Martina Dragone

Antimicrobial peptides (AMPs) represent promising scaffolds for the development of novel broad‐spectrum antiviral therapeutics. Based on structural modeling data, we applied a systematic sequence downsizing approach to the 20‐amino‐acid AMP Hs‐1 from Hypsiboas semilineatus to identify its minimal active region. A series of N‐ and C‐terminally truncated derivatives was synthesized to preserve the predicted amphipathic α‐helical core. Circular dichroism showed that the peptides adopt a random‐coil conformation in aqueous solution and transition to an α‐helical structure in hydrophobic environments, which is essential for membrane disruption. Antiviral screening against enveloped viruses revealed that the peptides interfere with the early stages of infection while maintaining a favorable safety profile. Notably, variant Hs‐1[7–20] showed a two‐to‐four‐fold potency enhancement over the parent peptide, indicating the successful removal of a detrimental N‐terminal segment. Further optimization via N‐terminal acetylation and C‐terminal amidation yielded Hs‐1[7–20]mod, which exhibited increased antiviral efficacy and serum stability, achieving low‐micromolar IC50 values (0.5–0.7 µM) under virus pretreatment conditions while maintaining low toxicity (CC50 > 100 µM). In conclusion, Hs‐1 was successfully optimized into a potent, stable, and safe antiviral candidate Hs‐1[7–20]mod.

Formulation strategies that modify the physicochemical behavior of herbicides may influence their environmental fate and toxicity. In this context, this study investigates the effect of β-cyclodextrin (β-CD) complexation on the solubility and aquatic toxicity of chlorpropham (CLP), monuron (MON), and propanil (PRO), herbicides still in use in different parts of the world and frequently detected in aquatic environments at concentrations ranging from ng/L to µg/L. The solubility enhancement mediated by β-cyclodextrin was explored using UV-Vis and NMR spectroscopy, evaluating the impact of complexation on herbicides’ water solubility. Ecotoxicological evaluations were performed in Raphidocelis subcapitata (R. subcapitata) and Brachionus calyciflorus (B. calyciflorus), representing primary producers and consumers. Acute toxicity in B. calyciflorus significantly increased following complexation, with LC50 values decreasing from 178.09 µM (CLP), 32.32 µM (MON), and 20.77 µM (PRO) to 4.89, 2.55, and 2.29 µM, respectively. Chronic exposure further confirmed heightened sensitivity in rotifers (EC50: 0.04 µM for β-CD: MON; 0.02 µM for β-CD:PRO). R. subcapitata exhibited higher sensitivity to CLP (EC50: 2.57 µM), consistent with its mitosis inhibition mechanism. Risk Quotient (RQ) analysis, based on current environmental concentrations, revealed an ecotoxicological concern for MON and PRO. Overall, our study indicates that although β-cyclodextrin enhances herbicides solubility, it may also increase their bioavailability and toxicity underlining the necessity to evaluate a novel formulation not only from the point of view of the efficacy enhancement.

The Ros/MucR family is constituted by proteins controlling the expression of genes crucial for the interaction with eukaryotic hosts. Ros/MucR family members were classified as H-NS-like proteins in α-proteobacteria, as they share fundamental features with H-NS proteins playing a pivotal role in controlling gene expression by structuring the bacterial genome. Here, we identified two new Ros/MucR family members in Sinorhizobium meliloti. They differ from classical MucR homologs since MucR2 lacks the circular oligomeric structure typical of other family members and MucR3 shows a concentration-dependent oligomerization ability with a low propensity to form circular particles, as shown by cryogenic electron microscopy. Moreover, MucR2 and MucR3 present a new zinc coordination sphere. The newly identified MucRs bind DNA, but lack the DNA bridging activity, which is crucial for structuring the bacterial genome. Using mass spectrometry, light scattering, NMR, EMSA and bridging assay, our study reports the first identification and characterization of two new MucRs and indicates that Ros/MucR family members control gene expression through distinct mechanisms. These results provide an important framework for future studies aimed at dissecting the interplay among MucR proteins and understanding how they can jointly orchestrate condition-dependent gene expression in bacterial species expressing multiple mucR homologous genes.

Models designed to study protein/metal ion interaction are helpful to provide insights into the rules of metal ion selectivity. In this context, the prokaryotic zinc finger family Ros/MucR offers an example of several naturally occurring homologues binding a structural zinc ion with coordination spheres characterized by different amino acid arrays. In particular, Ros87, the zinc binding domain of the protein Ros from A. tumefaciens, binds Zn(II) with a classical Cys ₂ His ₂ coordination sphere, but most of its homologues show a substitution of the second cysteine by an aspartate. In this study, the binding properties to Zn(II), Co(II) and Cd(II) of the protein Ros87‐C27D, Ros87 mutant with a CysAspHis ₂ coordination sphere, are investigated by means of UV–vis, CD and NMR spectroscopies. Dissociation constants, structural effects and the resulting mechanisms of folding are compared with the wild‐type protein bearing the classical Cys ₂ His ₂ coordination sphere. CysAspHis ₂ coordination sphere induces a two‐state mechanism of folding in the presence of all three different metals, while, differently in the case of Ros87 complexed to Zn(II) or Co(II), the presence of the second cysteine in the coordination sphere leads to the formation of a stable metal binding folding intermediate. Our study underlines how the interplay between the different metal ions and the coordinating amino acid sets is determinant in defining the different Kds and the folding pathway of a given protein.

The development of new formulations can be driven by the knowledge of host–guest complexes using cyclodextrins which have the ability to include guest molecules within their hydrophobic cavities, improving the physicochemical properties of the guest. To rationally explore new pesticide formulations, the effects of cyclodextrins on the properties of such guest molecules need to be explored. Imidacloprid is a neonicotinoid systemic insecticide used worldwide. In this study, the inclusion complexes of Imidacloprid (IMI) with β-cyclodextrin (β-CD) were prepared in the solid state by co-precipitation and the physical mixing method, with a stoichiometry of 1:1 and 1:2 molar ratios. The obtained products, Imidacloprid:β-cyclodextrin inclusion complex (IMI:β-CD), were characterized in the solid state by Fourier transform-infrared (FT-IR) spectroscopy and X-ray powder diffractometry (XRD). In solution, the 1:1 stoichiometry for the inclusion complexes was established by the Job plot method, and the binding constant of IMI:β-CD was determined by UV–vis titration. The toxicity was determined in producers and primary consumers of the freshwater trophic chain, the green alga Raphidocelis subcapitata and the rotifer Brachionus calyciflorus, respectively. The results indicated that Imidacloprid forms inclusion complexes with CDs showing improved physicochemical properties compared to free Imidacloprid. The formation of the inclusion complex reduced the chronic toxicity in rotifers when IMI concentrations were close to those of environmental concern (tenths/hundredths of micromoles/L). Therefore, CD inclusion complexes could provide important advantages to be considered for the future industrial production of new formulations.

Persistence and degradation are important factors in determining the safe use of such synthetic products, and numerous studies have been addressed to develop pesticide remediation methods aimed at ameliorating these features. In this frame, the use of different cyclodextrins (CDs) molecules has attracted considerable attention due to their well-known non-toxic nature, limited environmental impact, and capability to reduce the environmental and health risks of pesticides. CDs appear to be a valuable tool for the elimination of pesticides from polluted areas as well as for better pesticide formulations that positively influence their hydrolysis or degradation. The present work investigates the interaction between β-cyclodextrins and three commonly used pesticides (i.e., chlorpropham, monuron, and propanil) both in solution and in the solid state by means of UV-Vis, FT-IR, and X-ray powder diffractometry. We show that such interactions result in all three cases in the formation of inclusion complexes with a 1:1 stoichiometry and binding constants (K_b) of 369.9 M⁻¹ for chlorpropham, 292.3 M⁻¹ for monuron, and 298.3 M⁻¹ for propanil. We also report the energy-minimized structures in silico for each complex. Our data expand and complement the available literature data in indicating CDs as a low-cost and very effective tool capable of modulating the properties that determine the environmental fate of pesticides.

A strict interplay is known to involve copper and zinc in many cellular processes. For this reason, the results of copper’s interaction with zinc binding proteins are of great interest. For instance, copper interferences with the DNA-binding activity of zinc finger proteins are associated with the development of a variety of diseases. The biological impact of copper depends on the chemical properties of its two common oxidation states (Cu(I) and Cu(II)). In this framework, following the attention addressed to unveil the effect of metal ion replacement in zinc fingers and in zinc-containing proteins, we explore the effects of the Zn(II) to Cu(I) or Cu(II) replacement in the prokaryotic zinc finger domain. The prokaryotic zinc finger protein Ros, involved in the horizontal transfer of genes from A. tumefaciens to a host plant infected by it, belongs to a family of proteins, namely Ros/MucR, whose members have been recognized in different bacteria symbionts and pathogens of mammals and plants. Interestingly, the amino acids of the coordination sphere are poorly conserved in most of these proteins, although their sequence identity can be very high. In fact, some members of this family of proteins do not bind zinc or any other metal, but assume a 3D structure similar to that of Ros with the residues replacing the zinc ligands, forming a network of hydrogen bonds and hydrophobic interactions that surrogates the Zn-coordinating role. These peculiar features of the Ros ZF domain prompted us to study the metal ion replacement with ions that have different electronic configuration and ionic radius. The protein was intensely studied as a perfectly suited model of a metal-binding protein to study the effects of the metal ion replacement; it appeared to tolerate the Zn to Cd substitution, but not the replacement of the wildtype metal by Ni(II), Pb(II) and Hg(II). The structural characterization reported here gives a high-resolution description of the interaction of copper with Ros, demonstrating that copper, in both oxidation states, binds the protein, but the replacement does not give rise to a functional domain.