77 research outputs found
Apolipoprotein A-I associated amyloidoses: the intriguing case of a natively unfolded protein fragment.
No full textNon
Insights into the mechanism(s) of action and therapeutic applications of Esculentin-1a-derived antimicrobial peptides
No full textCationic α-helical antimicrobial peptides (AMPs) hold promise for treatment of the raising multi-drug resistant microbial infections, due to their broad spectrum of activity and membrane-perturbing mechanism of action. Compared to conventional antibiotics, these features make them newsworthy molecules that hardly induce microorganisms to acquire resistance to them. Among these pathogens, Pseudomonas aeruginosa is the most clinically relevant Gram-negative bacterium known to cause serious human infections, e.g. pneumoniae, especially in immune-compromised patients, such as cystic fibrosis (CF) sufferers and keratitis, associated to contact lens (CL) wear. This is due to the unique ability of this pathogen to adhere to different types of inert materials or biological tissues, and to grow in a more resistant and dangerous sessile life form, called biofilm. Recently, two Esculentin-1a-derived antimicrobial peptides i.e. Esc(1-21) and its D-amino acids containing Esc(1-21)-1c, [Esc peptides], have been fully characterized for their powerful antipseudomonal activity against both planktonic and biofilm forms. The diastereomer showed a higher bactericidal activity than the all-L isomer against the more dangerous Pseudomonas biofilm phenotype; a lower cytotoxicity and higher biostability. However, when tested in vitro against the free-living form of this pathogen, it displayed a weaker bactericidal effect. Here, to investigate the reason accounting for this discrepancy, mechanistic studies on intact bacterial cells were initially carried out. Then to further understand the effect of packing parameters, i.e. composition, charge, shape and negative intrinsic curvature of membrane phospholipids in the membrane-permeabilizing activity of Esc peptides, leakage assays and circular dichroism spectroscopy analysis were carried out. Our results have suggested that the weaker in vitro antibacterial activity of Esc(1-21)-1c on the planktonic phenotype of the Gram-negative bacterium P. aeruginosa is mainly correlated to a slighter ability in permeabilizing both outer and inner bacterial membranes. Notably, experiments with lipid vesicles have suggested that if electrostatic interactions between negatively-charged membrane phospholipids and positively-charged peptide molecules do play a crucial role in the peptides’ membrane perturbing activity, this latter is hampered by the bilayer structure packing parameters including hydrogen bonding and intrinsic curvature, associated to phosphatidylserine (PE), especially for the diastereomer compared to all-L parent peptide. In parallel, we explored the molecular mechanism underlying the biofilm inhibition activity of Esc peptides when used at dosages below the minimal growth inhibitory concentration (1/8 MIC), by studying the peptides’ effect on the expression of key genes involved in the bacterial virulence and motility, as well as the peptide’ interaction with the bacterial signaling nucleotide ppGpp. Our findings revealed that the two D-amino acids containing Esc(1-21)-1c, confer the peptide the ability to downregulate the expression of biofilm-associated genes, likely as a result of increased peptide stability and prolonged binding to ppGpp compared to the all-L peptide. Furthermore, we reported two different applicative strategies to ameliorate the biological properties of these two AMPs: (i) encapsulation in poly(lactide-co-glycolide) (PLGA) nanoparticles; and (ii) covalent conjugation to soft CLs. In the first case, to enhance the peptides’ bioavailability and to optimize their translocation to the target infectious site, Esc peptides were loaded into PLGA nanoparticles (NPs) engineered with polyvinyl alcohol (PVA). The peptides-loaded NPs were found to be more efficient in diffusing through artificial CF mucus and simulated bacterial extracellular matrix compared to the free peptides. Moreover, they were more efficient in inhibiting P. aeruginosa growth under both in vitro and in vivo conditions at long term. In the second case, Esc peptides were covalently immobilized to hydrogel soft CLs and tested for their ability to reduce bacterial colonization. The antimicrobial CLs were able to cause more than four log reduction in the number of bacterial cells within 20 min and to reduce bacterial adhesion to their surface in 24 hours. Finally, the ability of both peptides to limit the onset of microbial resistance was also evaluated by exposing Pseudomonas strains to multiple cycles of treatment at sub-MIC dosages. Interestingly, in contrast with conventional antibiotics, Esc peptides did not induce resistance in P. aeruginosa cells. Overall, besides providing knowledges on the molecular mode(s) of action the two esculentin-derived AMPs, our data suggest that Esc peptides, particularly Esc(1-21)-1c, have great potential to be developed as novel drugs for treatment and prevention of P. aeruginosa pneumonia and keratitis
Nuove combinazioni di molecole ad attività bioinsetticida per la protezione delle colture agraria
No full textInnovative molecular platforms for G-quadruplex stabilization
No full textInnovative molecular platforms for G-quadruplex stabilization
S. Di Ciolo,a B. Airoldi,a L.C.M. Pariani,a C. F. Morelli,a C. Giannini,a A. Silvani,a D. Malpicci,a E. Cariati,a E. Lucenti,b C. Platella,c D. Musumeci,c R. Gaglione, c A. Arciello,c D. Montesarchioc
aDipartimento di Chimica, Università degli studi di Milano, Milano
bCNR-SCITEC, Istituto di Scienze e Tecnologie Chimiche “Giulio Natta”, Milano
cDipartimento di Scienze Chimiche, Università di Napoli Federico II, Napoli
[email protected]
G-quadruplexes (G4s) have received considerable attention in recent years, due to evidence of their occurrence in living cells. 1Formed by stacks of guanine quartets, G4s belong to the wide class of non-canonical nucleic acid structures. Putative G4 forming sequences are distributed in genomic regions relevant for a number of pathologies, spanning from cancer to viral infections, where they can act as regulators of genetic information transfer.2 In this context, G4-targeting is regarded as a novel potential therapeutic approach for the treatment of associated diseases.3 Taking inspiration from the recent literature on G4 ligands4 and relying on the scaffold hopping strategy,5 we selected three different heterocyclic frameworks, potentially able to bind G4s by stacking the external G-tetrads. We functionalized these scaffolds with proper polar or positively charged flexible groups for binding to G4 loops/grooves, and/or with alkylating warheads for more selective covalent targeting. To investigate the G4 binding properties of the new molecules, in vitro biophysical studies were performed employing both telomeric and oncogene promoter G4-forming sequences. Interestingly, the investigated compounds showed potent G4-stabilizing properties and a certain preference for G4 over duplex DNA. Under preliminary in vitro studies, some ligands proved to inhibit cancer cells proliferation, not affecting healthy cells used as control.
References:
[1] S. Burge, G. N. Parkinson, P. Hazel, A. K. Todd, S. Neidle, Nucleic Acids Res. 2006, 34, 5402–5415.
[2] R. Hänsel-Hertsch, M. Di Antonio, S. Balasubramanian, Nat. Rev. Mol. Cell Biol. 2017, 18, 279–284.
[3] A. R. Duarte, E. Cadoni, A.S. Ressurreição, R. Moreira, A. Paulo ChemMedChem, 2018, 13, 869-893.
[4] M. P. O'Hagan, J. C. Morales, M. C. Galan, Eur. J. Org. Chem. 2019, 4995–5017.
[5] H. Sun,1, G. Tawa, A. Wallqvist Drug Discov Today. 2012 17(7-8), 310–324
Innovative molecular platforms for G-quadruplex stabilizatioN
No full textInnovative molecular platforms for G-quadruplex stabilization
S. Di Ciolo,a B. Airoldi,a L.C.M. Pariani,a C. F. Morelli,a C. Giannini,a A. Silvani,a D. Malpicci,a E. Cariati,a E. Lucenti,b C. Platella,c D. Musumeci,c R. Gaglione, c A. Arciello,c D. Montesarchioa
aDipartimento di Chimica, Università degli studi di Milano, Milano
bCNR-SCITEC, Istituto di Scienze e Tecnologie Chimiche “Giulio Natta”, Milano
cDipartimento di Scienze Chimiche, Università di Napoli Federico II, Napoli
e-mail: [email protected]
G-quadruplexes (G4s) have received considerable attention in recent years, due to evidence of their occurrence in living cells. 1Formed by stacks of guanine quartets, G4s belong to the wide class of non-canonical nucleic acid structures. Putative G4 forming sequences are distributed in genomic regions relevant for a number of pathologies, spanning from cancer to viral infections, where they can act as regulators of genetic information transfer.2 In this context, G4-targeting is regarded as a novel potential therapeutic approach for the treatment of associated diseases.3 Taking inspiration from the recent literature on G4 ligands4 and relying on the scaffold hopping strategy,5 we selected three different heterocyclic frameworks, potentially able to bind G4s by stacking the external G-tetrads. We functionalized these scaffolds with proper polar or positively charged flexible groups for binding to G4 loops/grooves, and/or with alkylating warheads for more selective covalent targeting. To investigate the G4 binding properties of the new molecules, in vitro biophysical studies were performed employing both telomeric and oncogene promoter G4-forming sequences. Interestingly, the investigated compounds showed potent G4-stabilizing properties and a certain preference for G4 over duplex DNA. Under preliminary in vitro studies, some ligands proved to inhibit cancer cells proliferation, not affecting healthy cells used as control.
References
[1] S. Burge, G. N. Parkinson, P. Hazel, A. K. Todd, S. Neidle, Nucleic Acids Res. 2006, 34, 5402–5415.
[2] R. Hänsel-Hertsch, M. Di Antonio, S. Balasubramanian, Nat. Rev. Mol. Cell Biol. 2017, 18, 279–284.
[3] A. R. Duarte, E. Cadoni, A.S. Ressurreição, R. Moreira, A. Paulo ChemMedChem, 2018, 13, 869-893.
[4] M. P. O'Hagan, J. C. Morales, M. C. Galan, Eur. J. Org. Chem. 2019, 4995–5017.
[5] H. Sun,1, G. Tawa, A. Wallqvist Drug Discov Today. 2012 17(7-8), 310–324
Benefits from dietary polyphenols for brain aging and Alzheimer's disease
No full textBrain aging and the most diffused neurodegenerative diseases of the elderly are characterized by oxidative damage, redox metals homeostasis impairment and inflammation. Food polyphenols can counteract these alterations in vitro and are therefore suggested to have potential anti-aging and brain-protective activities, as also indicated by the results of some epidemiological studies. Despite the huge and increasing amount of the in vitro studies trying to unravel the mechanisms of action of dietary polyphenols, the research in this field is still incomplete, and questions about bioavailability, biotransformation, synergism with other dietary factors, mechanisms of the antioxidant activity, risks inherent to their possible pro-oxidant activities are still unanswered. Most of all, the capacity of the majority of these compounds to cross the blood-brain barrier and reach brain is still unknown. This commentary discusses recent data on these aspects, particularly focusing on effects of curcumin, resveratrol and catechins on Alzheimer's disease. © 2008 Springer Science+Business Media, LLC
Enzymatically active fibrils generated by self-assembly of the ApoA-I fibrillogenic domain functionalized with a catalytic moiety.
No full textEnzymatically active fibrils were produced by self-assembly of a bifunctional chimeric protein, made up of a fibrillogenic and a catalytic moiety. For this purpose, the fibrillogenic domain of Apolipoprotein A-I (ApoA-I), a 93-residue polypeptide named [1-93]ApoA-I, was functionalized with the enzyme glutathione S-transferase (GST). The fusion protein GST-[1-93]ApoA-I was expressed, isolated to homogeneity and characterized. In the soluble form, GST-[1-93]ApoA-I was found to be fully active as a GST enzyme, and to have high propensity to self-aggregate. Upon incubation for 3 weeks at pH 6.4, insoluble aggregates were generated. Analyzed by AFM, they were found to contain fibrillar structures often organized into large fiber networks. Fibrils were loaded on the membrane of a microfiltration unit and tested for enzymatic activity by filtering the substrate through the fibrillar network. Fibrils were shown to be catalytically active, stable over time and reusable, as no loss of activity was detected when fibrils were repeatedly tested. Our findings suggest that catalytically active fibrils may be of interest for biocatalytic applications in nanobiotechnology
La co-produzione in piante transgeniche di tabacco di molecole, che inibiscono la sintesi della tripsina e l’attività triptica, conferisce tolleranza ad insetti e funghi patogeni
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