The Interaction of Metal Complexes with G-quadruplex DNA

Un approccio computazionale è stato proposto per lo studio dell’interazione di complessi metallici di basi di Schiff con DNA. Nel capitolo 2, è stato investigato il meccanismo di azione di complessi di Nichel(II), Rame(II) e Zinco(II) con B e G-quadruplex DNA. Il G-quadruplex è una conformazione non canonica adottata da particolari sequenze ricche in guanina. Recentemente, è stata dimostrata la sua esistenza in cellule umane, in regioni telomeriche e non telomeriche, ed è stato proposto come un possibile target per una nuova categoria di agenti antineoplastici. I capitoli successivi sono basati su dati raccolti durante due periodi di ricerca all’estero. Nel capitolo 3, basato sugli studi eseguiti presso l’Università tecnica di Braunschweig, verrà mostrato come i campi di forza, oggigiorno di uso comune in Chimica/Biofisica Computazionale, siano in grado di riprodurre correttamente la stabilità relativa di G-quadruplex modello. Inoltre, è stata studiata in dettaglio l’interazione di una classe di leganti organici, noti G-quadruplex binders, con un modello di quadruplex parallelo. L’approccio computazionale ha messo in evidenza l’importanza del considerare esplicitamente la protonazione dei leganti. Nel capitolo 4, risultato di una COST Short-Term Scientific Mission in Francia presso l’Universitè de Lorraine, è descritta la procedura usata per riprodurre gli spettri di Dicroismo Circolare delle principali conformazioni dei G-quadruplex.A computational approach was proposed to study the binding and the stability of metal complex-nucleic acid supramolecular systems. In particular, the interaction of transition metal complexes with DNA structures named “G-quadruplexes” was considered. G-quadruplex conformations are present in telomeres and several oncogenes and they are involved in the inhibition of telomerase, a protein responsible for immortalization of cancer cells. The main purpose of the project was then to provide a computational tool to design chemical compounds able to selectively stabilize G-quadruplex structures

Cancer-Related Mutations Alter RNA-Driven Functional Cross-Talk Underlying Premature-Messenger RNA Recognition by Splicing Factor SF3b

The pillar of faithful premature-messenger (pre-mRNA)splicingis the precise recognition of key intronic sequences by specific splicingfactors. The heptameric splicing factor 3b (SF3b) recognizes the branchpoint sequence (BPS), a key part of the 3 & PRIME; splice site. SF3bcontains SF3B1, a protein holding recurrent cancer-associated mutations.Among these, K700E, the most-frequent SF3B1 mutation, triggers aberrantsplicing, being primarily implicated in hematologic malignancies.Yet, K700E and the BPS recognition site are 60 & ANGS; apart, suggestingthe existence of an allosteric cross-talk between the two distal spots.Here, we couple molecular dynamics simulations and dynamical networktheory analysis to unlock the molecular terms underpinning the impactof SF3b splicing factor mutations on pre-mRNA selection. We establishthat by weakening and remodeling interactions of pre-mRNA with SF3b,K700E scrambles RNA-mediated allosteric cross-talk between the BPSand the mutation site. We propose that the altered allostery contributesto cancer-associated missplicing by mutated SF3B1. This finding broadensour comprehension of the elaborate mechanisms underlying pre-mRNAmetabolism in eukaryotes

Anticancer Activity, Reduction Mechanism and G-Quadruplex DNA Binding of a Redox-Activated Platinum(IV)–Salphen Complex

Aiming at reducing the unselective cytotoxicity of Pt(II) chemotherapeutics, a great deal of effort has been concentrated into the design of metal‐containing drugs with different anticancer mechanisms of action. Inert Pt(IV) prodrugs have been proposed to be a valid alternative as they are activated by reduction directly into the cell releasing active Pt(II) species. On the other hand, a promising strategy for designing metallodrugs is to explore new potential biological targets rather than canonical B‐DNA. G‐quadruplex nucleic acid, obtained by self‐assembly of guanine‐rich nucleic acid sequences, has recently been considered an attractive target for anticancer drug design. Therefore, compounds capable of binding and stabilizing this type of DNA structure would be greatly beneficial in anticancer therapy. Here, computational analysis reports the mechanism of action of a recently synthesized Pt(IV)–salphen complex conjugating the inertness of Pt(IV) prodrugs with the ability to bind G‐quadruplexes of the corresponding Pt(II) complex. The reduction mechanism of the Pt(IV) complex with a biological reducing agent was investigated in depth by means of DFT, whereas classical MD simulations were carried out to shed light into the binding mechanism of the released Pt(II) complex. The results show that the Pt(IV) prodrug may be reduced by both inner‐ and outer‐sphere mechanisms, and the active Pt(II) complex, as a function of its protonation state, stabilizes the G‐quadruplex DNA prevalently, either establishing π‐stacking nteractions with the terminal G‐tetrad or through electrostatic interactions along with H‐bonds formation

(Dipyrido[3,2-a:2',3'-c]phenazine)(glycinato)copper(II) perchlorate: A novel DNA-intercalator with anti-proliferative activity against thyroid cancer cell lines

A novel copper(II) heteroleptic complex of dipyrido[3,2-a:2′,3′-c]phenazine (dppz) and glycinato (gly) as chelating ancillary ligand, [Cu(dppz)(gly)]ClO4 (1), was synthesized and characterized. X-ray crystallography revealed that the coordination geometry of the cationic [Cu(dppz)(gly)]+ unit is hexacoordinated and shows a distorted octahedral coordination geometry in the solid state, with the N,N and N,O chelating atoms of dppz and glycinato, respectively, in the square plane and in which the planar units are connected in a monodimensional polymeric array by the apical copper coordination of the second carboxylic oxygen atom. Biological assays showed that 1 exhibits a remarkable anti-proliferative activity against the two human anaplastic thyroid cancer cell lines 8505c (BrafV600E/V600E) and SW1736 (BrafWT/V600E), in a dose- and time-dependent manner. In details, the IC50 after 48 h of drug exposure was 2.86±0.54 μM for SW1736 and 1.05±0.48 μM for 8505c. On the other hand, the IC50 shown by cis-diamminedichloroplatinum(II) (cisplatin) against the same cell lines was 2.50±0.40 μM and 6.03±0.78 μM, respectively. Optical microscopy observations, after 48 h of treatment, showed morphological cell changes typical of apoptosis, confirmed by DNA ladder assays. DNA interaction studies, performed by UV absorption spectrophotometry, circular dichroism and viscosimetry, clearly showed that [Cu(dppz)(gly)]ClO4 is a DNA-intercalator, with a DNA-binding constant, Kb, of 2.1×106 M−1, suggesting that the mechanism of the cytotoxic activity can be related to its DNA-binding

Antimicrobial and Antibiofilm Activity of a Recombinant Fragment of β-Thymosin of Sea Urchin Paracentrotus lividus

With the aim to obtain new antimicrobials against important pathogens such as Staphylococcus aureus and Pseudomonas aeruginosa, we focused on antimicrobial peptides (AMPs) from Echinoderms. An example of such peptides is Paracentrin 1 (SP1), a chemically synthesised peptide fragment of a sea urchin thymosin. In the present paper, we report on the biological activity of a Paracentrin 1 derivative obtained by recombination. The recombinant paracentrin RP1, in comparison to the synthetic SP1, is 22 amino acids longer and it was considerably more active against the planktonic forms of S. aureus ATCC 25923 and P. aeruginosa ATCC 15442 at concentrations of 50 µg/mL. Moreover, it was able to inhibit biofilm formation of staphylococcal and P. aeruginosa strains at concentrations equal to 5.0 and 10.7 µg/mL, respectively. Molecular dynamics (MD) simulations allowed to rationalise the results of the experimental investigations, providing atomistic insights on the binding of RP1 toward models of mammalian and bacterial cell membranes. Overall, the results obtained point out that RP1 shows a remarkable preference for bacterial membranes, in excellent agreement with the antibacterial activity, highlighting the promising potential of using the tested peptide as a template for the development of novel antimicrobial agents

Paracentrin 1, a synthetic antimicrobial peptide from the sea-urchin Paracentrotus lividus, interferes with staphylococcal and Pseudomonas aeruginosa biofilm formation

The rise of antibiotic-resistance as well as the reduction of investments by pharmaceutical companies in the development of new antibiotics have stimulated the investigation for alternative strategies to conventional antibiotics. Many antimicrobial peptides show a high specificity for prokaryotes and a low toxicity for eukaryotic cells and, due to their mode of action the development of resistance is considered unlikely. We recently characterised an antimicrobial peptide that was called Paracentrin 1 from the 5-kDa peptide fraction from the coelomocyte cytosol of the Paracentrotus lividus. In this study, the chemically synthesised Paracentrin 1, was tested for its antimicrobial and antibiofilm properties against reference strains of Gram positive and Gram negative. The Paracentrin 1 was active against planktonic form of staphylococcal strains (reference and isolates) and Pseudomonas aeruginosa ATCC 15442 at concentrations ranging from 12.5 to 6.2 mg/ml. The Paracentrin 1 was able to inhibit biofilm formation of staphylococcal and Pseudomonas aeruginosa strains at concentrations ranging from 3.1 to 0.75 mg/ml. We consider the tested peptide as a good starting molecule for novel synthetic derivatives with improved pharmaceutical potentia

Molecular basis for endocrine disruption by pesticides targeting aromatase and estrogen receptor

The intensive use of pesticides has led to their increasing presence in water, soil, and agricultural products. Mounting evidence indicates that some pesticides may be endocrine disrupting chemicals (EDCs), being therefore harmful for the human health and the environment. In this study, three pesticides, glyphosate, thiacloprid, and imidacloprid, were tested for their ability to interfere with estrogen biosynthesis and/or signaling, to evaluate their potential action as EDCs. Among the tested compounds, only glyphosate inhibited aromatase activity (up to 30%) via a non-competitive inhibition or a mixed inhibition mechanism depending on the concentration applied. Then, the ability of the three pesticides to induce an estrogenic activity was tested in MELN cells. When compared to 17\u3b2-estradiol, thiacloprid and imidacloprid induced an estrogenic activity at the highest concentrations tested with a relative potency of 5.4 7 10 1210 and 3.7 7 10 129, respectively. Molecular dynamics and docking simulations predicted the potential binding sites and the binding mode of the three pesticides on the structure of the two key targets, providing a rational for their mechanism as EDCs. The results demonstrate that the three pesticides are potential EDCs as glyphosate acts as an aromatase inhibitor, whereas imidacloprid and thiacloprid can interfere with estrogen induced signaling

A Novel Peptide with Antifungal Activity from Red Swamp Crayfish Procambarus clarkii

The defense system of freshwater crayfish Procambarus clarkii as a diversified source of bioactive molecules with antimicrobial properties was studied. Antimicrobial activity of two polypeptideenriched extracts obtained from hemocytes and hemolymph of P. clarkii were assessed against Gram positive (Staphylococcus aureus, Enterococcus faecalis) and Gram negative (Pseudomonas aeruginosa, Escherichia coli) bacteria and toward the yeast Candida albicans. The two peptide fractions showed interesting MIC values (ranging from 11 to 700 g/mL) against all tested pathogens. Polypeptideenriched extracts were further investigated using a high-resolution mass spectrometry and database search and 14 novel peptides were identified. Some peptides and their derivatives were chemically synthesized and tested in vitro against the bacterial and yeast pathogens. The analysis identified a synthetic derivative peptide, which showed an interesting antifungal (MIC and MFC equal to 31.2 g/mL and 62.5 g/mL, respectively) and antibiofilm (BIC50 equal to 23.2 g/mL) activities against Candida albicans and a low toxicity in human cells

Antimicrobial and Antibiofilm Activity of a Recombinant Fragment of β-Thymosin of Sea Urchin Paracentrotus lividus

With the aim to obtain new antimicrobials against important pathogens such as Staphylococcus aureus and Pseudomonas aeruginosa, we focused on antimicrobial peptides (AMPs) from Echinoderms. An example of such peptides is Paracentrin 1 (SP1), a chemically synthesised peptide fragment of a sea urchin thymosin. In the present paper, we report on the biological activity of a Paracentrin 1 derivative obtained by recombination. The recombinant paracentrin RP1, in comparison to the synthetic SP1, is 22 amino acids longer and it was considerably more active against the planktonic forms of S. aureus ATCC 25923 and P. aeruginosa ATCC 15442 at concentrations of 50 µg/mL. Moreover, it was able to inhibit biofilm formation of staphylococcal and P. aeruginosa strains at concentrations equal to 5.0 and 10.7 µg/mL, respectively. Molecular dynamics (MD) simulations allowed to rationalise the results of the experimental investigations, providing atomistic insights on the binding of RP1 toward models of mammalian and bacterial cell membranes. Overall, the results obtained point out that RP1 shows a remarkable preference for bacterial membranes, in excellent agreement with the antibacterial activity, highlighting the promising potential of using the tested peptide as a template for the development of novel antimicrobial agents

An omics perspective to the molecular mechanisms of anticancer metallo-drugs in the computational microscope era

Introduction: Metallo-drugs have attracted enormous interest for cancer treatment. The achievements of this drug-type are summarized by the success story of cisplatin. That being said, there have been many drawbacks with its clinical use, which prompted decades worth of research efforts to move towards safer and more effective agents, either containing platinum or different metals. Areas covered: In this review, the authors provide an atomistic picture of the molecular mechanisms involving selected metallo-drugs from structural and molecular simulation studies. They also provide an omics perspective, pointing out many unsettled aspects of the most relevant families of metallo-drugs at an epigenetic level. Expert opinion: Molecular simulations are able to provide detailed information at atomistic and temporal (ps) resolutions that are rarely accessible to experiments. The increasing accuracy of computational methods and the growing performance of computational platforms, allow us to mirror wet lab experiments in silico. Consequently, the molecular mechanisms of drugs action/failure can be directly viewed on a computer screen, like a 'computational microscope', allowing us to harness this knowledge for the design of the next-generation of metallo-drugs