Francesca Ghirga

A function-oriented synthesis (FOS) strategy guided the development of abietane-based derivatives as novel inhibitors of ArnT (4-amino-4-deoxy- l -arabinose transferase)-mediated colistin resistance in Pseudomonas aeruginosa .

Recent advancements in bioconjugation chemistry have increasingly focused on thiol-based strategies, offering reversible and stimuli-responsive mechanisms, particularly suited for biomedical applications. This review aims to provide a critical overview of the latest developments in thiol-containing linkers, such as disulfide bonds and photocleavable groups, emphasizing their role in enabling controllable and often reversible conjugation of biomolecules. The review will explore several applications, including peptide synthesis and peptide-stapling strategies, antibody-drug conjugates (ADCs), and responsive biomaterials, categorize key classes of cleavable thiol-based linkers, and analyze their mechanisms. Covering the literature from the past 15 years, focusing on innovations until 2024, this review addresses the chemical foundations and practical implementations of these systems, identifying current limitations and proposing future directions for designing selective, biocompatible, and functionally dynamic conjugation platforms.

Peptide nucleic acids (PNAs) feature a neutral peptide-like backbone, providing nuclease resistance and potential for precision medicine and diagnostics through specific DNA and RNA binding. However, their therapeutic use is hindered by poor solubility and cell permeability. In this study, we demonstrated that negatively charged PNAs can be readily loaded into the polycationic Humanized Archaeoglobus Ferritin, namely, PA3.2-HumAfFt bioconjugate system, following a divalent-cation-triggered oligomerization technique. The versatility of PNA chemistry enabled the production of synthetic nucleic acid homologues with varying lengths and charges, ranging from positive to negative. We evaluated the loading performance of HumAfFt with and without chemical modifications and investigated the release dynamics of PNAs under conditions simulating the intracellular environment. Our findings demonstrated the effective uptake, release, and biological activity of PNAs in cancer cells, notably silencing the GAPDH gene with good efficiency. This evaluation paves the way for optimizing PNA-based therapeutics and broadening their applications.

Limonium gmelini (Willd.) Kuntze, a plant widely used in traditional medicine, has garnered increasing attention for its diverse pharmacological activities, including anti-inflammatory, hepatoprotective, antioxidant, and antimicrobial effects. This study aimed to explore the chemical composition and biological activities of polysaccharides and polyphenolic compounds extracted from both the roots and aboveground parts of Limonium gmelini. Several methods of extraction, including ultrasound-assisted extraction (UAE), conventional maceration (CM), and supercritical fluid extraction (SFE), were employed to obtain bioactive fractions. Chemical profiling, primarily represented by monosaccharides and polyphenolic compounds, was characterized and analyzed using proton nuclear magnetic resonance spectroscopy (¹H-NMR) and gas chromatography-mass spectrometry (GC-MS) techniques. While polyphenol-rich fractions exhibited significant antibacterial activity, particularly against Staphylococcus epidermidis, polysaccharide-rich aqueous fractions showed minimal antibacterial activity. Among the methods, CM and UAE yielded higher polyphenol content, whereas SFE provided more selective extractions. Notably, methanolic SPE fractions derived from the roots were especially enriched in active polyphenols such as gallic acid, myricetin, and naringenin, and they exhibited the highest antibacterial activity against Staphylococcus epidermidis. In contrast, extracts from the aboveground parts showed more moderate activity and a partially different chemical profile. These findings underscore the importance of plant part selection and support the targeted use of root-derived polyphenol-enriched fractions from L. gmelini as promising candidates for the development of natural antibacterial agents. Further investigation is needed to isolate and validate the most active constituents for potential therapeutic applications.

Natural products (NPs) are highly profitable pharmacological tools due to their chemical diversity and ability to modulate biological systems. Accessing new chemical entities while retaining the biological relevance of natural chemotypes is a fundamental goal in the design of novel bioactive compounds. Notably, NPs have played a crucial role in understanding Hedgehog (HH) signalling and its pharmacological modulation in anticancer therapy. However, HH antagonists developed so far have shown several limitations, thus growing interest in the design of second‐generation HH inhibitors. Through smart manipulation of the NPs core‐scaffold, unprecedented and intriguing architectures have been achieved following different design strategies. This study reports the rational design and synthesis of a first and second generation of anthraquinone‐based hybrids by combining the rhein scaffold with variously substituted piperazine nuclei that are structurally similar to the active portion of known SMO antagonists, the main transducer of the HH pathway. A thorough functional and biological investigation identified RH2_2 and RH2_6 rhein‐based hybrids as valuable candidates for HH inhibition through SMO antagonism, with the consequent suppression of HH‐dependent tumour growth. These findings also corroborated the successful application of the NPs‐based hybrid design strategy in the development of novel NP‐based SMO antagonists.

HMGA proteins are intrinsically disordered (ID) chromatin architectural factors characterized by three DNA binding domains (AT-hooks) that allow them to bind into the DNA minor groove of AT-rich stretches. HMGA are functionally involved in regulating transcription, RNA processing, DNA repair, and chromatin remodeling and dynamics. These proteins are highly expressed and play essential functions during embryonic development. They are almost undetectable in adult tissues but are re-expressed at high levels in all cancers where they are involved in neoplastic transformation and cancer progression. We focused on identifying new small molecules capable of binding into the minor groove of AT-rich DNA sequences that could compete with HMGA for DNA binding and, thus, potentially interfere with their activities. Here, a docking-based virtual screening of a unique high diversity in-house library composed of around 1000 individual natural products identified 16 natural compounds as potential minor groove binders that could inhibit the interaction between HMGA and DNA. To verify the ability of these selected compounds to compete with HMGA proteins, we screened them using electrophoretic mobility shift assays. We identified Sorocein C, a Diels-Alder (D-A)-type adducts, isolated from Sorocea ilicifolia and Sorocea bonplandii with an HMGA/DNA-displacing activity and compared its activity with that of two structurally related compounds, Sorocein A and Sorocein B. All these compounds showed a cytotoxicity effect on cancer cells, suggesting that the Sorocein-structural family may provide new and yet unexplored chemotypes for the development of minor groove binders to be evaluated as anticancer agents.

One of the main problems in developing immunosensors featuring carbon nanotubes (CNTs) is immobilizing antibodies (Abs) onto the CNT surface to afford selective binding to target antigens (Ags). In this work, we developed a practical supramolecular Ab conjugation strategy based on resorc[4]arene modifiers. To improve the Ab orientation on the CNTs surface and optimizing the Ab/Ag interaction, we exploited the host‐guest approach by synthesizing two newly resorc[4]arene linkers R1 and R2 via well‐established procedures. The upper rim was decorated with eight methoxyl groups to promote selective recognition of the fragment crystallizable (F_c) region of the Ab. Moreover, the lower rim was functionalized with 3‐bromopropyloxy or 3‐azidopropiloxy substituents to bind the macrocycles on the multi‐walled carbon nanotubes (MWCNTs) surface. Accordingly, several chemical modifications of MWCNTs were evaluated. After the morphological and electrochemical characterization of nanomaterials, the resorc[4]arene‐modified MWCNTs were deposited onto a glassy carbon electrode surface to evaluate their potential applicability for label‐free immunosensor development. The most promising system showed an improved electrode active area (A_EL) of almost 20 % and a site‐oriented immobilization of the SARS‐CoV‐2 spike protein S1 antibody (Ab‐SPS1). The developed immunosensor revealed a good sensitivity (23.64 μA mL ng⁻¹ cm⁻²) towards the SPS1 antigen and a limit of detection (LOD) of 1.01 ng mL⁻¹.

Aiming to identify highly effective and selective G-quadruplex ligands as anticancer candidates, five natural compounds were investigated here, i.e., the alkaloids Canadine, D-Glaucine and Dicentrine, as well as the flavonoids Deguelin and Millettone, selected as analogs of compounds previously identified as promising G-quadruplex-targeting ligands. A preliminary screening with the G-quadruplex on the Controlled Pore Glass assay proved that, among the investigated compounds, Dicentrine is the most effective ligand of telomeric and oncogenic G-quadruplexes, also showing good G-quadruplex vs. duplex selectivity. In-depth studies in solution demonstrated the ability of Dicentrine to thermally stabilize telomeric and oncogenic G-quadruplexes without affecting the control duplex. Interestingly, it showed higher affinity for the investigated G-quadruplex structures over the control duplex (K_b~10⁶ vs. 10⁵ M⁻¹), with some preference for the telomeric over the oncogenic G-quadruplex model. Molecular dynamics simulations indicated that Dicentrine preferentially binds the G-quadruplex groove or the outer G-tetrad for the telomeric and oncogenic G-quadruplexes, respectively. Finally, biological assays proved that Dicentrine is highly effective in promoting potent and selective anticancer activity by inducing cell cycle arrest through apoptosis, preferentially targeting G-quadruplex structures localized at telomeres. Taken together, these data validate Dicentrine as a putative anticancer candidate drug selectively targeting cancer-related G-quadruplex structures.

In recent years, several efforts have been made to develop selective, sensitive, fast response, and miniaturized immunosensors with improved performance for the monitoring and screening of analytes in several matrices, significantly expanding the use of this technology in a broad range of applications. However, one of the main technical challenges in developing immunosensors is overcoming the complexity of binding antibodies (Abs) to the sensor surface. Most immobilizing approaches lead to a random orientation of Abs, resulting in lower binding site density and immunoaffinity. In this context, supramolecular chemistry has emerged as a suitable surface modification tool to achieve the preorganization of artificial receptors and to improve the functional properties of self-assembled monolayers. Herein, a supramolecular chemistry/nanotechnology-based platform was conceived to develop sensitive label-free electrochemical immunosensors, by using a resorcarene macrocycle as an artificial linker for the oriented antibody immobilization. To this aim, a water-soluble bifunctional resorc[4]arene architecture (RW) was rationally designed and synthesized to anchor gold-coated magnetic nanoparticles (Au@MNPs) and to maximize the amount of the active immobilized antibody (Ab) in the proper "end-on" orientation. The resulting supramolecular chemistry-modified nanoparticles, RW@Au@MNPs, were deposited onto graphite screen printed electrodes which were then employed to immobilize three different Abs. Furthermore, an immunosensor for atrazine (ATZ) analysis was realized and characterized by the differential pulse voltammetry technique to demonstrate the validity of the developed biosensing platform as a proof of concept for electrochemical immunosensors. The RW-based immunosensor improved Ab_ATZ loading on Au@MNPs and sensitivity toward ATZ by almost 1.5 times compared to the random platform. Particularly, the electrochemical characterization of the developed immunosensor displays a linearity range toward ATZ within 0.05-1.5 ng/mL, a limit of detection of 0.011 ng/ml, and good reproducibility and stability. The immunosensor was tested by analyzing spiked fortified water samples with a mean recovery ranging from 95.7 to 108.4%. The overall good analytical performances of this immunodevice suggest its application for the screening and monitoring of ATZ in real matrices. Therefore, the results highlighted the successful application of the resorc[4]arene-based sensor design strategy for developing sensitive electrochemical immunosensors with improved analytical performance and simplifying the Ab immobilization procedure.