Synthesis of obovatol and related neolignan analogues as α-glucosidase and α-amylase inhibitors.

Diabetes mellitus is a metabolic disease characterized by hyperglycemia, which can be counteracted by the inhibition of α-glucosidase (α-Glu) and α-amylase (α-Amy), enzymes responsible for the hydrolysis of carbohydrates. In recent decades, many natural compounds and their bioinspired analogues have been studied as α-Glu and α-Amy inhibitors. However, no studies have been devoted to the evaluation of α-Glu and α-Amy inhibition by the neolignan obovatol (1). In this work, we report the synthesis of 1 and a library of new analogues. The synthesis of these compounds was achieved by implementing methodologies based on: phenol allylation, Claisen/Cope rearrangements, methylation, Ullmann coupling, demethylation, phenol oxidation and Michael-type addition. Obovatol (1) and ten analogues were evaluated for their in vitro inhibitory activity towards α-Glu and α-Amy. Our investigation highlighted that the naturally occurring 1 and four neolignan analogues (11, 22, 26 and 27) were more effective inhibitors than the hypoglycemic drug acarbose (α-Amy: 34.6 µM; α-Glu: 248.3 µM) with IC5O value of 6.2-23.6 µM toward α-Amy and 39.8-124.6 µM toward α-Glu. Docking investigations validated the inhibition outcomes, highlighting optimal compatibility between synthesized neolignans and both the enzymes. Concurrently circular dichroism spectroscopy detected the conformational changes in α-Glu induced by its interaction with the studied neolignans. Detailed studies through fluorescence measurements and kinetics of α-Glu and α-Amy inhibition also indicated that 1, 11, 22, 26 and 27 have the greatest affinity for α-Glu and 1, 11 and 27 for α-Amy. Surface plasmon resonance imaging (SPRI) measurements confirmed that among the compounds studied, the neolignan 27 has the greater affinity for both enzymes, thus corroborating the results obtained by kinetics and fluorescence quenching. Finally, in vitro cytotoxicity of the investigated compounds was tested on human colon cancer cell line (HCT-116). All these results demonstrate that these obovatol-based neolignan analogues constitute promising candidates in the pursuit of developing novel hypoglycemic drugs.

Electronic Circular Dichroism Detects Conformational Changes Associated with Proteasome Gating Confirmed Using AFM Imaging.

Many chronic diseases, including cancer and neurodegeneration, are linked to proteasome dysregulation. Proteasome activity, essential for maintaining proteostasis in a cell, is controlled by the gating mechanism and its underlying conformational transitions. Thus, developing effective methods to detect gate-related specific proteasome conformations could be a significant contribution to rational drug design. Since the structural analysis suggests that gate opening is associated with a decrease in the content of α-helices and ß-sheets and an increase in random coil structures, we decided to explore the application of electronic circular dichroism (ECD) in the UV region to monitor the proteasome gating. A comparison of ECD spectra of wild type yeast 20S proteasome (predominantly closed) and an open-gate mutant (α3ΔN) revealed an increased intensity in the ECD band at 220 nm, which suggests increased contents of random coil and ß-turn structures. This observation was further supported by evaluating ECD spectra of human 20S treated with low concentration of SDS, known as a gate-opening reagent. Next, to evaluate the power of ECD to probe a ligand-induced gate status, we treated the proteasome with H2T4, a tetracationic porphyrin that we showed previously to induce large-scale protein conformational changes upon binding to h20S. H2T4 caused a significant increase in the ECD band at 220 nm, interpreted as an induced opening of the 20S gate. In parallel, we imaged the gate-harboring alpha ring of the 20S with AFM, a technique that we used previously to visualize the predominantly closed gate in latent human or yeast 20S and the open gate in α3ΔN mutant. The results were convergent with the ECD data and showed a marked decrease in the content of closed-gate conformation in the H2T4-treated h20S. Our findings provide compelling support for the use of ECD measurements to conveniently monitor proteasome conformational changes related to gating phenomena. We predict that the observed association of spectroscopic and structural results will help with efficient design and characterization of exogenous proteasome regulators.

Modulation of the 20S Proteasome Activity by Porphyrin Derivatives Is Steered through Their Charge Distribution.

Cationic porphyrins exhibit an amazing variety of binding modes and inhibition mechanisms of 20S proteasome. Depending on the spatial distribution of their electrostatic charges, they can occupy different sites on α rings of 20S proteasome by exploiting the structural code responsible for the interaction with regulatory proteins. Indeed, they can act as competitive or allosteric inhibitors by binding at the substrate gate or at the grooves between the α subunits, respectively. Moreover, the substitution of a charged moiety in the peripheral arm with a hydrophobic moiety revealed a "new" 20S functional state with higher substrate affinity and catalytic efficiency. In the present study, we expand our structure-activity relationship (SAR) analysis in order to further explore the potential of this versatile class of 20S modulators. Therefore, we have extended the study to additional macrocyclic compounds, displaying different structural features, comparing their interaction behavior on the 20S proteasome with previously investigated compounds. In particular, in order to evaluate how the introduction of a peptidic chain can affect the affinity and the interacting mechanism of porphyrins, we investigate the MTPyApi, a porphyrin derivatized with an Arg-Pro-rich antimicrobial peptide. Moreover, to unveil the role played by the porphyrin core, this was replaced with a corrole scaffold, a "contracted" version of the tetrapyrrolic ring due to the lack of a methine bridge. The analysis has been undertaken by means of integrated kinetic, Nuclear Magnetic Resonance, and computational studies. Finally, in order to assess a potential pharmacological significance of this type of investigation, a preliminary attempt has been performed to evaluate the biological effect of these molecules on MCF7 breast cancer cells in dark conditions, envisaging that porphyrins may indeed represent a powerful tool for the modulation of cellular proteostasis.

Carbon dots surface chemistry drives fluorescent properties: New tools to distinguish isobaric peptides.

The possibility to design rational carbon dots surface functionalization for specific analytical and bioanalytical applications is hindered by the lack of a full knowledge of the surface chemical features driving fluorescent properties. In this model study, we have synthesized four different peptides, three of which are isobaric and not distinguishable by common MSMS experiments. After having characterized the peptides conformations by CD analyses, we have covalently bonded all four peptides to carbon dots by using different experimental procedures, which produce different functional groups on the carbon dots surface. The peptide orientations obtained on the differently functionalized surface of the nanoparticles were different and produced different fluorescent responses. The reported results indicate the possibility to design amino and carboxyl enriched surface carbon dots to answer specific chemical requirements, paving the way for the use of these nanoparticles as a versatile and useful new chemical and biochemical tool.

Interactions Between Spermine-Derivatized Tentacle Porphyrins and The Human Telomeric DNA G-Quadruplex.

G-rich DNA sequences have the potential to fold into non-canonical G-Quadruplex (GQ) structures implicated in aging and human diseases, notably cancers. Because stabilization of GQs at telomeres and oncogene promoters may prevent cancer, there is an interest in developing small molecules that selectively target GQs. Herein, we investigate the interactions of meso-tetrakis-(4-carboxysperminephenyl)porphyrin (TCPPSpm4) and its Zn(II) derivative (ZnTCPPSpm4) with human telomeric DNA (Tel22) via UV-Vis, circular dichroism (CD), and fluorescence spectroscopies, resonance light scattering (RLS), and fluorescence resonance energy transfer (FRET) assays. UV-Vis titrations reveal binding constants of 4.7 × 106 and 1.4 × 107 M-1 and binding stoichiometry of 2⁻4:1 and 10⁻12:1 for TCPPSpm4 and ZnTCPPSpm4, respectively. High stoichiometry is supported by the Job plot data, CD titrations, and RLS data. FRET melting indicates that TCPPSpm4 stabilizes Tel22 by 36 ± 2 °C at 7.5 eq., and that ZnTCPPSpm4 stabilizes Tel22 by 33 ± 2 °C at ~20 eq.; at least 8 eq. of ZnTCPPSpm4 are required to achieve significant stabilization of Tel22, in agreement with its high binding stoichiometry. FRET competition studies show that both porphyrins are mildly selective for human telomeric GQ vs duplex DNA. Spectroscopic studies, combined, point to end-stacking and porphyrin self-association as major binding modes. This work advances our understanding of ligand interactions with GQ DNA.

Chirality Enhancement of Porphyrin Supramolecular Assembly Driven by a Template Preorganization Effect.

Cationic polylysine promotes, under neutral conditions, the spontaneous aggregation of opposite charged ZnTPPS in water. Spectroscopic investigations evidence a different preorganization of ZnTPPS onto the polypeptide matrix depending on the chain length. Spinodal decomposition theory in confined geometry is used to model this mechanism by considering the time evolution of a homogeneous distribution of randomly adsorbed particles (porphyrins) onto a rodlike polyelectrolyte (polymer) of variable length L.

Electrostatic Map Of Proteasome α-Rings Encodes The Design of Allosteric Porphyrin-Based Inhibitors Able To Affect 20S Conformation By Cooperative Binding.

The importance of allosteric proteasome inhibition in the treatment of cancer is becoming increasingly evident. Motivated by this urgent therapeutic need, we have recently identified cationic porphyrins as a highly versatile class of molecules able to regulate proteasome activity by interfering with gating mechanisms. In the present study, the mapping of electrostatic contacts bridging the regulatory particles with the α-rings of the human 20S proteasome led us to the identification of (meso-tetrakis(4-N-methylphenyl pyridyl)-porphyrin (pTMPyPP4) as a novel non-competitive inhibitor of human 20S proteasome. pTMPyPP4 inhibition mechanism implies a positive cooperative binding to proteasome, which disappears when a permanently open proteasome mutant (α-3ΔN) is used, supporting the hypothesis that the events associated with allosteric proteasome inhibition by pTMPyPP4 interfere with 20S gating and affect its "open-closed" equilibrium. Therefore, we propose that the spatial distribution of the negatively charged residues responsible for the interaction with regulatory particles at the α-ring surface of human 20S may be exploited as a blueprint for the design of allosteric proteasome regulators.

Hierarchical effect behind the supramolecular chirality of silver(I)-cysteine coordination polymers.

Cysteine is a sulfur-containing amino acid that easily coordinates to soft metal ions and grafts to noble metal surfaces. Recently, chiroptical activity of Ag(+)/cysteine coordination polymers has been widely studied, while, on the other hand, the appearance of a plasmon-enhanced circular dichroic signal (PECD) at the plasmonic spectral region (λ > 400 nm) has been observed for AgNPs capped with chiral sulfur-containing amino acids. These two events are both potentially exploited for sensing applications. However, the presence of Ag(+) ions in AgNP colloidal solution deals with the competition of cysteine grafting at the metal NP surface and/or metal ion coordination. Herein we demonstrate that the chiroptical activity observed by adding cysteine to AgNP colloids prepared by pulsed laser ablation in liquids (PLAL) is mainly related to the formation of CD-active Ag(+)/cysteine supramolecular polymers. The strict correlation between supramolecular chirality and hierarchical effects, driven by different chemical environments experienced by cysteine when different titration modalities are used, is pivotal to validate cysteine as a fast and reliable probe to characterize the surface oxidation of AgNPs prepared by pulsed laser ablation in liquids by varying the laser wavelengths.

Cationic porphyrins are reversible proteasome inhibitors.

The aim of this study is to verify if water-soluble porphyrins can be used as proteasome inhibitors. We have found that cationic porphyrins inhibit proteasome peptidase activities much more effectively than the corresponding anionic derivatives. The relevance of electrostatics in driving porphyin-proteasome interactions has been confirmed by the observation that the inhibitory efficiency of the cationic macrocycles decreases with the number of positive substituents. We have also investigated various metalloporphyrins, which differ due to the different propension of the central metal ion toward axial coordination. Our experimental results indicate that the naked cationic porphyrins are the most active in reversibly inhibiting the three main protease activities of the proteasome in the micromolar range. A spectroscopic characterization of porphyrin-proteasome interactions by UV-vis spectra parallels the results of inhibition assays: the higher the inhibitory effect the stronger the spectroscopic variations are. To interpret the action of porphyrins at a molecular level, we have performed calculations evidencing that cationic porphyrins may hinder the access to the canonical proteolytic site on the proteasome ß5 subunit. In particular, an inspection of the top-scoring docking modes shows that the tetracationic porphyrin blocks the catalytic pocket, close to the N termini of the ß5 proteasome subunit, more efficiently than its anionic counterpart. Proteasome inhibition activity of porphyrins unites their known anticancer properties making them suitable as a scaffold for the design of novel multitargeted molecules.