Solid-phase Peptide Synthesis Method Research Articles

Synthesis of Antioxidant Peptide SCAP1 (Leu-Ala-Asn-Ala-Lys)

SCAP1 is a pentapeptide isolated from the hydrolysate of oyster (Saccostrea cucullata), which has antioxidant and anticancer properties. This study aims to find a novel strategy to obtain antioxidant peptide SCAP1 (Leu-Ala-Asn-Ala-Lys) through chemical synthesis. This study reports the first synthesis of SCAP1 through the solid phase peptide synthesis (SPPS) method with Fmoc strategy on 2-chlorotrityl chloride resin. A HATU-HOAt coupling reagent was employed in all amide bond formations and 95% TFA in water was employed to release the desired peptide from the solid support. The crude peptide was purified using successive preparative-RP-MPLC and RP-HPLC to obtain the pure peptide. The synthesized peptide was confirmed by mass spectrometry, showing a correct molecular ion peak at m/z 516.3143 (M+H)+, referring to the desired SCAP1. 1H-NMR spectra of the peptide revealed protons signals consistent to the structure of SCAP1. During the synthesis, the coupling between Ala and Asn(Trt) was not straightforward, which led to a low yield of the final product. The use of Trt-based Asn was not a suitable option, as it could cause aggregation during the synthesis, eventually leading to a poor yield of the final product. SCAP1 was obtained as a white solid with a percent yield of 8.28%.

Abstract 1769: CSox-based sensors for continuous, homogeneous and quantitative monitoring of protein kinase and phosphatase activity

Abstract Introduction: Protein kinases are a diverse group of 518 enzymes whose dysregulation lies at the center of many diseases. Currently, 30% of all drug development efforts are focused on protein kinases. Although 41 drugs are approved and >120 in clinical trials, these are predominately ATP-competitive inhibitors. More recently, there has been an expanded focus on kinase inhibitors with different modes of action, where new tools are needed to effectively characterize inhibitor mechanism of action, predict drug potency and to drive decisions earlier in the drug development process. We developed a simple yet powerful method for the generation of peptide sensors that can be used for the continuous, quantitative and homogenous detection of kinase and phosphatase activity with recombinant enzymes and crude lysates to enable target discovery and drug development. Experimental Procedures: We harnessed chelation-enhanced fluorescence by combining next generation sulfonamido-oxine (Sox) chromophore technology with high-throughput solid-phase peptide synthesis methods to identify optimized sequences based on physiological substrates. Enzyme activity is monitored kinetically using fluorescence intensity (Ex/Em 360/485 nm) or in endpoint mode using Europium and time-resolved fluorescence (Ex/Em 360/620 nm). Results: We demonstrate the ability to rapidly identify novel optimized substrates, where performance measures included higher reaction rates, lower Km's, higher signal/background, increased sensitivity and specificity. We identified highly generic substrates (for robust detection of 80 Tyrosine kinases) and highly-selective substrates (for quantitative detection of targeted kinases in crude cell or tissue lysates for profiling, potency assessments and SAR). We have developed sensors to monitor activity of high-profile tyrosine kinases, including the EGFR and clinically-relevant mutants, JAK kinases, Tec-kinases, and, serine/threonine kinases, including CDK1-9, MAPK pathway (MAP4Ks, MAP3Ks, MAPKs & MAPKAPKs), PKR/EIF2AKs and PIM1. In addition, CSox-based phosphopeptide substrates are used to monitor protein phosphatases with specificity for tyrosine (PTP1B, SHP1/2) or serine/threonine (PP2A, PP2C, PHLPP). Conclusions: The generation of robust activity-based sensors, even where peptide assays previously weren’t available, opens new areas for effective drug discovery. The Sox-based kinetic assay format is ideal for elucidating drug mechanism of action, potency, and enzyme regulation. The PhosphoSens-Red endpoint format is ideal for HTS, SAR and profiling. Together, these formats can be applied across the entire target discovery and drug development workflow, providing a quantum improvement in performance and productivity needed to address the challenges and opportunities of next generation protein kinase and phosphatase inhibitors. Citation Format: Erik M. Schaefer, Susan Cornell-Kennon, Bill Lu. CSox-based sensors for continuous, homogeneous and quantitative monitoring of protein kinase and phosphatase activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1769.

Spontaneous Formation of Nanoparticles from Peptide-Vinyl Polymer Diblock Hybrids Prepared by RAFT Polymerization and Their Interactions with Cells.

Novel polymeric nanoparticles (NPs) with uniform sizes were prepared from peptide–vinyl polymer diblock hybrids by the self-organized precipitation method. Hybrid polymers of polystyrene (PSt) and tetrapeptide (cell-binding epitope RGDS, reverse SDGR, cationic KKKK, and anionic DDDD) were successfully synthesized by combining solid-phase peptide synthesis and reversible addition fragmentation chain transfer polymerization methods. Narrowly dispersed hybrid polymers (polydispersity index < 1.25, Mn 14 000–17 000) were obtained. Altering the preparation conditions easily tuned the size and size distribution of the NPs. When the ζ-potentials for the NP suspensions were measured at pH 6.0, the obtained values corresponded to the net charge of each peptide segment. More importantly, the NPs could encapsulate fluorescent Nile red (NR) and magnetic iron oxide NP (MNP), which might be suitable for fluorescent imaging and magnet-induced patterning of cells, respectively. The interactions of NPs with cells (NIH/3T3 fibroblast) and the magnetic effects were examined for NR/MNP-loaded PSt–RGDS and −SDGR NPs. Both NPs were readily incorporated into cells, but only NR/MNP-loaded PSt–RGDS NP showed magnetic responsiveness in cell adhesion and cultures.

Design and synthesis of rhamnose-modified exenatide conjugate by sortase A-mediated ligation

Exenatide modified with rhamnose as a hapten was designed and synthesized by Sortase A-mediated ligation. An Exenatide peptide analog comprised of 46 amino acids, including the sortase A recognition motif LPETG at the C-terminus, was synthesized by solid phase peptide synthesis method. A tri-glycine modified-rhamnose derivative was chemically synthesized in seven steps in good yield. The site-specific conjugation between them catalyzed by Sortase A completed in 3 h and the rhamnose-modified Exenatide conjugate was obtained after semi-preparative HPLC purification and characterized by MALDI-TOF MS. This method should be generally useful for the synthesis of other Exenatide analog for biological studies.

Synthesis and Antimicrobial Activity of a New Drug Based on a Retro-Analog of Cathelicidin—Polypeptide SE-33

Polypeptide SE-33 (SETRPVLNRLFDKIRQVIRKFEKGIKEKSKRFF), which is a retro analog of natural antimicrobial protein cathelicidin LL-37 (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES), was synthesized by the method of solid-phase peptide synthesis. Similar to the natural peptide, polypeptide SE-33 forms an amphipathic alpha helix but has an inverted amino acid sequence compared to cathelicidin. It has been shown the physicochemical properties of polypeptide SE-33 are similar to those of the natural compound. In vitro experiments have shown that polypeptide SE-33 exerts a bactericidal effect on the cells of Staphylococcus aureus Wood 46, which is comparable with the effect of cathelicidin LL-37, as well as pronounced antifungal activity against the clinical isolates of Candida albicans, Cryptococcus neoformans, Rhodotorula mucilaginosa, Trichosporon cutaneum, Geotrichum sp. The MICs of polypeptide SE-33 for different fungal strains were in the range of 31.2 to 1024 μg/mL. Polypeptide SE-33 demonstrated high activity in vivo in the model of vulvovaginal candidiasis (VVC) in mice comparable with that of pimafucin. In the absence of side effects and signs of pathology, polypeptide SE-33 in all doses tested (1, 10 and 50 mg/mL) statistically reduced the vaginal load of the mice compared to the placebo group. The pronounced antibacterial and antifungal activity of polypeptide SE-33, as well as the absence of a toxic effect in the VVC model in mice, suggest polypeptide SE-33 as a promising antimicrobial agent.

PET Imaging of HER2-Positive Tumors with Cu-64-Labeled Affibody Molecules.

Previous studies has demonstrated the utility of human epidermal growth factor receptor type 2 (HER2) as an attractive target for cancer molecular imaging and therapy. An affibody protein with strong binding affinity for HER2, ZHER2:342, has been reported. Various methods of chelator conjugation for radiolabeling HER2 affibody molecules have been described in the literature including N-terminal conjugation, C-terminal conjugation, and other methods. Cu-64 has recently been extensively evaluated due to its half-life, decay properties, and availability. Our goal was to optimize the radiolabeling method of this affibody molecule with Cu-64, and translate a positron emission tomography (PET) probe with the best in vivo performance to clinical PET imaging of HER2-positive cancers. In our study, three anti-HER2 affibody proteins-based PET probes were prepared, and their in vivo performance was evaluated in mice bearing HER2-positive subcutaneous SKOV3 tumors. The affibody analogues, Ac-Cys-ZHER2:342, Ac-ZHER2:342(Cys39), and Ac-ZHER2:342-Cys, were synthesized using the solid phase peptide synthesis method. The purified small proteins were site-specifically conjugated with the maleimide-functionalized chelator, 1,4,7,10-tetraazacyclododecane-1,4,7-tris- aceticacid-10-maleimidethylacetamide (maleimido-mono-amide-DOTA). The resulting DOTA-affibody conjugates were then radiolabeled with Cu-64. Cell uptake assay of the resulting PET probes, [64Cu]DOTA-Cys-ZHER2:342, [64Cu]DOTA-ZHER2:342(Cys39), and [64Cu]DOTA-ZHER2:342-Cys, was performed in HER2-positive human ovarian SKOV3 carcinoma cells at 4 and 37°C. The binding affinities of the radiolabeled peptides were tested by cell saturation assay using SKOV3 cells. PET imaging, biodistribution, and metabolic stability studies were performed in mice bearing SKOV3 tumors. Cell uptake assays showed high and specific uptake by incubation of Cu-64-labeled affibodies with SKOV3 cells. The affinities (KD) of the PET radio probes as tested by cell saturation analysis were in the low nanomolar range with the ranking of [64Cu]DOTA-Cys-ZHER2:342 (25.2 ± 9.2nM) ≈ [64Cu]DOTA-ZHER2:342-Cys (32.6 ± 14.7nM) > [64Cu]DOTA-ZHER2:342(Cys39) (77.6 ± 22.2nM). In vitro stability and in vivo metabolite analysis study revealed that all three probes were stable enough for in vivo imaging applications, while [64Cu]DOTA-Cys-ZHER2:342 showed the highest stability. In vivo small-animal PET further demonstrated fast tumor targeting, good tumor accumulation, and good tumor to normal tissue contrast of all three probes. For [64Cu]DOTA-Cys-ZHER2:342, [64Cu]DOTA-ZHER2:342(Cys39), and [64Cu]DOTA-ZHER2:342-Cys, tumor uptake at 24h are 4.0 ± 1.0% ID/g, 4.0 ± 0.8 %ID/g, and 4.3 ± 0.7 %ID/g, respectively (mean ± SD, n = 4). Co-injection of the probes with non-labeled anti-HER2 affibody proteins confirmed in vivo specificities of the compounds by tumor uptake reduction. The three Cu-64-labeled ZHER2:342 analogues all display excellent HER2 targeting ability and tumor PET imaging quality. Although varied in the position of the radiometal labeling of these three Cu-64-labeled ZHER2:342 analogues, there is no significant difference in tumor and normal tissue uptakes among the three probes. [64Cu]DOTA-Cys-ZHER2:342 stands out as the most superior PET probe because of its highest affinities and in vivo stability.

Protective action of a modified fragment of galanine in rats with doxorubicin-induced heart failure

The use of the anticancer drug doxorubicin (Dox) is limited due to its cardiotoxic effect. Using the method of automatic solid-phase peptide synthesis, we obtained a synthetic agonist of galanin receptors GalR1-3 [RAla14, His15]-galanine (2-15) (G), exhibiting cardioprotective properties. It was purified by high performance liquid chromatography (HPLC). The homogeneity and structure of the peptide was confirmed by HPLC, 1H-NMR spectroscopy and mass spectroscopy. The purpose of this study was to study the effect of G on the metabolism and cardiac function of rats with chronic heart failure (CHF) caused by Dox. Experiments were performed using male Wistar rats weighing 280-300 g. The control group of animals (C) was intraperitoneally treated with saline for 8 weeks; the doxorubicin group (D) of rats was intraperitoneally treated with Doх; the group of Doх + peptide G (D+G) received intraperitoneally injections of Doх and subcutaneously injections of peptide G; the peptide G group (G) was subcutaneously treated with G. At the beginning and at the end of the study, the concentration of thiobarbituric acid reactive substances (TBARS) and the activity of creatine kinase-MB (CK-MB) were determined in blood plasma; the animals were weighed, and cardiac function was assessed using echocardiography. At the end of the experiments, the hearts were used for determination of metabolites and assessment of oxidative phosphorylation in mitochondria. After 8-week treatment, animals of group D were characterized by severe heart failure, the lack of weight gain and an increase in plasma TBARS concentration and CK-MB activity. These disorders were accompanied by a decrease in the content of myocardial high-energy phosphates, a reduction inmitochondrial respiratory parameters, accumulation of lactate and glucose in the heart, and disturbances in the metabolism of alanine and glutamic and aspartic acids. Coadministration of G and Dox prevented the increase in plasma CK-MB activity and significantly reduced the plasma TBARS concentration. At the end of the experiments animals of group D+G had higher myocardial energy state and the respiratory control index of mitochondria than animals of group D, there was a decrease in anaerobic glycolysis and no changes in the amino acid content compared to the control. The peptide G significantly improved the parameters of cardiac function and caused weight gain in animals of group D+G in comparison with these parameters in group D. The obtained results demonstrate the ability of a novel agonist of galanin receptors GalR1-3 to attenuate Dox-indiced cardiotoxicity.

Au Hollow Nanorods-Chimeric Peptide Nanocarrier for NIR-II Photothermal Therapy and Real-time Apoptosis Imaging for Tumor Theranostics.

The strategy that combines photodynamic therapy (PDT) and photothermal therapy (PTT) is widely used to achieve strong antitumor efficiency. Since light in the NIR-II window possesses ideal penetration ability, developing NIR-II PTT and NIR-II light triggered photosensitizer release for combined PDT and PTT is very promising in nanomedicine.Methods: We develop a novel nanocarrier (termed AuHNRs-DTPP) by conjugating photosensitizer contained chimeric peptide (DTPP) to Au hollow nanorods (AuHNRs). AuHNRs was obtained by a Te-templated method with the assistance of L-cysteine. The chimeric peptide PpIX-PEG8-GGK(TPP)GRDEVDGC (DTPP) was obtained through a solid-phase peptide synthesis (SPPS) method.Results: Under the 1064 nm laser irradiation, the nanocarrier can accumulate heat quickly for efficient PTT, and then release activated photosensitizer for real-time apoptosis imaging. Thereafter, supplementary PDT can be conducted to kill tumor cells survived from the PTT, and meanwhile the normal tissue can be protected from photo-toxicity.Conclusion: This designed AuHNRs-DTPP nanocarrier with remarkable therapy effect, real-time apoptosis imaging ability and reduced skin damage is of great potential in nanomedicine application.

Dendrimeric Poly(Epsilon-Lysine) Delivery Systems for the Enhanced Permeability of Flurbiprofen across the Blood-Brain Barrier in Alzheimer's Disease.

Alzheimer’s disease (AD) is a progressive brain disorder and age-related disease characterised by abnormal accumulation of β-amyloid (Aβ). The development of drugs to combat AD is hampered by the lack of therapeutically-active molecules able to cross the blood-brain barrier (BBB). It is agreed that specifically-designed carriers, such as dendrimers, could support the drug penetration across the BBB. The aim of this study was to design biocompatible and biodegradable dendrimeric delivery systems able to carry Flurbiprofen (FP), as drug for AD treatment, across the BBB and liberate it at the target tissue. These dendrons were synthesised using solid-phase peptide synthesis method and characterised by mass spectrometry and fourier-transform infrared spectroscopy (FTIR). The results revealed successful synthesis of dendrons having FP been integrated during the synthesis at their branching ends. Cytotoxicity assays demonstrated the biocompatibility of the delivery systems, whereas HPLC analysis showed high percentages of permeability across an in vitro BBB model for FP-integrated dendrons. Results also revealed the efficiency of drug conjugates on the γ-secretase enzyme in target cells with evidence of eventual drug release by hydrolysis of the carrier. This study demonstrates that the coupling of FP to dendrimeric delivery systems can successfully be achieved during the synthesis of the poly(epsilon-lysine) macromolecules to improve the transport of the active drug across the BBB.

Synthesis of high functionality and quality mannose-grafted lipids to produce macrophage-targeted liposomes

The mannose receptor, which is responsible for tumor invasion, proliferation, and metastasis in the tumor microenvironment, is overexpressed in tumor-associated macrophages. Mannose is commonly applied to PEGylated liposomes in macrophage-targeted cancer therapy. To develop a high functionality and quality (HFQ) lipid for macrophage-targeted liposomes, we designed a novel mannosylated lipid with improved mannose receptor binding affinity using serine–glycine repeats (SG)n. We synthesized Man(S)-(SG)5-SSK-K(Pal)2 using only a fluorenylmethyloxycarbonyl (Fmoc) protecting group solid-phase peptide synthesis method, which produced a high-quality lipid at a moderately good yield. We then prepared Man-(SG)5/PEGylated liposomes using a post-insertion technique to insert Man(S)-(SG)5-SSK-K(Pal)2 into the PEGylated liposomes. In vitro cell investigations revealed that the Man-(SG)5/PEGylated liposomes effectively associated with mouse peritoneal macrophages by interacting with the mannose receptors. The results suggest that we produced a novel high-quality, highly functional mannosylated lipid that is suitable for clinical drug delivery applications.

Solid-Phase Synthesis of Peptide-Conjugated Perylene Diimide Bolaamphiphile and Its Application in Photodynamic Therapy

Here, we describe a rapid and efficient synthetic method of peptide-conjugated perylene diimide (P-PDI) using solid-phase peptide synthesis (SPPS). Due to severe insolubility of perylene dianhydride (PDA) as a starting material of perylene diimide (PDI), PDA was initially conjugated with amino acids to obtain soluble PDI derivatives. Target peptides were synthesized on a 2-chlorotrityl chloride resin using the SPPS method and then conjugated with the amino acid-appended PDI. Various conditions such as loading levels, reaction times and solvents were optimized for introducing the peptides to both sides of the amino acid-appended PDI. The final P-PDI was obtained with a maximum yield of 80% in 12 h. Its singlet oxygen-derived phototoxicity on cells was confirmed, which could be applicable to photodynamic therapy.

Design and synthesis of plant cyclopeptide Astin C analogues and investigation of their immunosuppressive activity

To further investigate on the structure-activity relationships of immunosuppressive Astin C, seventeen analogues 1–17 were designed and synthetized via amino acid substitution strategy by the solid-phase peptide synthesis method for the first time. In comparison with Astin C (IC50 = 12.6 ± 3.3 μM), only compounds 2 (IC50 = 38.4 ± 16.2 μM), 4 (IC50 = 51.8 ± 12.7 μM), 5 (IC50 = 65.2 ± 15.6 μM), and 8 (IC50 = 61.8 ± 12.4 μM) exhibited immunosuppressive activity in the Lymph node cells of mice. These results showed that the Astin C analogues containing D-amino acid residues, hydrophobic long-chain alkyl substituents, and aryl substituents performed better than those carrying hydrophilic amino acid residues and short-chain alkyl substituents. Moreover compounds 15, 16, and 17 had no immunosuppressive activity, which suggested that cis-3,4-dichlorinated proline played an important role in the immunosuppressive activity of Astin C.

Chemical synthesis and structural analysis of guanylate cyclase C agonist linaclotide

Guanylate cyclase C (GC-C) is an important receptor protein expressed by intestinal epithelial cells, and its dysregulation leads to severe intestinal diseases. Linaclotide is a 14-amino acid peptide approved by the FDA for the treatment of irritable bowel syndrome with constipation (IBS-C), which activates guanylate cyclase C to accelerate intestinal transit. Drug molecule design based on structural information plays a crucial role and the activity of linaclotide still need to improve, while the structure of linaclotide remains unknown. In this work, linaclotide and its d-enantiomer were obtained through Fmoc solid phase peptide synthesis method and co-crystalized through racemic crystallization. The crystal structure showed that linaclotide has a tight, three-beta turns structure immobilized by three pairs of disulfide bonds.

Discovery of a self-assembling and self-adjuvant lipopeptide as a saccharide-free peptide vaccine targeting EGFRvIII positive cutaneous melanoma.

Recently, tumor immunotherapy has achieved great progress in the treatment of hematological and solid neoplasms. The DC vaccines, KLH-conjugated vaccines or glycosylated peptide vaccines can efficiently induce immune responses against tumors. In the current study, we have discovered cholesteryl PADRE-EGFRvIII epitope-conjugated lipopeptide self-assembled micelles as a potential self-adjuvant vaccine against cutaneous melanoma. The lipopeptide vaccines were synthesized using a standard solid phase peptide synthesis method, and these vaccines could elicit both a humoral and a cellular immune response to EGFRvIII positive melanoma cells. Their high humoral immunoreaction stimulation properties in combination with their cytotoxic T-cell eliciting properties provide them with potent tumor inhibitory capacity. In therapeutic and preventive xenograft models of B16-EGFRvIII melanoma cells, the self-adjuvant lipopeptide vaccine micelles efficiently prevented tumor growth as well as tumorigenesis. Our results provide a novel platform for eliciting immune responses to non-antigenic cancer-related epitopes in peptide cancer vaccine discovery and development.

Design, Synthesis and Biological Evaluation of Novel Peptide-Like Analogues as Selective COX-2 Inhibitors.

A new series of peptide-like derivatives containing different aromatic amino acids and possessing pharmacophores of COX-2 inhibitors as SO2Me or N3 attached to the para position of an end phenyl ring was synthesized for evaluation as selective cyclooxygenase-2 (COX-2) inhibitors. The synthetic reactions were based on the solid phase peptide synthesis method using Wang resin. One of the analogues, i.e., compound 2d, as the representative of these series was recognized as the most effective and the highest selective COX-2 inhibitor with IC50 value of 0.08 μM and COX-2 selectivity index of 351.2, among the other synthesized compounds. Molecular docking study was operated to determine possible binding models of compound 2d to COX-2 enzyme. The study showed that the p-azido-phenyl fragment of 2d occupied inside the secondary COX-2 binding site (Arg513, and His90). The structure-activity relationships acquired disclosed that compound 2d with 4-(azido phenyl) group as pharmacophore and histidine as amino acid gives the essential geometry to provide inhibition of the COX-2 enzyme with high selectivity. Compound 2d can be a good candidate for the development of new hits of COX-2 inhibitors.

The toxicity study of synthesized inverse carnosine peptide analogues on HepG2 and HT-29 cells.

Objective:Cancer has risen as the main cause of diseases with the highest rate of mortality in the world. Drugs used in cancer, usually demonstrate side effects on normal tissues. On the other hand, anticancer small peptides, effective on target tissues, should be safe on healthy organs, as being naturally originated compounds. In addition, they may have good pharmacokinetic properties. carnosine, a natural dipeptide, has shown many biological functions, including anti-oxidant, anti-senescence, anti-inflammatory and anticancer activities. This study, with the aim of introducing new anticancer agents with better properties, is focused on the synthesis and cytotoxic evaluation of some peptide analogues of carnosine.Materials and Methods:The cytotoxic activity of the synthesized peptides, prepared by the solid-phase peptide synthesis method, was evaluated against two cell lines of HepG2 and HT-29 using MTT assay, lactate dehydrogenase (LDH) assay and flow cytometry analysis.Results:Linear and cyclic analogues of carnosine peptide showed cytotoxicity, demonstrated by several experiments, against HepG2 and HT-29 cell lines with mean IC50 values ranging from 9.81 to 16.23 µg/ml. Among the peptides, compounds 1c, 3c and 6b (linear analogue of 3c) showed a considerable toxic activity on the cancerous cell lines.Conclusion:The cyclic peptide analogues of carnosine with His-β-Ala-Pro-β-Ala-His (1c) and β-Ala-His-Pro-His-β-Ala (3c) sequences showed cytotoxic activity on cancerous cells of HepG2 and HT-29, better than carnosine, and thus can be good candidates to develop new anticancer agents. The mechanism of cytotoxicity may be through cell apoptosis.

Effects of N-terminal and C-terminal modification on cytotoxicity and cellular uptake of amphiphilic cell penetrating peptides

Purpose To assess the effect of “N-Acetylation and C-Amidation” on the cellular uptake, cytotoxicity and performance of amphiphilic cell penetrating peptides (CPP) loaded with methotrexate (MTX). Methods Several CPPs were synthesized by solid phase peptide synthesis method. Some of these sequences were modified with pyroglutamic acid at N-terminus and benzylamine or memantine at C-terminus. The resultant nanomaterials were prepared due to the physical linkage between CPPs and MTX. The internalization and cytotoxicity of both CPP-MTX bioconjugates and unmodified CPPs against MCF-7 human breast adenocarcinoma cells was evaluated. Results N-l and C-terminal modification did not alter the toxicity of CPPs. Physical linkage of CPPs with MTX resulted in a lower drug loading efficiency in comparison with chemically conjugated CPP-MTX bio-conjugates. Both nano-complexes increase the toxic effect of MTX on MCF-7 cells. Furthermore, N- and C-terminal modification may cause a tangible reduction in cellular uptake of CPPs. Conclusion In conclusion, it was shown that cytotoxicity of modified peptides which were physically linked with MTX, considerably higher than both physically loaded unmodified peptides and chemically conjugated peptides with MTX. Also, cell internalization was reduced after peptide end-protection. These findings confirmed the effectiveness of N- and C-terminal modifications on cell viability and CPPs internalization.

Synthesis and cellular characterization of various nano-assemblies of cell penetrating peptide-epirubicin-polyglutamate conjugates for the enhancement of antitumor activity

A new class of cell penetrating peptides (CPPs) named peptide amphiphile was designed to improve the intracellular uptake and the antitumor activity of epirubicin (EPR). Various amphiphilic CPPs were synthesized by solid phase peptide synthesis method and were chemically conjugated to EPR. Their corresponding nanoparticles (CPPs-E4 and CPPs-E8) were prepared via non-covalent binding of the peptides and polyanions. Cytotoxicity and anti-proliferative activity were evaluated by MTT assay. Cellular uptake was examined by flow cytometry and fluorescence microscopy. The CPPs exhibited slight cytotoxicity. Binding of polyglutamate to CPPs (CPPs-E4 and CPPs-E8 nanoparticles) decreased their cytotoxicity. CPPs-E8 nanoparticles showed lower cytotoxicity than CPPs-E4 nanoparticles. Cellular uptake of K3W4K3-E8, K2W4K2-E8 and W3K4W3-E8 reached 100% with no difference between each of the mentioned CPPs and its nanoparticles at 50 µM. The anti-proliferative activity of EPR was enhanced following conjugation to peptides and nanoparticles at 25 µM. CPPs-EPR-E4 and CPPs-E8-EPR nanoparticles displayed higher anti-proliferative activity than CPPs-EPR at 25 µM. CPPs-E8-EPR nanoparticles showed higher anti-proliferative activity than CPPs-E4-EPR. K3W4K3-E8-EPR nanoparticles exhibited the highest anti-proliferative activity at 25 µM. The synthesized peptide nanoparticles are proposed as suitable carriers for improving the intracellular delivery of EPR into tumor cells with low cytotoxicity and high antitumor activity.

Sequence-specific and Selective Recognition of Double-stranded RNAs over Single-stranded RNAs by Chemically Modified Peptide Nucleic Acids.

RNAs are emerging as important biomarkers and therapeutic targets. Thus, there is great potential in developing chemical probes and therapeutic ligands for the recognition of RNA sequence and structure. Chemically modified Peptide Nucleic Acid (PNA) oligomers have been recently developed that can recognize RNA duplexes in a sequence-specific manner. PNAs are chemically stable with a neutral peptide-like backbone. PNAs can be synthesized relatively easily by the manual Boc-chemistry solid-phase peptide synthesis method. PNAs are purified by reverse-phase HPLC, followed by molecular weight characterization by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF). Non-denaturing polyacrylamide gel electrophoresis (PAGE) technique facilitates the imaging of the triplex formation, because carefully designed free RNA duplex constructs and PNA bound triplexes often show different migration rates. Non-denaturing PAGE with ethidium bromide post staining is often an easy and informative technique for characterizing the binding affinities and specificities of PNA oligomers. Typically, multiple RNA hairpins or duplexes with single base pair mutations can be used to characterize PNA binding properties, such as binding affinities and specificities. 2-Aminopurine is an isomer of adenine (6-aminopurine); the 2-aminopurine fluorescence intensity is sensitive to local structural environment changes, and is suitable for the monitoring of triplex formation with the 2-aminopurine residue incorporated near the PNA binding site. 2-Aminopurine fluorescence titration can also be used to confirm the binding selectivity of modified PNAs towards targeted double-stranded RNAs (dsRNAs) over single-stranded RNAs (ssRNAs). UV-absorbance-detected thermal melting experiments allow the measurement of the thermal stability of PNA-RNA duplexes and PNA·RNA2 triplexes. Here, we describe the synthesis and purification of PNA oligomers incorporating modified residues, and describe biochemical and biophysical methods for characterization of the recognition of RNA duplexes by the modified PNAs.

Sequence-specific and Selective Recognition of Double-stranded RNAs over Single-stranded RNAs by Chemically Modified Peptide Nucleic Acids

RNAs are emerging as important biomarkers and therapeutic targets. Thus, there is great potential in developing chemical probes and therapeutic ligands for the recognition of RNA sequence and structure. Chemically modified Peptide Nucleic Acid (PNA) oligomers have been recently developed that can recognize RNA duplexes in a sequence-specific manner. PNAs are chemically stable with a neutral peptide-like backbone. PNAs can be synthesized relatively easily by the manual Boc-chemistry solid-phase peptide synthesis method. PNAs are purified by reverse-phase HPLC, followed by molecular weight characterization by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF). Non-denaturing polyacrylamide gel electrophoresis (PAGE) technique facilitates the imaging of the triplex formation, because carefully designed free RNA duplex constructs and PNA bound triplexes often show different migration rates. Non-denaturing PAGE with ethidium bromide post staining is often an easy and informative technique for characterizing the binding affinities and specificities of PNA oligomers. Typically, multiple RNA hairpins or duplexes with single base pair mutations can be used to characterize PNA binding properties, such as binding affinities and specificities. 2-Aminopurine is an isomer of adenine (6-aminopurine); the 2-aminopurine fluorescence intensity is sensitive to local structural environment changes, and is suitable for the monitoring of triplex formation with the 2-aminopurine residue incorporated near the PNA binding site. 2-Aminopurine fluorescence titration can also be used to confirm the binding selectivity of modified PNAs towards targeted double-stranded RNAs (dsRNAs) over single-stranded RNAs (ssRNAs). UV-absorbance-detected thermal melting experiments allow the measurement of the thermal stability of PNA-RNA duplexes and PNA·RNA2 triplexes. Here, we describe the synthesis and purification of PNA oligomers incorporating modified residues, and describe biochemical and biophysical methods for characterization of the recognition of RNA duplexes by the modified PNAs.