Angelo Spinello

Toward a rationale for the PTC124 (Ataluren) promoted readthrough of premature stop codons: a computational approach and GFP-reporter cell-based assay.

The presence in the mRNA of premature stop codons (PTCs) results in protein truncation responsible for several inherited (genetic) diseases. A well-known example of these diseases is cystic fibrosis (CF), where approximately 10% (worldwide) of patients have nonsense mutations in the CF transmembrane regulator (CFTR) gene. PTC124 (3-(5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl)-benzoic acid), also known as Ataluren, is a small molecule that has been suggested to allow PTC readthrough even though its target has yet to be identified. In the lack of a general consensus about its mechanism of action, we experimentally tested the ability of PTC124 to promote the readthrough of premature termination codons by using a new reporter. The reporter vector was based on a plasmid harboring the H2B histone coding sequence fused in frame with the green fluorescent protein (GFP) cDNA, and a TGA stop codon was introduced in the H2B-GFP gene by site-directed mutagenesis. Additionally, an unprecedented computational study on the putative supramolecular interaction between PTC124 and an 11-codon (33-nucleotides) sequence corresponding to a CFTR mRNA fragment containing a central UGA nonsense mutation showed a specific interaction between PTC124 and the UGA codon. Altogether, the H2B-GFP-opal based assay and the molecular dynamics (MD) simulation support the hypothesis that PTC124 is able to promote the specific readthrough of internal TGA premature stop codons.

Selective G-quadruplex stabilizers: Schiff-base metal complexes with anticancer activity

The affinity of three square-planar nickel(II) (1), copper(II) (2) and zinc(II) (3) Schiff-base complexes for wild-type human telomeric (h-Telo) and protooncogene c-myc G-quadruplex (G4) DNA was investigated by UV-visible absorption spectroscopy and circular dichroism. DNA-binding constants (Kb) were determined by spectrophotometric titrations for both G4-DNA and B-DNA. The results obtained point out that the three metal complexes selectively bind G4-DNA with higher affinity, up to two orders of magnitude, with respect to B-DNA. The nickel(II) complex 1 was found to be the most effective G4-DNA stabilizer and the Kb values decrease in the order 1 > 2 ≈ 3. Innovative computational investigations, consisting of molecular dynamics (MD) simulations followed by density functional theory/molecular mechanics (DFT/MM) calculations, provide atomistic support for the interpretation of the binding mechanism to G4-DNA by end stacking and also of the experimental affinity order. Interestingly, 1 is able to induce G4-DNA formation of h-Telo sequences, also in the absence of K+ cations. This last result is nicely confirmed and highlighted by polymerase chain reaction (PCR) stop assays, which show the ability of the title compounds to induce and stabilize G4 structures inhibiting the amplification of PCR products. Finally, compounds 1–3 showed concentration and time-dependent cytotoxicity towards HeLa and MCF-7 human cancer cell lines, inducing significant effects on cell cycle distribution with G2/M arrest in HeLa cells and G0/G1 arrest in MCF-7 cells. Overall, the PCR inhibition and anticancer activity of the three compounds decreases in the same order 1 > 2 ≈ 3, in excellent correlation with the G4-DNA-binding affinity, implying that G4-DNA is the biotarget for their biological activity.

A theoretical and experimental investigation of the spectroscopic properties of a DNA-intercalator salphen-type Zn(II) complex.

The photophysical and DNA-binding properties of the cationic zinc(II) complex of 5-triethylammonium methyl salicylidene ortho-phenylenediiminato (ZnL(2+)) were investigated by a combination of experimental and theoretical methods. DFT calculations were performed on both the ground and the first excited states of ZnL(2+) and on its possible mono- and dioxidation products, both in vacuo and in selected solvents mimicked by the polarizable continuum model. Comparison of the calculated absorption and fluorescence transitions with the corresponding experimental data led to the conclusion that visible light induces a two-electron photooxidation process located on the phenylenediiminato ligand. Kinetic measurements, performed by monitoring absorbance changes over time in several solvents, are in agreement with a slow unimolecular photooxidation process, which is faster in water and slower in less polar solvents. Moreover, structural details of ZnL-DNA binding were obtained by DFT calculations on the intercalation complexes between ZnL and the d(ApT)2 and d(GpC)2 dinucleoside monophosphate duplexes. Two main complementary binding interactions are proposed: 1) intercalation of the central phenyl ring of the ligand between the stacked DNA base pairs; 2) external electrostatic attraction between the negatively charged phosphate groups and the two cationic triethylammonium groups of the Schiff-base ligand. Such suggestions are supported by fluorescence titrations performed on the ZnL/DNA system at different ionic strengths and temperatures. In particular, the values of the DNA-binding constants obtained at different temperatures provided the enthalpic and entropic contributions to the binding and confirmed that two competitive mechanisms, namely, intercalation and external interaction, are involved. The two mechanisms are coexistent at room temperature under physiological conditions.

Circular Dichroism of DNA G-Quadruplexes: Combining Modeling and Spectroscopy To Unravel Complex Structures

We report on the comparison between the computational and experimental determination of electronic circular dichroism spectra of different guanine quadruplexes obtained from human telomeric sequences. In particular the difference between parallel, antiparallel, and hybrid structures is evidenced, as well as the induction of transitions between the polymorphs depending on the solution environment. Extensive molecular dynamics simulations (MD) are used to probe the conformational space of the different quadruplexes, and subsequently state-of-the-art hybrid quantum mechanics/molecular mechanics (QM/MM) techniques coupled with excitonic semiempirical Hamiltonian are used to simulate the macromolecular induced circular dichroism. The coupling of spectroscopy and molecular simulation allows an efficient one-to-one mapping between structures and optical properties, offering a way to disentangle the rich, yet complicated, quantity of information embedded in circular dichroism spectra. We show that our methodology is robust and efficient and allows us to take into account subtle conformational changes. As such, it could be used as an efficient tool to investigate structural modification upon DNA/drug interactions.

Enhancement of premature stop codon readthrough in the CFTR gene by Ataluren (PTC124) derivatives

Premature stop codons are the result of nonsense mutations occurring within the coding sequence of a gene. These mutations lead to the synthesis of a truncated protein and are responsible for several genetic diseases. A potential pharmacological approach to treat these diseases is to promote the translational readthrough of premature stop codons by small molecules aiming to restore the full-length protein. The compound PTC124 (Ataluren) was reported to promote the readthrough of the premature UGA stop codon, although its activity was questioned. The potential interaction of PTC124 with mutated mRNA was recently suggested by molecular dynamics (MD) studies highlighting the importance of H-bonding and stacking π-π interactions. To improve the readthrough activity we changed the fluorine number and position in the PTC124 fluoroaryl moiety. The readthrough ability of these PTC124 derivatives was tested in human cells harboring reporter plasmids with premature stop codons in H2BGFP and FLuc genes as well as in cystic fibrosis (CF) IB3.1 cells with a nonsense mutation. Maintaining low toxicity, three of these molecules showed higher efficacy than PTC124 in the readthrough of the UGA premature stop codon and in recovering the expression of the CFTR protein in IB3.1 cells from cystic fibrosis patient. Molecular dynamics simulations performed with mutated CFTR mRNA fragments and active or inactive derivatives are in agreement with the suggested interaction of PTC124 with mRNA.

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 characterized 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 synthesized 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 potential.

The inhibition of glycerol permeation through aquaglyceroporin-3 induced by mercury(II): A molecular dynamics study.

Mercurial compounds are known to inhibit water permeation through aquaporins (AQPs). Although in the last years some hypotheses were proposed, the exact mechanism of inhibition is still an open question and even less is known about the inhibition of the glycerol permeation through aquaglyceroporins. Molecular dynamics (MD) simulations of human aquaporin-3 (AQP3) have been performed up to 200ns in the presence of Hg(2+) ions. For the first time, we have observed the unbiased passage of a glycerol molecule from the extracellular to cytosolic side. Moreover, the presence of Hg(2+) ions covalently bound to Cys40 leads to a collapse of the aromatic/arginine selectivity filter (ar/R SF), blocking the passage of both glycerol and water. Interestingly, the local conformational changes of the protein follow mercury coordination by water and by aminoacidic donor atoms. Overall, the obtained results are important to improve the design of selective AQP inhibitors for future therapeutic and imaging applications.

Quaternary structures of GroEL and naïve-Hsp60 chaperonins in solution: A combined SAXS-MD study

The quaternary structures of bacterial GroEL and human naive-Hsp60 chaperonins in physiological conditions have been investigated by an innovative approach based on a combination of synchrotron Small Angle X-ray Scattering (SAXS) in-solution experiments and molecular dynamics (MD) simulations. Low-resolution SAXS experiments over large and highly symmetric oligomers are analyzed on the basis of the high-resolution structure of the asymmetric protein monomers, provided by MD. The results reveal remarkable differences between the solution and the crystallographic structure of GroEL and between the solution structures of GroEL and of its human homologue Hsp60.

The Right Answer for the Right Electrostatics: Force Field Methods Are Able to Describe Relative Energies of DNA Guanine Quadruplexes.

Different force fields and approximate density functional theory were applied in order to study the rotamer space of the telomeric G-quadruplex DNA. While some force fields show an erratic behavior when it comes to the reproduction of the higher-order DNA conformer space, OPLS and MMFF implementations are able to reproduce the experimentally known energy order. The stabilizing effect of the AA (anti-anti) versus SA (syn-anti) conformer is analyzed applying mechanical bond strength descriptors (compliance constants). The fact that we observe the correct energy order using appropriate force fields is in contrast with results previously reported, which suggested the general inappropriateness of force fields for the description of G-quadruplex structures.

Metal Ions and Metal Complexes in Alzheimer’s Disease

BACKGROUND Alzheimers disease (AD) is the most common form of dementia that seriously affects daily life. Even if AD pathogenesis is still subject of debate, it is generally accepted that cerebral cortex plaques formed by aggregated amyloid-β (Aβ) peptides can be considered a characteristic pathological hallmark. It is well known that metal ions play an important role in the aggregation process of Aβ. METHODS This review focuses on the anti-Aβ aggregation activity of chelating ligands as well as on the use of metal complexes as diagnostic probes and as potential drugs. CONCLUSION While chelating agents, such as curcumin or flavonoid derivatives, are currently used to capture metal ions responsible for Aβ aggregation, the potential application of platinum, ruthenium and cobalt complexes, among others, of several heterocyclic ligands, represents a promising new strategy to fight AD.