Twisted nanoribbons from a RGD-bearing cholic acid derivative.

In light of the biomedical interest for self-assembling amphiphiles bearing the tripeptide Arg-Gly-Gly (RGD), a cholic acid derivative was synthesized by introducing an aromatic moiety on the steroidal skeleton and the RGD sequence on the carboxylic function of its chain 17-24, thus forming a peptide amphiphile with the unconventional rigid amphiphilic structure of bile salts. In aqueous solution, the compound self-assembled into long twisted ribbons characterized by a very low degree of polydispersity in terms of width (≈25nm), thickness (≈4.5nm) and pitch (≈145nm). It was proposed that in the ribbon the molecules are arranged in a bilayer structure with the aromatic moieties in the interior, strongly involved in the intermolecular interaction, whereas the RGD residues are located at the bilayer-water interface. The nanostructure is significantly different from those generally provided by RGD-containing amphiphiles with the conventional peptide-tail structure, for which fibers with a circular cross-section were observed, and successfully tested as scaffolds for tissue regeneration. From previous work on the use of this kind of nanostructures, it is known that features like morphology, rigidity, epitope spacing and periodicity are important factors that dramatically affect cell adhesion and signaling. Within this context, the reported results demonstrate that bile salt-based peptide surfactants are promising building blocks in the preparation of non-trivial RGD-decorated nanoaggregates with well-defined morphologies and epitope distributions.

PEGylated ß-Sheet Breaker Peptides as Inhibitors of ß-Amyloid Fibrillization.

Three PEGylated ß-sheet breaker peptides are designed as new inhibitors of ß-amyloid fibrillization. The peptide Ac-Leu-Pro-Phe-Phe-Asp-NH2 , considered the lead compound, and hexamers in which taurine and ß-alanine substitute the acetyl group, are conjugated to poly(ethylene glycol); this conjugates self-assemble into nanoparticles. The activity of the PEGylated peptides as inhibitors of amyloid fibrillization are tested in vitro using circular dichroism spectroscopy and scanning electron microscopy. The experimental results indicate that PEGylation does not impair the ability of the ß-sheet breaker peptides to inhibit fibrillogenesis in vitro. Moreover, microscopy images of ß-amyloid incubated for 6 days with the taurine-containing peptide, suggest that this conjugate has major anti-fibrillogenesis activity and demonstrate the important role of the sulfonamide function against the amyloid aggregation.

IKKα inibition by a glucosamine derivative enhances Maspin expression in osteosarcoma cell line.

Chronic inflammation has been associated to cancer development by the alteration of several inflammatory pathways, such as Nuclear Factor-κB pathway. In particular, IκB kinase α (IKKα), one of two catalytic subunit of IKK complex, has been described to be associated to cancer progression and metastasis in a number of cancers. The molecular mechanism by which IKKα affects cancer progression is not yet completely clarified, anyway an association between IKKα and the expression of Maspin (Mammary Serine Protease Inhibitor or SerpinB5), a tumor suppressor protein, has been described. IKKα shuttles between cytoplasm and nucleus, and when is localized into the nuclei, IKKα regulates the expression of several genes, among them Maspin gene, whose expression is repressed by high amount of nuclear IKKα. Considering that high levels of Maspin have been associated with reduced metastatic progression, it could be hypothesized that the repression of IKKα nuclear translocation could be associated with the repression of metastatic phenotype. The present study is aimed to explore the ability of a glucosamine derivative, 2-(N-Carbobenzyloxy)l-phenylalanylamido-2-deoxy-ß-d-glucose (NCPA), synthesized in our laboratory, to stimulate the production of Maspin in an osteosarcoma cell line, 143B. Immunofluorescence and Western blotting experiments showed that NCPA is able to inhibit IKKα nuclear translocation, and to stimulate Maspin production. Moreover, in association with stimulation of Maspin production we found the decrease of ß1 Integrin expression, the down-regulation of metalloproteases MMP-9 and MMP-13 production and cell migration inhibition. Taking in account that ß1 Integrin and MMP-9 and -13 have been correlated with the invasiveness of osteosarcoma, considering that NCPA affects the invasiveness of 143B cell line, we suggest that this molecule could affect the osteosarcoma metastatic ability.

Self-assembly and drug release study of linear l,d-oligopeptide-poly(ethylene glycol) conjugates.

The preparation and structural organisation of new bioinspired nanomaterials based on regular alternating enantiomeric sequence of tetra- and hexapeptides end-linked to poly(ethylene glycol) (PEG) is reported. The peptide moiety is composed of two or three repeats of l-Ala-d-Val units while the PEG has a molecular weight of 2kDa. The self-assembling properties of the two conjugates depend significantly on the length of the peptide. Nanoparticles with different sizes and morphologies are formed, the structural properties of which are compared with the previously studied l-Ala-d-Val octapeptide conjugate that self-assembles into rod-like nanoparticles. The aggregation properties were studied by NMR, circular dichroism, fluorescence spectroscopies and dynamic light scattering. The morphology and size of the nanoparticles were assessed by scanning electron microscopy and dynamic light scattering. The loading and release of a model drug were also investigated. This study demonstrates that, by changing the length of the peptide, it is possible to modulate the self-assembly and loading properties of peptide-PEG conjugates.

Neuroprotective Effect of Brassica oleracea Sprouts Crude Juice in a Cellular Model of Alzheimer's Disease.

ß-Amyloid peptide (Aß) aberrant production and aggregation are major factors implicated in the pathogenesis of Alzheimer's disease (AD), causing neuronal death via oxidative stress. Several studies have highlighted the importance of polyphenolic antioxidant compounds in the treatment of AD, but complex food matrices, characterized by a different relative content of these phytochemicals, have been neglected. In the present study, we analyzed the protective effect on SH-SY5Y cells treated with the fragment Aß 25-35 by two crude juices of broccoli sprouts containing different amounts of phenolic compounds as a result of different growth conditions. Both juices protected against Aß-induced cytotoxicity and apoptotic cell death as evidenced by cell viability, nuclear chromatin condensation, and apoptotic body formation measurements. These effects were mediated by the modulation of the mitochondrial function and of the HSP70 gene transcription and expression. Furthermore, the juices upregulated the intracellular glutathione content and mRNA levels or activity of antioxidant enzymes such as heme oxygenase-1, thioredoxin, thioredoxin reductase, and NAD(P)H: quinone oxidoreductase 1 via activation of NF-E2-related factor 2 (Nrf2). Although the effects of the two juices were similar, the juice enriched in phenolic compounds showed a greater efficacy in inducing the activation of the Nrf2 signalling pathway.

ß-sheet interfering molecules acting against ß-amyloid aggregation and fibrillogenesis.

ß-Sheet aggregates and amyloid fibrils rising from conformational changes of proteins are observed in several pathological human conditions. These structures are organized in ß-strands that can reciprocally interact by hydrophobic and π-π interactions. The amyloid aggregates can give rise to pathological conditions through complex biochemical mechanisms whose physico-chemical nature has been understood in recent times. This review focuses on the various classes of natural and synthetic small molecules able to act against ß-amyloid fibrillogenesis and toxicity that may represent new pharmacological tools in Alzheimer's diseases. Some peptides, named 'ß-sheet breaker peptides', are able to hamper amyloid aggregation and fibrillogenesis by interfering with and destabilizing the non native ß-sheet structures. Other natural compounds, like polyphenols or indolic molecules such as melatonin, can interfere with ß-amyloid peptide pathogenicity by inhibiting aggregation and counteracting oxidative stress that is a key hallmark in Alzheimer's disease.

Computational and experimental studies on ß-sheet breakers targeting Aß1-40 fibrils.

In this work we present and compare the results of extensive molecular dynamics simulations of model systems comprising an Aß1-40 peptide in water in interaction with short peptides (ß-sheet breakers) mimicking the 17-21 region of the Aß1-40 sequence. Various systems differing in the customized ß-sheet breaker structure have been studied. Specifically we have considered three kinds of ß-sheet breakers, namely Ac-LPFFD-NH2 and two variants thereof, one obtained by substituting the acetyl group with the sulfonic amino acid taurine (Tau-LPFFD-NH2) and a second novel one in which the aspartic acid is substituted by an asparagine (Ac-LPFFN-NH2). Thioflavin T fluorescence, circular dichroism, and mass spectrometry experiments have been performed indicating that ß-sheet breakers are able to inhibit in vitro fibril formation and prevent the ß sheet folding of portions of the Aß1-40 peptide. We show that molecular dynamics simulations and far UV circular dichroism provide consistent evidence that the new Ac-LPFFN-NH2 ß-sheet breaker is more effective than the other two in stabilizing the native α-helix structure of Aß1-40. In agreement with these results thioflavin T fluorescence experiments confirm the higher efficiency in inhibiting Aß1-40 aggregation. Furthermore, mass spectrometry data and molecular dynamics simulations consistently identified the 17-21 Aß1-40 portion as the location of the interaction region between peptide and the Ac-LPFFN-NH2 ß-sheet breaker.

PARP-1 modulates amyloid beta peptide-induced neuronal damage.

Amyloid beta peptide (Aß) causes neurodegeneration by several mechanisms including oxidative stress, which is known to induce DNA damage with the consequent activation of poly (ADP-ribose) polymerase (PARP-1). To elucidate the role of PARP-1 in the neurodegenerative process, SH-SY5Y neuroblastoma cells were treated with Aß25-35 fragment in the presence or absence of MC2050, a new PARP-1 inhibitor. Aß25-35 induces an enhancement of PARP activity which is prevented by cell pre-treatment with MC2050. These data were confirmed by measuring PARP-1 activity in CHO cells transfected with amylod precursor protein and in vivo in brains specimens of TgCRND8 transgenic mice overproducing the amyloid peptide. Following Aß25-35 exposure a significant increase in intracellular ROS was observed. These data were supported by the finding that Aß25-35 induces DNA damage which in turn activates PARP-1. Challenge with Aß25-35 is also able to activate NF-kB via PARP-1, as demonstrated by NF-kB impairment upon MC2050 treatment. Moreover, Aß25-35 via PARP-1 induces a significant increase in the p53 protein level and a parallel decrease in the anti-apoptotic Bcl-2 protein. These overall data support the hypothesis of PARP-1 involvment in cellular responses induced by Aß and hence a possible rationale for the implication of PARP-1 in neurodegeneration is discussed.

Inhibition of amyloid peptide fragment Aß25-35 fibrillogenesis and toxicity by N-terminal ß-amino acid-containing esapeptides: is taurine moiety essential for in vivo effects?

We report the synthesis and fibrillogenesis inhibiting activity of the new peptide derivatives 1-4, related to the pentapeptide Ac-LPFFD-NH(2) (iAß5p), proposed by Soto and co-workers and widely recognized as one of the most active ß-sheet breaker agents. The Aß(25-35) fragment of the parent full-length Aß(1-42) was used as fibrillogenesis model. The activity of peptide derivatives 1-4 was tested in vitro by thioflavin T binding assay, far UV CD spectroscopy, and scanning electron microscopy. Their ability to hinder the toxic effect of Aß(25-35) in vivo was studied by monitoring the viability of human SH-SY5Y neuroblastoma cells and the prevention of superoxide anion radical release from BV2 microglial cells. The results point to a favourable role in the fibrillogenesis inhibitory activity of the sulphonamide junction for compounds 1 and 2, containing an N,N-dimethyltaurine and a taurine amino-terminal moiety, respectively. Furthermore, compounds 1 and 2 show a significant protective effect on cell viability, rescuing the cells from the toxicity exerted by Aß(25-35) treatment.

Synthesis and activity of endomorphin-2 and morphiceptin analogues with proline surrogates in position 2.

The opioid agonists endomorphins (Tyr-Pro-Trp-Phe-NH(2); EM1 and Tyr-Pro-Phe-Phe-NH(2); EM2) and morphiceptin (Tyr-Pro-Phe-Pro-NH(2)) exhibit an extremely high selectivity for mu-opioid receptor. Here a series of novel EM2 and morphiceptin analogues containing in place of the proline at position 2 the S and R residues of beta-homologues of proline (HPro), of 2-pyrrolidinemethanesulphonic acid (HPrs) and of 3-pyrrolidinesulphonic acid (betaPrs) have been synthesized and their binding affinity and functional activity have been investigated. The highest micro-receptor affinity is shown by [(S)betaPrs(2)]EM2 analogue (6e) which represents the first example of a beta-sulphonamido analogue in the field of opioid peptides.

A peptidyl-glucosamine derivative affects IKKalpha kinase activity in human chondrocytes.

INTRODUCTION: Nuclear factor-kappaB (NF-kappaB) transcription factor regulates several cell signaling pathways, such as differentiation and inflammation, which are both altered in osteoarthritis. Inhibitor kappaB kinase (IKK)alpha and IKKbeta are kinases involved in the activation of the NF-kappaB transcription factor. The aim of the present study was to determine the effects of glucosamine (GlcN), which is administered in the treatment of osteoarthritis, and of its 2-(N-Acetyl)-L-phenylalanylamido-2-deoxy-beta-D-glucose (NAPA) derivative on IKK kinases and, consequently, on NF-kappaB activation in human chondrocytes. METHODS: The human chondrosarcoma cell line HTB-94 and human primary chondrocytes were stimulated with tumor necrosis factor (TNF)alpha after pre-treatment with GlcN or NAPA. Gene mRNA expression level was evaluated by real-time PCR. Inhibitor kappaB protein (IkappaB)alpha phosphorylation and p65 nuclear re-localization were analyzed by Western blotting; IKKalpha nuclear re-localization was also investigated by immunocytochemistry and Western blotting. IKK kinase activity was studied by in vitro kinase assay. RESULTS: After TNFalpha stimulation, the mRNA expression level of some of the genes under NF-kappaB control, such as interleukin (IL)-6 and IL-8, increased, while treatment with GlcN and NAPA reverted the effect. We investigated the possibility that GlcN and NAPA inhibit IKK kinase activity and found that NAPA inhibits the IKKalpha kinase activity, whereas GlcN does not. Interestingly, both GlcN and NAPA inhibit IKKalpha nuclear re-localization. CONCLUSIONS: Our results demonstrate that glucosamine and its peptidyl derivative can interfere with NF-kappaB signaling pathway by inhibiting IKKalpha activity in human chondrocytes. However, the mechanism of action of the two molecules is not completely overlapping. While NAPA can both specifically inhibit the IKKalpha kinase activity and IKKalpha nuclear re-localization, GlcN only acts on IKKalpha nuclear re-localization.

Indolylarylsulfones bearing natural and unnatural amino acids. Discovery of potent inhibitors of HIV-1 non-nucleoside wild type and resistant mutant strains reverse transcriptase and coxsackie B4 virus.

New potent indolylarylsulfone (IAS) HIV-1 NNRTIs were obtained by coupling natural and unnatural amino acids to the 2-carboxamide and introducing different electron-withdrawing substituents at position 4 and 5 of the indole nucleus. The new IASs inhibited the HIV-1 replication in human T-lymphocyte (CEM) cells at low/subnanomolar concentration and were weakly cytostatic. Against the mutant L100I, K103N, and Y181C RT HIV-1 strains in CEM cells, sulfones 3, 4, 19, 27, and 31 were comparable to EFV. The new IASs were inhibitors to Coxsackie B4 virus at low micromolar (2-9 microM) concentrations. Superimposition of PLANTS docked conformations of IASs 19 and 9 revealed different hydrophobic interactions of the 3,5-dimethylphenyl group, for which a staking interaction with Tyr181 aromatic side chain was observed. The binding mode of 19 was not affected by the L100I mutation and was consistent with the interactions reported for the WT strain.

Synthesis and activity of fibrillogenesis peptide inhibitors related to the 17-21 beta-amyloid sequence.

Peptide derivatives 1-5, incorporating synthetic non-proteinogenic amino acids, related to the beta-amyloid 17-21 fragment of the amyloidogenic Abeta(1-40), and the N-protected decapeptide 6, corresponding to a dimeric sequence of the same fragment, have been synthesized. These compounds were designed by using Soto's pentapeptide Ac-LPFFD-NH(2) (iAbeta5p) as lead compound. Their activity as inhibitors of fibrillogenesis and stability against enzymatic degradation have been determined. Compounds 1, 5 and 6 are potent inhibitors in comparison to the lead compound. Exposure to chymotrypsin of peptide derivatives 1-5, all containing unnatural amino acids, shows increased stability as compared with iAbeta5p and 6. Conformational properties of the new compounds have been determined by CD and FT-IR spectroscopies.

Synthesis and bioactivity of chemotactic tetrapeptides: fMLF-OMe analogues incorporating spacer aminoacids at the lateral positions.

A small library of N-For and N-Boc tetrapeptidic analogues of the chemotactic tripeptide For-Met-Leu-Phe-OMe (fMLF-OMe), obtained by incorporating three different spacer aminoacids (Gly, betaAla and Pro) between the native residues of Met and Leu (N-For- and N-Boc-Met-Xaa-Leu-Phe-OMe; Xaa2 series) and Leu and Phe (N-For- and N-Boc-Met-Leu-Xaa-Phe-OMe; Xaa3 series), have been synthesized and examined for their biological activity as agonists and antagonists. Chemotaxis, lysozyme release and superoxide anion production have been measured. All the N-For analogues maintain good to moderate chemotactic activity with the betaAla3 15 model reaching the maximum value. All the N-Boc tetrapeptides are efficient chemotactic antagonists. Conversely, with the exception of the moderate antagonistic activity exhibited by the N-Boc Xaa2 models against lysozyme release, all the other N-Boc analogues do not show significant activity against both superoxide anion and lysozyme release.

Characterization of mutants of Sulfolobus solfataricus signature amidase able to hydrolyse R-ketoprofen amide.

The amidase from Sulfolobus solfataricus enantioselectively hydrolyzes S-ketoprofen amide to its corresponding acid. We identify three independent SsAH mutants that hydrolyze R-ketoprofen amide and built computational models of their three-dimensional structure. Interestingly the mutations do not specifically affect residues near the active site, or directly interacting with the substrate.

Effects of intra-articular administration of glucosamine and a peptidyl-glucosamine derivative in a rabbit model of experimental osteoarthritis: a pilot study.

The aim of this pilot study was to analyze the effects of glucosamine (GlcN) and its N-acetyl-phenylalanine derivative (NAPA) in Vitamin A model of osteoarthritis (OA) in rabbits. GlcN or NAPA or saline solution was intra-articularly administered in rabbit OA knees. Histological analysis revealed that treatment with GlcN or NAPA was associated with more homogeneous chondrocyte cellularity, absence of fissures and fragmentation and more intense staining of the matrix with Alcian Blue compared to the articular surfaces of the knees treated with saline solution. Comparative in vitro study performed on rabbit primary chondrocytes revealed that GlcN and NAPA were also able to counteract the IL-1beta-upregulation of genes coding for metalloproteases and inflammatory cytokines. Our preliminary in vivo and in vitro studies suggest that GlcN and NAPA could play a disease-modifying protective role in OA by an anti-catabolic effect and an anti-inflammatory activity on chondrocytes.

Alpha-peptide/beta-sulfonamidopeptide hybrids: analogs of the chemotactic agent for-Met-Leu-Phe-OMe.

In order to gain information on the activity shown by alpha-peptide/beta-sulfonamidopeptide hybrid analogs of the potent chemotactic agent fMLF-OMe, a structure-activity study is reported on N-Boc- and N-formyl tripeptide models containing an aminoalkanesulfonic acid as central residue. Directed migration (chemotaxis), superoxide anion production, and lysozyme release have been measured. The biochemical functions and the conformational properties of the new compounds are discussed and related to previously studied models containing beta-residues.

beta-Sulfonamido gonadotropin-releasing hormone analogs: synthesis and evaluation of several parent hormone properties.

With the aim of producing long-acting analogs of gonadotropin releasing hormone (GnRH), four analogs, containing -X(6) (aa)psi(CH(2)SO(2)NH)-Leu(7) building unit (X(aa)=Gly, Ala, Val or Phe), and a reduced-size analog [Des-Tyr(5)]-GnRH which includes the unit Phe(5)psi(CH(2)SO(2)NH)-Leu(6), and [beta-Ala(6)]-GnRH were synthesized. The peptides were evaluated for their capacity to induce LH-release from rat pituitary cells and to withstand proteolysis by pituitary-derived enzymes, compared with the parent peptide GnRH. Albeit stable toward enzymatic degradation, the sulfonamido containing peptides were only marginally bioactive. [beta-Ala(6)]-GnRH, however, induced LH-release and bound to pituitary receptors nearly as efficiently as GnRH. This analog was also highly stable toward proteolysis suggesting that it may serve as a long-acting GnRH-analog.

Beta-peptido sulfonamides: for-Met-Leu-Phe-OMe analogues containing taurine and chiral beta-amino-ethanesulfonic acid residues.

A series of new beta-peptido sulfonamides, related to the chemotactic tripeptide fMLF-OMe, has been synthesized. The examined 1a,b-7a,b models contain the achiral -HN-(CH(2))(2)-SO(2)- taurine (Tau) residue or the chiral -HN-CH(nBu)-CH(2)-SO(2)- and -HN-CH(iBu)-CH(2)-SO(2)- residues, corresponding to the beta-aminocarboxylic acid counterparts beta(3)-HNle and beta(3)-HLeu, respectively. The biological activity of the new analogues has been determined on human neutrophils and compared with that of the reference ligand as well as that of the previously studied related models. The results are analyzed in terms of structure-activity relationships. The conformational preferences of the new tripeptides 1b and 2b, containing a central chiral beta-amino-ethanesulfonic acid residue, have also been discussed.