Bifunctional Peptide Research Articles

Screening of bacteria-binding peptides and one-pot ZnO surface modification for bacterial cell entrapment.

Short functional peptides are promising materials for use as targeting recognition probes. Toll-like receptor 4 (TLR4) plays an essential role in pathogen recognition and in activation of innate immunity. Here, the TLR4 amino acid sequence was used to screen for bacterial cell binding peptides using a peptide array. Several octamer peptides, including GRHIFWRR, demonstrated binding to Escherichia coli as well as lipopolysaccharides. Linking this peptide with the ZnO-binding peptide HKVAPR, creates a bi-functional peptide capable of one-step ZnO surface modification for bacterial cell entrapment. Ten-fold increase in entrapment of E. coli was observed using the bi-functional peptide. The screened peptides and the simple strategy for nanomaterial surface functionalization can be employed for various biotechnological applications including bacterial cell entrapment onto ZnO surfaces.

Identification and characterization of novel enhanced cell penetrating peptides for anti-cancer cargo delivery.

Cell penetrating peptides (CPP) are able cross the membrane and to transport cargos, presenting a great potential in drug delivery and diagnosis. In this paper, we have identified novel natural or synthetic CPPs. We have validated their rapid and efficient time and dose-dependent penetration, the absence of toxicity, the intracellular localization and the stability to proteases degradation, one of the main bottlenecks of peptides. Moreover, we have associate a cargo (an interfering peptide blocking the association of the serine/threonine phosphatase PP2A to its inhibitor, the oncogene SET) to the new generated shuttles and showed that they new bi-functional peptides keep the original properties of the shuttle and, in addition, are able to induce apoptosis due to the properties of the cargo. The CPPs identified in this study have promising perspectives for future anti-cancer drug delivery.

Modification of Degradable Nonviral Delivery Vehicle With a Novel Bifunctional Peptide to Enhance Transfection In Vivo

To increase in vivo DNA transfection efficiency of a nonviral delivery vehicle, its tumor targeting and nuclear delivery ability was improved. A novel bifunctional peptide tLyP-1-NLS (named K12) was prepared by coupling a tumor-targeting peptide (tLyP-1) with a nuclear localization signal (NLS), and then was used to modify a degradable polyethyleneimine (PEI) derivative called "N-octyl-N-quaternary chitosan (OTMCS)-PEI". The carrier OTMCS-PEI-K12 was characterized in terms of the physicochemical properties, in vitro gene transfection and antitumor effect in vivo. OTMCS-PEI-K12 showed good suitability, stability and transfection capacity in vitro on the premise of noncytotoxicity. OTMCS-PEI-K12/pING4 complexes induced extensive apoptosis of tumor tissues and shrunk the tumor volume of mice noticeably in vivo. This study offers a way to enhance in vivo transfection of a nonviral carrier.

Pharmacodynamics in Alzheimer’s disease model rats of a bifunctional peptide with the potential to accelerate the degradation and reduce the toxicity of amyloid β-Cu fibrils

We have focused on accelerating the degradation of fAβ-Cu as well as synthetically reducing the ROS toxicity by GR, and, consequently, its benefits in vivo. The bifunctional peptide GR can not only disaggregate fAβ-Cu into smaller fragments to facilitate uptake and degradation by PC12 cell, but also suppresses the ROS generated by fAβ-Cu. Thus, the viability of PC12 cell treated with fAβ-Cu has increased from 38% to 70% after GR administration, overwhelming GGH (46%) and RR (48%). The in vivo studies have revealed that GR improves the spatial memory ability and reduce the amount of senile plaques within brain of AD model rats. Thus, we suppose the bifunctional inhibitor GR has good application prospects in the treatment of AD treatment.

Proximity-Induced Reactivity and Product Selectivity with a Rationally Designed Bifunctional Peptide Catalyst

Cooperative catalytic systems are making significant advances in modern organic synthesis due to the potential to combine multiple catalytic cycles or enable enzyme-like proximity effects. We report the rational design of a bifunctional helical peptide catalyst that displays an imidazolidinone catalyst in close proximity to a thiourea binding site and enables proximity-enhanced reactivity and selectivity. The helical structure of the peptide and the binding of both reactants are shown to be essential for enhanced reactivity in Diels–Alder and indole alkylation reactions, and up to 28 000 catalyst turnovers are achieved. A variety of Lewis basic functional groups facilitate binding and proximity-enhanced reactivity, and product selectivity is observed that cannot be achieved in the absence of the peptide template.

Synthesis of a Bifunctional Peptide Inhibitor-IgG1 Fc Fusion That Suppresses Experimental Autoimmune Encephalomyelitis.

Multiple sclerosis (MS) is a neurodegenerative disease that is estimated to affect over 2.3 million people worldwide. The exact cause for this disease is unknown but involves immune system attack and destruction of the myelin protein surrounding the neurons in the central nervous system. One promising class of compounds that selectively prevent the activation of immune cells involved in the pathway leading to myelin destruction are bifunctional peptide inhibitors (BPIs). Treatment with BPIs reduces neurodegenerative symptoms in experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. In this work, as an effort to further improve the bioactivity of BPIs, BPI peptides were conjugated to the N- and C-termini of the fragment crystallizable (Fc) region of the human IgG1 antibody. Initially, the two peptides were conjugated to IgG1 Fc using recombinant DNA technology. However, expression in yeast resulted in low yields and one of the peptides being heavily proteolyzed. To circumvent this problem, the poorly expressed peptide was instead produced by solid phase peptide synthesis and conjugated enzymatically using a sortase-mediated ligation. The sortase-mediated method showed near-complete conjugation yield as observed by SDS-PAGE and mass spectrometry in small-scale reactions. This method was scaled up to obtain sufficient quantities for testing the BPI-Fc fusion in mice induced with EAE. Compared to the PBS-treated control, mice treated with the BPI-Fc fusion showed significantly reduced disease symptoms, did not experience weight loss, and showed reduced de-myelination. These results demonstrate that the BPI peptides were highly active at suppressing EAE when conjugated to the large Fc scaffold in this manner.

Epratuzumab and Blinatumomab as Therapeutic Antibodies for Treatment of Pediatric Acute Lymphoblastic Leukemia: Current Status and Future Perspectives.

More than 85% of children affected by acute lymphoblastic leukemia (ALL) are successfully treated; however relapse remains a remarkable clinical concern, with 50-60% of relapsing patients facing a fatal outcome. Management of relapsed patients includes standardized intensive risk-adapted regimens based on conventional drugs, and hematopoietic stem cells transplantation for patients with unfavourable features. Biological drugs, in particular the monoclonal antibody epratuzumab and the bi-functional recombinant single chain peptide blinatumomab, have been recently recognized as novel potential agents to be integrated in salvage ALL therapy to further improve rescue outcome. A systematic search of peer-reviewed scientific literature and clinical trials in public databases has been carried out. Both clinical and pre-clinical studies have been included to summarize recent evidence on epratuzumab and blinatumomab for salvage ALL therapy. Sixty-two papers and 25 clinical trials were included. Although not all patients responded properly to these agents, their use in relapsed and refractory pediatric ALL seems promising. Phase 3 studies have recently begun and more consistent results about epratuzumab and blinatumomab safety and efficacy in comparison to conventional therapies are expected in the next years. Epratuzumab seems safe in the dosing scheme proposed in ALL, but its efficacy over the conventional chemotherapy is still questionable. Blinatumomab has shown promising results in high risk cases such as elder adult patients and conclusive studies on pediatric ALL are needed. Patient inter-individual variability to these agents has not been investigated in depth, but this issue needs to be addressed, in particular for blinatumomab.

Evaluation of strategically-designed bifunctional peptide coating by QCM-D and osteoblast behaviors on titanium

Arginine-glycine-aspartic acid (RGD) peptide is the most widely employed motif as biomimetic coating on titanium (Ti) surface to accelerate osseointegration between Ti implants and bone tissue. Ti binding motif (TBP) has the ability of specifically recognizing and binding onto Ti surface via electrostatically interaction with TiO2 layer. Here, TBP was introduced to connect RGD with different design strategies to form three chimeric dual-targeting peptides. Quartz crystal microbalance with dissipation monitoring (QCM-D) was used to monitor the real-time absorbance of these peptides on Ti plates in molecular level as well as to estimate the properties of peptide films. In addition, MC3T3-E1 cell behaviors on Ti plates coated with these peptides were evaluated in adhesion, spreading and proliferation under mechanical loading condition which simulated the Ti implants load carrying in oral cavity.

Bifunctional opioid/nociceptin hybrid KGNOP1 effectively attenuates pain-related behaviour in a rat model of neuropathy

A bifunctional peptide containing an opioid and nociceptin receptor-binding pharmacophore, H-Dmt-D-Arg-Aba-β-Ala-Arg-Tyr-Tyr-Arg-Ile-Lys-NH2 (KGNOP1), was tested for its analgesic properties when administered intrathecally in naïve and chronic constriction injury (CCI)-exposed rats with neuropathy-like symptoms. KGNOP1 significantly increased the acute pain threshold, as measured by the tail-flick test, and also increased the threshold of a painful reaction to mechanical and thermal stimuli in CCI-exposed rats. Both of the effects could be blocked by pre-administration of [Nphe1]-Nociceptin (1-13)-NH2 (NPhe) or naloxone, antagonists for nociceptin and opioid receptors, respectively. This led us to conclude that KGNOP1 acts as a dual opioid and nociceptin receptor agonist in vivo. The analgesic effect of KGNOP1 proved to be more powerful than clinical drugs such as morphine and buprenorphine. Repeated daily intrathecal injections of KGNOP1 led to the development of analgesic tolerance, with the antiallodynic action being completely abolished on day 6. Nevertheless, the development of tolerance to the antihyperalgesic effect was delayed in comparison to morphine, which lost its efficacy as measured by the cold plate test after 3days of daily intrathecal administration, whereas KGNOP1 was efficient up to day 6. A single intrathecal injection of morphine to KGNOP1-tolerant rats did not raise the pain threshold in any of the behavioural tests; in contrast, a single intrathecal dose of KGNOP1 significantly suppressed allodynia and hyperalgesia in morphine-tolerant rats.

Peptide-directed synthesis of fluorescent gold nanoparticles for mitochondria-targeted confocal imaging of temperature

Gold nanoparticles (AuNPs) with strong red fluorescence (peaking at 657 nm under 365 nm excitation) were synthesized with the assistance of a bifunctional peptide. The peptide was rationally designed with two functional regions: region 1 (CCY) for synthesizing AuNPs and region 2 (MLRAALSTARRGPRLSRLL) for targeting mitochondria. The resulting AuNPs show excellent stability, low toxicity, good biocompatibility, as can target mitochondria. The AuNPs exhibit a temperature dependent fluorescence with high sensitivity and good reversibility. Fluorescence intensity drops to 56% of its initial value on elevating temperature from 20 to 55 °C, while the quantum yield drops from 7.1% to 3.9% and fluorescence lifetime (which is 12.2 μs at 20 °C) drops to 8.96 μs at 55 °C. The AuNPs are shown to be viable mitochondrial thermometers for use in HeLa cells.

Targeting Prostate Cancer with a Combination of WNT Inhibitors and a Bi-functional Peptide.

Prostate cancer is the most common cancer in the Western world. A bi-functional peptide was combined with wingless-related integration site (WNT) inhibitors to determine if there is an additive therapeutic effect when they are used against prostate cancer, since their efficacy has already been proven when used alone. A bi-functional peptide (TP-LYT) was designed with a target domain (LTVSPWY) and a lytic domain (KLAKLAK)2, and a second peptide with the same lytic domain but a random sequence instead of the target domain was used as a negative control. Two different WNT inhibitors were used, ethacrynic acid and ciclopiroxolamine. They were tested on prostate cancer cells using the WST-8 assay. A synergistic effect of peptides and WNT inhibitors was demonstrated, increasing the toxicity against cancer cells. Our findings potentially allow safer treatment since lower concentrations of WNT inhibitors can be used in combination with this bi-functional peptide.

Cryptic bioactivity capacitated by synthetic hybrid plant peptides

Evolution often diversifies a peptide hormone family into multiple subfamilies, which exert distinct activities by exclusive interaction with specific receptors. Here we show that systematic swapping of pre-existing variation in a subfamily of plant CLE peptide hormones leads to a synthetic bifunctional peptide that exerts activities beyond the original subfamily by interacting with multiple receptors. This approach provides new insights into the complexity and specificity of peptide signalling.

Electronic Detection of Single Cancer Cells with Graphene Field Effect Transistors

Here, we present electronic-type biosensors for ultra-sensitive detection of pancreatic cancer cells, based on graphene field effect transistors. The devices was decorated with bi-functional pyrene-iRGD peptides in which the pyrene moiety binds to the graphene though pi-pi interaction and the iRGD peptides binds to neuropilin receptors in the pancreatic cancer cells. Real-time electronic detection enabled detecting few cancer cells in the presence of the human serum, which is a critical for the early detection of cancer diagnosis and treatments. We acknowledge the support of ND EPSCoR New Faculty Award and ND NASA EPSCoR RID Grant.

Self-Assembled Bifunctional Peptide as Effective Drug Delivery Vector with Powerful Antitumor Activity.

E‐cadherin/catenin complex is crucial for cancer cell migration and invasion. The histidine‐alanine‐valine (HAV) sequence has been shown to inhibit a variety of cadherin‐based functions. In this study, by fusing HAV and the classical tumor‐targeting Arg‐Gly‐Asp (RGD) motif and Asn‐Gly‐Arg (NGR) motif to the apoptosis‐inducing peptide sequence‐AVPIAQK, a bifunctional peptide has been constructed with enhanced tumor targeting and apoptosis effects. This peptide is further processed as a nanoscale vector to encapsulate the hydrophobic drug docetaxel (DOC). Bioimaging analysis shows that peptide nanoparticles can penetrate into xenograft tumor cells with a significantly long retention in tumors and high tumor targeting specificity. In vivo, DOC/peptide NPs are substantially more effective at inhibiting tumor growth and prolonging survival compared with DOC control. Together, the findings of this study suggest that DOC/peptide NPs may have promising applications in pulmonary carcinoma therapy.

Kunitz-Type Peptide HCRG21 from the Sea Anemone Heteractis crispa Is a Full Antagonist of the TRPV1 Receptor.

Sea anemone venoms comprise multifarious peptides modulating biological targets such as ion channels or receptors. The sequence of a new Kunitz-type peptide, HCRG21, belonging to the Heteractis crispa RG (HCRG) peptide subfamily was deduced on the basis of the gene sequence obtained from the Heteractis crispa cDNA. HCRG21 shares high structural homology with Kunitz-type peptides APHC1–APHC3 from H. crispa, and clusters with the peptides from so named “analgesic cluster” of the HCGS peptide subfamily but forms a separate branch on the NJ-phylogenetic tree. Three unique point substitutions at the N-terminus of the molecule, Arg1, Gly2, and Ser5, distinguish HCRG21 from other peptides of this cluster. The trypsin inhibitory activity of recombinant HCRG21 (rHCRG21) was comparable with the activity of peptides from the same cluster. Inhibition constants for trypsin and α-chymotrypsin were 1.0 × 10−7 and 7.0 × 10−7 M, respectively. Electrophysiological experiments revealed that rHCRG21 inhibits 95% of the capsaicin-induced current through transient receptor potential family member vanilloid 1 (TRPV1) and has a half-maximal inhibitory concentration of 6.9 ± 0.4 μM. Moreover, rHCRG21 is the first full peptide TRPV1 inhibitor, although displaying lower affinity for its receptor in comparison with other known ligands. Macromolecular docking and full atom Molecular Dynamics (MD) simulations of the rHCRG21–TRPV1 complex allow hypothesizing the existence of two feasible, intra- and extracellular, molecular mechanisms of blocking. These data provide valuable insights in the structural and functional relationships and pharmacological potential of bifunctional Kunitz-type peptides.

Self-Assembled Amyloid Peptides with Arg-Gly-Asp (RGD) Motifs As Scaffolds for Tissue Engineering.

Self-assembled peptides gain increasing interest as biocompatible and biodegradable scaffolds for tissue engineering. Rationally designed self-assembling building blocks that carry cell adhesion motifs such as Arg-Gly-Asp (RGD) are especially attractive. We have used a combination of theoretical and experimental approaches toward such rational designs, especially focusing on modular designs that consist of a central ultrashort amphiphilic motif derived from the adenovirus fiber shaft. In this study, we rationally designed RGDSGAITIGC, a bifunctional self-assembling amyloid peptide which encompasses cell adhesion and potential cysteine-mediated functionalization properties through the incorporation of an RGD sequence motif and a cysteine residue at the N- and C- terminal end, respectively. We performed replica exchange MD simulations that suggested that the key factor determining cell adhesion is the total solvent accessibility of the RGD motif and also that the C-terminal cysteine is adequately exposed. The designer peptides self-assembled into fibers that are structurally characterized with Transmission Electron Microscopy, Scanning Electron Microscopy and X-ray fiber diffraction. Furthermore, they supported cell adhesion and proliferation of a model cell line. We consider that the current bifunctional properties of the RGDSGAITIGC fibril-forming peptide can be exploited to fabricate novel biomaterials with promising biomedical applications. Such short self-assembling peptides that are amenable to computational design offer open-ended possibilities toward multifunctional tissue engineering scaffolds of the future.

Target-specific delivery of siRNA into hepatoma cells' cytoplasm by bifunctional carrier peptide.

RNA interference (RNAi) is among the most potential approach for the therapy of hepatocellular carcinoma and the major barrier hindering siRNA therapeutics is the low efficiency of delivery to the desired cells. The current study aimed at developing a novel peptide for more efficient hepatoma targeted siRNA delivery, by combining luteinizing hormone-releasing hormone with hepatoma targeting specificity and MPG△NLS with cytoplasm-delivery tendency. The developed bifunctional peptide LHRH-MPG△NLS and siRNA were mixed together and resulted in LHRH-MPG△NLS/siRNA polyplexes through self-assembly. The polyplexes were characterized by agarose gel retardation and dynamic light scatting analysis. Hepatoma targeting specificity was analyzed with the GE IN Cell Analyzer 2000 High-Content Cellular Analysis System after cell transfection, and the effect of RNA interference was detected by RT-PCR. The results demonstrated that LHRH-MPG△NLS was able to assemble with siRNA to form stable and nano-sized peptide/siRNA polyplexes, which could inhibit the expression of the target gene and was essentially non-cytotoxic, as compared with the commercial transfection reagent lipofectamine 2000.

High yield synthesis of cyclic analogues of antibacterial peptides P-113 by Sortase A-mediated ligation and their conformation studies

P-113 is a fragment of natural occurring peptide Histatin 5 found in human saliva. This peptide exhibited broad spectrum of antibacterial and antifungal biological activities. In this study, bifunctional P-113 peptides 2–5 were designed as Sortase A substrates and synthesized by solid support peptide synthesis, where the N-terminus were equipped with glycine and its analogues, and C-terminus were extended with LPETGGS, respectively. Under Sortase A catalyzed condition, head to tail cyclization products 7–10 were afforded in yields from 76% to 93%. The conformation insights of linear peptides 2–5 and cyclic analogues 7–10 in aqueous buffers and in trifluroethanol (TFE) analyzed by circular dichroism (CD) suggested that α-helix structures were produced progressively in hydrophobic environment independent of the cyclization, which displayed the similar behavior as parent peptide P-113.

A non-viral suicide gene delivery system traversing the blood brain barrier for non-invasive glioma targeting treatment

Herpes simplex virus type I thymidine kinase gene (HSV-TK) in viral vector is a promising strategy against glioblastoma multiforme (GBM). However, the biosafety risk restricts its application in clinic. In this work, poly (l-lysine)-grafted polyethylenimine (PEI-PLL), which combines the high transfection efficiency of polyethylenimine and the good biodegradability of poly (l-lysine), was adopted as the non-viral vector backbone. Angiopep-2, a blood brain barrier (BBB) crossing and glioma targeting bifunctional peptide was conjugated on PEI-PLL via polyethyleneglycol (PEG) and designated as PPA. The optimal transfection ratio of PPA/DNA complexes nanoparticles (PPA NPs) was firstly characterized. Next, the glioma targeting of the PPA NPs was confirmed through cellular uptake and transfection analysis. The in vivo imaging studies demonstrated that the PPA NPs could not only penetrate BBB but also accumulate in striatum and cortex via systemic administration. Moreover, the PPA/HSV-TK NPs showed remarkably anti-glioma effect and survival benefit in an invasive orthotopic human GBM mouse model through inhibiting proliferation and inducing apoptosis (p<0.05 vs control). This study firstly illustrated that the cationic polymer PPA could be exploited as an efficient gene vector to cross the BBB, and innovatively provided a potential non-viral nanomedicine for noninvasive suicide gene therapy in the glioma treatment.

Protein-Nanoparticle Hydrogels That Self-assemble in Response to Peptide-Based Molecular Recognition.

Recently, supramolecular hydrogels assembled through nonspecific interactions between polymers and nanoparticles (termed PNP systems) were reported to have rapid shear-thinning and self-healing properties amenable for cell-delivery applications in regenerative medicine. Here, we introduce protein engineering concepts into the design of a new family of PNP hydrogels to enable direct control over the polymer-nanoparticle interactions using peptide-based molecular recognition motifs. Specifically, we have designed a bifunctional peptide that induces supramolecular hydrogel assembly between hydroxy apatite nanoparticles and an engineered, recombinant protein. We demonstrate that this supramolecular assembly critically requires molecular recognition, as no assembly is observed in the presence of control peptides with a scrambled amino acid sequence. Titration of the bifunctional peptide enables direct control over the number of physical cross-links within the system and hence the resulting hydrogel mechanical properties. As with previous PNP systems, these materials are rapidly shear-thinning and self-healing. As proof-of-concept, we demonstrate that these materials are suitable for therapeutic cell delivery applications in a preclinical murine calvarial defect model.