Phage-displayed Peptide Library Research Articles

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.

Short- and long-term effect of colorectal cancer targeting peptides conjugated to gold nanoparticles in rats' liver and colon after single exposure.

Peptides play important roles in the diagnosis, prognostic predictors, and treatment of various kinds of cancer. Peptides (p.C, p.L and p.14), derived from the phage display peptide libraries, specifically binds to colorectal cancer (CRC) cells in vitro. To allow tumor specificity and selectivity for in vivo diagnosis of CRC, biotinylated p.C, p.L and p.14 were conjugated to AuNPs (14nm) via the biotin-streptavidin interaction. Male Wistar rats were intravenously injected with a single dose (100µg/kg body weight) of AuNPs (citrate-AuNPs, PEG-AuNPs, p.C-PEG-, p.L-PEG- and p.14-PEG-AuNPs). Animals were monitored for behavioral changes, and sacrificed either 14days or 84days post-injection. Biochemical changes, oxidative stress, and histology of the liver and colon were assessed. No significant changes were noted in the rats injected with all the AuNPs, except p.L-PEG-AuNPs that caused significant toxicity (p < 0.05) 14days post-exposure when compared to control group, as evidenced by increased relative liver weight, increased malondialdehyde levels and histological changes in the liver. These changes, however, returned to normalcy 84days post-injection. It can be concluded, based on these findings, that p.L induced a transient toxicity in rats after a single intravenous injection, and can therefore be considered non-toxic long-term after a single exposure.

Chemical Modification of Phage-Displayed Helix-Loop-Helix Peptides to Construct Kinase-Focused Libraries.

Conformationally constrained peptides hold promise as molecular tools in chemical biology and as a new modality in drug discovery. The construction and screening of a target‐focused library could be a promising approach for the generation of de novo ligands or inhibitors against target proteins. Here, we have prepared a protein kinase‐focused library by chemically modifying helix‐loop‐helix (HLH) peptides displayed on phage and subsequently tethered to adenosine. The library was screened against aurora kinase A (AurA). The selected HLH peptide Bip‐3 retained the α‐helical structure and bound to AurA with a K D value of 13.7 μM. Bip‐3 and the adenosine‐tethered peptide Bip‐3‐Adc provided IC50 values of 103 μM and 7.7 μM, respectively, suggesting that Bip‐3‐Adc bivalently inhibited AurA. In addition, the selectivity of Bip‐3‐Adc to several protein kinases was tested, and was highest against AurA. These results demonstrate that chemical modification can enable the construction of a kinase‐focused library of phage‐displayed HLH peptides.

Synthetic polypeptide crotamine: characterization as a myotoxin and as a target of combinatorial peptides.

Crotamine is a rattlesnake-derived toxin that causes fast-twitch muscle paralysis. As a cell-penetrating polypeptide, crotamine has been investigated as an experimental anti-cancer and immunotherapeutic agent. We hypothesized that molecules targeting crotamine could be designed to study its function and intervene in its adverse activities. Here, we characterize synthetic crotamine and show that, like the venom-purified toxin, it induces hindlimb muscle paralysis by affecting muscle contraction and inhibits KCNA3 (Kv1.3) channels. Synthetic crotamine, labeled with a fluorophore, displayed cell penetration, subcellular myofiber distribution, ability to induce myonecrosis, and bind to DNA and heparin. Here, we used this functionally validated synthetic polypeptide to screen a combinatorial phage display library for crotamine-binding cyclic peptides. Selection for tryptophan-rich peptides was observed, binding of which to crotamine was confirmed by ELISA and gel shift assays. One of the peptides (CVWSFWGMYC), synthesized chemically, was shown to bind both synthetic and natural crotamine and to block crotamine-DNA binding. In summary, our study establishes a functional synthetic substitute to the venom-derived toxin and identifies peptides that could further be developed as probes to target crotamine. KEY MESSAGES: Synthetic crotamine was characterized as a functional substitute for venom-derived crotamine based on myotoxic effects. A combinatorial peptide library was screened for crotamine-binding peptides. Tryptophan-rich peptides were shown to bind to crotamine and interfere with its DNA binding. Crotamine myofiber distribution and affinity for tryptophan-rich peptides provide insights on its mechanism of action.

Development of peptides targeting receptor binding site of the envelope glycoprotein to contain the West Nile virus infection

West Nile virus (WNV), re-emerging neurotropic flavivirus, can cross the blood–brain barrier (BBB) and cause fatal encephalitis and meningitis. Infection of the human brain microvascular endothelial cells (hBMECs), building blocks of the BBB, represents the pivotal step in neuroinvasion. Domain III (DIII) of the envelope (E) glycoprotein is a key receptor-binding domain, thus, it is an attractive target for anti-flavivirus strategies. Here, two combinatorial phage display peptide libraries, Ph.D.-C7C and Ph.D.-12, were panned against receptor-binding site (RBS) on DIII to isolate peptides that could block DIII. From series of pannings, nine peptides (seven 7-mer cyclic and two 12-mer linear) were selected and overexpressed in E. coli SHuffle T5. Presence of disulfide bond in 7-mer peptides was confirmed with thiol-reactive maleimide labeling. Except for linear peptide 19 (HYSWSWIAYSPG), all peptides proved to be DIII binders. Among all peptides, 4 cyclic peptides (CTKTDVHFC, CIHSSTRAC, CTYENHRTC, and CLAQSHPLC) showed significant blocking of the interaction between DIII and hBMECs, and ability to neutralize infection in cultured cells. None of these peptides showed toxic or hemolytic activity. Peptides identified in this study may serve as potential candidates for the development of novel antiviral therapeutics against WNV.

Quantitative proteomic analysis for evaluating affinity isolation of extracellular vesicles

Absolute quantification with mass spectrometry and isotope labeled internal standards has found broad applications in biomedical research. In the present research, it was used for developing and evaluating a new affinity-based approach to isolate extracellular vesicles (EVs) from human plasma. First, a phage display peptide library was screened against EVs as a bait and absolute quantification of multiple proteins helped to select the best bait available. Then, absolute quantification was used to evaluate the efficiency of affinity chromatography on peptide-Sepharose. In summary, we have demonstrated that peptides with affinity to EVs selected from phage library screening can be valuable ligands for EVs isolation. SignificanceExtracellular vesicles (EVs) have an important role in intercellular communication for all cell types. This makes EVs a promising new type of therapeutics capable to deliver drugs to specific sites with no off-target side effects. However, their isolation, and correct assignment of their biological function and properties remains an obscure field of research. In this study, we proposed to use MRM quantitation of a pattern of EVs and non-EVs proteins to develop a purification protocol based on affinity peptides selected from phage library screening. MRM quantification of EVs proteins can also help in identifying those that are subpopulation specific markers for further target-specific isolation.

Recombinant Peptide Mimetic NanoLuc Tracer for Sensitive Immunodetection of Mycophenolic Acid.

Mycophenolic acid (MPA) is an immunosuppressant drug commonly used to prevent organ rejection in transplanted patients. MPA monitoring is of great interest due to its small therapeutic window. In this work, a phage-displayed peptide library was used to select cyclic peptides that bind to the MPA-specific recombinant antibody fragment (Fab) and mimic the behavior of MPA. After biopanning, several phage-displayed peptides were isolated and tested to confirm their epitope-mimicking nature in phage-based competitive immunoassays. After identifying the best MPA mimetic (ACEGLYAHWC with a disulfide constrained loop), several immunoassay approaches were tested, and a recombinant fusion protein containing the peptide sequence with a bioluminescent enzyme, NanoLuc, was developed. The recombinant fusion enabled its direct use as the tracer in competitive immunoassays without the need for secondary antibodies or further labeling. A bioluminescent sensor, using streptavidin-coupled magnetic beads for the immobilization of the biotinylated Fab antibody, enabled the detection of MPA with a detection limit of 0.26 ng mL–1 and an IC50 of 2.9 ± 0.5 ng mL–1. The biosensor showed good selectivity toward MPA and was applied to the analysis of the immunosuppressive drug in clinical samples, of both healthy and MPA-treated patients, followed by validation by liquid chromatography coupled to diode array detection.

Abstract 1445: A nuclear localizing peptide that targets glioblastoma

Abstract Glioblastoma (GBM) is one of the most aggressive and fatal primary brain neoplasms with a median survival of less than 15 months even after current standard of cares including surgical resection, radiotherapy and systemic therapy. Although systemic anticancer agents, such as temozolomide, are used as the standard of care for GBM, therapeutic efficacy is limited due to the off-target systemic toxicity of traditional chemotherapeutics. Targeted systemic therapy is emerging as an important treatment strategy for cancer therapy. Phage display peptide library was used to discover peptides that undergo endocytosis in GBM. We have identified a novel peptide that accumulates intracellularly in multiple GBM cell lines. Intracellular localization was quantified by FACS analysis of the FITC-labeled peptide. As compared to untreated GBM cells, D54, U251 and SF295 human GBM cell lines treated with 5.55 µM of peptide-FITC conjugate showed a significant FITC-positive shift on FACS analysis with 83%, 85% and 71% of cells internalizing the peptide, respectively. Confocal laser scanning microscopy experiments further confirm the intracellular targeting of the FITC-peptide in D54, U251 and SF295 GBM cell lines with associated peptide nuclear localization. Further studies are underway to elucidate the microscopic biodistribution of the nuclear homing peptide (NHP) in GBM mouse models. This work is significant as a novel cell surface, intracellular or nuclear binding partner may be identified for peptide-targeted cancer drug therapy. Furthermore, the NHP has potential to be translated to the clinic as targeted therapy for GBM patients in the form of a peptide-drug conjugate. Citation Format: Calvin D. Lewis, Abhay Singh, Vaishali Kapoor, Dennis E. Hallahan. A nuclear localizing peptide that targets glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1445.

Abstract 1446: GRP78 targeting peptides deliver liposomal doxorubicin specifically to cancer and enhance the efficacy of radiation therapy

Abstract Traditional radiation therapy is often associated with significant toxicity to normal cells that could limit cancer therapy's success. The greater understanding of the molecular differences between cancer cells and normal cells has led to targeted therapies in cancer treatment. We discovered the radiation-inducible expression of Glucose regulated protein 78kDa (GRP78) in cancer using phage-display peptide libraries. In this study, we screened a peptide library and discovered novel peptides targeting GRP78. The top ten peptides were further evaluated for their affinity of binding to recombinant GRP78 using surface plasmon resonance technology. Since GRP78 is induced on cancer cells' surface, we irradiated the lung cancer cell lines A549 and H460 to evaluate peptide binding. Flow cytometry analysis of Dylight 488 conjugated peptides led to identifying peptides A and K with the highest binding level to irradiated A549 and H460. Peptides A and K were compared for their efficacy to deliver liposomal doxorubicin specifically to tumors. Copper-free click-chemistry was used to conjugate peptides to the liposome surface. Whole-body near-infrared imaging was performed to evaluate the biodistribution of the peptide conjugated liposomes in nude mice bearing A549 and H460 tumors. Both peptides A and K specifically delivered liposomes to the tumors compared to the non-targeted liposomes. In pilot tumor growth experiments, we found that GRP78 targeting peptide conjugated liposomes delayed the growth of tumors in nude mice compared to non-targeted liposomes. The inhibition of tumor growth was further enhanced in combination with radiation, indicating efficient delivery of the radiation-sensitizer doxorubicin specifically to tumors. Currently, studies are underway to evaluate the microscopic biodistribution of doxorubicin in tumor sections. Mass spectrometry analysis of various organs will be performed to determine the pharmacokinetics of liposomal doxorubicin delivered by GRP78 targeting peptides. In conclusion, our novel GRP78 targeting peptides specifically deliver liposomal doxorubicin to tumors. Doxorubicin encapsulated in GRP78 targeting peptide conjugated liposomes enhances the tumor growth inhibitory activity of radiation therapy. Specific delivery of radiation-sensitizing drugs encapsulated in a liposome by peptides that target radiation-inducible GRP78 is a promising therapeutic strategy for various cancers. Citation Format: Abhay K. Singh, Chandresh Shyam, Calvin Lewis, Wendy Zhang, Sapna Deore, Vaishali Kapoor, Dennis E. Hallahan. GRP78 targeting peptides deliver liposomal doxorubicin specifically to cancer and enhance the efficacy of radiation therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1446.

The identification of a B-cell epitope in bovine viral diarrhea virus (BVDV) core protein based on a mimotope obtained from a phage-displayed peptide library

Bovine pestivirus A and B, previously known as bovine viral diarrhea virus (BVDV)-1 and 2, respectively, are important pathogens of cattle worldwide, which causes significant economic losses. B-cell epitopes in BVDV glycoprotein E2 and nonstructural protein NS2/3 have been extensively identified. In this study, we screened a 12-mer phage display peptide library using commercial goat anti-BVDV serum, and identified a mimotope “LTPHKHHKHLHA” referred to as P3. With sequence alignment, a putative B-cell epitope “77ESRKKLEKALLA88” termed as P3-BVDV1/2 residing in BVDV core protein was identified. The synthesized peptides of both P3 and P3-BVDV1/2 show strong reactivity with BVDV serum in immune blot assay. Immunization of mice with these individual peptides leads to the production of antibody that cannot neutralize virus infectivity. Thus for the first time we identified a B-cell epitope, “77ESRKKLEKALLA88”, in BVDV core protein. Interestingly, the epitope was highly conserved in Pestivirus A, B, C, D, as well as emerging Pestivirus E and I, but highly variable in Pestiviruses H, G, F, and J, as well as unclassified Pestivirus originated from non-ruminant animals. Whether this putative B-cell epitope is implicated in pestivirus pathogenesis or evolution needs further investigations once large numbers of isolates are available in the future.

Identification of COVID-19 B-cell epitopes with phage-displayed peptide library

BackgroundCoronavirus disease 19 (COVID-19) first appeared in the city of Wuhan, in the Hubei province of China. Since its emergence, the COVID-19-causing virus, SARS-CoV-2, has been rapidly transmitted around the globe, overwhelming the medical care systems in many countries and leading to more than 3.3 million deaths. Identification of immunological epitopes on the virus would be highly useful for the development of diagnostic tools and vaccines that will be critical to limiting further spread of COVID-19.MethodsTo find disease-specific B-cell epitopes that correspond to or mimic natural epitopes, we used phage display technology to determine the targets of specific antibodies present in the sera of immune-responsive COVID-19 patients. Enzyme-linked immunosorbent assays were further applied to assess competitive antibody binding and serological detection. VaxiJen, BepiPred-2.0 and DiscoTope 2.0 were utilized for B-cell epitope prediction. PyMOL was used for protein structural analysis.Results36 enriched peptides were identified by biopanning with antibodies from two COVID-19 patients; the peptides 4 motifs with consensus residues corresponding to two potential B-cell epitopes on SARS-CoV-2 viral proteins. The putative epitopes and hit peptides were then synthesized for validation by competitive antibody binding and serological detection.ConclusionsThe identified B-cell epitopes on SARS-CoV-2 may aid investigations into COVID-19 pathogenesis and facilitate the development of epitope-based serological diagnostics and vaccines.

Identification of a Glypican-3 Binding Peptide From a Phage-Displayed Peptide Library for PET Imaging of Hepatocellular Carcinoma.

Tumor-targeting peptides functioned as molecular probes are essential for multi-modality imaging and molecular-targeting therapy in caner theronostics. Here, we performed a phage-displayed bio-panning to identify a specific binding peptide targeting Glypican-3 (GPC-3), a promising biomarker in hepatocellular carcinoma (HCC). After screening in the cyclic peptide library, a candidate peptide named F3, was isolated and showed specific binding to HCC cell lines. In a bio-distribution study, higher accumulation of F3 peptide was observed in HepG-2 tumors compared to PC-3 tumors in xenograft models. After labeling with radioactive 68Ga, the F3 peptide tracer enabled the specific detection of tumors in HCC tumor models with PET imaging. More importantly, the expression of GPC-3 in human tissue samples may be distinguished by an F3 fluorescent peptide probe indicating its potential for clinical application. This cyclic peptide targeting GPC-3 has been validated, and may be an alternative to serve as an imaging probe or a targeting domain in the drug conjugate.

Combining biological and chemical diversity.

Phage display enables screening of billions of peptides comprised mainly of natural amino acids. Now, a method to attach and encode a range of structurally diverse compounds has been reported. This method can expand the chemical space covered by phage display peptide libraries.

Induction of anti-Zearalenone immune response with mimotopes identified from a phage display peptide library

Zearalenone (ZEN), a type of non-steroidal estrogenic mycotoxin, is mainly produced by several species of Fusarium molds. It is ubiquitous in contaminated grains and grain products all over the world, posing a serious threat to animal and human health. This study aims to screen the mimotopes of ZEN from a phage display random 12-mer peptide library and explore their immunogenicity. A monoclonal antibody (mAb) against ZEN was employed as the target for mimotope selection from a phage display random peptide library. After four rounds of panning, six mimotopes that could specifically bind to ZEN mAb were obtained. In order to explore the immunogenicity of these mimotopes, Balb/c mice were immunized with phages Z8, Z21, Z35, Z8:Z21:Z35(1:1:1) and the conjugate of ZEN-bovine serum albumin (ZEN-BSA), respectively. The titers of antibodies in the mice immunized with mimotopes were 1:3200 (Z8), 1:3200 (Z21), 1:6400 (Z35), 1:6400 (1:1:1 mixture of Z8, Z21 and Z35), and the binding between serum antibodies and ZEN-OVA could be blocked by ZEN standards. These results demonstrated that the mimotopes of ZEN could induce specific antibodies against ZEN, suggesting that these displayed peptides were immunogenic.

Screening and Identification of a Specific Binding Peptide to Ovarian Cancer Cells from a Phage-Displayed Peptide Library

To select specific binding peptides for imaging and detection of human ovarian cancer. The phage 12-mer peptide library was used to select specific phage clones to ovarian cancer cells. After four rounds of biopanning, the binding specificity of randomly selected phage clones to ovarian cancer cells was determined by enzyme-linked immunosorbent assay (ELISA). DNA sequencing and homology analysis were performed on specifically bound phages. The binding ability of the selected peptides to SKOV3 cells was confirmed by fluorescence microscopy and flow cytometry. After four rounds of optimized biological panning, phage recovery was 34-fold higher than that of the first round, and the specific phage clones bound to SKOV3 cells were significantly enriched. A total of 32 positive phage clones were preliminarily identified by ELISA from 54 randomly selected clones, and the positive rate was 59.3%. S36 was identified as the clone with best affinity to SKOV3 cells via fluorescence microscopy and flow cytometry. A representative clone of OSP2, S36 is expected to be an effective probe for diagnosis and treatment of ovarian cancer.

Heat shock protein A4L is a potent autoantigen for testicular autoimmunity in mice

Experimental autoimmune orchitis (EAO) may be used as a model to investigate immunological infertility in men. Murine EAO is induced via immunization with auto-immunogenic antigens (AIAgs) from testicular germ cells (TGCs). CD4 + T cells play a crucial role in EAO induction. However, whether AIAgs induce an immune response remains unclear. We aimed to identify self-antigens that induce EAO by screening a phage display library of random TGC peptides using IgG from EAO-induced A/J mice. Twenty TGC-specific AIAgs were detected, and G protein-coupled receptor kinase 2 interacting protein-1 (GIT1) and heat shock protein A4L (HSPA4L) were identified as candidate AIAgs that induce EAO. Immunization with GIT1 or HSPA4L, emulsified in complete Freund’s adjuvant, resulted in 66 % or 100 % incidence of EAO, respectively, indicating that HSPA4L is a most potent AIAg that induces EAO in mice. These findings may expectedly help improve the diagnostic procedures and treatment of immunological infertility in men.

Neutralizing antibody PR8-23 targets the footprint of the sialoglycan receptor binding site of H1N1 hemagglutinin.

Influenza virus cause seasonal influenza epidemic and seriously sporadic influenza pandemic outbreaks. Hemagglutinin (HA) is an important target in the therapeutic treatment and diagnostic detection of the influenza virus. Variation in the sialic acid receptor binding site leads to strain-specific binding and results in different binding modes to the host receptors. Here, we evaluated the neutralizing activity and hemagglutination inhibition activity of a prepared murine anti-H1N1 monoclonal antibody PR8-23. Then we identified the epitope peptide of antibody PR8-23 by phage display technique from phage display peptide libraries. The identified epitope, 63-IAPLQLGKCNIA-74, containing two α-helix and two β-fold located at the footprint of the sialoglycan receptor on the RBS in the globular head domain of HA. It broads the growing arsenal of motifs for the amino acids on the globular head domain of HA in sialic acid receptor binding site and neutralizing antibody production.

Phage display identification of immunodominant epitopes and autoantibodies in autoimmune diseases

Phage display represents an invaluable tool to study autoimmune diseases. The side effects of immunosuppressive drugs for the treatment of autoimmune diseases raise awareness of the need to explore alternative therapeutic approaches such as antibodies and peptides. Therefore, phage display is an important technique for generating such molecules, so the purpose of this review is to determine the potential advantages of this technique in the research of autoimmune diseases. Many studies have also demonstrated the efficacy of phage display in identifying immunodominant epitopes of autoimmune diseases such as Goodpasture disease, immunologic thrombocytopenia, and systemic lupus erythematosus. Phage display peptide libraries have been screened with immunopurified autoantibodies from patients with autoimmune diseases. This makes it possible to more precisely locate the autoantibody binding sites, reveal a possible epitope sharing between the host and microbe, and identify a motif that mimics an antigenic structure such as that of dsDNA. Several studies have been conducted that have investigated the effectiveness of phage display in isolating autoantibody repertoires of autoantibodies against human epitopes. This allows the identification and design of antibody fragments (e.g., Fab, scFv, sdAb) that could block the binding of autoantibodies such as the deposition of IgG in the kidney and reduce the clinical signs of disease. In conclusion, phage display helps identify common epitopes and hotspot residues that can be potential therapeutic targets for the treatment of autoimmune diseases. This leads to a better understanding of the immunopathogenesis of autoimmune diseases and the development of more specific therapeutic strategies.

Specific EtMIC3-binding peptides inhibit Eimeria tenella sporozoites entry into host cells

Avian coccidiosis caused by Eimeria leads to huge economic losses on the global poultry industry. In this study, microneme adhesive repeat regions (MARR) bc1 of E. tenella microneme protein 3 (EtMIC3-bc1) was used as ligand, and peptides binding to EtMIC3 were screened from a phage display peptide library. The positive phage clones were checked by enzyme-linked immunosorbent assay (ELISA). Competitive ELISA was applied to further verify the binding capability between the positive phages and recombinant EtMIC3-bc1 protein or sporozoites protein. The inhibitory effects of target peptides on sporozoites invasion of MDBK cells were measured in vitro. Chickens were orally administrated with target positive phages and the protective effects against homologous challenge were evaluated. The model of three-dimensional (3D) structure for EtMIC3-bc1 was conducted, and molecular docking between target peptides and EtMIC3-bc1 model was analyzed. The results demonstrated that three selected positive phages specifically bind to EtMIC3-bc1 protein. The three peptides A, D and W effectively inhibited invasion of MDBK cells by sporozoites, showing inhibited ratio of 71.8%, 54.6% and 20.8%, respectively. Chickens in the group orally inoculated with phages A displayed more protective efficacies against homologous challenge than other groups. Molecular docking showed that amino acids in three peptides, especially in peptide A, insert into the hydrophobic groove of EtMIC3-bc1 protein, and bind to EtMIC3-bc1 through intermolecular hydrogen bonds. Taken together, the results suggest EtMIC3-binding peptides inhibit sporozoites entry into host cells. This study provides new idea for exploring novel strategies against coccidiosis.

Searching for Peptide Inhibitors of T Regulatory Cell Activity by Targeting Specific Domains of FOXP3 Transcription Factor.

(1) Background: The ability of cancer cells to evade the immune system is due in part to their capacity to induce and recruit T regulatory cells (Tregs) to the tumor microenvironment. Strategies proposed to improve antitumor immunity by depleting Tregs generally lack specificity and raise the possibility of autoimmunity. Therefore, we propose to control Tregs by their functional inactivation rather than depletion. Tregs are characterized by the expression of the Forkhead box protein 3 (FOXP3) transcription factor, which is considered their “master regulator”. Its interaction with DNA is assisted primarily by its interaction with other proteins in the so-called “Foxp3 interactome”, which elicits much of the characteristic Treg cell transcriptional signature. We speculated that the disruption of such a protein complex by using synthetic peptides able to bind Foxp3 might have an impact on the functionality of Treg cells and thus have a therapeutic potential in cancer treatment. (2) Methods: By using a phage-displayed peptide library, or short synthetic peptides encompassing Foxp3 fragments, or by studying the crystal structure of the Foxp3:NFAT complex, we have identified a series of peptides that are able to bind Foxp3 and inhibit Treg activity. (3) Results: We identified some peptides encompassing fragments of the leuzin zipper or the C terminal domain of Foxp3 with the capacity to inhibit Treg activity in vitro. The acetylation/amidation of linear peptides, head-to-tail cyclization, the incorporation of non-natural aminoacids, or the incorporation of cell-penetrating peptide motifs increased in some cases the Foxp3 binding capacity and Treg inhibitory activity of the identified peptides. Some of them have shown antitumoral activity in vivo. (4) Conclusions: Synthetic peptides constitute an alternative to inhibit Foxp3 protein–protein interactions intracellularly and impair Treg immunosuppressive activity. These peptides might be considered as potential hit compounds on the design of new immunotherapeutic approaches against cancer.