Editorial: Special Issue "Galectins: Structure, Function and Therapeutic Inhibitors".

Galectins, ß-galactoside-binding proteins, play relevant roles in different biological processes; therefore, they are becoming emerging targets for diagnostic and therapeutic approaches [...].

Biophysical and Structural Characterization of the Interaction between Human Galectin-3 and the Lipopolysaccharide from Pseudomonas aeruginosa.

Given the significant involvement of galectins in the development of numerous diseases, the aim of the following work is to further study the interaction between galectin-3 (Gal3) and the LPS from Pseudomonas aeruginosa. This manuscript focused on the study of the interaction of the carbohydrate recognition domain of Gal3 with the LPS from Pseudomonas aeruginosa by means of different complementary methodologies, such as circular dichroism; spectrofluorimetry; dynamic and static light scattering and evaluation of the impact of Gal3 on the redox potential membranes of Escherichia coli and P. aeruginosa cells, as well as ITC and NMR studies. This thorough investigation reinforces the hypothesis of an interaction between Gal3 and LPS, unraveling the structural details and providing valuable insights into the formation of these intricate molecular complexes. Taken together, these achievements could potentially prompt the design of therapeutic drugs useful for the development of agonists and/or antagonists for LPS receptors such as galectins as adjunctive therapy for P. aeruginosa.

Identification of Mortalin as the Main Interactor of Mycalin A, a Poly-Brominated C-15 Acetogenin Sponge Metabolite, by MS-Based Proteomics.

Mycalin A (MA) is a polybrominated C-15 acetogenin isolated from the marine sponge Mycale rotalis. Since this substance displays a strong antiproliferative bioactivity towards some tumour cells, we have now directed our studies towards the elucidation of the MA interactome through functional proteomic approaches, (DARTS and t-LIP-MS). DARTS experiments were performed on Hela cell lysates with the purpose of identifying MA main target protein(s); t-LiP-MS was then applied for an in-depth investigation of the MA-target protein interaction. Both these techniques exploit limited proteolysis coupled with MS analysis. To corroborate LiP data, molecular docking studies were performed on the complexes. Finally, biological and SPR analysis were conducted to explore the effect of the binding. Mortalin (GRP75) was identified as the MA's main interactor. This protein belongs to the Hsp70 family and has garnered significant attention due to its involvement in certain forms of cancer. Specifically, its overexpression in cancer cells appears to hinder the pro-apoptotic function of p53, one of its client proteins, because it becomes sequestered in the cytoplasm. Our research, therefore, has been focused on the possibility that MA might prevent this sequestration, promoting the re-localization of p53 to the nucleus and facilitating the apoptosis of tumor cells.

Synthesis of Brominated Lactones Related to Mycalin A: Selective Antiproliferative Activity on Metastatic Melanoma Cells and Inhibition of the Cell Migration.

Starting from D-xylonolactone and D-ribonolactone, several five-membered bromolactones, related to the C1-C5 portion of mycalin A lactone, have been synthesized. The bromination of D-ribonolactone with HBr/AcOH, without a subsequent transesterification step, has been studied for the first time, giving us most of the acetylated lactones investigated in the present study. For each compound, where possible, both the C-3 alcohol and the corresponding acetate were prepared. Evaluation of their anti-tumor activity showed that all the acetates possess a good cytotoxicity towards human melanoma (A375), human cervical adenocarcinoma (HeLa) and human metastatic melanoma (WM266) cancer cells, comparable or even higher than that displayed by the original mycalin A lactone. Lactone acetates derived from D-ribonolactone showed the higher selectivity of action, exhibiting a strong cytotoxicity on all the tested tumor cells but only a limited toxicity on healthy human dermal fibroblast (HDF) cells, used as a control. Wound healing assays showed that two of these substances inhibit the migration of the WM266 cells.

Exploring the Molecular Interactions of Symmetrical and Unsymmetrical Selenoglycosides with Human Galectin-1 and Galectin-3.

Galectins (Gals) are small cytosolic proteins that bind ß-galactoside residues via their evolutionarily conserved carbohydrate recognition domain. Their dysregulation has been shown to be associated with many diseases. Consequently, targeting galectins for clinical applications has become increasingly relevant to develop tailored inhibitors selectively for one galectin. Accordingly, binding studies providing the molecular details of the interaction between galectin and inhibitor may be useful for the rational design of potent and selective antagonists. Gal-1 and Gal-3 are among the best-studied galectins, mainly for their roles in cancer progression; therefore, the molecular details of their interaction with inhibitors are demanded. This work gains more value by focusing on the interaction between Gal-1 and Gal-3 with the selenylated analogue of the Gal inhibitor thiodigalactose, characterized by a selenoglycoside bond (SeDG), and with unsymmetrical diglycosyl selenides (unsym(Se). Gal-1 and Gal-3 were produced heterologously and biophysically characterized. Interaction studies were performed by ITC, NMR spectroscopy, and MD simulation, and thermodynamic values were discussed and integrated with spectroscopic and computational results. The 3D complexes involving SeDG when interacting with Gal-1 and Gal-3 were depicted. Overall, the collected results will help identify hot spots for the design of new, better performing, and more specific Gal inhibitors.

Phosphodiester Silybin Dimers Powerful Radical Scavengers: A Antiproliferative Activity on Different Cancer Cell Lines.

Silibinin is the main biologically active component of silymarin extract and consists of a mixture 1:1 of two diastereoisomeric flavonolignans, namely silybin A (1a) and silybin B (1b), which we call here silybins. Despite the high interest in the activity of this flavonolignan, there are still few studies that give due attention to the role of its stereochemistry and, there is still today a strong need to investigate in this area. In this regard, here we report a study concerning the radical scavenger ability and the antiproliferative activity on different cell lines, both of silybins and phosphodiester-linked silybin dimers. An efficient synthetic strategy to obtain silybin dimers in an optical pure form (6aa, 6ab and 6bb) starting from a suitable building block of silybin A and silybin B, obtained by us from natural extract silibinin, was proposed. New dimers show strong antioxidant properties, determined through hydroxyl radical (HO●) scavenging ability, comparable to the value reported for known potent antioxidants such as quercetin. A preliminary screening was performed by treating cells with 10 and 50 µM concentrations for 48 h to identify the most sensitive cell lines. The results show that silibinin compounds were active on Jurkat, A375, WM266, and HeLa, but at the tested concentrations, they did not interfere with the growth of PANC, MCF-7, HDF or U87. In particular, both monomers (1a and 1b) and dimers (6aa, 6ab and 6bb) present selective anti-proliferative activity towards leukemia cells in the mid-micromolar range and are poorly active on normal cells. They exhibit different mechanisms of action in fact all the cells treated with the 1a and 1b go completely into apoptosis, whereas only part of the cells treated with 6aa and 6ab were found to be in apoptosis.

Design, Synthesis, and Anticancer Activity of a Selenium-Containing Galectin-3 and Galectin-9N Inhibitor.

Galectins are soluble ß-D-galactoside-binding proteins whose implication in cancer progression and disease outcome makes them prominent targets for therapeutic intervention. In this frame, the development of small inhibitors that block selectively the activity of galectins represents an important strategy for cancer therapy which is, however, still relatively underdeveloped. To this end, we designed here a rationally and efficiently novel diglycosylated compound, characterized by a selenoglycoside bond and the presence of a lipophilic benzyl group at both saccharide residues. The relatively high binding affinity of the new compound to the carbohydrate recognition domain of two galectins, galectin 3 and galectin 9, its good antiproliferative and anti-migration activity towards melanoma cells, as well as its anti-angiogenesis properties, pave the way for its further development as an anticancer agent.

Screening a Molecular Fragment Library to Modulate the PED/PEA15-Phospholipase D1 Interaction in Cellular Lysate Environments.

The overexpression of PED/PEA15, the phosphoprotein enriched in diabetes/phosphoprotein enriched in the astrocytes 15 protein (here referred simply to as PED), observed in some forms of type II diabetes, reduces the transport of insulin-stimulated glucose by binding to the phospholipase D1 (PLD1). The inhibition of the PED/PLD1 interaction was shown to restore basal glucose transport, indicating PED as a pharmacological target for the development of drugs capable of improving insulin sensitivity and glucose tolerance. We here report the identification and selection of PED ligands by means of NMR screening of a library of small organic molecules, NMR characterization of the PED/PLD1 interaction in lysates of cells expressing PLD1, and modulation of such interactions using BPH03, the best selected ligand. Overall, we complement the available literature data by providing detailed information on the structural determinants of the PED/PLD1 interaction in a cellular lysate environment and indicate BPH03 as a precious scaffold for the development of novel compounds that are able to modulate such interactions with possible therapeutic applications in type II diabetes.

More Is Always Better Than One: The N-Terminal Domain of the Spike Protein as Another Emerging Target for Hampering the SARS-CoV-2 Attachment to Host Cells.

Although the approved vaccines are proving to be of utmost importance in containing the Coronavirus disease 2019 (COVID-19) threat, they will hardly be resolutive as new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, a single-stranded RNA virus) variants might be insensitive to the immune response they induce. In this scenario, developing an effective therapy is still a dire need. Different targets for therapeutic antibodies and diagnostics have been identified, among which the SARS-CoV-2 spike (S) glycoprotein, particularly its receptor-binding domain, has been defined as crucial. In this context, we aim to focus attention also on the role played by the S N-terminal domain (S1-NTD) in the virus attachment, already recognized as a valuable target for neutralizing antibodies, in particular, building on a cavity mapping indicating the presence of two druggable pockets and on the recent literature hypothesizing the presence of a ganglioside-binding domain. In this perspective, we aim at proposing S1-NTD as a putative target for designing small molecules hopefully able to hamper the SARS-CoV-2 attachment to host cells.

A novel approach for studying receptor-ligand interactions on living cells surface by using NUS/T1ρ-NMR methodologies combined with computational techniques: The RGDechi15D-αvß5 integrin complex.

Structural investigations of receptor-ligand interactions on living cells surface by high-resolution Nuclear Magnetic Resonance (NMR) are problematic due to their short lifetime, which often prevents the acquisition of experiments longer than few hours. To overcome these limitations, we developed an on-cell NMR-based approach for exploring the molecular determinants driving the receptor-ligand recognition mechanism under native conditions. Our method relies on the combination of high-resolution structural and dynamics NMR data with Molecular Dynamics simulations and Molecular Docking studies. The key point of our strategy is the use of Non Uniform Sampling (NUS) and T1ρ-NMR techniques to collect atomic-resolution structural and dynamics information on the receptor-ligand interactions with living cells, that can be used as conformational constraints in computational studies. In fact, the application of these two NMR methodologies allows to record spectra with high S/N ratio and resolution within the lifetime of cells. In particular, 2D NUS [1H-1H] trNOESY spectra are used to explore the ligand conformational changes induced by receptor binding; whereas T1ρ-based experiments are applied to characterize the ligand binding epitope by defining two parameters: T1ρ Attenuation factor and T1ρ Binding Effect. This approach has been tested to characterize the molecular determinants regulating the recognition mechanism of αvß5-integrin by a selective cyclic binder peptide named RGDechi15D. Our data demonstrate that the developed strategy represents an alternative in-cell NMR tool for studying, at atomic resolution, receptor-ligand recognition mechanism on living cells surface. Additionally, our application may be extremely useful for screening of the interaction profiling of drugs with their therapeutic targets in their native cellular environment.

Tautomeric and conformational switching in a new versatile N-rich heterocyclic ligand.

A new N-rich triazolo-triazole derivative, 4-methyl-7-(pyrazin-2-yl)-2H-[1,2,4]triazolo[3,2-c][1,2,4]triazole (C8H7N7), bearing a pyrazine residue at 7-position of the triazolo-triazole bicycle, was synthesized, and its acid-base and metal coordination properties were evaluated in solution. The results showed amphoteric behavior and the formation of stable complexes with Cu(ii) and Zn(ii) in pH intervals in which the ligand is neutral or deprotonated. Computational studies were performed in order to evaluate the stability of the different tautomers/conformers of the ligand, and the proton position in the neutral and acidic forms. Single crystal X-ray analysis of the free neutral ligand (2H/s-trans tautomer/conformer), and of its singly protonated (2H-3H/s-trans), doubly protonated (2H-3H-7H/s-trans) and deprotonated forms showed that the influence of the pyrazine ring on the triazolo-triazole system is mainly as electron withdrawing and chelating group, and proton acceptor. Different coordination modes have been evidenced for the neutral and deprotonated ligand. Upon metal coordination, the neutral ligand switches from 2H/s-trans to 3H/s-cis tautomer/conformer forming five-membered chelate rings, while the anionic deprotonated ligand forms six-membered chelate rings in the s-trans conformation. Altogether, five different tautomers/conformers of the ligand were isolated and characterized. In vitro tests confirmed the general antiproliferative activity of triazolo-triazole compounds and the importance of substitution in position 7 for their selectivity.

Synthesis, Antiproliferative Activity, and DNA Binding Studies of Nucleoamino Acid-Containing Pt(II) Complexes.

We here report our studies on the reaction with the platinum(II) ion of a nucleoamino acid constituted by the l-2,3-diaminopropanoic acid linked to the thymine nucleobase through a methylenecarbonyl linker. The obtained new platinum complexes, characterized by spectroscopic and mass spectrometric techniques, were envisaged to exploit synergistic effects due to the presence of both the platinum center and the nucleoamino acid moiety. The latter can be potentially useful to protect the complexes from early deactivation, as well as to facilitate their cell internalization. The biological activity of the complexes in terms of antiproliferative effects was evaluated in vitro on different cancer cell lines and healthy cells, showing the best results on human cervical adenocarcinoma (HeLa) cells along with good selectivity for cancer over normal cells. In contrast, the metal-free nucleoamino acid did not show any cytotoxicity on both normal and cancer cell lines. Finally, the ability of the novel Pt(II) complexes to bind various DNA model systems was investigated by circular dichroism (CD) spectroscopy and polyacrylamide gel electrophoresis analyses proving that the newly obtained compounds can potentially target DNA, similarly to other well-known anticancer Pt complexes, with a peculiar G-quadruplex vs. duplex selectivity.

Toward G-Quadruplex-Based Anticancer Agents: Biophysical and Biological Studies of Novel AS1411 Derivatives.

Certain G-quadruplex forming guanine-rich oligonucleotides (GROs), including AS1411, are endowed with cancer-selective antiproliferative activity. They are known to bind to nucleolin protein, resulting in the inhibition of nucleolin-mediated phenomena. However, multiple nucleolin-independent biological effects of GROs have also been reported, allowing them to be considered promising candidates for multi-targeted cancer therapy. Herein, with the aim of optimizing AS1411 structural features to find GROs with improved anticancer properties, we have studied a small library of AS1411 derivatives differing in the sequence length and base composition. The AS1411 derivatives were characterized by using circular dichroism and nuclear magnetic resonance spectroscopies and then investigated for their enzymatic resistance in serum and nuclear extract, as well as for their ability to bind nucleolin, inhibit topoisomerase I, and affect the viability of MCF-7 human breast adenocarcinoma cells. All derivatives showed higher thermal stability and inhibitory effect against topoisomerase I than AS1411. In addition, most of them showed an improved antiproliferative activity on MCF-7 cells compared to AS1411 despite a weaker binding to nucleolin. Our results support the hypothesis that the antiproliferative properties of GROs are due to multi-targeted effects.

Selective Targeting of αvß5 Integrin in HepG2 Cell Line by RGDechi15D Peptide.

Recently, the research community has become increasingly concerned with the receptor αvß5, a member of the well-known integrin family. Different ongoing studies have evidenced that αvß5 integrin regulates not only physiological processes but also a wide array of pathological events, suggesting the receptor as a valuable biomarker to specifically target for therapeutic/diagnostic purposes. Remarkably, in some tumors the involvement of the receptor in cell proliferation, tumor dissemination and angiogenesis is well-documented. In this scenario, the availability of a selective αvß5 antagonist without 'off-target' protein effects may improve survival rate in patients with highly aggressive tumors, such as hepatocellular carcinoma. We recently reported a cyclic peptide, RGDechi15D, obtained by structure-activity studies. To our knowledge it represents the first peptide-based molecule reported in the literature able to specifically bind αvß5 integrin and not cross react with αvß3. Here we demonstrated the ability of the peptide to diminish both adhesion and invasion of HepG2 cells, an in vitro model system for hepatocellular carcinoma, to reduce the cell proliferation through an apoptotic process, and to interfere with the PI3K pathway. The peptide, also decreases the formation of new vessels in endothelial cells. Taken together these results indicate that the peptide can be considered a promising molecule with properties suited to be assessed in the future for its validation as a selective therapeutic/diagnostic weapon in hepatocarcinoma.

Antiproliferative Activity of Mycalin A and Its Analogues on Human Skin Melanoma and Human Cervical Cancer Cells.

Mycalin A, a polybrominated C15 acetogenin isolated from the encrusting sponge Mycale rotalis, displays an antiproliferative activity on human melanoma (A375) and cervical adenocarcinoma (HeLa) cells and induces cell death by an apoptotic mechanism. Various analogues and degraded derivatives of the natural substance have been prepared. A modification of the left-hand part of the molecule generates the most active substances. A structurally simplified lactone derivative of mycalin A, lacking the C1-C3 side chain, is the most active among the synthesized compounds exhibiting a strong cytotoxicity on both A375 and HeLa cells but not but not on human dermal fibroblast (HDF) used as healthy cells. Further evidence on a recently discovered chlorochromateperiodate-catalyzed process, used to oxidise mycalin A, have been collected.

A Multi-Targeting Approach to Fight SARS-CoV-2 Attachment.

The public health has declared an international state of emergency due to the spread of a new coronavirus (SARS-CoV-2) representing a real pandemic threat so that to find potential therapeutic agents is a dire need. To this aim, the SARS-CoV-2 spike (S) glycoprotein represents a crucial target for vaccines, therapeutic antibodies, and diagnostics. Since virus binding to ACE-2 alone could not be sufficient to justify such severe infection, in order to facilitate medical countermeasure development and to search for new targets, two further regions of S protein have been taken into consideration here. One is represented by the recently identified ganglioside binding site, exactly localized in our study in the galectin-like domain, and the other one by the putative integrin binding sites contained in the RBD. We propose that a cooperating therapy using inhibitors against multiple targets altogether i.e., ACE2, integrins and sugars could be definitely more effective.

Probing the DNA Reactivity and the Anticancer Properties of a Novel Tubercidin-Pt(II) Complex.

Herein, we reported on the synthesis of a novel Pt(II) neutral complex having as ligand the nucleoside tubercidin, a potent anti-tumor agent extracted from the bacterium Streptomyces Tubercidicus. In detail, the chelation of the metal by a diamine linker installed at C6 purine position of tubercidin assured the introduction of a cisplatin-like unit in the molecular scaffold. The behavior of the synthesized complex with a double-strand DNA model was monitored by CD spectroscopy and compared with that of cisplatin and tubercidin. In addition, the cell viability was evaluated against HeLa, A375 and WM266 human cancer cell lines using the MTT test. Lastly, the results of the apoptotic assay (FITC Annexin V) performed on the HeLa cancer cell line are also reported.

Bio-Inspired Dual-Selective BCL-2/c-MYC G-Quadruplex Binders: Design, Synthesis, and Anticancer Activity of Drug-like Imidazo[2,1-i]purine Derivatives.

In the search for new drug-like selective G-quadruplex binders, a bioinspired design focused on the use of nucleobases as synthons in a multicomponent reaction was herein proved to be viable and successful. Hence, a new class of multifunctionalized imidazo[2,1-i]purine derivatives, easily synthesized with a convergent approach, allowed for the identification of the first dual BCL2/c-MYC gene promoter G-quadruplex ligand. Biophysical studies involving circular dichroism melting experiments, microscale thermophoresis measurements, NMR titrations, and computational docking calculations, as well as biological investigations including cytotoxicity and apoptotic assays, and quantitative polymerase chain reaction and Western blot analyses, were performed to assess the potency and to characterize the binding mode of the newly identified lead compound. The absence of toxicity toward normal cells, together with the small molecular weight (≅500 Da), the water solubility, the ease of functionalization, and the selectivity profile, are promising and desirable features to develop G-quadruplex binders as safe and effective anticancer agents.

Synthesis, DNA binding studies, and antiproliferative activity of novel Pt(II)-complexes with an L-alanyl-based ligand.

An artificial alanine-based amino acid {(S)-2-amino-3-[4-propyl-3-(thiophen-2-yl)-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-1-yl]propanoic acid, here named TioxAla}, bearing a substituted triazolyl-thione group on the side chain and able to bind RNA biomedical targets, was here chosen as a valuable scaffold for the synthesis of new platinum complexes with potential dual action owing to the concomitant presence of the metal centre and the amino acid moiety. Three new platinum complexes, obtained from the reaction of TioxAla with K2PtCl4, were characterized by mass spectrometry, nuclear magnetic resonance and UV-vis spectroscopy: one compound (Pt1, bis-{(S)-2-amino-3-[4-propyl-3-(thiophen-2-yl)-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-1-yl]propanoate-O,S} platinum(II)) consisted of two amino acid units coordinating the Pt(II) ion; the other two, Pt2 [potassium dichloro-{(S)-2-amino-3-[4-propyl-3-(thiophen-2-yl)-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-1-yl]propanoate (O,S)} platinum(II)] and Pt3 [potassium dichloro-{(S)-2-amino-3-[4-propyl-3-(thiophen-2-yl)-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-1-yl]propanoate (O,N)} platinum(II)], were isomers bearing one TioxAla unit, and two chlorides as Pt-ligands. Pt coordination involved preferentially the amino, carboxylic and thione functions of TioxAla. By preliminary antiproliferative assays, a moderate cytotoxic activity on cancer cells was observed only for Pt2 and Pt3, while no anticancer activity was found for both the chloride-free complex (Pt1) and TioxAla. This cytotoxicity, however lower than that of cisplatin, well correlated with the marked ability, here found only for Pt2 and Pt3 complexes, to bind DNA sequences either in random coil or in structured forms (duplex and G-quadruplex), as verified by spectroscopic and spectrometric analysis.