Pep5-based antitumor peptides containing multifunctional fragments with enhanced activity and synergistic effect.

In this study, we designed a series of hybrid peptides based on pep5-TAT (P-05), comprising antitumor segment (pep5), endosomal escape segment ((LLHH)3) and cell penetrating/membrane disrupting segment (TAT, R9, sC182). These peptides exhibited remarkable antitumor activity towards tumor cells (HepG2, A549). Among them, the IC50 values of peptide P-09 were 4.0 and 4.8 times lower than those of P-05 in HepG2 and A549 cells, respectively. It was proved that P-09 could enter tumor cells through endocytosis and direct penetration and induce the apoptosis and necrosis. The antitumor effects were attributed to the synergistic effect of membrane disruption and proteasome inhibition, which occurred during and after the cellular entry, respectively. The whole process was accompanied by excessive ROS production. In vivo, P-09 exhibited enhanced ability to inhibit the growth of HepG2 subcutaneous tumor xenografts than P-05 in nude mice. In brief, this work provided valuable insights into the design of peptide-based antitumor agents with synergistic antitumor effects.

Design of artificial α-helical peptides targeting both gp41 deep pocket and subpocket as potent HIV-1 fusion inhibitors.

Both the deep pocket region and its neighboring subpocket site on the N-trimer of HIV-1 gp41 protein can serve as targets for the development of HIV-1 entry inhibitors. Pocket-binding domain (PBD)-containing peptides with the potential to inhibit HIV-1 fusion through targeting the deep pocket have been extensively exploited. However, using an artificial peptide strategy, we herein report the design of α-helical lipopeptides with non-native protein sequences as HIV-1 fusion inhibitors that can occupy both gp41 deep cavity and subpocket sites. The most active compound, PP24C, inhibited HIV-1 replication, including T20-resistant HIV-1 mutants, at low nanomolar level. Biophysical approaches revealed that both the artificial α-helical peptide P35A4 and its cholesterol-tagged peptide PP24C could bind to T21 peptide used as a target surrogate comprising both pockets. Our study offers a new template for the design of artificial anti-HIV-1 therapeutics and highlights the novel concept of peptide secondary structure-based virus fusion inhibitors.

Substance P containing peptide gene delivery vectors for specifically transfecting glioma cells mediated by a neurokinin-1 receptor.

Gene therapy provides a promising treatment for glioblastoma multiforme, which mainly depends on two key aspects, crossing the blood brain barrier (BBB) effectively and transfecting target cells selectively. In this work, we reported a series of peptide-based vectors for transfecting glioma cells specifically consisting of several functional segments including a cell-penetrating peptide, targeting segment substance P (SP), an endosomal escape segment, a PEG linker and a stearyl moiety. The conformations and DNA-loading capacities of peptide vectors and the self-assembly behaviors of peptide/pGL3 complexes were characterized. The in vitro gene transfection was evaluated in U87, 293T-NK1R, and normal 293T cell lines. The transfection efficiency ratio of P-02 (SP-PEG4-K(C18)-(LLHH)3-R9) to Lipo2000 in the U87 cell line was about 36% higher than that in the 293T cell line. The neurokinin-1 receptor (NK1R) in U87 cells mediated the transfection process via interactions with the ligand SP in peptide vectors. The mechanism of NK1R mediated transfection was demonstrated by the use of gene-modified 293T cells expressing NK1R, as well as the gene transfection in the presence of free SP. Besides, P-02 could promote the pGL3 plasmids to cross the BBB model in vitro and achieved the EGFP gene transfection in the brain of zebrafish successfully. The designed peptide vectors, owing to their specific transfection capacity in glioma cells, provide a potential approach for glioblastoma multiforme gene therapy.

The effect of structural modification of antimicrobial peptides on their antimicrobial activity, hemolytic activity, and plasma stability.

In this article, a series of modifications were made on an antimicrobial peptide F2,5,12 W, including altering the amino acid sequence, introducing cysteine and other typical amino acids, developing peptide dimers via disulfide bonds, and conjugating with mPEG, in order to enhance the antimicrobial activity, plasma stability, and reduce the hemolytic activity of peptides. The results showed that mPEG conjugation could significantly improve the plasma stability and reduce the hemolytic activity of peptides, while the antimicrobial activity decreased meanwhile. However, altering the sequence of the peptide without changing its amino acid composition had little impact on its antimicrobial activity and plasma stability. The introduction of cysteine enhanced the plasma stability of peptides conspicuously, but at the same time, the increased hydrophobicity of peptides increased their hemolysis. The antimicrobial mechanism and cytotoxicity of the peptides with relatively high antimicrobial activity were also studied. In general, this study provided some ideas for the rational design and structure optimization of antimicrobial peptides.

Inhibition of SARS-CoV-2 (previously 2019-nCoV) infection by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion.

The recent outbreak of coronavirus disease (COVID-19) caused by SARS-CoV-2 infection in Wuhan, China has posed a serious threat to global public health. To develop specific anti-coronavirus therapeutics and prophylactics, the molecular mechanism that underlies viral infection must first be defined. Therefore, we herein established a SARS-CoV-2 spike (S) protein-mediated cell-cell fusion assay and found that SARS-CoV-2 showed a superior plasma membrane fusion capacity compared to that of SARS-CoV. We solved the X-ray crystal structure of six-helical bundle (6-HB) core of the HR1 and HR2 domains in the SARS-CoV-2 S protein S2 subunit, revealing that several mutated amino acid residues in the HR1 domain may be associated with enhanced interactions with the HR2 domain. We previously developed a pan-coronavirus fusion inhibitor, EK1, which targeted the HR1 domain and could inhibit infection by divergent human coronaviruses tested, including SARS-CoV and MERS-CoV. Here we generated a series of lipopeptides derived from EK1 and found that EK1C4 was the most potent fusion inhibitor against SARS-CoV-2 S protein-mediated membrane fusion and pseudovirus infection with IC50s of 1.3 and 15.8 nM, about 241- and 149-fold more potent than the original EK1 peptide, respectively. EK1C4 was also highly effective against membrane fusion and infection of other human coronavirus pseudoviruses tested, including SARS-CoV and MERS-CoV, as well as SARSr-CoVs, and potently inhibited the replication of 5 live human coronaviruses examined, including SARS-CoV-2. Intranasal application of EK1C4 before or after challenge with HCoV-OC43 protected mice from infection, suggesting that EK1C4 could be used for prevention and treatment of infection by the currently circulating SARS-CoV-2 and other emerging SARSr-CoVs.

Polyion complexes of a cationic antimicrobial peptide as a potential systemically administered antibiotic.

Antimicrobial peptides (AMPs) are regarded as next-generation antibiotics to replace conventional antibiotics due to their rapid and broad-spectrum antimicrobial properties and far less sensitivity to the development of pathogen resistance. However, they are susceptible to proteolysis in vivo by endogenous or bacterial proteases as well as induce the lysis of red blood cells, which prevent their intravenous applications. In this work, polyion complex (PIC) micelles of the cationic AMP MSI-78 and the anionic copolymer methoxy poly(ethylene glycol)-b-poly(α-glutamic acid) (mPEG-b-PGlu) were prepared to develop novel antimicrobial agents for potential application in vivo. With an increase in molar ratio of mPEG-b-PGlu to MSI-78, the complexation ability of the PIC micelles increased. FITC-labeled MSI-78 showed a sustained release from the PIC micelles. More importantly, these PIC micelles greatly decreased the hemolytic toxicity of MSI-78 to human red blood cells, without influencing its antimicrobial activity. Thus, this approach could be used as a suitable in vivo delivery method of AMPs in the future.

Design, synthesis, and activity evaluation of novel erythropoietin mimetic peptides.

The approval of the erythropoietin (EPO) mimetic peptide drug peginesatide in 2012 was a breakthrough for the treatment of secondary anemia. However, due to severe allergic reactions, peginesatide was recalled a year later. In this study, 12 novel peptides were designed and synthesized by substituting specific amino acids of the monomeric peptide in peginesatide, with the aim of obtaining new EPO mimetic peptides with higher activities and lower side effects than the parent compound. Their cell proliferation activities were evaluated, and the structure-activity relationships were analyzed. Five compounds had equal cell proliferation activity to the control peptide. Among them, one compound showed a higher in vivo activity than the control peptide, with no obvious side effects.

Enhanced gene transfection efficiency by use of peptide vectors containing laminin receptor-targeting sequence YIGSR.

This study presents the design and evaluation of a series of multifunctional peptides and their gene delivery abilities. The peptide sequences contained a cell-penetrating segment, six continuous histidine residues, a stearyl moiety and a laminin receptor-targeting segment. The YIGSR segment promoted cellular uptake through the interaction with laminin receptors on the surface of cells, which resulted in a great improvement in gene transfection efficiency. The conformation, particle size and zeta potential of peptide/DNA complexes were characterized via circular dichroism and dynamic light scattering. Their gene transfection efficiency was investigated by fluorescence-activated cell sorting and confocal microscopy. The transfection efficiency of the designed peptide vectors was higher than that of Lipo 2000. The peptide TAT-H6-K(C18)-YIGSR displayed transfection efficiencies at N/P ratios of 6, which was 3.5 and 7 times higher than that of Lipo 2000 in B16F10 and 293T cells, respectively. All peptides exhibited lower cytotoxicity than Lipo 2000 in B16F10 and 293T cells. In summary, the designed YIGSR-containing multifunctional peptide gene vectors promoted cellular uptake and gene transfection. Their in vivo transfection ability was investigated in zebrafish, and the transfection efficiency was determined by confocal microscopy and bioluminescence imaging. The peptide vectors, owing to their relatively short sequences and ease of functionalization, offer a promising approach for gene delivery because of their low cytotoxicity and high transfection efficiency.

The structure and configuration changes of multifunctional peptide vectors enhance gene delivery efficiency.

We designed a series of peptide vectors that contain functional fragments with the goal of enhancing cellular internalization and gene transfection efficiency. The functional fragments included a cell-penetrating peptide (R9), a cationic amphiphilic α-helical peptide [(LLKK)3-H6 or (LLHH)3], a stearyl moiety, and cysteine residues. Vectors were also synthesized with D-type amino acids to improve their proteolytic stability. The conformations, particle sizes, and zeta potentials for complexes of these peptides with pGL3 plasmid DNA were characterized by circular dichroism and dynamic light scattering. In addition, cellular uptake of the peptide/DNA complexes and gene transfection efficiency were investigated with fluorescence-activated cell sorting and confocal laser-scanning microscopy. Greater transfection efficiency was achieved with the vectors containing the R9 segment, and the efficiency was greater than Lipo2000. In addition, the D-type C18-c(llkk)3ch6-r9 had about 7 times and 5.5 times the transfection efficiency of Lipo2000 in 293T cells and NIH-3T3 cells at the N/P ratio of 6, respectively. Overall, the multifunctional peptide gene vectors containing the R9 segment exhibited enhanced cellular internalization, a high gene transfection efficiency, and low cytotoxicity.

Histidine-enriched multifunctional peptide vectors with enhanced cellular uptake and endosomal escape for gene delivery.

Peptide vectors offer a promising gene delivery approach because of their biocompatibility and ease of functionalization. This article describes the design and evaluation of a series of multifunctional peptides and their gene delivery abilities. The peptides were composed of a cell-penetrating segment, stearyl moiety, cationic amphiphilic α-helical segment, and cysteine and histidine residues. The proton sponge effect of histidine residues at low pH and the α-helical conformation should improve endosomal escape. Inclusion of d-type amino acids should improve proteolytic stability. The conformation, particle size and zeta potential of peptide/DNA complexes were characterized by circular dichroism and dynamic light scattering. Gene transfection efficiency was investigated by fluorescence-activated cell sorting and confocal microscopy. Transfection efficiencies of the designed peptide vectors were better than those of C18-C(LLKK)3C-TAT and Lipo2000. d-Type peptide C18-c(llhh)3c-tat showed three times higher transfection efficiency at N/P ratios of 6 and 8 than Lipo2000 in NIH-3T3 and 293T cells. All peptides showed lower cytotoxicity than Lipo2000 in NIH-3T3 and 293T cells. In the presence of trypsin or serum in vitro, d-type peptides showed better stability than l-type peptides. Overall, the designed histidine-enriched multifunctional peptide gene vectors promoted cellular uptake, endosomal escape and gene transfection.

Design, synthesis, and evaluation of water-soluble morpholino-decorated paclitaxel prodrugs with remarkably decreased toxicity.

Novel water-soluble paclitaxel prodrugs were designed and synthesized by introducing morpholino groups through different linkers. These derivatives showed 400-20,000-times greater water solubility than paclitaxel as well as comparable activity in MCF-7 and HeLa cell lines. The prodrug PM4 was tested in the S-180 tumor mouse model, with paclitaxel as the positive control. The results showed that PM4 had comparable antitumor activity as paclitaxel, with tumor inhibition of 54% versus 56%, and remarkably decreased toxicity. The survival rate of treated mice was 8/8 in the PM4 group, compared to 3/8 in the paclitaxel group.

Gonadotropin-releasing hormone receptor-targeted paclitaxel-degarelix conjugate: synthesis and in vitro evaluation.

To increase the selectivity of chemotherapeutic agents, receptor-mediated tumor-targeting approaches have been developed. Here, degarelix [Ac-D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(Cbm)-Leu-ILys-Pro-D-Ala-NH2], a gonadotropin-releasing hormone antagonist, was employed as a targeting moiety for paclitaxel (PTX). Five PTX-degarelix conjugates were synthesized, in which PTX was attached via disulfide bond to the different position in the degarelix sequence. All of the PTX-degarelix conjugates exhibited a half-life greater than 10 h determined in human serum. A fluorometric imaging plate reader assay showed that the conjugates LK-MY-9 and LK-MY-10 had an antagonism efficacy similar to that of degarelix. The in vitro cytostatic effects of the conjugates were determined by a (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) assay, and the 50% inhibitory concentration value of the conjugates on 3T3 mouse embryonic fibroblast cells were one order of magnitude higher than the 50% inhibitory concentration values of the conjugates on MCF-7 human breast cancer cells and HT-29 human colon cancer cells. Receptor saturation tests further demonstrated that pre-incubation of the cells with degarelix reduced the efficacy of LK-MY-10 in a concentration-dependent manner. In conclusion, degarelix is a valid and stable moiety that has great potential for targeting chemotherapy drugs.

CHP1002, a novel andrographolide derivative, inhibits pro-inflammatory inducible nitric oxide synthase and cyclooxygenase-2 expressions in RAW264.7 macrophages via up-regulation of heme oxygenase-1 expression.

Andrographolides, a type of diterpene lactone, are widely known to have anti-inflammatory and anti-oxidative properties. CHP1002, a synthetic derivative of andrographolide, has similar anti-inflammatory action in mouse ear swelling test and rat paw edema test. In the present study, the mechanism of anti-inflammatory effects of CHP1002 was investigated in RAW264.7 macrophages. CHP1002 potently suppressed inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expressions in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. CHP1002 reduced the production of iNOS-derived nitric oxide (NO) and COX-2-derived prostaglandin E2 (PGE2). CHP1002 induced heme oxygenase-1 (HO-1) expression via activation of extracellular signal-regulated kinase (ERK) and NF-E2 related factor 2 transcription factor (Nrf2). Down-regulation of LPS-induced iNOS and COX-2 expressions was partially reversed by the HO-1 inhibitor zinc protoporphyrin (ZnPP). In addition, CHP1002 significantly attenuated LPS-induced TNF-α, IL-1ß and IL-6 production. CHP1002 effectively induced HO-1 and was capable of inhibiting some macrophage-derived pro-inflammatory mediators, which may be closely correlated with its anti-inflammatory action.