P53 Peptide Research Articles

Allergen-specific immunotherapy by recombinant Der P1 allergen-derived peptide-based vaccine in an allergic mouse model.

Increased IgE levels have made house dust mite allergens one of the most frequent causes of allergies worldwide. Treatment reduces the IgE antibodies and types two cytokines, namely interleukin-4 (IL-4) and IL-13. Although existing treatments significantly reduce IgE or IL-4/IL-13, they are very costly. This study aimed to construct a recombinant protein derived from rDer p1 peptides in the form of an immunotherapy approach and to measure the response of IgE and IgG antibodies. The proteins were isolated, purified, and evaluated using the SDS-PAGE and Bradford test and confirmed by using Western blot. To evaluate immunotherapy efficiency, 24 BALB/C mice were sensitized intraperitoneally with house dust mites (HDM) adsorbed to Aluminum hydroxide (Alum) and randomly divided into four groups of six: control sensitized, HDM extract, rDer p1, and DpTTDp vaccine. To immunization, four groups of random mice were each treated with phosphate-buffered saline, 100 μg of rDer p1 protein, DpTTDp, or HDM extract, every 3 days. Direct ELISA determined HDM-specific IgG and IgE subclasses. Data were analyzed in SPSS and Graph pad prism software. Values of p < .05 were considered significant. After immunization of mice, the rDer P1 and recombinant vaccine like HDM extract increased IgG antibody titer and decreased IgE-dependent reactivity in allergic mice to rDer P1. Also, the levels of inflammatory IL-4 and IL-13 cytokines as allergic stimulants decreased. The use of present available recombinant proteins is considered a viable, cost-effective, and long-term option for providing effective HDM allergy immunotherapy vaccines without side effects.

Exploring the Effect of Hydrocarbon Cross-linkers on the Structure and Binding of Stapled p53 peptide

Exploring the Effect of Hydrocarbon Cross-linkers on the Structure and Binding of Stapled p53 peptide

Lactobacillus paracasei L9 affects disease progression in experimental autoimmune neuritis by regulating intestinal flora structure and arginine metabolism

BackgroundAutoimmune neuropathies are common peripheral nervous system (PNS) disorders. Environmental influences and dietary components are known to affect the course of autoimmune diseases. Intestinal microorganisms can be dynamically regulated through diet, and this study combines intestinal microorganisms with diseases to open up new therapeutic ideas.MethodsIn Lewis rats, a model of EAN was established with P0 peptide, Lactobacillus were used as treatment, serum T-cell ratio, inflammatory factors, sciatic neuropathological changes, and pathological inflammatory effects on intestinal mucosa were detected, and fecal metabolomics and 16 s microbiome analysis were performed to further explore the mechanism.ResultsIn the EAN rat model, Lactobacillus paracasei L9 (LP) could dynamically regulate the CD4+/CD8+T balance in serum, reduce serum IL-1, IL-6 and TNF-α expression levels, improve sciatic nerve demyelination and inflammatory infiltration, and reduce nervous system score. In the rat model of EAN, intestinal mucosa was damaged. Occludin and ZO-1 were downregulated. IL-1, TNF-α and Reg3γ were upregulated. LP gavage induced intestinal mucosa recovery; occludin and ZO-1 upregulation; IL-1, TNF-α and Reg3γ downregulation. Finally, metabolomics and 16 s microbiome analysis were performed, and differential metabolites were enriched with an important metabolic pathway, arginine and proline metabolism.ConclusionLP improved EAN in rats by influencing intestinal community and the lysine and proline metabolism.

Sel1-like proteins and peptides are the major Oxalobacter formigenes-derived factors stimulating oxalate transport by human intestinal epithelial cells.

Kidney stones (KSs) are very common, excruciating, and associated with tremendous healthcare cost, chronic kidney disease (CKD), and kidney failure (KF). Most KSs are composed of calcium oxalate and small increases in urinary oxalate concentration significantly enhance the stone risk. Oxalate also potentially contributes to CKD progression, kidney disease-associated cardiovascular diseases, and poor renal allograft survival. This emphasizes the urgent need for plasma and urinary oxalate lowering therapies, which can be achieved by enhancing enteric oxalate secretion. We previously identified Oxalobacter formigenes (O. formigenes)-derived factors secreted in its culture-conditioned medium (CM), which stimulate oxalate transport by human intestinal Caco2-BBE (C2) cells and reduce urinary oxalate excretion in hyperoxaluric mice by enhancing colonic oxalate secretion. Given their remarkable therapeutic potential, we now identified Sel1-like proteins as the major O. formigenes-derived secreted factors using mass spectrometry and functional assays. Crystal structures for six proteins were determined to confirm structures and better understand functions. OxBSel1-14-derived small peptides P8 and P9 were identified as the major factors, with P8 + 9 closely recapitulating the CM's effects, acting through the oxalate transporters SLC26A2 and SLC26A6 and PKA activation. Besides C2 cells, P8 + 9 also stimulate oxalate transport by human ileal and colonic organoids, confirming that they work in human tissues. In conclusion, P8 and P9 peptides are identified as the major O. formigenes-derived secreted factors and they have significant therapeutic potential for hyperoxalemia, hyperoxaluria, and related disorders, impacting the outcomes of patients suffering from KSs, enteric hyperoxaluria, primary hyperoxaluria, CKD, KF, and renal transplant recipients.NEW & NOTEWORTHY We previously identified Oxalobacter formigenes-derived secreted factors stimulating oxalate transport by human intestinal epithelial cells in vitro and reducing urinary oxalate excretion in hyperoxaluric mice by enhancing colonic oxalate secretion. We now identified Sel1-like proteins and small peptides as the major secreted factors and they have significant therapeutic potential for hyperoxalemia and hyperoxaluria, impacting the outcomes of patients suffering from kidney stones, primary and secondary hyperoxaluria, chronic kidney disease, kidney failure, and renal transplant recipients.

Preclinical Assessment of EPO-Derived Peptide JM4 in Experimental Autoimmune Neuritis (P10-5.008)

<h3>Objective:</h3> Determine clinical efficacy of JM4, a synthetic erythropoietin-derived 19-mer peptide in experimental autoimmune neuritis (EAN). <h3>Background:</h3> Guillain-Barré syndrome, an acute inflammatory demyelinating polyneuropathy (AIDP), is an autoimmune disease causing injuries to myelinated nerves. Mild AIDP cases result in temporary motor and sensory deficits, but severe AIDP cases can also cause dysautonomia and respiratory failure leading to disability or death. Treatments include intravenous immune globulin or plasma exchange, but only 30–40% of patients experience clinical improvement. More effective interventions are needed to improve AIDP clinical outcomes. JM4 is a 19-mer peptide derived from human erythropoietin, which does not have an unwanted side effect of erythropoiesis. We demonstrated JM4 therapy had prolonged therapeutic benefits in an experimental autoimmune encephalomyelitis (EAE) model. Since inflammatory mechanisms underlying EAE are shared with EAN, an animal model for AIDP, we reasoned JM4 would also have clinical benefit in EAN. <h3>Design/Methods:</h3> EAN was induced in C57BL/6 mouse and in Lewis rat using murine peripheral nerve myelin P0 antigen and bovine myelin P2 peptide, respectively. EAN clinical scoring paradigm was used to assess neurological deficits. At deficit onset, EAN mice were administered JM4 intravenously (IV) for 10 days and EAN rats were administered JM4 IV for 3 days followed by intraperitoneal injection for 7 days. Sciatic nerves from EAN rodents were stained with Luxol fast blue to assess demyelination and with cresyl violet to assess mononuclear cell infiltration. <h3>Results:</h3> JM4 treatment reduced peak EAN clinical scores compared to sham treatment. Based on staining analysis, EAN induction caused mononuclear cell infiltration and demyelination in the sciatic nerve. <h3>Conclusions:</h3> Preliminary clinical scoring data from rodent models suggest JM4 may have therapeutic benefits in EAN models. Further studies are needed to correlate improvements in clinical scoring with histological analysis of demyelination, mononuclear cell infiltration and neuroinflammatory markers in EAN models. <b>Disclosure:</b> Dr. Arismendi has nothing to disclose. The institution of Dr. Choi has received research support from Department of Veterans Affairs. Dr. Husar has nothing to disclose. Peter C. Dowling, MD has nothing to disclose. Dr. Lu has nothing to disclose.

1177 Treatment of mice with mutant p53 peptide results in a cell-mediated immune response

1177 Treatment of mice with mutant p53 peptide results in a cell-mediated immune response

Computational Study of Driving Forces in ATSP, PDIQ, and P53 Peptide Binding: C═O···C═O Tetrel Bonding Interactions at Work.

Understanding the molecular interactions that drive peptide folding is crucial to chemistry and biology. In this study, we analyzed the role of CO···CO tetrel bonding (TtB) interactions in the folding mechanism of three different peptides (ATSP, pDIQ, and p53), which exhibit a different propensity to fold in an α helix motif. To achieve this goal, we used both a recently developed Bayesian inference approach (MELDxMD) and Quantum Mechanics (QM) calculations at the RI-MP2/def2-TZVP level of theory. These techniques allowed us to study the folding process and to evaluate the strength of the CO···CO TtBs as well as the synergies between TtBs and hydrogen-bonding (HB) interactions. We believe that the results derived from our study will be helpful for those scientists working in computational biology, peptide chemistry, and structural biology.

Biomimetic Silica Particles with Self-Loading BMP-2 Knuckle Epitope Peptide and Its Delivery for Bone Regeneration.

Biomimetic silica deposition is an in-situ immobilization method for bioactive molecules under biocompatible conditions. The osteoinductive P4 peptide derived from the knuckle epitope of bone morphogenetic protein (BMP), which binds to BMP receptor-II (BMPRII), has been newly found to contain silica formation ability. We found that the two lysine residues at the N-terminus of P4 played a vital role in silica deposition. The P4 peptide co-precipitated with silica during P4-mediated silicification, yielding P4/silica hybrid particles (P4@Si) with a high loading efficiency of 87%. P4 was released from P4@Si at a constant rate for over 250 h, representing a zero-order kinetic model. In flow cytometric analysis, P4@Si showed a 1.5-fold increase in the delivery capacity to MC3T3 E1 cells than the free form of P4. Furthermore, P4 was found anchored to hydroxyapatite (HA) through a hexa-glutamate tag, followed by P4-mediated silicification, yielding P4@Si coated HA. This suggested a superior osteoinductive potential compared to silica or P4 alone coated HA in the in vitro study. In conclusion, the co-delivery of the osteoinductive P4 peptide and silica by P4-mediated silica deposition is an efficient method for capturing and delivering its molecules and inducing synergistic osteogenesis.

Identification of a peptide ligand for human ALDH3A1 through peptide phage display: Prediction and characterization of protein interaction sites and inhibition of ALDH3A1 enzymatic activity.

Aldehyde dehydrogenase 3A1 (ALDH3A1) by oxidizing medium chain aldehydes to their corresponding carboxylic acids, is involved in the detoxification of toxic byproducts and is considered to play an important role in antioxidant cellular defense. ALDH3A1 has been implicated in various other functions such as cell proliferation, cell cycle regulation, and DNA damage response. Recently, it has been identified as a putative biomarker of prostate, gastric, and lung cancer stem cell phenotype. Although ALDH3A1 has multifaceted functions in both normal and cancer homeostasis, its modes of action are currently unknown. To this end, we utilized a random 12-mer peptide phage display library to identify efficiently human ALDH3A1-interacting peptides. One prevailing peptide (P1) was systematically demonstrated to interact with the protein of interest, which was further validated in vitro by peptide ELISA. Bioinformatic analysis indicated two putative P1 binding sites on the protein surface implying biomedical potential and potent inhibitory activity of the P1 peptide on hALDH3A1 activity was demonstrated by enzymatic studies. Furthermore, in search of potential hALDH3A1 interacting players, a BLASTp search demonstrated that no protein in the database includes the full-length amino acid sequence of P1, but identified a list of proteins containing parts of the P1 sequence, which may prove potential hALDH3A1 interacting partners. Among them, Protein Kinase C Binding Protein 1 and General Transcription Factor II-I are candidates of high interest due to their cellular localization and function. To conclude, this study identifies a novel peptide with potential biomedical applications and further suggests a list of protein candidates be explored as possible hALDH3A1-interacting partners in future studies.

Fingolimod ameliorates chronic experimental autoimmune neuritis by modulating inflammatory cytokines and Akt/mTOR/NF-κB signaling.

Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is an immune-mediated disease that targets the myelin sheaths of the peripheral nerves. Fingolimod is a sphingosine 1 phosphate (S1P) receptor antagonist with a high affinity for S1P receptors through the Akt-mTOR pathway, and prior research has suggested that it might be helpful in autoimmune illnesses. Chronic experimental autoimmune neuritis (c-EAN) was induced by immunizing Lewis rats with the S-palm P0(180-199) peptide, and then the treatment group was intraperitoneally injected with fingolimod (1mg/kg) daily. Hematoxylin and eosin staining was used to assess the severity of nerve injury. Immunohistochemistry staining showed that fingolimod's anti-inflammatory effects on c-EAN rats might be realized through the NF-κB signaling pathway. Tumor necrosis factor-α (TNF-α), interferon-γ (INF-γ), interleukin-1beta (IL-1β), interleukin 6 (IL-6), inducible nitric oxide synthase (iNOS), and intercellular adhesion molecule-1 (ICAM-1) were measured to evaluate the inflammation levels, and pAkt, p-S6, and p-p65 were used to measure the abundance of downstream activation markers to determine whether the Akt/mTOR/NF-κB signaling pathway was activated in the c-EAN model. Fingolimod treatment reduced the inflammatory reaction and the expression of NF-κB in sciatic nerves. It also decreased the mRNA levels of the proinflammatory cytokines TNF-α, IFN-γ, IL-1β, IL-6, iNOS, and ICAM-1 and pAkt, p-S6, and p-p65, representing the Akt/mTOR/NF-κB signaling pathway. Our data showed that fingolimod could improve the disease course, alleviate the decrease in inflammation, and reduce proinflammatory cytokines through the Akt/mTOR/NF-κB axis in c-EAN rats, which could be beneficial for the development of CIDP-related research.

Antifungal Peptide SP1 Damages Polysaccharide Capsule of Cryptococcus neoformans and Enhances Phagocytosis of Macrophages.

Cryptococcus neoformans is a fungal pathogen which causes nearly half a million deaths worldwide each year. Under host-relevant conditions, it produces a characteristic polysaccharide capsule. The polysaccharide capsule is one of the main virulence factors of C. neoformans, which involves antiphagocytosis and immune responses of the host to cause a lack of an immune. Meanwhile, the polysaccharide capsule is a promising drug target because of the absence of analogs in the host. Here, we demonstrate that antifungal peptide SP1, which is derived from the N terminus of Saccharomyces cerevisiae GAPDH (glyceraldehyde-3-phosphate dehydrogenase), disrupts the polysaccharide capsule of C. neoformans H99. The mechanism is possibly due to the interaction of SP1 with glucuronoxylomannan (GXM). Disruption of the polysaccharide capsule enhances the adhesion and phagocytosis of C. neoformans H99 by macrophages and reduces the replication of C. neoformans H99 within macrophages. Additionally, SP1 exhibits antifungal activity against cryptococcal biofilms associated with the capsular polysaccharides. These findings suggest the potential of SP1 as a drug candidate for the treatment of cryptococcosis. IMPORTANCE C. neoformans is an opportunistic pathogen that causes invasive infections with a high mortality rate. Currently, the clinical drugs available for the treatment of cryptococcosis are limited to amphotericin B, azoles, and flucytosine. Amphotericin is nephrotoxic, and the widespread use of azoles and 5-flucytosine has led to a rapid development of drug resistance in C. neoformans. There is an urgent need to develop new and effective anticryptococcal drugs. Targeting virulence factors is a novel strategy for developing antifungal drugs. The antifungal peptide SP1 is capable of disrupting the polysaccharide capsule, which is a principal virulence factor of C. neoformans. Studying the mechanism by which SP1 damages the polysaccharide capsule and investigating the potential benefits of SP1 in removing C. neoformans from the host provides baseline data to develop a therapeutic strategy against refractory cryptococcal infections. This strategy would involve both inhibiting virulence factors and directly killing C. neoformans cells.

Design and synthesis of novel N-terminal peptides of integrin and aminopeptidase are new finding for anticancer activity

The short peptides, containing the amino acid sequence asparagine-glycine-arginine (NGR) and arginine-glycine-aspartic acid (RGD), possess the strong binding ability to N (APN/CD13) aminopeptidase receptor and integrin proteins involved in antitumor properties are overexpressed. A novel short N-terminal modified hexapeptides P1 and P2 was designed and synthesized using the Fmoc-chemistry solid phase peptide synthesis protocol. Notably, the cytotoxicity of the MTT assay demonstrated the viability of normal and cancer cells up to lower peptide concentrations. Interestingly, both peptides show good anticancer activities against the four cancer cells and normal cells namely, Hep-2, HepG2, MCF-7, A375, and Vero and compared with standard drugs, doxorubicin and paclitaxel. Additionally, in silico studies were applied to predict the binding sites and binding orientation of the peptides for potential anticancer targets. Steady-state fluorescence measurements showed that peptide P1 exhibits preferential interactions with POPC/POPG anionic bilayers rather than the zwitterionic POPC lipid bilayers and peptide P2, did not show any preferential interaction with lipids bilayers. But impressively, peptide P2 shows anticancer activity due to the NGR/RGD motif. Circular dichroism studies demonstrated that the peptide's secondary structure changes only minimally upon binding to the anionic lipid bilayers.

Effect of Newly Synthesized Structures of Peptides on the Stability of the Monolayers Formed.

The aim of the study was to evaluate the effect of the peptide structure (WKWK)2-KWKWK-NH2, P4 (C12)2-KKKK-NH2, P5 (KWK)2-KWWW-NH2, P6 (KK)2-KWWW-NH2 on their physicochemical properties. The thermogravimetric method (TG/DTG) was used, which made it possible to observe the course of chemical reactions and phase transformations occurring during the heating of solid samples. Based on the DSC curves, the enthalpy of the processes occurring in the peptides was determined. The influence of the chemical structure of this group of compounds on their film-forming properties was determined using the Langmuir-Wilhelmy trough method and was followed by molecular dynamics simulation. Evaluated peptides showed high thermal stability and the first significant mass loss occurred only at about 230 °C and 350 °C. The analysis of the compressibility coefficient of individual peptides indicates that all formed peptide monolayers were in the expanded liquid phase. Their maximum compressibility factor was less than 50.0 mN/m. Its highest value of 42.7 mN/m was achieved in a monolayer made of P4. The results obtained in molecular dynamic simulation indicate that non-polar side chains played an important role in the properties of the P4 monolayer, and the same applies to P5, except that a spherical effect was observed here. A slightly different behavior was observed for the P6 and P2 peptide systems, where the type of amino acids present had an influence. The obtained results indicate that the structure of the peptide affected its physicochemical and layer-forming properties.

Interaction of Amphipathic Peptide from Influenza Virus M1 Protein with Mitochondrial Cytochrome Oxidase.

The Bile Acid Binding Site (BABS) of cytochrome oxidase (CcO) binds numerous amphipathic ligands. To determine which of the BABS-lining residues are critical for interaction, we used the peptide P4 and its derivatives A1-A4. P4 is composed of two flexibly bound modified α-helices from the M1 protein of the influenza virus, each containing a cholesterol-recognizing CRAC motif. The effect of the peptides on the activity of CcO was studied in solution and in membranes. The secondary structure of the peptides was examined by molecular dynamics, circular dichroism spectroscopy, and testing the ability to form membrane pores. P4 was found to suppress the oxidase but not the peroxidase activity of solubilized CcO. The Ki(app) is linearly dependent on the dodecyl-maltoside (DM) concentration, indicating that DM and P4 compete in a 1:1 ratio. The true Ki is 3 μM. The deoxycholate-induced increase in Ki(app) points to a competition between P4 and deoxycholate. A1 and A4 inhibit solubilized CcO with Ki(app)~20 μM at 1 mM DM. A2 and A3 hardly inhibit CcO either in solution or in membranes. The mitochondrial membrane-bound CcO retains sensitivity to P4 and A4 but acquires resistance to A1. We associate the inhibitory effect of P4 with its binding to BABS and dysfunction of the proton channel K. Trp residue is critical for inhibition. The resistance of the membrane-bound enzyme to inhibition may be due to the disordered secondary structure of the inhibitory peptide.

Sustained and Targeted Delivery of Self-Assembled Doxorubicin Nonapeptides Using pH-Responsive Hydrogels for Osteosarcoma Chemotherapy

While chemotherapeutic agents have particularly potent effects in many types of cancer, their clinical applications are still far from satisfactory due to off-target drug exposure, chemotherapy resistance, and adverse effects, especially in osteosarcoma. Therefore, it is clinically promising to construct a novel tumor-targeted drug delivery system to control drug release and alleviate side effects. In this study, a pH-responsive nonapeptide hydrogel was designed and fabricated for the tumor-targeted drug delivery of doxorubicin (DOX). Using a solid-phase synthesis method, a nonapeptide named P1 peptide that is structurally akin to surfactant-like peptides (SLPs) due to its hydrophobic tail and hydrophilic head was synthesized. The physicochemical properties of the P1 hydrogel were characterized via encapsulation capacity, transmission electron microscopy (TEM), circular dichroism (CD), zeta potential, rheological analysis, and drug release studies. We also used in vitro and in vivo experiments to investigate the cytocompatibility and tumor inhibitory efficacy of the drug-loaded peptide hydrogel. The P1 peptide could self-assemble into biodegradable hydrogels under neutral conditions, and the prepared drug-loaded hydrogels exhibited good injectability and biocompatibility. The in vitro drug release studies showed that DOX-P1 hydrogels had high sensitivity to acidic conditions (pH 5.8 versus 7.4, up to 3.6-fold). Furthermore, the in vivo experiments demonstrated that the DOX-P1 hydrogel could not only amplify the therapeutic effect but also increase DOX accumulation at the tumor site. Our study proposes a promising approach to designing a pH-responsive hydrogel with controlled doxorubicin-release action based on self-assembled nonapeptides for targeted chemotherapy.

Biodistribution and Adjuvant Effect of an Intranasal Vaccine Based on Chitosan Nanoparticles against Paracoccidioidomycosis

Paracoccidioidomycosis (PCM) is a fungal infection caused by the thermodimorphic Paracoccidioides sp. PCM mainly affects the lungs, but, if it is not contained by the immune response, the disease can spread systemically. An immune response derived predominantly from Th1 and Th17 T cell subsets facilitates the elimination of Paracoccidioides cells. In the present work, we evaluated the biodistribution of a prototype vaccine based on the immunodominant and protective P. brasiliensis P10 peptide within chitosan nanoparticles in BALB/c mice infected with P. brasiliensis strain 18 (Pb18). The generated fluorescent (FITC or Cy5.5) or non-fluorescent chitosan nanoparticles ranged in diameter from 230 to 350 nm, and both displayed a Z potential of +20 mV. Most chitosan nanoparticles were found in the upper airway, with smaller amounts localized in the trachea and lungs. The nanoparticles complexed or associated with the P10 peptide were able to reduce the fungal load, and the use of the chitosan nanoparticles reduced the necessary number of doses to achieve fungal reduction. Both vaccines were able to induce a Th1 and Th17 immune response. These data demonstrates that the chitosan P10 nanoparticles are an excellent candidate vaccine for the treatment of PCM.

Towards Radiolabeled EGFR-Specific Peptides: Alternatives to GE11.

The human epidermal growth factor receptor (EGFR) is closely related to several cancer-promoting processes and overexpressed on a variety of tumor types, rendering it an important target structure for the imaging and therapy of several malignancies. To date, approaches to develop peptidic radioligands able to specifically address and visualize EGFR-positive tumors have been of limited success. Most of the attempts were based on the lead GE11, as this peptide was previously described to be a highly potent EGFR-specific agent. However, since it has recently been shown that GE11 exhibits an insufficient affinity to the EGFR in monomeric form to be suitable as a basis for the development of tracers based on it, in the present work we investigated which other peptides might be suitable as lead structures for the development of EGFR-specific peptidic radiotracers. For this purpose, we developed 68Ga-labeled radioligands based on the peptides D4, P1, P2, CPP, QRH, EGBP and Pep11, having been described before as EGFR-specific. In addition, we also tested three truncated versions of the endogenous EGFR ligand hEGF (human epidermal growth factor) with respect to their ability to specifically target the EGFR with high affinity. Therefore, chelator-modified labeling precursors of the mentioned peptides were synthesized, radiolabeled with 68Ga and the obtained radioligands were evaluated for their hydrophilicity/lipophilicity, stability against degradation by human serum peptidases, in vitro tumor cell uptake, and receptor affinity in competitive displacement experiments on EGFR-positive A431 cells. Although all NODA-GA-modified (NODA-GA: (1,4,7-triazacyclononane-4,7-diyl)diacetic acid-1-glutaric acid) labeling precursors could be obtained more or less efficient in yields between 5 and 74%, the 68Ga-radiolabeling proved to be unsuccessful for two of the three truncated versions of hEGF ([68Ga]Ga-8 and [68Ga]Ga-9), producing several side-products. For the other agents [68Ga]Ga-1-[68Ga]Ga-7, [68Ga]Ga-10 and [68Ga]Ga-11, high radiochemical yields and purities of ≥98% and molar activities of up to 114 GBq/µmol were obtained. In the assay investigating the radiopeptide susceptibilities against serum peptidase degradation, the EGBP-based agent demonstrated a limited stability with a half-life of only 66.4 ± 3.0 min, whereas the other tracers showed considerably higher stabilities of up to an 8000 min half-life. Finally, all radiotracer candidates were evaluated in terms of tumor cell internalization and receptor binding potential on EGFR-positive A431 cell. In these experiments, all developed agents failed to show an EGFR-specific tumor cell uptake or a relevant EGFR-affinity. By contrast, the positive controls tested under identical conditions, [125I]I-hEGF and hEGF demonstrated the expected high EGFR-specific tumor cell uptake (33.6% after 1 h, being reduced to 1.9% under blocking conditions) and affinity (IC50 value of 15.2 ± 3.3 nM). Thus, these results indicate that none of the previously described peptidic agents developed for EGFR targeting appears to be a reasonable choice as a lead structure for the development of radiopeptides for targeting of EGFR-positive tumors. Likewise, the tested truncated variants of the endogenous hEGF do not seem to be promising alternatives for this purpose.

Investigation of the potential off-target effects on Munc 18 (p67) involved in synaptic vesical transport due to the interaction with peptide p5.

SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins and SM (Sec1/Munc-18 like) proteins are involved in the docking of vesicles with the target membranes followed by the release of neurotransmitters in the brain. The Syntaxin protein plays a crucial role in the SNARE complex formation. Munc 18 (p67) is known to regulate the different functions of these SNARE complexes by binding with different conformations of Syntaxin. It has also been established that the absence of p67 results in neuronal death. Our previous work studied the effect of the peptide p5, a promising drug candidate, on CDK5-p25, an established Alzheimer's disease pathological complex. In vivo and in vitro data shows that p5 selectively inhibits the pathological complex CDK5-p25 without affecting CDK5 physiology in presence of p67. This study focuses on p67-p5 interaction and on the characterization of any conformational changes induced by it, especially in the Syntaxin binding site (the cleft region between domains 1 and 3a). This provides insights into the potential off-target effects caused by p5 due to a potential inhibition of SNARE complex formation. Our Monte Carlo/Molecular dynamics method (MCMD) predicted nine different binding modes, four of which were analyzed previously and indicated conformational changes induced in the Syntaxin binding site at the important functional domains 3a and 2. Herein we investigate the remaining five p67-p5 modes in order to fully understand the interaction dynamics, and the relevant results are presented.

Structure-Activity Relationship Studies of Antimalarial Plasmodium Proteasome Inhibitors─Part II.

With increasing reports of resistance to artemisinins and artemisinin-combination therapies, targeting the Plasmodium proteasome is a promising strategy for antimalarial development. We recently reported a highly selective Plasmodium falciparum proteasome inhibitor with anti-malarial activity in the humanized mouse model. To balance the permeability of the series of macrocycles with other drug-like properties, we conducted further structure-activity relationship studies on a biphenyl ether-tethered macrocyclic scaffold. Extensive SAR studies around the P1, P3, and P5 groups and peptide backbone identified compound TDI-8414. TDI-8414 showed nanomolar antiparasitic activity, no toxicity to HepG2 cells, high selectivity against the Plasmodium proteasome over the human constitutive proteasome and immunoproteasome, improved solubility and PAMPA permeability, and enhanced metabolic stability in microsomes and plasma of both humans and mice.

Single-cell transcriptomes and T cell receptors of vaccine-expanded apolipoprotein B-specific T cells.

Atherosclerotic cardiovascular diseases are the major cause of death worldwide. CD4 T cells responding to Apolipoprotein B (ApoB), the core protein of most lipoproteins, have been identified as critical disease modulators. In healthy individuals, ApoB-reactive (ApoB+) CD4 T cells are mostly regulatory T cells (Tregs), which exert anti-inflammatory effects. Yet, they may obtain pro-inflammatory features and thus become proatherogenic. Evidence from animal studies suggests that vaccination against certain major histocompatibility complex (MHC) II-binding ApoB peptides induces an expansion of ApoB+ Tregs and thus confers atheroprotection. To date, in-depth phenotyping of vaccine-expanded ApoB+ T cells has not yet been performed. To this end, we vaccinated C57BL/6J mice with the ApoB-peptide P6 (ApoB978-993 TGAYSNASSTESASY) and performed single-cell RNA sequencing of tetramer-sorted P6+ T cells. P6+ cells were clonally expanded (one major, two minor clones) and formed a transcriptional cluster distinct from clusters mainly containing non-expanded P6+ and P6- cells. Transcriptomic profiling revealed that most expanded P6+ cells had a strong Treg signature and highly expressed genes mediating suppressive functions. Yet, some expanded P6+ cells only had a residual Treg signature and expressed genes related to T helper 1 (TH1) cells, which are proatherogenic. Modeling the T cell receptor (TCR) and P6:MHC-II interaction showed that only three amino acid residues in the α and β chain contact the P6 peptide in the MHC-II groove and thus determine the specificity of this TCR to P6. Our data begin to reveal the vaccination-induced response to an ApoB epitope.