefore, a new reliable diagnostic test is required. Our study aimed to select new diagnostic markers for dirofilariasis with potential application in diagnostics. We focused on single epitopes to ensure high specificity of diagnosis and avoid cross-reactivity with the other parasite infections common in dogs. Using phage display technology and 12-mer peptides library, we selected epitopes highly reactive with IgG from sera of infected dogs. Additionally, our study presents the possibility of detecting D. repens specific cell-free DNA in dogs with no microfilaria but high IgG and IgM antibody levels against parasite somatic antigen.

Is Cys(MTSL) the Best α-Amino Acid Residue to Electron Spin Labeling of Synthetically Accessible Peptide Molecules with Nitroxides?

Electron paramagnetic resonance spectroscopy, particularly its pulse technique double electron–electron resonance (DEER) (also termed PELDOR), is rapidly becoming an extremely useful tool for the experimental determination of side chain-to-side chain distances between free radicals in molecules fundamental for life, such as polypeptides. Among appropriate probes, the most popular are undoubtedly nitroxide electron spin labels. In this context, suitable biosynthetically derived, helical regions of proteins, along with synthetic peptides with amphiphilic properties and antibacterial activities, are the most extensively investigated compounds. A strict requirement for a precise distance measurement has been identified in a minimal dynamic flexibility of the two nitroxide-bearing α-amino acid side chains. To this end, in this study, we have experimentally compared in detail the side-chain mobility properties of the two currently most widely utilized residues, namely, Cys(MTSL) and 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC). In particular, two double-labeled, chemically synthesized 20-mer peptide molecules have been adopted as appropriate templates for our investigation on the determination of the model intramolecular separations. These double-Cys(MTSL) and double-TOAC compounds are both analogues of the almost completely rigid backbone peptide ruler which we have envisaged and 3D structurally analyzed as our original, unlabeled compound. Here, we have clearly found that the TOAC side-chain labels are largely more 3D structurally restricted than the MTSL labels. From this result, we conclude that the TOAC residue offers more precise information than the Cys(MTSL) residue on the side chain-to-side chain distance distribution in synthetically accessible peptide molecules.

Chemical Synthesis of a Functional Fluorescent-Tagged α-Bungarotoxin.

α-bungarotoxin is a large, 74 amino acid toxin containing five disulphide bridges, initially identified in the venom of Bungarus multicinctus snake. Like most large toxins, chemical synthesis of α-bungarotoxin is challenging, explaining why all previous reports use purified or recombinant α-bungarotoxin. However, only chemical synthesis allows easy insertion of non-natural amino acids or new chemical functionalities. Herein, we describe a procedure for the chemical synthesis of a fluorescent-tagged α-bungarotoxin. The full-length peptide was designed to include an alkyne function at the amino-terminus through the addition of a pentynoic acid linker. Chemical synthesis of α-bungarotoxin requires hydrazide-based coupling of three peptide fragments in successive steps. After completion of the oxidative folding, an azide-modified Cy5 fluorophore was coupled by click chemistry onto the toxin. Next, we determined the efficacy of the fluorescent-tagged α-bungarotoxin to block acetylcholine (ACh)-mediated currents in response to muscle nicotinic receptor activation in TE671 cells. Using automated patch-clamp recordings, we demonstrate that fluorescent synthetic α-bungarotoxin has the expected nanomolar affinity for the nicotinic receptor. The blocking effect of fluorescent α-bungarotoxin could be displaced by incubation with a 20-mer peptide mimicking the α-bungarotoxin binding site. In addition, TE671 cells could be labelled with fluorescent toxin, as witnessed by confocal microscopy, and this labelling was partially displaced by the 20-mer competitive peptide. We thus demonstrate that synthetic fluorescent-tagged α-bungarotoxin preserves excellent properties for binding onto muscle nicotinic receptors.

Distinct Dibasic Cleavage Specificities of Neuropeptide-Producing Cathepsin L and Cathepsin V Cysteine Proteases Compared to PC1/3 and PC2 Serine Proteases.

Neuropeptides, functioning as peptide neurotransmitters and hormones, are generated from proneuropeptide precursors by proteolytic processing at dibasic residue sites (i.e., KR, RK, KK, RR). The cysteine proteases cathepsin L and cathepsin V, combined with the serine proteases proprotein convertases 1 and 2 (PC1/3 and PC2), participate in proneuropeptide processing to generate active neuropeptides. To compare the dibasic cleavage properties of these proteases, this study conducted global, unbiased substrate profiling of these processing proteases using a diverse peptide library in multiplex substrate profiling by mass spectrometry (MSP-MS) assays. MSP-MS utilizes a library of 228 14-mer peptides designed to contain all possible protease cleavage sites, including the dibasic residue sites of KR, RK, KK, and RR. The comprehensive MSP-MS analyses demonstrated that cathepsin L and cathepsin V cleave at the N-terminal side and between the dibasic residues (e.g., ↓K↓R, ↓R↓K, and K↓K), with a preference for hydrophobic residues at the P2 position of the cleavage site. In contrast, the serine proteases PC1/3 and PC2 displayed cleavage at the C-terminal side of dibasic residues of a few peptide substrates. Further analyses with a series of dipeptide-AMC and tripeptide-AMC substrates containing variant dibasic sites with hydrophobic P2 residues indicated the preferences of cathepsin L and cathepsin V to cleave between dibasic residue sites with preferences for flanking hydrophobic residues at the P2 position consisting of Leu, Trp, Phe, and Tyr. Such hydrophobic amino acids reside in numerous proneuropeptides such as pro-NPY and proenkephalin that are known to be processed by cathepsin L. Notably, cathepsin L displayed the highest specific activity that was 10-, 64-, and 1268-fold greater than cathepsin V, PC1/3, and PC2, respectively. Peptide-AMC substrates with dibasic residues confirmed that PC1/3 and P2 cleaved almost exclusively at the C-terminal side of dibasic residues. These data demonstrate distinct dibasic cleavage site properties and a broad range of proteolytic activities of cathepsin L and cathepsin V, compared to PC1/3 and PC2, which participate in producing neuropeptides for cell-cell communication.

Identification of β-aminopyrrolidine containing peptides as β-amyloid aggregation inhibitors for Alzheimer's disease.

Alzheimer's disease (AD) is caused by a series of events initiated by the production and aggregation of the amyloid β-protein (Aβ). In the early stages of the disease, Aβ is released in a soluble form then progressively forms oligomeric, multimeric, and fibrillar aggregates, triggering neurodegeneration. Thus, development of inhibitors that initiate reverse Aβ aggregation is thought to be a logical approach in treating AD. In this context, we developed β-aminopyrrolidine containing 12 mer peptide 3 which is very potent in inhibiting the Aβ aggregation and also reducing Aβ(42)-induced cytotoxicity.

A novel process driving Alzheimer's disease validated in a mouse model: Therapeutic potential.

IntroductionThe neuronal mechanism driving Alzheimer's disease (AD) is incompletely understood.MethodsImmunohistochemistry, pharmacology, biochemistry, and behavioral testing are employed in two pathological contexts—AD and a transgenic mouse model—to investigate T14, a 14mer peptide, as a key signaling molecule in the neuropathology.ResultsT14 increases in AD brains as the disease progresses and is conspicuous in 5XFAD mice, where its immunoreactivity corresponds to that seen in AD: neurons immunoreactive for T14 in proximity to T14‐immunoreactive plaques. NBP14 is a cyclized version of T14, which dose‐dependently displaces binding of its linear counterpart to alpha‐7 nicotinic receptors in AD brains. In 5XFAD mice, intranasal NBP14 for 14 weeks decreases brain amyloid and restores novel object recognition to that in wild‐types.DiscussionThese findings indicate that the T14 system, for which the signaling pathway is described here, contributes to the neuropathological process and that NBP14 warrants consideration for its therapeutic potential.

Membrane Bound Peroxiredoxin-1 Serves as a Biomarker for In Vivo Detection of Sessile Serrated Adenomas.

Aim: Sessile serrated adenomas (SSAs) are premalignant lesions driven by the BRAFV600E mutation to give rise to colorectal cancers (CRCs). They are often missed during white light colonoscopy because of their subtle appearance. Previously, a fluorescently labeled 7mer peptide KCCFPAQ was shown to detect SSAs in vivo. We aim to identify the target of this peptide. Results: Peroxiredoxin-1 (Prdx1) was identified as the binding partner of the peptide ligand. In vitro binding assays and immunofluorescence staining of human colon specimens ex vivo supported this result. Prdx1 was overexpressed on the membrane of cells with the BRAFV600E mutation, and this effect was dependent on oxidative stress. RKO cells harboring the BRAFV600E mutation and human SSA specimens showed higher oxidative stress as well as elevated levels of Prdx1 on the cell membrane. Innovation and Conclusion: These results suggest that Prdx1 is overexpressed on the cell surface in the presence of oxidative stress and can serve as an imaging biomarker for in vivo detection of SSAs. Antioxid. Redox Signal. 36, 39-56.

Identifying specific matrix metalloproteinase-2-inhibiting peptides through phage display-based subtractive screening.

Gelatinases A and B, which are members of the matrix metalloproteinase (MMP) family, play essential roles in cancer development and metastasis, as they can break down basal membranes. Therefore, the determination and inhibition of gelatinases is essential for cancer treatment. Peptides that can specifically block each gelatinase may, therefore, be useful for cancer treatment. In this study, subtractive panning was carried out using a 12-mer peptide library to identify peptides that block gelatinase A activity (MMP-2), which is a key pharmacological target. Using this method, 17 unique peptide sequences were determined. MMP-2 inhibition by these peptides was evaluated through zymogram analyses, which revealed that four peptides inhibited MMP-2 activity by at least 65%. These four peptides were synthesized and used for in vitro wound healing using human umbilical vein endothelial cells, and two peptides, AOMP12 and AOMP29, were found to inhibit wound healing by 40%. These peptides are, thus, potential candidates for MMP-2 inhibition for cancer treatment. Furthermore, our findings suggest that our substractive biopanning screening method is a suitable strategy for identifying peptides that selectively inhibit MMP-2.

Mycobacterium tuberculosis Endonuclease VIII 2 (Nei2) forms a prereplicative BER complex with DnaN: Identification, characterization, and disruption of complex formation.

Mycobacterium tuberculosis Nei2 (Rv3297) is a BER glycosylase that removes oxidized base lesions from ssDNA and replication fork-mimicking substrates. We show that Endonuclease VIII 2 (Nei2) forms a BER complex with the β-clamp (DnaN, Rv0002) with a KD of 170nM. The Nei2-β-clamp interactions enhance Nei2's activities up to several folds. SEC analysis shows that one molecule of Nei2 binds to a single β-clamp dimer. Nei2 interacts with subsites I and II of the β-clamp via a noncanonical 223 QGCRRCGTLIAY239 Clamp Interacting Protein (CIP) motif in the C-terminal zinc-finger domain, which was previously shown by us to be dispensable for intrinsic Nei2 activity. The 12-mer peptide alone exhibited a KD of 10.28nM, suggesting that the motif is a key mediator of Nei2-β-clamp interactions. Finally, we identified inhibitors of Nei2-β-clamp interactions using rational methods, in vitro disruption, and SPR assays after querying a database of natural products. We found that Tubulosine, Fumitremorgin C, Toyocamycin, and Aleuritic acid exhibit IC50 values of 94.47, 83.49, 109.7, and 71.49µM, respectively. They act by disrupting Nei2-β-clamp interactions and do not affect intrinsic Nei2 activity. Among other things, the present study gives insights into the role of Nei2 in bacterial prereplicative BER.

A colorimetric sandwich-type bioassay for SARS-CoV-2 using a hACE2-based affinity peptide pair

The metallopeptidase angiotensin-converting enzyme 2 (ACE2) is the SARS-CoV-2 receptor required for viral entry based on its specific recognition of the spike protein receptor binding domain (S_RBD) on SARS-CoV-2. We constructed a human ACE2 (hACE2)-based peptide pair by ligating discontinuous key residues involved in the hACE2–S_RBD interaction. We firstly performed in silico simulations to identify a 12-mer and 15-mer peptide pair with capability to bind to the SARS-CoV-2 S_RBD via different binding sites. Then, the bio-layer interferometry validated the specific interactions between the peptides and S_RBD, with affinities at the nanomolar level. Lastly, we developed a colorimetric sandwich-type bioassay based on S_RBD-specific peptide-modified gold nanoparticles and found the colorimetric bioassay offered fast (<30 min), simple, and sensitive detection of S_RBD protein at levels as low as 0.01 nM (0.26 ng mL-1) in SARS-CoV-2. The linear signals ranging from 105 to 107 virus copies mL-1 were achieved in typical types of environmental waters spiked with lysed SARS-CoV-2 pseudovirus. The technology can serve as a beneficial supplement to the routine virus nucleic acid detection in environment media and wastewater treatment.

FinDr: A web server for in silico D-peptide ligand identification

In the rapidly expanding field of peptide therapeutics, the short in vivo half-life of peptides represents a considerable limitation for drug action. D-peptides, consisting entirely of the dextrorotatory enantiomers of naturally occurring levorotatory amino acids (AAs), do not suffer from these shortcomings as they are intrinsically resistant to proteolytic degradation, resulting in a favourable pharmacokinetic profile. To experimentally identify D-peptide binders to interesting therapeutic targets, so-called mirror-image phage display is typically performed, whereby the target is synthesized in D-form and L-peptide binders are screened as in conventional phage display. This technique is extremely powerful, but it requires the synthesis of the target in D-form, which is challenging for large proteins. Here we present finDr, a novel web server for the computational identification and optimization of D-peptide ligands to any protein structure (https://findr.biologie.uni-freiburg.de/). finDr performs molecular docking to virtually screen a library of helical 12-mer peptides extracted from the RCSB Protein Data Bank (PDB) for their ability to bind to the target. In a separate, heuristic approach to search the chemical space of 12-mer peptides, finDr executes a customizable evolutionary algorithm (EA) for the de novo identification or optimization of D-peptide ligands. As a proof of principle, we demonstrate the validity of our approach to predict optimal binders to the pharmacologically relevant target phenol soluble modulin alpha 3 (PSMα3), a toxin of methicillin-resistant Staphylococcus aureus (MRSA). We validate the predictions using in vitro binding assays, supporting the success of this approach. Compared to conventional methods, finDr provides a low cost and easy-to-use alternative for the identification of D-peptide ligands against protein targets of choice without size limitation. We believe finDr will facilitate D-peptide discovery with implications in biotechnology and biomedicine.

A short hepatitis C virus NS5A peptide expression by AAV vector modulates human T cell activation and reduces vector immunogenicity

Viral vector-mediated gene therapies have the potential to treat many human diseases; however, host immune responses against the vector and/or the transgene pose a safety risk to the patients and can negatively impact product efficacy. Thus, novel strategies to reduce vector immunogenicity are critical for the advancement of these therapies. T cell activation (TCA) is required for the development of immune responses during gene therapy. We hypothesized that modulation of TCA by incorporating a novel viral immunomodulatory factor into a viral vector may reduce unwanted TCA and immune responses during gene therapy. To test this hypothesis, we identified an immunomodulatory domain of the hepatitis C virus (HCV) NS protein 5A (NS5A) protein and studied the effect of viral vectors expressing NS5A peptide on TCA. Lentiviral vector-mediated expression of a short 20-mer peptide derived from the NS5A protein in human T cells was sufficient to inhibit TCA. Synthetic 20-mer NS5A peptide also inhibited TCA in primary human T cells. Mechanistically, the NS5A protein interacted with Lck and inhibited proximal TCR signaling. Importantly, NS5A peptide expression did not cause global T cell signaling dysfunction as distal T cell signaling was not inhibited. Finally, recombinant adeno-associated virus (AAV) vector expressing the 20-mer NS5A peptide reduced both the recall antigen and the TCR-mediated activation of human T cells and did not cause global T cell signaling dysfunction. Together, these data suggest that expression of a 20-mer NS5A peptide by an AAV vector may reduce unwanted TCA and may contribute to lower vector immunogenicity during gene therapy.

Factor VIII Epitope Analysis Using a Random Peptide Phage-Display Library Approach in the Sippet Cohort

Background Inhibitor development is the most severe complication of hemophilia A care, and is associated with increased morbidity and mortality.Aims The aim of this study was to use a novel epitope mapping method to explore the factor VIII (FVIII)-specific epitope profile in the SIPPET cohort population.Methods The population consisted of 122 previously untreated patients with severe hemophilia A that were followed-up for 50 days of exposure to FVIII. Sampling was performed before FVIII treatment and at the end of the follow-up. The outcome was inhibitor development. The FVIII-specific IgG epitope repertoire was assessed by means of a novel high-throughput epitope mapping technique using a random peptide phage-display library. Using this assay, a set of affinity-selected 12-mer peptide sequences (also called mimotopes) that were strongly bound by FVIII-specific antibodies were identified. These mimotopes were clustered on the basis of sequence similarity and a consensus motif was generated for each mimotope cluster. Discriminative performance of these mimotope clusters was assessed by ROC analysis. Mimotope clusters were mapped onto the 3D structure of a B-domain deleted FVIII model using a B-cell epitope prediction algorithm (Mapitope).Results The FVIII-specific antibody response is polyclonal with several mimotope clusters. The most predominant mimotope clusters in inhibitor patients were mapped to the heavy chain of the FVIII molecule. Using plasma samples taken before exposure to FVIII, three mimotopes (with the consensus motifs “QM”, “PSLxWK” and “SWPHxxxxK”) were identified that predicted inhibitor development (with an AUC of 0.76, 0.80 and 0.76 respectively).Conclusion Information on immunodominant epitope clusters can be used to generate novel, less immunogenic FVIII proteins and set up diagnostic tests that predict the risk of inhibitor development before starting treatment with FVIII. [Display omitted] DisclosuresPalm: Protobios LLC: Current Employment, Patents & Royalties: Inventor of the patent application (PCT Application No. US/14079626) filed by Protobios that covers the use of phage display method to manipulate and monitor humoral immunity.. Palla: Pfizer: Other: Travel support; Kedrion: Other: Travel support; Novonordisk: Speakers Bureau. Peyvandi: Roche: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Sobi: Consultancy, Honoraria; Takeda: Consultancy, Honoraria.