Molecular structure and antibacterialactivity of degradation products from cephalexin solutions submitted to thermal and photolytic stress

Cephalexin is a beta-lactam antibiotic of the first generation of cephalosporins which is very effective against various bacterial infections. In this work, we investigate the structure and antibacterial activity of cephalexin solutions submitted to forced degradation under heat stress and photolytic irradiation. A combination of analytical techniques gathering LC/ESI-MS and NMR spectroscopy allowed us to identify different chemical species amongst the byproducts, revealing that photolysis via UV light leads to significant amounts of oxidized species that conserve the dihydrothiazine ring adjacent to the beta-lactam ring. In contrast, thermodegradation induces the rupture of the bioactive moiety possibly with the production of cephalosporinic acid and deaminated species, which are inactive to bacteria. Microbiological analyses using E. coli as a model organism indicated that the antimicrobial capacity of samples submitted to thermolysis is suppressed while solutions submitted to irradiation with UVA light preserve their bactericidal power. Atomic force microscopy showed that cells incubated with photodegraded cephalexin are much longer than those incubated with the undegraded antibiotic, indicating that byproducts from photolysis inhibit septum formation and likely affect the action of penicillin-binding protein 3 in the divisome of E. coli cells.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)19/20907-

Modification of the linker amino acid in the cell-penetrating peptide NickFect55 leads to enhanced pDNA transfection for in vivo applications

Despite numerous efforts over the last three decades, nucleic acid-based therapeutics still lack delivery platforms in the clinical stage. Cell-penetrating peptides (CPPs) may offer solutions as potential delivery vectors. We have previously shown that designing a “kinked” structure in the peptide backbone resulted in a CPP with efficient in vitro transfection properties. Further optimization of the charge distribution in the C-terminal part of the peptide led to potent in vivo activity with the resultant CPP NickFect55 (NF55). Currently, the impact of the linker amino acid was further investigated in the CPP NF55, with the aim to discover potential transfection reagents for in vivo application. Taking into account the expression of the delivered reporter in the lung tissue of mice, and the cell transfection in the human lung adenocarcinoma cell line, the new peptides NF55-Dap and NF55-Dab* have a high potential for delivering nucleic acid-based therapeutics to treat lung associated diseases, such as adenocarcinoma.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)19/20907-72022/3056-

A biophysical study of DNA condensation mediated by histones and protamines

The compaction of long DNA strands into confined spaces such as the nuclei of eukaryotic cells is an essential phenomenon towards the emergence of elaborated forms of life. Histones and protamines are the major nucleoproteins involved in this task participating in the formation of chromatin in somatic and germinative cells, respectively. In addition to a fundamental understanding of critical biological processes, DNA condensation also holds strong potential in biotechnology. Herein, we investigate the mesoscale structure of complexes formed between DNA and histones or protamines. A sophisticated set of biophysical methods encompassing steady-state fluorimetry, small-angle X-ray scattering and infrared nano spectroscopy was used to unveil both the self-assembly and molecular interactions of these complexes. We explored the fluorescence of a molecular rotor, thioflavin T, to investigate the accessibility of ligands in the inter-base environment of DNA strands. AFM-based infrared spectroscopy was used for the first time to probe the vibrational signature of individual DNA/nucleoprotein nano assemblies and disclose secondary-structure features. Our results show that protamines form highly compact structures in which DNA folding hinders access to the inter-base spacing. These assemblies exhibit diversified secondary-structure conformations, with the presence of -sheets stabilizing the packing. In contrast, histone-based complexes are characterized by fibrillar nano assemblies exhibiting larger inter strands separations and access to guest molecules that intercalate between bases. The findings presented here may help the understanding of DNA condensation mediated by these two major nucleoproteins and may assist the optimization of gene vehicles based on these promising nano assemblies.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)FAPESP, 19/20907-

Amyloid-like self-assembly of a hydrophobic cell-penetrating peptide and its use as a carrier for nucleic acids

Cell-penetrating peptides (CPPs) are a topic subject potentially exploitable for creating new nanotherapeutics for the delivery of bioactive loads. These compounds are often classified into three major categories according to their physicochemical characteristics: cationic, amphiphilic, and hydrophobic. Among them, the group of hydrophobic CPPs has received increasing attention in recent years due to toxicity concerns posed by highly cationic CPPs. The hexapeptide PFVYLI (P: proline, F: phenylalanine, V: valine, Y: tyrosine, L: leucine and I: isoleucine), a fragment derived from the C-terminal portion of α1-antitrypsin, is a prototypal example of hydrophobic CPP. This sequence shows reduced cytotoxicity, capacity of nuclear localization, and its small size readily hints suitability as a building block to construct nanostructured materials. In this study, we examine the self-assembling properties of PFVYLI and investigate its ability to form non-covalent complexes with nucleic acids. By using a combination of biophysical tools including synchrotron small-angle X-ray scattering and atomic force microscopy-based infrared spectroscopy, we discovered that this CPP self-assembles into discrete nanofibrils with remarkable amyloidogenic features. Over the course of days, these fibrils coalesce into rod-like crystals that easily reach the micrometer range. Despite lacking cationic residues in the composition, PFVYLI forms non-covalent complexes with nucleic acids that retain -sheet pairing found in amyloid aggregates. In vitro vectorization experiments performed with double-stranded DNA fragments indicate that complexes promote the internalization of nucleic acids, revealing that tropism toward cell membranes is preserved upon complexation. On the other hand, transfection assays with splice-correction oligonucleotides (SCOs) for luciferase expression show limited bioactivity across a narrow concentration window, suggesting that propensity to form amyloidogenic aggregates may trigger endosomal entrapment. We anticipate that the findings presented here open perspectives for using this archetypical hydrophobic CPP in the fabrication of nanostructured scaffolds, which potentially integrate properties of amyloids and translocation capabilities of CPPs.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)19/20907-

Aggregation limiting cell-penetrating peptides derived from protein signal sequences

Alzheimer’s disease (AD) is the most common neurodegenerative disease (ND) and the leading cause of dementia. It is characterized by non-linear, genetic-driven pathophysiological dynamics with high heterogeneity in the biological alterations and the causes of the disease. One of the hallmarks of the AD is the progression of plaques of aggregated amyloid-β (Aβ) or neurofibrillary tangles of Tau. Currently there is no efficient treatment for the AD. Nevertheless, several breakthroughs in revealing the mechanisms behind progression of the AD have led to the discovery of possible therapeutic targets. Some of these include the reduction in inflammation in the brain, and, although highly debated, limiting of the aggregation of the Aβ. In this work we show that similarly to the Neural cell adhesion molecule 1 (NCAM1) signal sequence, other Aβ interacting protein sequences, especially derived from Transthyretin, can be used successfully to reduce or target the amyloid aggregation/aggregates in vitro. The modified signal peptides with cell-penetrating properties reduce the Aβ aggregation and are predicted to have anti-inflammatory properties. Furthermore, we show that by expressing the Aβ-EGFP fusion protein, we can efficiently assess the potential for reduction in aggregation, and the CPP properties of peptides in mammalian cells.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)19/20907-72022/3056-

Structure optimization of lipopeptide assemblies for aldol reactions in an aqueous medium

Four amphiphilic peptides were synthesized, characterized, and evaluated regarding their efficiency in the catalysis of direct aldol reactions in water. The lipopeptides differ by having a double lipid chain and a guanidinium pyrrole group functionalizing one Lys side chain. All the samples are composed of the amino acids L-proline (P), L-arginine (R), or L-lysine (K) functionalized with the cationic guanidiniocarbonyl pyrrole unit (GCP), L-tryptophan (W), and L-glycine (G), covalently linked to one or two long aliphatic chains, leading to surfactant-like designs with controlled proline protonation state and different stereoselectivity. Critical aggregation concentrations (cac) were higher in the presence of the GCP group, suggesting that self-assembly depends on charge distribution along the peptide backbone. Cryogenic Transmission Electron Microscopy (Cryo-TEM) and Small Angle X-ray Scattering (SAXS) showed a rich polymorphism including spherical, cylindrical, and bilayer structures. Molecular dynamics simulations performed to assess the lipopeptide polymorphs revealed an excellent agreement with core-shell arrangements derived from SAXS data and provided an atomistic view of the changes incurred by modifying head groups and lipid chains. The resulting nanostructures behaved as excellent catalysts for aldol condensation reactions, in which superior conversions (>99%), high diastereoselectivities (ds = 94:6), and enantioselectivities (ee = 92%) were obtained. Our findings contribute to elucidate the effect of nanoscale organization of lipopeptide assemblies in the catalysis of aldol reactions in an aqueous environment.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)19/20907-

ZnO Nanoparticle/Poly(vinyl alcohol) Nanocomposites via Microwave-Assisted Sol–Gel Synthesis for Structural Materials, UV Shielding, and Antimicrobial Activity

Polymer nanocomposites based on poly(vinyl alcohol) (PVA) and ZnO hold a privileged position in the development of organic/inorganic hybrid multifunctional materials for applications ranging from food packing to biotechnological platforms. However, a remarkable drawback is that most of the currently available synthetic routes are based on approaches that are both time- and energy-consuming and often lead to heterogeneous polymer films that require compatibilizers to disperse inorganic nanoparticles into the organic matrix. In this work, we present a route for synthesizing ZnO_PVA nano-composite films through a sol−gel strategy that uses microwaves as a heat source and PVA as a reactant. We show that nanocomposites produced using this approach exhibit enhanced mechanical properties, UV shielding capabilities, and antimicrobial activity and potentialize their application in the production of antibacterial films against Gram-positive and Gram-negative strains. We show that these properties are easily modulated by controlling the synthesis parameters, such as the irradiation time and power, and the use of PVA excludes the need for compatibilizers since it simultaneously behaves as the polymer matrix and a mediator for in situ synthesis of nanostructured ZnO clusters. The method presented here is straightforward, inexpensive, and applied to other polyols to enhance the functionalities of materials based on these compounds.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)19/20907-