FITC-labeled Peptide Research Articles

Abstract 3111: Preclinical development of a peptide-based immune-surveillance PET tracer for immuno-oncology stratification

Abstract Introduction: Cancer immunotherapies, such as immune checkpoint inhibitors (ICIs), have become an integral part of cancer treatment. However, significant differences in their effectiveness persist among individual patients. This variability partly stems from poorly immunogenic tumors, referred to as "cold" tumors, characterized by low levels of tumor-infiltrating leukocytes (TILs). Studies indicate that lower levels of TILs, specifically CD4+ and CD8+ T-cells, lead to reduced efficacy of certain immunotherapies. Therefore, the imaging of these cell types could be invaluable in screening cancer patients to predict the effectiveness of a prescribed immunotherapeutic agent before and during treatment. CD4+ T-cells, identified by their high expression of CD4 receptors, could serve as a potential molecular imaging target for an immuno-oncology relevant PET tracer. For this purpose a peptide-based PET tracer offer several beneficial characteristics such as relatively low circulation time, cell penetration, and low development costs. Methods: All peptides were identified through peptide phage display (PPD) and commercially synthesized (>95% purity). These peptides underwent screening for their binding affinity to recombinant human CD4 and a negative control protein using surface plasmon resonance (SPR) on a Biacore X100. Peptides exhibiting specific binding to CD4, as determined by SPR, were further selected for FITC-labeling (>95% purity). FITC-labeled peptides were applied in fluorescence-based cell assays conducted on the SUP-T1 (CRL-1942) cell line and an established SUP-T1 CD4 knockout (KO) cell line. Flow cytometry assays were performed using a Fortessa X-20, while live cell imaging was conducted on either a LSM980 or LSM900 confocal microscopy. Simultaneously, an in silico docking method was employed, utilizing ColabFold—an online complex predictor powered by AlphaFold2—for CD4-peptide docking analysis. Results: Several peptides successfully identified via PPD exhibited binding to recombinant human CD4 as shown by SPR screening. The FITC-labeling and assessment against SUP-T1 and corresponding CD4 KO cell line unveiled specific CD4 binding within a subset of peptides, as observed in both flow cytometry and confocal microscopy. In vitro data was compared to CD4-peptide docking results to create a list of peptides for future in vivo studies. Conclusions: Multiple peptides successfully identified via PPD showed robust in vitro CD4 binding through SPR screening. Select peptides also demonstrated specific CD4 binding in cell assays, corroborated by docking results. Future prospective for the high performing peptides include in vivo PET/CT Ga-68 or Cu-64 imaging in tumors with high and low CD4+ T-cell infiltration. Citation Format: Anders W. Sinkjaer, Danna K. Sell, Ida M. Dawoodi, Babak Bakhshinejad, Camilla Stavnsbjerg, Andreas Kjaer. Preclinical development of a peptide-based immune-surveillance PET tracer for immuno-oncology stratification [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3111.

Screening high affinity monoclonal antibody producing hybridomas using a graphene oxide-based fluorescence biosensor

Screening monoclonal antibody (mAb) producing hybridomas by ELISA using microplates is time-consuming and expensive. The combination of biosensing and flow cytometry represents a promising rapid, high-throughput method for screening mAb producing clones. Based on the assembly of graphene oxide (GO) and fluorescein isothiocyanate (FITC)-labeled peptide that included the N-terminal portion of selenoprotein K (SELENOK) antigen epitope, we designed a novel GO fluorescence biosensor for screening high affinity mAb producing hybridomas. The FITC-labeled peptide was strongly adsorbed on the surface of GO, which effectively quenched the FITC fluorescence. The specific SELENOK mAb competitively bound to FITC-Peptide, resulting in FITC-Peptide release from the GO surface and fluorescence recovery. This sensor was successfully utilized to sensitively detect SELENOK mAb with low detection limit of 0.25 U. In addition, we employed this FITC-Peptide/GO biosensor to in situ visualizing and high-throughput screening SELENOK mAb hybridomas (SMAHs) at the single-cell level. The proposed sensor is simple, relatively inexpensive, highly specific, and applicable for cell sort assay, demonstrating the effective use for peptide-antibody interactions in the specific screening for other mAb hybridomas libraries.

FITC-Labeled RGD Peptides as Novel Contrast Agents for Functional Fluorescent Angiographic Detection of Retinal and Choroidal Neovascularization.

The development of choroidal neovascularization (CNV) is a crucial factor in the pathophysiology and prognosis of exudative age-related macular degeneration (AMD). Therefore, the detection of CNV is essential for establishing an appropriate diagnosis and treatment plan. Current ophthalmic imaging techniques, such as fundus fluorescent angiography and optical coherence tomography, have limitations in accurately visualizing CNV lesions and expressing CNV activity, owing to issues such as excessive dye leakage with pooling and the inability to provide functional information. Here, using the arginine-glycine-aspartic acid (RGD) peptide's affinity for integrin αvβ3, which is expressed in the neovascular endothelial cells in ocular tissues, we propose the use of fluorescein isothiocyanate (FITC)-labeled RGD peptide as a novel dye for effective molecular imaging of CNV. FITC-labeled RGD peptides (FITC-RGD2), prepared by bioconjugation of one FITC molecule with two RGD peptides, demonstrated better visualization and precise localization of CNV lesions than conventional fluorescein dyes in laser-induced CNV rodent models, as assessed using various imaging techniques, including a commercially available clinical fundus camera (Optos). These results suggest that FITC-RGD2 can serve as an effective novel dye for the diagnosis of neovascular retinal diseases, including AMD, by enabling early detection and treatment of disease occurrence and recurrence after treatment.

Identification and validation of Sertoli cell homing peptides as molecular steering for testis targeted drug delivery

The testicle, an organ privileged with immunity because of Blood-Testis Barrier (BTB), poses a major impediment to developing and delivering drugs to the testes. These problems can be prevented by targeting testicular cells using specific ligands, such as homing peptides. This is the first study to demonstrate the successful selection of Sertoli cell homing peptides using a phage display peptide library. The identification of peptides is performed with Sanger sequencing and high-throughput NGS. The Sertoli cell and testis targeting potential of the SCHP1 and SCHP2 was confirmed using confocal microscopy and flow cytometry of the FITC-labelled peptides and in vivo bio-distribution of the corresponding Cy5.5-tagged peptides. Secondary structures were predicted in the setting of different polarity by circular dichroism. The results suggest that SCHP1 and SCHP2 can effectively target Sertoli cells. In vivo bio-distribution in mouse models indicated significantly higher uptake of SCHP1 and SCHP2 by testes compared with the heart, brain, and spleen. SCHP1 and SCHP2 can be adopted as molecular steering for targeted male contraceptive delivery, treatment of testicular cancer, and male infertility. Further development of the peptides into peptidomimetics may increase their stability, and information on the molecular targets of these peptides may reveal their therapeutic potential.

First identification of Nocardia seriolae GapA adhesion function and its three B-cell epitopes with cell-binding activity.

Fish nocardiosis mainly caused by Nocardia seriolae (N. seriolae) is a serious threat to aquaculture. Bacterial adhesion to host cells mediated by adhesin is an initial step of pathogenesis. But it is not clear whether glyceraldehyde-3-phosphate dehydrogenase (GapA) is an adhesin of N. seriolae. Here, recombinant GapA protein (rGapA) was prokaryotic expressed, and its role in the bacterial adhesion to Ctenopharyngodon idella kidney cells was investigated by indirect immunofluorescence, protein-binding assay and adhesion inhibition assay. The results showed that an obvious green fluorescence was observed on the surface of the cells co-incubated with rGapA protein; the cytomembrane proteins of the cells pretreated with rGapA could react with anti-rGapA antibody; and the antibody significantly inhibited the adhesion ability of the bacteria. Subsequently, B-cell linear epitopes of GapA protein were identified by using a immunoinformatics approach combined with peptide ELISA and Western blot for the first time. It was found that four predicted epitopes (Ep58-69 , Ep139-150 , Ep186-197 , Ep318-329 ) could all react with anti-rGapA antibody and obviously inhibit the immunoreactivity between rGapA and anti-rGapA antibody, and they were confirmed as indeed B-cell linear epitopes of the protein. Furthermore, flow cytometry analysis found the percentage of positive cells co-incubated with FITC-labelled epitope peptides (Ep139-150 , Ep186-197 , Ep318-329 ) was significantly higher than those in the FITC-labelled Ep58-69 , unrelated control peptide and cell control. Collectively, GapA is an adhesin of N. seriolae, and epitope peptides (Ep139-150 , Ep186-197 , Ep318-329 ) possess cell-binding activity, which are potential candidates for developing a multiple epitopes-based adhesin vaccine against fish nocardiosis.

Non-canonical proline-tyrosine interactions with multiple host proteins regulate Ebola virus infection.

The Ebola virus VP30 protein interacts with the viral nucleoprotein and with host protein RBBP6 via PPxPxY motifs that adopt non-canonical orientations, as compared to other proline-rich motifs. An affinity tag-purification mass spectrometry approach identified additional PPxPxY-containing host proteins hnRNP L, hnRNPUL1, and PEG10, as VP30 interactors. hnRNP L and PEG10, like RBBP6, inhibit viral RNA synthesis and EBOV infection, whereas hnRNPUL1 enhances. RBBP6 and hnRNP L modulate VP30 phosphorylation, increase viral transcription, andexert additive effects on viral RNA synthesis. PEG10 has more modest inhibitory effects on EBOV replication. hnRNPUL1 positively affects viral RNA synthesis but in a VP30-independent manner. Binding studies demonstrate variable capacity of the PPxPxY motifs from these proteins to bind VP30, define PxPPPPxY as an optimal binding motif, and identify the fifth proline and the tyrosine as most critical for interaction. Competition binding and hydrogen-deuterium exchange mass spectrometry studies demonstrate that each protein binds a similar interface on VP30. VP30 therefore presents a novel proline recognition domain that is targeted by multiple host proteins to modulate viral transcription.

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.

Competitive and noncompetitive fluorescence polarization immunoassays for the detection of benzothiostrobin using FITC-labeled dendrimer-like peptides

Peptides obtained from phage display libraries are valuable reagents for small-molecule immunoassays. However, their application in fluorescence polarization immunoassays (FPIAs) is limited by phage particles. Here, monomer, dendrimer-like dimer, tetramer peptidomimetic and anti-immunocomplex tracers were designed and synthesized using lysine as special scaffolds and spacers to develop competitive and noncompetitive FPIAs for benzothiostrobin. The affinity between tracers and monoclonal antibodies or immunocomplexes increased with the tracer valence. A higher signal-to-noise ratio and sensitivity could be generated in the FPIAs based on tetramer tracers. The sensitivities of competitive (50% inhibitory concentration) and noncompetitive (50% saturation concentration) FPIAs were 19.71 ± 4.65 and 40.43 ± 2.73 ng mL−1, respectively. The spiked recoveries were 78.3%−105.2% with relative standard deviations (RSDs) of 0.7%−15.4% for the competitive FPIA, while 78.7%−115.3% with RSDs of 0.7%−12.5% for the noncompetitive FPIA. The amounts of benzothiostrobin in rice detected by the FPIAs were consistent with those detected by high performance liquid chromatography.

Design and screening of a novel neuropilin-1 targeted penetrating peptide for anti-angiogenic therapy in glioma

AimsThis study aimed to design and screen a dual functional fusion peptide that could penetrate the blood-brain barrier and target neuropilin 1 (NRP1) overexpressed in vascular endothelial cells for the anti-angiogenesis of glioma treatment. Main methodsAt the cellular level, the in vitro anti-angiogenic activity of six NRP1 targeting peptides was screened by testing the ability to inhibit the proliferation and tube formation of HUVECs. Then, the in vitro anti-angiogenic activity of two fusion peptides containing different linkers was screened by testing the ability to inhibit HUVECs proliferation, tube formation and migration. The effect of fusion peptide on VEGFR2 related signal pathway was confirmed by Western-blotting. Surface plasmon resonance technology was used to detect the affinity of the fusion peptide to NRP1. The ability of FITC-labeled peptides to penetrate cells was confirmed by cell uptake assay. By establishing an orthotopic glioma model, we evaluated the ability of FITC-labeled peptides to penetrate the blood-brain barrier and their anti-glioma growth activity in vivo. Key findingsWe found that NRP1 targeting peptide RP7 and linker cysteine were the most suitable key components in the fusion peptide. We also found that the fusion peptide Tat-C-RP7 we constructed had the strongest ability to penetrate the blood-brain barrier and anti-angiogenic activity in vitro and in vivo. SignificanceAt present, NRP1 targeting peptide as a drug delivery tool and molecular probe seems to have received more attention. We constructed a fusion peptide Tat-C-RP7 with strong anti-angiogenic activity for the treatment of glioma.

Identification of a novel cell-penetrating peptide derived from the capsid protein of chicken anemia virus and its application in gene delivery.

Cell membranes are a great obstacle for entrance of gene therapeutic agents. Cell-penetrating peptides (CPPs) have been proven as a promising gene delivery tool. However, the early TAT peptide derived from the HIV transcription activator protein has been proven that the sequence contains Furin protease cleaved motifs which limited the TAT application in delivery of exogenous active molecules. In the present study, through the bioinformatics and experimental approach, we have identified a novel CPP derived from the N terminus of VP1 protein of chicken anemia virus (CAV), designated as CVP1-N2, which is rich in arginine residues and contains α-helical structure. Then, the ability of CVP1-N2 cell penetrating was detected using confocal imaging and flow cytometry. FITC-labeled CVP1-N2 peptide could rapidly internalize into different types of live cells with dose dependence and without cytotoxic effects by MTT assay. Surprisingly, CVP1-N2 with a pattern of nuclear sub-location has shown the higher uptake efficiency than TAT. At 10, 1, and 0.1 μM, the mean relative internalization of CVP1-N2 was respectively 1.08-, 12-, and 75-fold higher than that of CVP1, as well as 1.6-, 56-, and 75-fold higher than that of TAT. Moreover, using endocytic inhibitors along with low-temperature stress validated that the CVP1-N2 internalization route is direct translocation pathway. Finally, the capacity of CVP1-N2 for delivery of gene into cells was determined, where it was able to carry red fluorescent protein (RFP) and apoptin genes into cells respectively and induce the apoptosis. All these data indicate that CVP1-N2 could be used as a novel gene delivery vehicle for gene therapy in the future. KEY POINTS: • 1CVP1-N2 was identified as a novel more efficient cell-penetrating peptide. • 2. CVP1-N2 localized to the nucleus through the direct transduction pathway. • 3. CVP1-N2 was able to deliver the apoptin gene into HCT116 cells and induce apoptosis.

Molecular interaction of an antagonistic amylin analog with the extracellular domain of receptor activity-modifying protein 2 assessed by fluorescence polarization

The peptide hormone amylin receptor is a complex of the calcitonin receptor (CTR) and an accessory protein called receptor activity-modifying proteins (RAMPs). The soluble extracellular domain (ECD) of CTR is an important binding site of peptide hormone calcitonin. RAMPs also have an ECD and the association of CTR ECD with RAMP ECD enhances the affinity of peptide hormone amylin. However, the mechanism of how RAMP ECD association enhances amylin affinity remains elusive. Here, we report evidence supporting direct molecular interaction between an antagonistic amylin analog AC413 and RAMP2 ECD. We measured FITC-labeled peptide affinity for purified receptor ECD using fluorescence polarization (FP). We first found that RAMP2 ECD addition to maltose-binding protein (MBP)-tagged CTR ECD and an engineered MBP-tagged RAMP2 ECD-CTR ECD fusion protein (MBP-RAMP2-CTR ECD fusion) enhanced AC413 affinity. This suggests that these recombinant ECD systems represent functional amylin receptors. Interestingly, AC413 C-terminal residue Tyr25 (Y25) to Pro mutation eliminated its selective affinity for the MBP-RAMP2-CTR ECD fusion suggesting the critical role of the AC413 C-terminal residue in amylin receptor selectivity. Our structural model of the RAMP2 ECD:CTR ECD complex predicted molecular interaction of AC413 C-terminal residue Y25 with RAMP2 Glu101 (E101). Our FP peptide-binding assay showed that the RAMP2 E101A mutation of MBP-RAMP2-CTR ECD fusion decreased AC413 affinity by 7-fold, while the affinity of AC413 with the Y25P mutation was minimally changed. Consistently, AC413 binding affinity for the MBP-free RAMP2-CTR ECD fusion protein was also markedly decreased by the RAMP2 E101A mutation, while the affinity of AC413 with the Y25P mutation was moderately decreased. Together, our results support the molecular interaction between the AC413 C-terminal residue Y25 and RAMP2 E101 expanding our understanding of how the accessory protein RAMP2 enhances affinity of peptide hormone amylin for its receptor.

Identification of peptides interfering with the LRRK2/PP1 interaction.

Serine/threonine phosphatases are responsible for modulating the activities of the protein kinases implicated in the development of several pathologies. Here we identified by a PEP-scan approach a peptide of LRRK2, a Parkinson’s disease associated protein, interacting with the phosphatase PP1. In order to study its biological activity, the peptide was fused via its N-terminal to an optimized cell penetrating peptide. We synthesized from the original peptide five interfering peptides and identified two (Mut3DPT-LRRK2-Short and Mut3DPT-LRRK2-Long) able to disrupt the LRRK2/PP1 interaction by competition in anti-LRRK2 immunoprecipitates. Using FITC-labelled peptides, we confirmed their internalization into cell lines as well as into primary cells obtained from healthy or ill human donors. We confirmed by ELISA test the association of Mut3DPT-LRRK2-Long peptide to purified PP1 protein. The peptides Mut3DPT-LRRK2-5 to 8 with either N or C-terminal deletions were not able to disrupt the association LRRK2/PP1 nor to associate with purified PP1 protein. The interfering sequences blocking the PP1/LRRK2 interaction were also fused to a shuttle peptide able to cross the blood brain barrier and showed that the newly generated peptides BBB-LRRK2-Short and BBB-LRRK2-Long were highly resistant to protease degradation. Furthermore, they blocked PP1/LRRK2 interaction and they penetrated into cells. Hence, these newly generated peptides can be employed as new tools in the investigation of the role of the LRRK2/PP1 interaction in normal and pathological conditions.

Immune Regulation, but Not Antibacterial Activity, Is a Crucial Function of Hepcidins in Resistance against Pathogenic Bacteria in Nile Tilapia (Oreochromis niloticus Linn.).

In this study, the functions of a recombinant propeptide (rProOn-Hep1) and the synthetic FITC-labelled mature peptides sMatOn-Hep1 and sMatOn-Hep2 were analyzed. Moreover, sMatOn-Hep1 and sMatOn-Hep2 were mildly detected in the lymphocytes of peripheral blood mononuclear cells (PBMCs) and strongly detected in head kidney macrophages. The in vitro binding and antibacterial activities of these peptides were slightly effective against several pathogenic bacteria. Immune regulation by sMatOn-Hep1 was also analyzed, and only sMatOn-Hep1 significantly enhanced the phagocytic index in vitro (p < 0.05). Interestingly, intraperitoneal injection of sMatOn-Hep1 (10 or 100 µg) significantly elevated the phagocytic activity, phagocytic index, and lysozyme activity and clearly decreased the iron ion levels in the livers of the treated fish (p < 0.05). Additionally, sMatOn-Hep1 enhanced the expression levels of CC and CXC chemokines, transferrin and both On-Hep genes in the liver, spleen and head kidney, for 1–96 h after injection, but did not properly protect the experimental fish from S. agalactiae infection after 7 days of treatment. However, the injection of S. agalactiae and On-Heps indicated that 100 μg of sMatOn-Hep1 was very effective, while 100 μg of rProOn-Hep1 and sMatOn-Hep2 demonstrated moderate protection. Therefore, On-Hep is a crucial iron-regulating molecule and a key immune regulator of disease resistance in Nile tilapia.

Ultra-Compact Microsystems-Based Confocal Endomicroscope.

Point-of-care medical diagnosis demands immediate feedback on tissue pathology. Confocal endomicroscopy can provide real-time in vivo images with histology-like features. The working channel in medical endoscopes are becoming smaller in dimension. Microsystems methods can produce tiny mechanical scanners. We demonstrate a flexible fiber instrument for in vivo imaging as an endoscope accessory. The optical path is folded on-axis to reduce length while allowing the beam to expand and achieve a numerical aperture of 0.41. A high-speed parametric resonance mirror produces large deflection angles > 13°, and is mounted on a 2 mm diameter chip designed with clamp structures for reduced space. A compact lens assembly provides diffraction-limited lateral and axial resolution of 1.5 and [Formula: see text], respectively. A working distance of [Formula: see text] and field-of-view of [Formula: see text] m are achieved. Miniature apparatus is fabricated to assemble and align the scanhead components. The optics and scanner are packaged in a distal tip with 2.4 mm diameter and 10 mm rigid length. These dimensions allow the endomicroscope to pass forward easily through the 2.8 mm diameter working channel in medical endoscopes commonly used in clinical practice. Fluorescence images are collected in vivo at 10 frames per second in the colon of genetically-engineered mice that spontaneously develop adenomas. A FITC-labeled peptide heterodimer is administered intravenously to provide specific contrast. Sub-cellular structures are visualized to distinguish pre-malignant from normal mucosa. These results demonstrate use of microsystems methods to produce an ultra-compact instrument with sufficiently small dimensions for broad use.

Cadmium association with DREAM promotes DREAM interactions with intracellular partners in a similar manner to its physiological ligand, calcium.

Cd2+ exposure has been associated with neurodegenerative diseases and other pathologies, but the underlying mechanism through which it exerts toxic effects remain unresolved. Using calorimetric and spectroscopic techniques, we show that Cd2+ binds to EF-hands in DREAM (downstream regulatory element antagonist modulator) with an equilibrium dissociation constant of 89 ± 10 nM, which is superior to that determined for Ca2+ (Kd = 1000 nM). Analogous to Ca2+ binding, Cd2+ binding triggers changes in the protein secondary and tertiary structure, including increased exposure of the hydrophobic cavities, as determined using a fluorescent probe, 1-anilinonaphthalene-8-sulfonic acid. In addition, we demonstrate that Cd2+ binding modulates DREAM interactions with FITC-labeled peptides that mimic binding sites of DREAM effector proteins; helix-9 of presenilin-1, and site-1 and site 2 of potassium voltage channel 4.3 (residues 2-22 and 70-90, respectively). Cd2+ association with DREAM increases its affinity for helix 9 of presenilin roughly 30-times compared to metal-free DREAM. The DREAM affinity for site-1 and site 2 is elevated approximately 7 and 15 times, respectively, in the presence of Cd2+. The above results suggest that DREAM and probably other members of the neuronal calcium sensor family bind Cd2+ with an affinity that is superior to that for Ca2+ and the interactions between toxic Cd2+ and DREAM and other neuronal calcium sensors provide novel insight into the molecular mechanism of Cd2+ neurotoxicity.

Versatile Layer-By-Layer Highly Stable Multilayer Films: Study of the Loading and Release of FITC-Labeled Short Peptide in the Drug Delivery Field.

A viable short FITC-peptide immobilization is the most essential step in the fabrication of multilayer films based on FITC-peptide. These functional multilayer films have potential applications in drug delivery, medical therapy, and so forth. These FITC-peptides films needed to be handled with a lot of care and precision due to their sensitive nature. In this study, a general immobilization method is reported for the purpose of stabilizing various kinds of peptides at the interfacial regions. Utilizing Mesoporous silica nanoparticles can help in the preservation of these FITC-peptides by embedding themselves into these covalently cross-linked multilayers. This basic outlook of the multilayer films is potent enough and could be reused as a positive substrate. The spatio-temporal retention property of peptides can be modulated by varying the number of capping layers. The release speed of guest molecules such as tyrosine within FITC-peptide or/and adamantane (Ad)-in short peptides could also be fine-tuned by the specific arrangements of the multilayers of mesoporous silica nanoparticles (MSNs) and hyaluronic acid- cyclodextrin (HA-CD) multilayer films.

Small Peptide Ligands for Targeting EGFR in Triple Negative Breast Cancer Cells

The efficacy of chemotherapy for cancer treatment can be increased by targeted drug delivery to the cancer cells. This is particularly important for triple negative breast cancer (TNBC) for which chemotherapy is a major form of treatment. Here we designed and screened a library of 30 peptides starting with a previously reported epidermal growth factor receptor (EGFR) targeting peptide GE11 (YHWYGYTPQNVI). A direct peptide array-whole cell binding assay, where the peptides are conjugated to a cellulose membrane, was used to identify four peptides with enhanced binding to TNBC cells. Next, the four peptides were synthesized as FITC-labelled soluble peptides to study their direct uptake by TNBC cells using flow cytometry. The results showed that peptide analogue 22 had several fold higher uptake by the TNBC cells compared to the lead peptide GE11. The specific uptake of the peptide analogue 22 was confirmed by competition experiment using pure EGF protein. Further, peptide 22 showed dose dependent uptake by the TNBC MDA-MB-231 cells (105) with uptake saturating at around 2 μM peptide concentration. Thus, peptide 22 is a promising EGFR specific TNBC cell binding peptide that can be conjugated directly to a chemotherapeutic drug or to nanoparticles for targeted drug delivery to enhance the efficacy of chemotherapy for TNBC treatment.

Synthetic bovine lactoferrin peptide Lfampin kills Entamoeba histolytica trophozoites by necrosis and resolves amoebic intracecal infection in mice.

Amoebiasis caused by the protozoan parasite Entamoeba histolytica remains a public health problem in developing countries, making the identification of new anti-amoebic compounds a continuing priority. Previously, we have shown that lactoferrin (Lf) and several Lf-derived peptides exhibit in vitro anti-amoebic activity independently of their iron-binding activity. Here, we evaluated the amoebicidal effect of synthetic Lf-derived peptides Lfcin-B, Lfcin 17-30, and Lfampin, analyzed the mechanism of death induced by the peptides and determined their therapeutic effects on murine intestinal amoebiasis. MTT assays in trophozoite cultures of E. histolytica exposed to each peptide (1–1000 μM) showed that Lfampin is far more amoebicidal than Lfcins. Lfampin killed 80% of trophozoites at doses higher than 100 μM in 24 h, and FACs analysis using Annexin V/propidium iodide showed that death occurred mainly by necrosis. In contrast, Lfcin-B and Lfcin 17-30 appeared to have no significant effect on amoebic viability. FACs and confocal microscopy analysis using FITC-labeled peptides showed that all three peptides are internalized by the amoeba mainly using receptor (PI3K signaling) and actin-dependent pathways but independent of clathrin. Docking studies identified cholesterol in the amoeba’s plasma membrane as a possible target of Lfampin. Oral treatment of intracecally infected mice with the abovementioned peptides at 10 mg/kg for 4 days showed that Lfampin resolved 100% of the cases of intestinal amoebiasis, whereas Lfcin 17-30 and Lfcin-B were effective in resolving infection in 80 and 70% of cases, respectively. These data show that although synthetic bovine Lf-derived peptides exhibit varying amoebicidal potentials in vitro, they do resolve murine intestinal amoebiasis efficiently, suggesting that they may be useful as a therapeutic treatment.

Development of a Pancreatic Cancer Specific Binding Peptide Using Phage Display

Pancreatic cancer has a very poor prognosis, and early diagnosis is a way to increase the survival rate of patients. The purpose of this study was to develop pancreatic cancer-specific peptides for imaging studies. Three pancreatic cancer cell lines, MIA PaCa-2, UACC-462, and BxPC-3, and a control cell line, CCD841, were used. Biopannings were performed on MIA PaCa-2 using a phage display library. After this, the peptides were synthesized and labeled with fluorescein isothiocyanate (FITC). Immunocytochemistry (ICC), enzyme-linked immunosorbent assay (ELISA), and fluorescence- activated cell sorter (FACS) were performed to examine the specific binding. To examine its therapeutic applications, a photosensitizer, chlorin e6 (Ce6), was conjugated on the peptide and photodynamic therapy was performed. Cell survival was investigated using a [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide] assay. After three biopannings, the phages were amplified from 1.4×104 to 3.2×105 plaque-forming units. The most strongly binding phage was selected from the ELISA and ICC results. FITC-labeled peptide, M5, in the three pancreatic cancer cell lines showed significantly higher immunofluorescence in the ICC experiments than that of CCD841. The higher binding ability to MIA PaCa-2 cells was confirmed from FACS analysis, which showed a right shift compared to CCD841. M5 bound to Ce6 showed a significantly lower cell survival rate than that of Ce6 alone in photodynamic therapy, which was observed consistently as a change in the tumor size and fluorescence intensity in MIA PaCa-2 cell-implanted animal models. This study showed that the noble peptide, M5, binds specifically to the pancreatic cancer cell line, MIA PaCa-2. The M5 peptide has potential use in future optical diagnostic and therapeutic purposes.

Identification of Receptor Ligands in Apo B100 Reveals Potential Functional Domains.

LDL, VLDL and other members of the low-density lipoparticles (LLPs) enter cells through a large family of receptors. The actual receptor ligand(s) in apolipoprotein B100, one of the main proteins of LLP, remain(s) unknown. The objective of this study was to identify true receptor ligand(s) in apo B100, a molecule of 4563 residues. Apo B100 contains 33 analogues of Cardin-Weintraub arginine/lysine-based receptor ligand motifs and shares key lysine motifs and sequence similarity with the LDL receptor-associated protein, MESD, and heat shock proteins. Eleven FITC-labeled synthetic peptides of 21-42 residues, with at least one ligand, were tested for binding and internalization using HeLa cells. All peptides bind but display different binding capacities and patterns. Peptides B0013, B0582, B2366, and B2932 mediate endocytosis and appear in distinct sites in the cytoplasm. B0708 and B3181 bind and remain on the cell surface as aggregates/clusters. Peptides B3119 (Site A) and B3347 (Site B), the putative ligands, showed low binding and no cell entry capacity. Apo B100 regions in this study share similarities with related proteins of known function including chaperone proteins and Apo BEC stimulating protein, and not directly related proteins, e.g., the DNA-binding domain of interferon regulatory factors, MSX2-interacting protein, and snake venom Zinc metalloproteinase-disintegrin-like proteins.