Blocking Peptides Research Articles

Peptide Inhibitors of Kv1.5: An Option for the Treatment of Atrial Fibrillation.

The human voltage gated potassium channel Kv1.5 that conducts the IKur current is a key determinant of the atrial action potential. Its mutations have been linked to hereditary forms of atrial fibrillation (AF), and the channel is an attractive target for the management of AF. The development of IKur blockers to treat AF resulted in small molecule Kv1.5 inhibitors. The selectivity of the blocker for the target channel plays an important role in the potential therapeutic application of the drug candidate: the higher the selectivity, the lower the risk of side effects. In this respect, small molecule inhibitors of Kv1.5 are compromised due to their limited selectivity. A wide range of peptide toxins from venomous animals are targeting ion channels, including mammalian channels. These peptides usually have a much larger interacting surface with the ion channel compared to small molecule inhibitors and thus, generally confer higher selectivity to the peptide blockers. We found two peptides in the literature, which inhibited IKur: Ts6 and Osu1. Their affinity and selectivity for Kv1.5 can be improved by rational drug design in which their amino acid sequences could be modified in a targeted way guided by in silico docking experiments.

Blockade of Microglial Kv1.3 Potassium Channels by the Peptide HsTX1[R14A] Attenuates Lipopolysaccharide-mediated Neuroinflammation

The expression of voltage-gated potassium Kv1.3 channels is increased in activated microglia, with non-selective blockade reported to attenuate microglial-mediated neuroinflammation. In this study, we evaluated the impact of a potent and selective peptidic blocker of Kv1.3 channels, HsTX1[R14A], on microglial-mediated neuroinflammation in vitro and in vivo. Treatment with both 0.1 and 1 µg/mL lipopolysaccharide (LPS) significantly (p < 0.05) increased Kv1.3 abundance on the surface of BV-2 microglia in association with increased levels of mRNA for tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6). The increased transcription of TNF-α and IL-6 was significantly attenuated (by 24.9 and 20.2%, respectively) by HsTX1[R14A] (100 nM). The concomitant increase in TNF-α and IL-6 release from BV-2 microglia was significantly attenuated by HsTX1[R14A] by 10.7 and 12.6%, respectively. In LPS-treated primary mouse microglia, the levels of TNF-α and nitric oxide were also attenuated by HsTX1[R14A] (26.1 and 20.4%, respectively). In an LPS-induced mouse model of neuroinflammation, both an immediate and delayed subcutaneous dose of HsTX1[R14A] (2 mg/kg) significantly reduced plasma and brain levels of the pro-inflammatory mediators TNF-α, IL-1β and IL-6, with no impact on the anti-inflammatory IL-10. These results demonstrate that HsTX1[R14A] is a promising therapeutic candidate for the treatment of diseases with a neuroinflammatory component.

Evaluation of Efficient Non-reducing Enzymatic and Chemical Ligation Strategies for Complex Disulfide-Rich Peptides.

Double-knotted peptides identified in venoms and synthetic bivalent peptide constructs targeting ion channels are emerging tools for the study of ion channel pharmacology and physiology. These highly complex and disulfide-rich peptides contain two individual cystine knots, each comprising six cysteines and three disulfide bonds. Until now, native double-knotted peptides, such as Hi1a and DkTx, have only been isolated from venom or produced recombinantly, whereas engineered double-knotted peptides have successfully been produced through enzymatic ligation using sortase A to form a seamless amide bond at the ligation site between two knotted toxins, and by alkyne/azide click chemistry, joining two peptide knots via a triazole linkage. To further pursue these double-knotted peptides as pharmacological tools or probes for therapeutically relevant ion channels, we sought to identify a robust methodology resulting in a high yield product that lends itself to rapid production and facile mutational studies. In this study, we evaluated the ligation efficiency of enzymatic (sortase A5°, butelase 1, wild-type OaAEP 1, C247A-OaAEP 1, and peptiligase) and mild chemical approaches (α-ketoacid-hydroxylamine, KAHA) for forming a native amide bond linking the toxins while maintaining the native disulfide connectivity of each pre-folded peptide. We used two NaV1.7 inhibitors: PaurTx3, a spider-derived gating modifier peptide, and KIIIA, a small cone snail-derived pore blocker peptide, which have previously been shown to increase affinity and inhibitory potency on hNaV1.7 when ligated together. Correctly folded peptides were successfully ligated in varying yields, without disulfide bond shuffling or reduction, with sortase A5° being the most efficient, resulting in 60% ligation conversion within 15 min. In addition, electrophysiology studies demonstrated that for these two peptides, the amino acid composition of the linker did not affect the activity of the double-knotted peptides. This study demonstrates the powerful application of enzymes in efficiently ligating complex disulfide-rich peptides, paving the way for facile production of double-knotted peptides.

Peptide Blocking Self-Polymerization of Extracellular Calcium-Sensing Receptor Attenuates Hypoxia-Induced Pulmonary Hypertension.

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Delivery of the VIVIT Peptide to Human Glioma Cells to Interfere with Calcineurin-NFAT Signaling.

The activation of NFAT (nuclear factor of activated T cells) transcription factors by calcium-dependent phosphatase calcineurin is a key step in controlling T cell activation and plays a vital role during carcinogenesis. NFATs are overexpressed in many cancers, including the most common primary brain tumor, gliomas. In the present study, we demonstrate the expression of NFATs and NFAT-driven transcription in several human glioma cells. We used a VIVIT peptide for interference in calcineurin binding to NFAT via a conserved PxIxIT motif. VIVIT was expressed as a fusion protein with a green fluorescent protein (VIVIT-GFP) or conjugated to cell-penetrating peptides (CPP), Sim-2 or 11R. We analyzed the NFAT expression, phosphorylation, subcellular localization and their transcriptional activity in cells treated with peptides. Overexpression of VIVIT-GFP decreased the NFAT-driven activity and inhibited the transcription of endogenous NFAT-target genes. These effects were not reproduced with synthetic peptides: Sim2-VIVIT did not show any activity, and 11R-VIVIT did not inhibit NFAT signaling in glioma cells. The presence of two calcineurin docking sites in NFATc3 might require dual-specificity blocking peptides. The cell-penetrating peptides Sim-2 or 11R linked to VIVIT did not improve its action making it unsuitable for evaluating NFAT dependent events in glioma cells with high expression of NFATc3.

Abstract 1913: Pre-existing HSV-1 Immunity enhances anti-cancer efficacy of a novel immune stimulating oncolytic virus - VG161

Abstract It is not clear if pre-existing anti-virus immunity or multicycle treatment of oncolytic virus (OV) can affect the treatment outcome. This study is to investigate the impact of pre-existing anti Herpes Simplex Virus type-1 (HSV-1) immunity or multicycle OV treatment on the efficacy of VG161, a novel HSV-1 OV armed with IL12, IL15 with its receptor α unit (IL15RA), and a PD-L1 peptide blocker (PDL1B). VG161 has entered phase I trial in Australia and China. Seventy Balb/c mice were divided into 2 groups. One group (35 mice) was subcutaneously immunized by mVG161 (a version of VG161 expresses mouse IL12, human IL15 and PDL1B) on day 1 (5x105 PFU/mouse) followed by a boost on day 7. The other group (35 mice) was non-immunized. On day 7 post-boost, the blood was collected from tail to determine anti-HSV antibody. At the same time, all 70 mice were subcutaneously implanted with CT26 mouse colon cancer cells. Once the tumors reached 100 mm3, 20 mice (10 from each group) were treated with intra-tumoral (IT) injection of either mVG161 at a dose of 5×106 PFU/mouse or Vehicle, once a day for 5 days. Hepatic cancer (HC) PDX tissues were dissected into pieces of 27 mm3 and subcutaneously implanted into humanized mice established by transplantation of 1×104 human CD34 stem cells (HLA matched with PDX). When the tumor reached 100-150 mm3, animals were divided into 4 groups (10 mice/group): vehicle control, positive control (oxaliplatin 5 mg/kg and gemcitabine 100 mg/kg), and 2 groups treated with VG161 at high dose (7.75×106 PFU) or low dose (3.10×105 PFU). The OV treatments were given for 3 cycles with IT injection of VG161 once a day for day 1-5 of each cycle. Tumor, spleen, and peripheral blood mononuclear cells were collected for further analysis. All HSV pre-immunized mice but not naïve mice were HSV-1 antibody positive. By day 19 post-treatment, mVG161 significantly inhibited CT26 tumor growth in both HSV-1 pre-immunized and non-immunized groups, while all control mice were euthanized due to tumor size reached 2000 mm3. Starting from day 27, tumors grew back in non-immunized group but not in pre-immunized group, resulting in a significant difference of tumor size between the 2 groups on day 31 to 39 (p&amp;lt;0.01). As compared to the negative controls, HC PDX tumors in both treatment groups were significantly smaller with percentage of T/C less than 40% on day 72. The median time for 3-fold increase of tumor size is 41.5 days (95%CI: 32-58) in the vehicle control, 74 days (95%CI: 51-undefined) in high dose group, and not reached in low dose group (p&amp;lt;0.01). Repeated treatment (2-3 cycles) increased the anti-cancer efficacy of VG161. The anti-cancer efficacy of VG161 can be enhanced by pre-immunization of HSV-1 or multicycle administration when the virus is given by IT, suggesting potential clinical benefits of HSV-1 based OV therapy in HSV sero-positive patients and multicycle administration of VG161 for long term maintenance treatment. Citation Format: Jun Ding, Jason Sun, Yana Murad, Erica Lee, Ismael Samudio, William Jia, Ronghua Zhao. Pre-existing HSV-1 Immunity enhances anti-cancer efficacy of a novel immune stimulating oncolytic virus - VG161 [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 1913.

Abstract 2848: Neomorphic cell-cell adhesion reprogramming facilitates metastasis of ESR1 mutant breast cancer

Abstract Background: Hotspot estrogen receptor-α (ER/ERα/ESR1) mutations occur in 30-40% endocrine resistant ER+ breast cancer and are associated with worse outcome. How these mutations facilitate metastasis remains ambiguous. It is necessary to identify a clear mechanism for therapeutic intervention. Methods: ESR1 mutations were detected by ddPCR. Transcriptome data were derived from cell lines and clinical samples. Y537S and D538G genome-edited MCF7 and T47D cell lines were used for in vitro phenotypic characterization. Cell-cell adhesive properties were assessed using calcein-labelled adhesion, spontaneous cell aggregation and ibidi microfluidic assays. Altered cell-cell adhesion genes were validated using qRT-PCR, immunoblot and immunostaining. Desmosome blocking peptides and carbenoxolone were used for blockade of adhesion. ER cistromes were profiled by ChIP-seq. Tail vein injection was performed on athymic nude mice to evaluate metastasis in vivo. Circulating tumor cell (CTC) clustering propensity in vivo was assessed via intracardiac injection followed by CTC microfilter capture. CTC-cluster gene signatures were generated from ER+ breast cancer CTC RNA-seq data. Results: ESR1 mutations were significantly enriched in distant metastases (12/48) vs local (0/27) recurrences, confirming their critical role in promoting metastasis. Transcriptomic analysis revealed altered cell-cell interaction pathways in ESR1 mutant tumors. ESR1 mutant cells formed more compact spheroids in suspension culture, and exhibited stronger cell-cell adhesion in static condition. Under microfluidic condition with physiological shear stress, mutant ESR1 cells derived more and larger clusters, which were prominently preserved from pre-existing clusters. This effect was correlated with increased expression of desmosome and gap junction genes in mutant cells and pharmacological blockade significantly reduced the enhanced cell-cell adhesion. ER ChIP-seq revealed no de novo mutant ER binding sites at the loci of the target genes, suggesting indirect regulation by mutant ER. This was exemplified by a secondary regulation from cFos/AP1 signaling of GJA1 expression, and an epigenetic regulation at DSC1/DSG1 loci through enhanced H3K4me2 and H3K27ac modification. In vivo studies showed ESR1 mutant cells derived more distant metastases, and MCF7 Y537S cells formed larger CTC clusters with increased compactness compared to WT cells. Finally, ER+ CTC-cluster signatures were enriched in ESR1 mutant tumors, suggesting their unique dependence on this pathway during metastasis. Conclusion: Hotspot ESR1 mutations induce expression of multiple desmosome and gap junction genes and confer enhanced cell-cell adhesion, which facilitates breast cancer metastasis via increased CTCs clustering propensity. These findings provide insights to the development of drugs targeting gap junction in ER mutant tumors. Citation Format: Yang Wu, Zheqi Li, Amir Bahreini, Jian Chen, Ye Qin, Kevin M. Levine, Nilgun Tasdemir, Nolan M. Priedigkeit, Li Zhu, George C. Tseng, Yu Jiang, Benjamin Troness, Laki Buluwela, Simak Ali, Spencer Arnesen, Jason Gertz, Ben Ho Park, Jennifer M. Atkinson, Dorraya El-Ashry, Adrian V. Lee, Steffi Oesterreich. Neomorphic cell-cell adhesion reprogramming facilitates metastasis of ESR1 mutant breast cancer [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 2848.

TRPV1 Blocker HCRG21 Suppresses TNF-α Production and Prevents the Development of Edema and Hypersensitivity in Carrageenan-Induced Acute Local Inflammation.

Currently the TRPV1 (transient receptor potential vanilloid type 1) channel is considered to be one of the main targets for pro-inflammatory mediators including TNF-α. Similarly, the inhibition of TRPV1 activity in the peripheral nervous system affects pro-inflammatory mediator production and enhances analgesia in total. In this study, the analgesic and anti-inflammatory effects of HCRG21, the first peptide blocker of TRPV1, were demonstrated in a mice model of carrageenan-induced paw edema. HCRG21 in doses of 0.1 and 1 mg/kg inhibited edema formation compared to the control, demonstrated complete edema disappearance in 24 h in a dose of 1 mg/kg, and effectively reduced the productionof TNF-α in both doses examined. ELISA analysis of blood taken 24 h after carrageenan administration showed a dramatic cytokine value decrease to 25 pg/mL by HCRG21 versus 100 pg/mL in the negative control group, which was less than the TNF-α level in the intact group (40 pg/mL). The HCRG21 demonstrated potent analgesic effects on the models of mechanical and thermal hyperalgesia in carrageenan-induced paw edema. The HCRG21 relief effect was comparable to that of indomethacin taken orally in a dose of 5 mg/kg, but was superior to this nonsteroidal anti-inflammatory drug (NSAID) in duration (which lasted 24 h) in the mechanical sensitivity experiment. The results confirm the existence of a close relationship between TRPV1 activity and TNF-α production once again, and prove the superior pharmacological potential of TRPV1 blockers and the HCRG21 peptide in particular.

The Entry Blocker Peptide Produced in Chlamydomonas reinhardtii Inhibits Influenza Viral Replication in vitro.

This year, a respiratory virus caused an emergency pandemic alert in health services around the world, showing the need for biotechnological approaches to fight these diseases. The influenza virus is one of the main viral agents that generate pandemic outbreaks. Currently, the majority of co-circulating influenza A virus (IAV) strains are adamantine‐ and oseltamivir-resistant strains, and the challenge is to find new antivirals for more efficient treatments. The antiviral entry blocker (EB) peptide is a promising candidate for blocking the virus entry into cells. The aim of this research was to express the EB peptide in the microalgae Chlamydomonas reinhardtii and test its antiviral activity against IAV in vitro. The EB peptide nucleotide sequence was introduced into the nuclear genome of microalgae using Agrobacterium tumefaciens transformation. The EB peptide amount produced in transformed microalgae was 4.99 ± 0.067% of the total soluble protein. In hemagglutination inhibition assays using influenza A/H1N1 pdm and influenza A H1N1/Virginia/ATCC/2009 strains, we reported that the EB peptide extract from the microalgae showed 100-fold higher efficiency than the EB synthetic peptide. In addition, both the EB peptide extract and synthetic peptide inhibited viral replication in MDCK cells (IC50 = 20.7 nM and IC50 = 754.4 nM, respectively); however, the EB peptide extract showed a 32-fold higher antiviral effectiveness than the synthetic peptide against influenza A/H1N1 pdm. Extracts from untransformed and transformed microalgae and synthetic peptide did not show cytotoxic effect on MDCK cell monolayers. Thus, C. reinhardtii may be a fast, safe, and effective expression platform for production of peptides with significant antiviral activity and can be used as a prophylactic treatment to reduce viral propagation.

Molecular basis for higher affinity of SARS‐CoV‐2 spike RBD for human ACE2 receptor

Severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) has caused substantially more infections, deaths, and economic disruptions than the 2002‐2003 SARS‐CoV. The key to understanding SARS‐CoV‐2's higher infectivity lies partly in its host receptor recognition mechanism. Experiments show that the human angiotensin converting enzyme 2 (ACE2) protein, which serves as the primary receptor for both CoVs, binds to the receptor binding domain (RBD) of CoV‐2's spike protein stronger than SARS‐CoV's spike RBD. The molecular basis for this difference in binding affinity, however, remains unexplained from X‐ray structures. To go beyond insights gained from X‐ray structures and investigate the role of thermal fluctuations in structure, we employ all‐atom molecular dynamics simulations. Microseconds‐long simulations reveal that while CoV and CoV‐2 spike‐ACE2 interfaces have similar conformational binding modes, CoV‐2 spike interacts with ACE2 via a larger combinatorics of polar contacts, and on average, makes 45% more polar contacts. Correlation analysis and thermodynamic calculations indicate that these differences in the density and dynamics of polar contacts arise from differences in spatial arrangements of interfacial residues, and dynamical coupling between interfacial and non‐interfacial residues. These results recommend that ongoing efforts to design spike‐ACE2 peptide blockers will benefit from incorporating dynamical information as well as allosteric coupling effects.

Discovery of Selective Transforming Growth Factor β Type II Receptor Inhibitors as Antifibrosis Agents.

Historically, modulation of transforming growth factor β (TGF-β) signaling has been deemed a rational strategy to treat many disorders, though few successful examples have been reported to date. This difficulty could be partially attributed to the challenges of achieving good specificity over many closely related enzymes that are implicated in distinct phenotypes in organ development and in tissue homeostasis. Recently, fresolimumab and disitertide, two peptidic TGF-β blockers, demonstrated significant therapeutic effects toward human skin fibrosis. Therefore, the selective blockage of TGF-β signaling assures a viable treatment option for fibrotic skin disorders such as systemic sclerosis (SSc). In this report, we disclose selective TGF-β type II receptor (TGF-βRII) inhibitors that exhibited high functional selectivity in cell-based assays. The representative compound 29 attenuated collagen type I alpha 1 chain (COL1A1) expression in a mouse fibrosis model, which suggests that selective inhibition of TGF-βRII-dependent signaling could be a new treatment for fibrotic disorders.

Basic and clinical science posters: Basic science of diabetes complications

Aims: Glucose evoked changes in profibrotic transforming growth factor-beta1 (TGFs1), increase connexin hemichannel mediated ATP release in kidney proximal tubule cells. With ATP linked to inflammation, we evaluated the efficacy of hemichannel blocker Peptide 5 in negating ATP induced inflammatory cytokine secretion in proximal tubule cells. Methods: Primary human proximal tubular epithelial cells (hPTECs), and immortalised human kidney (HK2) cells were treated with TGFs1 (10ng/ml) ± Peptide5 (25µM) for 48hrs. Carboxyfluorescein (200µM) dye uptake and ATP biosensing determined hemichannel activity and ATP release respectively. Proteome arrays determined cytokine secretion. Results: In TGFs1 treated hPTECs, carboxyfluorescein dye uptake decreased from 347.4±11.9%, (P<0.001, N=3) to 158.6±7.9% when co-incubated with Peptide 5. Parallel to this, ATP release increased from 0.16±0.1µM to 2.5±0.14µM (P<0.001, N=3) in TGF1 treated cells as compared to control. Peptide 5 significantly blunted this response, to 0.62±0.17µM (P<0.001, N=3). A proteome array determined the impact of Peptide 5 on secretion of 105 candidate chemokines and cytokines in TGFs1 treated primary cells. As an example, Peptide 5 significantly restored secretion of adiponectin from 163±8.0% (P<0.001, N=4) to 136±6.1% (P<0.05, N=4), granulocyte-macrophage colony-stimulating factor from 183±8.0% (P<0.001, N=4) to 111.3±5.9% (P<0.001, N=4) and interleukin 1-beta from 189±14.9 (P<0.001, N=4) to 109±9.6% (P<0.001, N=4) in TGF-s1 treated hPTECs, as compared to control. Conclusion: Glucose evoked changes in TGFs1 increase hemichannel mediated ATP release. Hemichannel blocker; Peptide 5 blocks ATP release and downstream cytokine and chemokine secretion, suggesting that Peptide 5 may be of future therapeutic potential in targeting inflammation of the diabetic kidney.

Collagen I Modifies Connexin-43 Hemichannel Activity via Integrin α2β1 Binding in TGFβ1-Evoked Renal Tubular Epithelial Cells.

Chronic Kidney Disease (CKD) is associated with sustained inflammation and progressive fibrosis, changes that have been linked to altered connexin hemichannel-mediated release of adenosine triphosphate (ATP). Kidney fibrosis develops in response to increased deposition of extracellular matrix (ECM), and up-regulation of collagen I is an early marker of renal disease. With ECM remodeling known to promote a loss of epithelial stability, in the current study we used a clonal human kidney (HK2) model of proximal tubular epithelial cells to determine if collagen I modulates changes in cell function, via connexin-43 (Cx43) hemichannel ATP release. HK2 cells were cultured on collagen I and treated with the beta 1 isoform of the pro-fibrotic cytokine transforming growth factor (TGFβ1) ± the Cx43 mimetic Peptide 5 and/or an anti-integrin α2β1 neutralizing antibody. Phase microscopy and immunocytochemistry observed changes in cell morphology and cytoskeletal reorganization, whilst immunoblotting and ELISA identified changes in protein expression and secretion. Carboxyfluorescein dye uptake and biosensing measured hemichannel activity and ATP release. A Cytoselect extracellular matrix adhesion assay assessed changes in cell-substrate interactions. Collagen I and TGFβ1 synergistically evoked increased hemichannel activity and ATP release. This was paralleled by changes to markers of tubular injury, partly mediated by integrin α2β1/integrin-like kinase signaling. The co-incubation of the hemichannel blocker Peptide 5, reduced collagen I/TGFβ1 induced alterations and inhibited a positive feedforward loop between Cx43/ATP release/collagen I. This study highlights a role for collagen I in regulating connexin-mediated hemichannel activity through integrin α2β1 signaling, ahead of establishing Peptide 5 as a potential intervention.

Lipopolysaccharide influences the plasma and brain pharmacokinetics of subcutaneously-administered HsTX1[R14A], a KV1.3-blocking peptide

KV1.3 is a voltage-gated potassium channel that is upregulated in neuroinflammatory conditions, such as Alzheimer's disease and Parkinson's disease. HsTX1[R14A] is a potent and selective peptide blocker of KV1.3 with the potential to block microglial KV1.3, but its brain uptake is expected to be limited owing to the restrictive nature of the blood-brain barrier. To assess its peripheral and brain exposure, a LC-MS/MS assay was developed to quantify HsTX1[R14A] concentrations in mouse plasma and brain homogenate that was reliable and reproducible in the range of 6.7–66.7 nM (r2 = 0.9765) and 15–150 pmol/g (r2 = 0.9984), respectively. To assess if neuroinflammation affected HsTX1[R14A] disposition, C57BL/6 mice were administered HsTX1[R14A] subcutaneously (2 mg/kg) 24 h after an intraperitoneal dose of Escherichia coli lipopolysaccharide (LPS), which is commonly used to induce neuroinflammation; brain and plasma concentrations of HsTX1[R14A] were then quantified over 120 min. LPS treatment significantly retarded the decline in HsTX1[R14A] plasma concentrations, presumably as a result of reducing renal clearance, and led to substantial brain uptake of HsTX1[R14A], presumably through disruption of brain inter-endothelial tight junctions. This study suggests that HsTX1[R14A] may reach microglia in sufficient concentrations to block KV1.3 in neuroinflammatory conditions, and therefore has the potential to reduce neurodegenerative diseases.

N-Terminal Tagging with GFP Enhances Selectivity of Agitoxin 2 to Kv1.3-Channel Binding Site.

Recently developed fluorescent protein-scorpion toxin chimeras (FP-Tx) show blocking activities for potassium voltage-gated channels of Kv1 family and retain almost fully pharmacological profiles of the parental peptide toxins (Kuzmenkov et al., Sci Rep. 2016, 6, 33314). Here we report on N-terminally green fluorescent protein (GFP)-tagged agitoxin 2 (GFP-L2-AgTx2) with high affinity and selectivity for the binding site of Kv1.3 channel involved in the pathogenesis of various (primarily of autoimmune origin) diseases. The basis for this selectivity relates to N-terminal location of GFP, since transposition of GFP to the C-terminus of AgTx2 recovered specific interactions with the Kv1.1 and Kv1.6 binding sites. Competitive binding experiments revealed that the binding site of GFP-L2-AgTx2 overlaps that of charybdotoxin, kaliotoxin 1, and agitoxin 2, the known Kv1.3-channel pore blockers. GFP-L2-AgTx2 was demonstrated to be applicable as a fluorescent probe to search for Kv1.3 pore blockers among individual compounds and in complex mixtures, to measure blocker affinities, and to visualize Kv1.3 distribution at the plasma membrane of Kv1.3-expressing HEK293 cells. Our studies show that definite combinations of fluorescent proteins and peptide blockers can result in considerable modulation of the natural blocker-channel binding profile yielding selective fluorescent ligands of certain channels.

Synthetic Antibody Mimics Based on Cancer-Targeting Immunostimulatory Peptides.

De novo cancer-targeting immunostimulatory peptides have been designed and developed as synthetic antibody mimics. A series of bifunctional peptides incorporating NKp30-binding and NK-cell-activating domains were synthesized as linear dimers and then extended into branching trimeric peptides by the incorporation of GRP78-targeting and tumor-cell-binding sequences. A selected trimeric peptide from this small set of peptides displayed binding capabilities on GRP78+ HepG2 and A549 target cells. Cell binding diminished in the presence of an anti-GRP78 peptide blocker, thus suggesting GRP78-binding dependence. Similarly, the selected trimeric peptide was also found to exhibit NK cell binding in an NKp30-dependent manner, which translated into NK cell activation as indicated by cytokine secretion. In co-culture, fluorescence microscopy revealed that the target GFP-expressing A549 cells were visibly associated with the effector NK cells when pre-activated with lead trimeric peptide. Accordingly, A549 cells were found to be compromised, as evidenced by the loss of GFP signal and notable detection of early-/late-stage apoptosis. Investigation of the immunological markers related to toxicity revealed detectable secretion of pro-inflammatory cytokines and chemokines, including IFN-γ, TNF-α, and IL-8. Furthermore, administration of peptide-activated NK cells into A549-tumor-bearing mice resulted in a consistent decrease in tumor growth when compared to the untreated control group. Taken together, the identification of a lead trimeric peptide capable of targeting and activating NK cells' immunotoxicity directly towards GRP78+ /B7H6- tumors provides a novel proof-of-concept for the development of cancer-targeting immunostimulatory peptide ligands that mimic antibody-targeting and -activating functions related to cancer immunotherapy applications.

The biologically functional identification of a novel TIM3-binding peptide P26 in vitro and in vivo

PurposeRecent studies have shown that TIM3 plays an important role in T-cell failure, which is closely related to the resistance to anti-programmed cell death protein 1 (PD-1) treatment. However, there have been no reports on the application of peptide blockers to TIM3. In this study, we endeavored to identify the in vitro and in vivo anti-tumor activities of a TIM3-targeting peptide screened from the phage peptide library.MethodsPhage display peptide library technology, surface plasmon resonance, flow cytometry, and mixed lymphocyte reaction were utilized to screen and demonstrate the bioactivities of P26, a TIM3-targeting peptide. Meanwhile, tumor growth assay was performed to evaluate the anti-tumor effect of P26.ResultsIn terms of affinity, we demonstrated that P26 specifically binds to TIM3 at the cellular and molecular levels, which therefore blocks the interaction between TIM3 and Galectin-9 (Gal-9) and competes with Gal-9 to bind TIM3. Additionally, P26 significantly increases T-cell activity and elevates IFN-γ and IL-2 levels in a dose-dependent manner. Notably, P26 also counteracts Gal-9-mediated T-cell suppression. More importantly, P26 can inhibit growth of MC38-hPD-L1 tumor in mice.ConclusionsP26, as a novel TIM3-binding peptide, has the ideal bioactivity connecting to TIM3 and the potential prospect of application in immunotherapy as an alternative or adjuvant to existing agents.

CD47/SIRPα blocking peptide identification and synergistic effect with irradiation for cancer immunotherapy

BackgroundImmunotherapy has achieved remarkable advances via a variety of strategies against tumor cells that evade immune surveillance. As important innate immune cells, macrophages play important roles in maintaining homeostasis, preventing...

NGF induces pulmonary arterial hyperreactivity through Connexin 43 increased expression

We have previously shown that the nerve growth factor (NGF) plays a critical role in pathophysiology of pulmonary hypertension (PH) by promoting pulmonary arterial (PA) inflammation, remodelling and hyperreactivity. To characterize mechanisms activated by NGF to induce PA hyperreactivity, focusing on Connexin 43 (Cx43), a gap junction protein essential for vascular reactivity. Expression of NGF and its TrkA receptor were evaluated by Western blotting in human PA smooth muscle cells (hPASMC) from controls or from patients suffering from idiopathic pulmonary arterial hypertension (IPAH). In cells treated or not with NGF (0.1-100 ng/ml, 24 h), Cx43 expression and localisation were evaluated by Western blotting, immunofluorescence and cell surface biotinylation. Gap junction activity was assessed by Lucifer yellow (LY) dye transfer assay. Contractions of control rat PA were induced by phenylephrine (PHE, 10-10-10-4 M) in absence or presence of NGF (100 ng/ml, 24 h). The role of Cx43 in NGF-induced rat PA reactivity was evaluated by use of 43Gap26 (Cx43 blocking peptide, 300 μM), or an anti-Cx43 siRNA (10nmol IV). NGF and TrkA expression were increased in hPASMC from IPAH patients compared to controls. NGF increased Cx43 expression in hPASMC from both controls and IPAH patients, but with a higher sensitivity in IPAH cells (maximum at 1 ng/ml vs. 100 ng/ml). This increase was abolished after treatment with K252a (TrkA kinase inhibitor). In control hPASMC, NGF increased Cx43 plasma membrane localisation and LY diffusion speed through Gap junctions. NGF-induced PA hyperreactivity to PHE was inhibited ex vivo by treatment with 43Gap26 and in vivo after administration of Cx43 siRNA. NGF pathway is unregulated in IPAH cells. NGF increases Cx43 PA expression through activation of its TrkA receptor. Cx43 increased expression at hPASMC plasma membrane upregulates gap junction activity, thus promoting NGF-induced PA hyperreactivity.

Detailed epitope mapping of neutralizing anti-drug antibodies against recombinant α-galactosidase A in patients with Fabry disease

BackgroundFabry disease (FD) is a lysosomal storage disease, treatable by enzyme replacement therapy (ERT) that substitutes deficient α-galactosidase A (AGAL). The formation of neutralizing anti-drug antibodies (ADA) inhibiting AGAL activity is associated with disease progression in affected male patients. In the current study, we performed a detailed epitope mapping of ADAs from antibody-positive males against infused AGAL. MethodsA detailed epitope mapping for 34 male FD patients with neutralizing ADAs against AGAL was performed. Based on this data, in silico analyses were used to identify potential epitope clusters and mapped surface-located or buried epitopes. ELISA-based assays against α-galactosidase B (NAGA) were performed to identify ADAs that potentially recognize shared epitopes of AGAL and NAGA. A subset of 20 patients was analyzed to assess if NAGA-recognizing ADAs against AGAL might affect long-term outcomes under ERT. ResultsThirty percent of the AGAL active site was recognized by patients' ADAs. No differences between buried and surface-located epitopes were observed. Dependent on the epitopes, ADAs against AGAL were also able to recognize human NAGA. Patients with NAGA recognizing anti-AGAL antibodies presented with lower plasma NAGA activities. The presence of NAGA-recognizing ADAs had no effect on disease progression. ConclusionIn conclusion, our current data underline previous reports demonstrating a large variation of antibody epitopes against AGAL. Detailed epitope mapping in affected patients might be the first step for the generation of patient-specific blocking peptides and/or immune adsorption columns for an individually tailored anti-antibody strategy.