Synthesis of oligotuftsin-based branched oligopeptide conjugates for chemotactic drug targeting.

The synthesis and chemotactic properties of a new class of branched oligopeptide-based conjugates are described. Tetratuftsin derivatives containing chemotactic formyl tripeptides (For-MLF, For-NleLF or For-MMM) in branches were prepared by stepwise solid-phase peptide synthesis. The influence of the composition and ionic charge of the carrier-branched oligopeptide on the chemotactic behaviour of the conjugate was studied in Tetrahymena pyriformis. Conjugates with methotrexate (Mtx) as a drug component was also prepared. For this, a GFLGC spacer, cleavable by cathepsin B, was used. The spacer with N-terminal methotrexate was coupled to the chloroacetylated chemotactic carrier molecule by thioether bond formation. The chemotactic activity and cytotoxity of Mtx conjugates were also studied.     

Beta-cyclodextrin/epoxysuccinyl peptide conjugates: a new drug targeting system for tumor cells

Beta-cyclodextrin is known to form inclusion complexes with hydrophobic drugs. Several tumor cell lines are known to secrete and/or contain membrane-associated cathepsin B which is possibly involved in invasion and metastasis. Based on these information, our recently developed endo-epoxysuccinyl peptide inhibitor MeO-Gly-Gly-Leu-(2S,3S)-tEps-Leu-Pro-OH for cathepsin B was conjugated with beta-cyclodextrin to obtain a site-directed drug carrier system. Furthermore, the conjugate, was shown to form an inclusion complex with the cytotoxic drug methotrexate.     

Synthesis and bioevaluation of aryl-guanidino polyamine conjugates targeting the polyamine transporter

Aryl-guanidino polyamine conjugates were prepared to evaluate their recognition for polyamine transporter (PAT) via a-difluoromethylornithine (DFMO) and spermidine (SPD)-treated B16 cell lines. The potent synergistic effects of DFMO on guanidino polyamine conjugates indicated that the presence of DFMO strongly facilitates the transport of conjugates into cells via PAT on cell membrane. The apoptotic mechanisms of triamine conjugates 10 and 1 (with and without guanidine groups) revealed that they induced apoptosis of Hela cells through the mitochondrial pathway associated with lysosomes, while DFMO strongly synergizes the function of 10 without changing the apoptotic route. Taken together, guanidino polyamine conjugates can target PAT for transport as normal polyamine ones, and the presence of guanidine in the polyamine vectors does not seem to alter the cellular targets of the conjugates, which may depend mainly on the pharmacophore.     

Synthesis and properties of ester-linked peptide nucleic acid prodrug conjugates

A Boc-protected amino acid containing an ester function, 2-([N-Boc-glycyl]oxymethyl)benzoic acid, has been synthesized and incorporated into peptide nucleic acid (PNA) oligomers. In model experiments it is found that the ester is fairly stable in aqueous solution at pH 7.4 and 37 degrees C (t(1/2) = 6 h), whereas it is rapidly cleaved in mouse serum and in kidney and liver homogenates (t(1/2) = 0.1-0.5 min). Furthermore, ester-linked fatty acid PNA conjugates targeted to an aberrant splice site in luciferase mRNA were prepared and shown to be twice as potent for inducing active luciferase as the corresponding conjugate not containing the linker. Thus, a PNA prodrug approach may be useful for both ex vivo as well as in vivo applications.     

Synthesis and evaluation of redox-sensitive gonadotropin-releasing hormone receptor-targeting peptide conjugates

Lytic peptides have been demonstrated to exhibit obvious advantages in cancer therapy with binding ability toward tumor cells via electrostatic attractions, which are lack of active targeting and aggregation to tumor tissue. In the present study, five conjugated lytic peptides were redesigned and constructed to target gonadotropin releasing hormone receptors (GnRHr), meanwhile, the disulfide bridge was introduced to achieve redox sensitive delivery based on the experience from the preliminary work of lytic peptides P3 and P7. YX-1, was considered to be the most promising for in-depth study. YX-1 possessed high potency (IC₅₀ = 3.16 ± 0.3 μM), low hemolytic effect, and cell membrane permeability in human A2780 ovarian cancer cells. Moreover, YX-1 had prominent pro-apoptotic activity by activating the mitochondria–cytochrome c–caspase apoptotic pathway. The study yielded the conjugate YX-1 with superior properties for antineoplastic activity, which makes it a promising potential candidate for targeting cancer therapy.     

Synthesis of radiometal-labeled and fluorescent cell-permeating peptide-PNA conjugates for targeting the bcl-2 proto-oncogene

The B-cell lymphoma/leukemia-2 (bcl-2) proto-oncogene has been associated with the transformation of benign lesions to malignancy, disease progression, poor prognosis, reduced survival, and development of resistance to radiation and chemotherapy in many types of cancer. The objective of this work was to synthesize an antisense peptide nucleic acid (PNA) complementary to the first six codons of the bcl-2 open reading frame, conjugated to a membrane-permeating peptide for intracellular delivery, and modified with a bifunctional chelating agent for targeting imaging and therapeutic radiometals to tumors overexpressing bcl-2. Four peptide-PNA constructs were synthesized by a combination of manual and automated stepwise elongation techniques, including bcl-2 antisense conjugates and nonsense conjugates with no complementarity to any known mammalian gene or DNA sequence. The PNA sequences were synthesized manually by solid-phase 9-fluorenylmethoxycarbonyl (Fmoc) techniques. Then a fully protected lysine monomer, modified with 1,4,7,10-tetraazacyclododecane-N,N',N',N'"-tetraacetic acid (DOTA) for radiometal chelation, was coupled manually to each PNA sequence. Synthesis of the DOTA-PNA conjugates was followed by automated elongation with a peptide sequence (PTD-4-glycine, PTD-4-G), known to mediate cellular internalization of impermeable effector molecules, or its retro-inverso analogue (ri-PTD-4-G). Preparation of the four conjugates required an innovative synthetic strategy, using mild acid conditions to generate hydrophobic, partially deprotected intermediates. These intermediates were purified by semipreparative reversed-phase HPLC and completely deprotected to yield pure peptide-PNA conjugates in 6% to 9% overall yield. Using modifications of this synthetic strategy, the ri-PTD-4-G conjugate of bcl-2 antisense PNA was prepared using a lysine derivative of tetramethylrhodamine (TMR) for fluorescence microscopy. Plasma stability studies showed that (111)In-DOTA-labeled ri-PTD-4-G-anti-bcl-2 PNA was stable for 168 h at 37 degrees C, unlike the conjugate containing the parent peptide sequence. Scanning confocal fluorescence microscopy of TMR-labeled ri-PTD-4-G-anti-bcl-2 PNA in Raji lymphoma cells demonstrated that the retro-inverso peptide was active in membrane permeation and mediated cellular internalization of the antisense PNA into the cytoplasm, where high concentrations of bcl-2 mRNA are expected to be present.     

一种拮抗HIV-1并靶向DC的融合基因的设计及表达鉴定

艾滋病病毒 (Human immunodeficiency virus,HIV) 通过与靶细胞膜的融合感染宿主细胞,研究表明阻断HIV与受体靶分子的结合可以阻止HIV进入宿主细胞,抑制HIV病毒的感染。设计合成了一个包含CD4和CCR5与HIV-1结合的主要功能结构区,及Flt3-L和Mip-3α分子的融合基因,构建了2个融合基因的真核表达载体pABK-CKR5-CD4/Flt3L-Mip3α (pABK-HIV-MF) 和pABK-CKR5-CD4 (pABK-HIV-MT),在人胚肾293细胞中进行了表达。RT-PCR、细胞免疫荧光技术、ELISA和Western blotting检测结果表明融合基因在真核细胞中获得了正确的表达,这为进一步研究其对于HIV-1的拮抗并靶向树突状细胞 (DC) 清除研究奠定了基础。     

Synthesis of peptide conjugates with vitamins for induction of antigen‐specific immunotolerance

In this report, we designed conjugates of an antigen peptide with the immunosuppressive vitamins all‐trans retinoic acid (ATRA) and vitamin D3 for efficient induction of antigen‐specific immunotolerance. We established a synthetic scheme for the preparation of the peptide‐vitamin conjugates, which the chemically unstable vitamins tolerated. Among the obtained conjugates, the ATRA conjugate successfully suppressed inflammatory effects in macrophages and dendritic cells and induced antigen presentation in dendritic cells. This synthetic method of conjugate is conceivably applicable to other antigen peptides for induction of antigen‐specific immunotolerance.     

New somatostatin-drug conjugates for effective targeting pancreatic cancer

Pancreatic cancer poorly responds to available drugs, and finding novel approaches to target this cancer type is of high significance. Here, based on a common property of pancreatic cancer cells to express somatostatin receptors (SSTR), we designed drug conjugates with novel somatostatin-derived cyclic peptides (SSTp) with broad selectivity towards SSTR types to facilitate drug targeting of the pancreatic cancer cells specifically. Uptake of our newly designed SSTps was facilitated by SSTRs expressed in the pancreatic cancers, including SSTR2, SSTR3, SSTR4 and SSTR5. Three major drugs were conjugated to our best SSTps that served as delivery vehicles, including Camptothecin (CPT), Combretastatin-4A (COMB) and Azatoxin (AZA). All designed drug conjugates demonstrated penetration to pancreatic cancer cell lines, and significant toxicity towards them. Furthermore, the drug conjugates specifically accumulated in tumors in the animal xenograft model, though some accumulation was also seen in kidney. Overall these findings lay the basis for development of novel drug series that could target the fatal pancreatic cancer.     

Liver cell specific targeting of peptide nucleic acid oligomers

Chimeric molecules consisting of peptide nucleic acid (PNA) and lactose have been synthesized to test the hypothesis that lactose moieties can promote cell-specific uptake of PNAs. We find that lactose modified PNAs rapidly enter liver-derived HepG2 cells while unmodified PNAs do not and that lactose modified PNAs can inhibit cellular telomerase.     

Synthesis and splice-redirecting activity of branched, arginine-rich peptide dendrimer conjugates of peptide nucleic acid oligonucleotides

Arginine-rich cell-penetrating peptides have found excellent utility in cell and in vivo models for enhancement of delivery of attached charge-neutral PNA or PMO oligonucleotides. We report the synthesis of dendrimeric peptides containing 2- or 4-branched arms each having one or more R-Ahx-R motifs and their disulfide conjugation to a PNA705 splice-redirecting oligonucleotide. Conjugates were assayed in a HeLa pLuc705 cell assay for luciferase up-regulation and splicing redirection. Whereas 8-Arg branched peptide-PNA conjugates showed poor activity compared to a linear (R-Ahx-R)(4)-PNA conjugate, 2-branched and some 4-branched 12 and 16 Arg peptide-PNA conjugates showed activity similar to that of the corresponding linear peptide-PNA conjugates. Many of the 12- and 16-Arg conjugates retained significant activity in the presence of serum. Evidence showed that biological activity in HeLa pLuc705 cells of the PNA conjugates of branched and linear (R-Ahx-R) peptides is associated with an energy-dependent uptake pathway, predominantly clathrin-dependent, but also with some caveolae dependence.     

Screening and optimization of ligand conjugates for lysosomal targeting

The use of lysosome-targeted liposomes may significantly improve the delivery of therapeutic enzymes and chaperones into lysosomes for the treatment of lysosomal storage disorders. The aim of this research was to synthesize new potentially lysosomotropic ligands on a base of Neutral Red and rhodamine B and to study their ability to enhance specific lysosomal delivery of surface-modified liposomes loaded with a model compound, fluorescein isothiocyanate-dextran (FD). The delivery of these liposomes and their content to lysosomes in HeLa cells was investigated by confocal immunofluorescent microscopy, subcellular fractionation, and flow cytometry. Confocal microscopy demonstrated that liposomes modified with derivatives of rhodamine B provide a good rate of colocalization with the specific lysosomal markers. The comparison of fluorescence of FD in lysosomes isolated by subcellular fractionation also showed that the efficiency of lysosomal delivery of the liposomal load by liposomes modified with some of synthesized ligands was significantly higher compared to that with plain liposomes. These results were additionally confirmed by flow cytometry of the intact cells treated with liposomes loaded with 5-dodecanoylaminofluorescein di-β-d-galactopyranoside, a specific substrate for the intralysosomal β-galactosidase, using a number of cell lines, including macrophages with induced phenotype of lysosomal enzyme deficiency; two of the synthesized ligands-rhodamine B DSPE-PEG(2k)-amide and 6-(3-(DSPE-PEG(2k))-thioureido) rhodamine B-demonstrated enhanced lysosomal delivery, in some cases, higher than that for commercially available rhodamine B octadecyl ester, with the best results (the enhancement of the lysosomal delivery up to 75% greater in comparison to plain liposomes) shown for the cells with induced lysosomal enzyme deficiency phenotype. Use of liposomes modified with rhodamine B derivatives may be advantageous for the development of drug delivery systems for the treatment of lysosome-associated disorders.     

Charge-directed targeting of antimicrobial protein-nanoparticle conjugates

Use of antimicrobial enzymes covalently attached to nanoparticles is of great interest as an antibiotic-free approach to treat microbial infections. Intrinsic properties of nanoparticles can also be used to add functionality to their conjugates with biomolecules. Here, we show in a model system that nanoparticle charge can be used to enhance delivery and increase bactericidal activity of an antimicrobial enzyme, lysozyme. Hen egg lysozyme was covalently attached to two types of polystyrene latex nanoparticles: positively charged, containing aliphatic amine surface groups, and negatively charged, containing sulfate and chloromethyl surface groups. In the case of bacterial lysis assay with a Gram-positive bacteria Micrococcus lysodeikticus, activity of lysozyme conjugated to positively charged nanoparticles was approximately twice as large as that of free lysozyme, while lysozyme conjugated to negatively charged nanoparticles showed little detectable activity. At the same time, when assayed using a low-molecular weight oligosaccharide substrate, lysozyme attached to both positively and negatively charged nanoparticles showed slightly lower activity than free enzyme. A possible explanation of these results is that lysozyme attached to negatively charged nanoparticles cannot be effectively targeted to the bacteria because of the electrostatic Coulombic repulsion from the negatively charged bacterial cell walls, whereas lysozyme conjugated to positively charged nanoparticles was targeted better than free enzyme due to stronger electrostatic attraction to bacteria. Zeta potential measurements confirmed the validity of this hypothesis. Thus, nanoparticle charge is an important factor that can be used to control targeting and activity of protein-nanoparticle conjugates.     

Synthesis of oligonucleotide-peptide conjugates carrying the c-myc peptide epitope as recognition system

Oligonucleotide-peptide conjugates 1-3 were prepared by sequential addition of the appropriate Boc-protected amino acids, followed by nucleoside phosphoramidites in the same support. These molecules are designed to be used for triplex formation and for the directed assembly of nanomaterials. The structures of the desired oligonucleotide-peptide conjugates were confirmed by mass spectrometry on small oligonucleotide-peptide conjugates, by gel electrophoresis, and by hybridization with complementary oligonucleotides. Oligonucleotides carrying the c-myc peptide were specifically recognized by the anti-c-myc monoclonal antibody.     

The cationic antimicrobial peptide LL-37 modulates dendritic cell differentiation and dendritic cell-induced T cell polarization

Dendritic cells (DC) are instrumental in orchestrating an appropriately polarized Th cell response to pathogens. DC exhibit considerable phenotypic and functional plasticity, influenced by lineage, Ag engagement, and the environment in which they develop and mature. In this study, we identify the human cationic peptide LL-37, found in abundance at sites of inflammation, as a potent modifier of DC differentiation, bridging innate and adaptive immune responses. LL-37-derived DC displayed significantly up-regulated endocytic capacity, modified phagocytic receptor expression and function, up-regulated costimulatory molecule expression, enhanced secretion of Th-1 inducing cytokines, and promoted Th1 responses in vitro. LL-37 may be an attractive therapeutic candidate for manipulating T cell polarization by DC.     

Multiple continuous-flow solid-phase peptide synthesis. Synthesis of an HIV antigenic peptide and its omission analogues

Multiple continuous-flow solid-phase peptide synthesis was performed on a standard polystyrene-based resin under low-pressure conditions using a simple manually operated synthesizer. Stable-flow resin-packed columns were prepared in small polypropylene flow reactors, adjustable for volume. The concurrent synthesis of 10 peptides was carried out in flow reactors concatenated together; solvents and reactants were passed through this set of columns using moderate overpressure. One decapeptide, H-Val-Tyr-Tyr-Arg-Asp-Ser-Arg-Asn-Pro-Leu-NH2, containing an antigenic determinant of the p31 protein product of the pol gene of the human immunodeficiency virus, and its nine omission analogues were synthesized.     

Synthesis of a disulfide-linked octameric peptide construct carrying three different antigenic determinants

In an effort to develop peptide vaccines against the influenza virus, we have successfully synthesized a disulfide-linked octameric homodimer that bears four copies of the influenza virus M2 protein ectodomain as well as two copies each of T-helper cell hemagglutinin epitopes, the I-E(d) restricted S1 and the I-A(d) restricted S2 fragments. Peptide attachment was via intermolecular disulfide formation from free sulfhydryl-bearing cysteine derivatives in solution. This reaction was efficient only when the amino-group of the cysteine was Fmoc-protected.     

Folic acid conjugates with photosensitizers for cancer targeting in photodynamic therapy: Synthesis and photophysical properties

Recent researches in photodynamic therapy have focused on novel techniques to enhance tumour targeting of anticancer drugs and photosensitizers. Coupling a photosensitizer with folic acid could allow more effective targeting of folate receptors which are over-expressed on the surface of many tumour cells. In this study, different folic acid–OEG-conjugated photosensitizers were synthesized, characterized and their photophysical properties were evaluated. The introduction of an OEG does not significantly improve the hydrophilicity of the FA–porphyrin. All the FA-targeted photosensitizers present good to very good photophysical properties. The best one appears to be Ce6. Molar extinction coefficient, fluorescence and singlet oxygen quantum yields were determined and were compared to the corresponding photosensitizer alone.     

Role of dendritic cell targeting in Venezuelan equine encephalitis virus pathogenesis

The initial steps of Venezuelan equine encephalitis virus (VEE) spread from inoculation in the skin to the draining lymph node have been characterized. By using green fluorescent protein and immunocytochemistry, dendritic cells in the draining lymph node were determined to be the primary target of VEE infection in the first 48 h following inoculation. VEE viral replicon particles, which can undergo only one round of infection, identified Langerhans cells to be the initial set of cells infected by VEE directly following inoculation. These cells are resident dendritic cells in the skin, which migrate to the draining lymph node following activation. A point mutation in the E2 glycoprotein gene of VEE that renders the virus avirulent and compromises its ability to spread beyond the draining lymph blocked the appearance of virally infected dendritic cells in the lymph node in vivo. A second-site suppressor mutation that restores viral spread to lymphoid tissues and partially restore virulence likewise restored the ability of VEE to infect dendritic cells in vivo.