γ sulphate PNA (PNA S): Highly Selective DNA Binding Molecule Showing Promising Antigene Activity

Peptide Nucleic Acids (PNAs), nucleic acid analogues showing high stability to enzyme degradation and strong affinity and specificity of binding toward DNA and RNA are widely investigated as tools to interfere in gene expression. Several studies have been focused on PNA analogues with modifications on the backbone and bases in the attempt to overcome solubility, uptake and aggregation issues. γ PNAs, PNA derivatives having a substituent in the γ position of the backbone show interesting properties in terms of secondary structure and affinity of binding toward complementary nucleic acids. In this paper we illustrate our results obtained on new analogues, bearing a sulphate in the γ position of the backbone, developed to be more DNA-like in terms of polarity and charge. The synthesis of monomers and oligomers is described. NMR studies on the conformational properties of monomers and studies on the secondary structure of single strands and triplexes are reported. Furthermore the hybrid stability and the effect of mismatches on the stability have also been investigated. Finally, the ability of the new analogue to work as antigene, interfering with the transcription of the ErbB2 gene on a human cell line overexpressing ErbB2 (SKBR3), assessed by FACS and qPCR, is described

MucR binds multiple target sites in the promoter of its own gene and is a heat-stable protein: Is MucR a H-NS-like protein?

The protein MucR from Brucella spp. is involved in the expression regulation of genes necessary for host interaction and infection. MucR is a member of the Ros/MucR family, which comprises prokaryotic zinc-finger proteins and includes Ros from Agrobacterium tumefaciens and the Ml proteins from Mesorhizobium loti. MucR from Brucella spp. can regulate the expression of virulence genes and repress its own gene expression. Despite the well-known role played by MucR in the repression of its own gene, no target sequence has yet been identified in the mucR promoter gene. In this study, we provide the first evidence that MucR from Brucella abortus binds more than one target site in the promoter region of its own gene, suggesting a molecular mechanism by which this protein represses its own expression. Furthermore, a circular dichroism analysis reveals that MucR is a heat-stable protein. Overall, the results of this study suggest that MucR might resemble a H-NS protein

High-Resolution Conformational Analysis of RGDechi-Derived Peptides Based on a Combination of NMR Spectroscopy and MD Simulations

The crucial role of integrin in pathological processes such as tumor progression and metastasis formation has inspired intense efforts to design novel pharmaceutical agents modulating integrin functions in order to provide new tools for potential therapies. In the past decade, we have investigated the biological proprieties of the chimeric peptide RGDechi, containing a cyclic RGD motif linked to an echistatin C-terminal fragment, able to specifically recognize αvβ3 without cross reacting with αvβ5 and αIIbβ3 integrin. Additionally, we have demonstrated using two RGDechi-derived peptides, called RGDechi1-14 and ψRGDechi, that chemical modifications introduced in the C-terminal part of the peptide alter or abolish the binding to the αvβ3 integrin. Here, to shed light on the structural and dynamical determinants involved in the integrin recognition mechanism, we investigate the effects of the chemical modifications by exploring the conformational space sampled by RGDechi1-14 and ψRGDechi using an integrated natural-abundance NMR/MD approach. Our data demonstrate that the flexibility of the RGD-containing cycle is driven by the echistatin C-terminal region of the RGDechi peptide through a coupling mechanism between the N- and C-terminal regions

Interaction between fac-[Re(H2O)3(CO)3]+ ion and histidine in aqueous solution

The chemistry of Rhenium(I) has assumed recently an important role for the synthesis of 186,188Re radiopharmaceuticals. Furthermore, the spectral properties of Rhenium(I) tricarbonyl complexes have been demonstrated to have applications as fluorochromes in fluorescence microscopy. The organometallic complex fac-[Re(H2O)3(CO)3]+ is taken into account due to its simple synthesis and its complexing properties towards biological molecules. Although numerous studies have been carried out on complexes with organic ligands in the solid state, there is little information on the speciation of Rhenium(I) in aqueous solution. By the presence of histidine residues in many biological systems, this work concerns the complexation of the Re(CO)3+ core with histidine in solutions 0.1 M NaClO4, at 25°C by potentiometric measurements by a glass electrode in the range of pH 2-10. Order to do this is necessary to establish hydrolytic equilibria of tricabonyl Rhenium(I). Data processing suggests the formation of the two hydrolytic species: Re(OH)(CO)3 and Re(OH)2(CO)3

Folding mechanisms steer the amyloid fibril formation propensity of highly homologous proteins

Significant advances in the understanding of themolecular determinants of fibrillogenesis can be expected from comparative studies of the aggregation propensities of proteins with highly homologous structures but different folding pathways. Here,we fully characterize, bymeans of stopped-flow, T-jump, CD and DSC experiments, the unfolding mechanisms of three highly homologous proteins, zinc binding Ros87 and Ml153–149 and zinc-lacking Ml452–151. The results indicate that the three proteins significantly differ in terms of stability and (un)folding mechanisms. Particularly, Ros87 and Ml153–149 appear to be much more stable to guanidine denaturation and are characterized by folding mechanisms including the presence of an intermediate. On the other hand, metal lacking Ml452–151 folds according to a classic two-state model. Successively, we have monitored the capabilities of Ros87, Ml452–151 and Ml153–149 to form amyloid fibrils under native conditions. Particularly, we show, by CD, fluorescence, DLS, TEM and SEM experiments, that after 168 hours, amyloid formation of Ros87 has started, while Ml153–149 has formed only amorphous aggregates and Ml452–151 is still monomeric in solution. This study shows how metal binding can influence protein folding pathways and thereby control conformational accessibility to aggregation-prone states, which in turn changes aggregation kinetics, shedding light on the role of metal ions in the development of protein deposition disease

Polypseudorotaxanes of Pluronic® F127 with Combinations of α- and β-Cyclodextrins for Topical Formulation of Acyclovir

Acyclovir (ACV) is one of the most used antiviral drugs for the treatment of herpes simplex virus infections and other relevant mucosal infections caused by viruses. Nevertheless, the low water solubility of ACV limits both its bioavailability and antiviral performance. The combination of block copolymer micelles and cyclodextrins (CDs) may result in polypseudorotaxanes with tunable drug solubilizing and gelling properties. However, the simultaneous addition of various CDs has barely been investigated yet. The aim of this work was to design and characterize ternary combinations of Pluronic® F127 (PF127), αCD and βCD in terms of polypseudorotaxane formation, rheological behavior, and ACV solubilization ability and controlled release. The formation of polypseudorotaxanes between PF127 and the CDs was confirmed by FT-IR spectroscopy, X-ray diffraction, and NMR spectroscopy. The effects of αCD/βCD concentration range (0–7% w/w) on copolymer (6.5% w/w) gel features were evaluated at 20 and 37 °C by rheological studies, resulting in changes of the copolymer gelling properties. PF127 with αCD/βCD improved the solubilization of ACV, maintaining the biocompatibility (hen’s egg test on the chorio-allantoic membrane). In addition, the gels were able to sustain acyclovir delivery. The formulation prepared with similar proportions of αCD and βCD provided a slower and more constant release. The results obtained suggest that the combination of Pluronic with αCD/βCD mixtures can be a valuable approach to tune the rheological features and drug release profiles from these supramolecular gelsThis research was funded by MIUR PRIN [20157WZM8A] (Italy), MINECO [SAF2017-83118-R] (Spain), Agencia Estatal de Investigación (AEI, Spain), Xunta de Galicia [ED431C 2016/008; AEMAT ED431E 2018/08] and European Regional Development Fund (FEDER)S

fac-[Re(H2O)3(CO)3]+Complexed with Histidine and Imidazole in Aqueous Solution: Speciation, Affinity and Binding Features

The use of isotopes of transition metal has opened many perspectives in the development of novel compounds utilizable for therapeutic or diagnostic purposes. In order to be exploited, though, they must be stabilized by chelating systems in coordination complexes. The tricarbonyl complexes of Technetium(I) and Rhenium(I) have assumed important roles in these fields as they are readily conjugated to biomolecules to form stable probe useful in the targeting approach. In this context the amino acid histidine represents a valuable ligand for bioconjugation. Since little is reported on the chemistry and speciation of Re(I) and Tc(I) in aqueous solution, here, we analyze the interaction of fac-[Re(H2O)3(CO)3]+ with histidine or with the simple imidazole ring by means of potentiometric and spectroscopic techniques. We describe in details the equilibria that arise in solution, calculate the constants of each equilibrium reaction and report coordination properties and atomic details of the formed complexes. Our data represent a valuable contribute towards the exploitation in nuclear medicine of histidine containing biomolecules complexing Tc (I) and Re(I)

Host and Viral Zinc-Finger Proteins in COVID-19

An unprecedented effort to tackle the ongoing COVID-19 pandemic has characterized the activity of the global scientific community over the last two years. Hundreds of published studies have focused on the comprehension of the immune response to the virus and on the definition of the functional role of SARS-CoV-2 proteins. Proteins containing zinc fingers, both belonging to SARS-CoV-2 or to the host, play critical roles in COVID-19 participating in antiviral defenses and regulation of viral life cycle. Differentially expressed zinc finger proteins and their distinct activities could thus be important in determining the severity of the disease and represent important targets for drug development. Therefore, we here review the mechanisms of action of host and viral zinc finger proteins in COVID-19 as a contribution to the comprehension of the disease and also highlight strategies for therapeutic developments