Identification and evaluation of antiviral activity of novel compounds targeting SARS-CoV-2 virus by enzymatic and antiviral assays, and computational analysis.

The viral genome of the SARS-CoV-2 coronavirus, the aetiologic agent of COVID-19, encodes structural, non-structural, and accessory proteins. Most of these components undergo rapid genetic variations, though to a lesser extent the essential viral proteases. Consequently, the protease and/or deubiquitinase activities of the cysteine proteases Mpro and PLpro became attractive targets for the design of antiviral agents. Here, we develop and evaluate new bis(benzylidene)cyclohexanones (BBC) and identify potential antiviral compounds. Three compounds were found to be effective in reducing the SARS-CoV-2 load, with EC50 values in the low micromolar concentration range. However, these compounds also exhibited inhibitory activity IC50 against PLpro at approximately 10-fold higher micromolar concentrations. Although originally developed as PLpro inhibitors, the comparison between IC50 and EC50 of BBC indicates that the mechanism of their in vitro antiviral activity is probably not directly related to inhibition of viral cysteine proteases. In conclusion, our study has identified new potential noncytotoxic antiviral compounds suitable for in vivo testing and further improvement.

On the Cholesterol Raising Effect of Coffee Diterpenes Cafestol and 16-O-Methylcafestol: Interaction with Farnesoid X Receptor.

The diterpene cafestol represents the most potent cholesterol-elevating compound known in the human diet, being responsible for more than 80% of the effect of coffee on serum lipids, with a mechanism still not fully clarified. In the present study, the interaction of cafestol and 16-O-methylcafestol with the stabilized ligand-binding domain (LBD) of the Farnesoid X Receptor was evaluated by fluorescence and circular dichroism. Fluorescence quenching was observed with both cafestol and 16-O-methylcafestol due to an interaction occurring in the close environment of the tryptophan W454 residue of the protein, as confirmed by docking and molecular dynamics. A conformational change of the protein was also observed by circular dichroism, particularly for cafestol. These results provide evidence at the molecular level of the interactions of FXR with the coffee diterpenes, confirming that cafestol can act as an agonist of FXR, causing an enhancement of the cholesterol level in blood serum.

Designing New Hybrid Antibiotics: Proline-Rich Antimicrobial Peptides Conjugated to the Aminoglycoside Tobramycin.

Resistance to aminoglycoside antibiotics is a serious problem, typically arising from inactivating enzymes, reduced uptake, or increased efflux in the important pathogens for which they are used as treatment. Conjugating aminoglycosides to proline-rich antimicrobial peptides (PrAMPs), which also target ribosomes and have a distinct bacterial uptake mechanism, might mutually benefit their individual activities. To this aim we have developed a strategy for noninvasively modifying tobramycin to link it to a Cys residue and through this covalently link it to a Cys-modified PrAMP by formation of a disulfide bond. Reduction of this bridge in the bacterial cytosol should release the individual antimicrobial moieties. We found that the conjugation of tobramycin to the well-characterized N-terminal PrAMP fragment Bac7(1-35) resulted in a potent antimicrobial capable of inactivating not only tobramycin-resistant bacterial strains but also those less susceptible to the PrAMP. To a certain extent, this activity also extends to the shorter and otherwise poorly active fragment Bac7(1-15). Although the mechanism that allows the conjugate to act when its individual components do not is as yet unclear, results are very promising and suggest this may be a way of resensitizing pathogens that have developed resistance to the antibiotic.

Fluorescent Imprinted Nanoparticles for Sensing of Chlorogenic Acid in Coffee Extracts.

Green coffee beans are particularly rich in chlorogenic acids (CGAs), and their identification and quantification are usually performed by HPLC, coupled with mass spectrometry (LC-MS). Although there are a few examples of molecularly imprinted polymers (MIPs) for chlorogenic acid (5-CQA) recognition present in the literature, none of them are based on optical fluorescence, which is very interesting given its great sensitivity. In the present manuscript, fluorescent polymeric imprinted nanoparticles were synthetized following the non-covalent approach using hydrogenated 5-O-caffeoylquinic acid (H-5-CQA) as the template. The capability of the polymer to bind 5-CQA was evaluated by HPLC and fluorescence. A real sample of coffee extract was also analyzed to verify the selectivity of the polymer. Polymer fMIP01, containing 4-vinylpyridine and a naphtalimide derivative as monomers, showed a good response to the fluorescence quenching in the range 39 µM-80 mM. In the real sample, fMIP01 was able to selectively bind 5-CQA, while caffeine was not recognized. To demonstrate this, there is a promising system that can be exploited in the design of an optical sensor for 5-CQA detection. Polymer fMIP01 was immobilized by physical entrapment on a functionalized glass surface, showing a quenching of fluorescence with an increase of the CGA concentration between 156 µM and 40 mM.

Oleocanthal Quantification Using 1H NMR Spectroscopy and Polyphenols HPLC Analysis of Olive Oil from the Bianchera/Belica Cultivar.

The cultivar Bianchera is an autochthonous variety from the eastern part of northern Italy, but it is also cultivated in the Slovenian and Croatian peninsula of Istria where it is named Belica (Slovenia) and Bjelica (Croatia). The properties of oleocanthal, a natural anti-inflammatory ibuprofen-like compound found in commercial monocultivar extra virgin olive oils, were determined by means of both quantitative 1H NMR (qNMR) and HPLC analyses, where qNMR was identified as a rapid and reliable method for determining the oleocanthal content. The total phenolic content (TPC) was determined by means of the Folin-Ciocalteau method and the major phenols present in the olive oils were also quantified by means of HPLC analyses. All these analyses confirmed that the cultivar Bianchera was very rich in polyphenols and satisfied the health claim provided by the EU Commission Regulation on the polyphenols content of olive oils and their beneficial effects on human health.

Interaction of the Coffee Diterpenes Cafestol and 16-O-Methyl-Cafestol Palmitates with Serum Albumins.

The main coffee diterpenes cafestol, kahweol, and 16-O-methylcafestol, present in the bean lipid fraction, are mostly esterified with fatty acids. They are believed to induce dyslipidaemia and hypercholesterolemia when taken with certain types of coffee brews. The study of their binding to serum albumins could help explain their interactions with biologically active xenobiotics. We investigated the interactions occurring between cafestol and 16-O-methylcafestol palmitates with Bovine Serum Albumin (BSA), Human Serum Albumin (HSA), and Fatty Free Human Serum Albumin (ffHSA) by means of circular dichroism and fluorimetry. Circular Dichroism (CD) revealed a slight change (up to 3%) in the secondary structure of fatty-free human albumin in the presence of the diterpene esters, suggesting that the aliphatic chain of the palmitate partly occupies one of the fatty acid sites of the protein. A warfarin displacement experiment was performed to identify the binding site, which is probably close but not coincident with Sudlow site I, as the affinity for warfarin is enhanced. Fluorescence quenching titrations revealed a complex behaviour, with Stern-Volmer constants in the order of 103-104 Lmol-1. A model of the HSA-warfarin-cafestol palmitate complex was obtained by docking, and the most favourable solution was found with the terpene palmitate chain inside the FA4 fatty acid site and the cafestol moiety fronting warfarin at the interface with site I.

Hydroxycinnamoyl Amino Acids Conjugates: A Chiral Pool to Distinguish Commercially Exploited Coffea spp.

The synthesis of five hydroxycinnamoyl amides (HCAs) was accomplished and their identification and quantification in the green coffee bean samples of Coffea arabica, Coffea canephora, and Coffea liberica was performed. The HCAs p-coumaroyl-N-tyrosine 1b, caffeoyl-N-phenylalanine 2b, caffeoyl-N-tyrosine 3b, and p-coumaroyl-N-tryptophan 4b were characteristic of the C. canephora species while caffeoyl-N-tryptophan 5b was present in both C. canephora and C. arabica, but with higher content in C. canephora. The HCAs presence was also analyzed in C. liberica for the first time and none of the targeted compounds was found, indicating that this species is very similar to C. arabica species. Between C. canephora samples from various origins, significant differences were observed regarding the presence of all the HCAs, with C. canephora from Tanzania containing all five derivatives.

One Pot Synthesis of Micromolar BACE-1 Inhibitors Based on the Dihydropyrimidinone Scaffold and Their Thia and Imino Analogues.

A library of dihydropyrimidinones was synthesized via a "one-pot" three component Biginelli reaction using different aldehydes in combination with ß-dicarbonyl compounds and urea. Selected 2-thiooxo and 2-imino analogs were also obtained with the Biginelli reaction from thiourea and guanidine hydrochloride, respectively. The products were screened in vitro for their ß-secretase inhibitory activity. The majority of the compounds resulted to be active, with IC50 in the range 100 nM-50 µM.

New heteroaryl carbamates: Synthesis and biological screening in vitro and in mammalian cells of wild-type and mutant HIV-protease inhibitors.

New heteroaryl HIV-protease inhibitors bearing a carbamoyl spacer were synthesized in few steps and high yield, from commercially available homochiral epoxides. Different substitution patterns were introduced onto a given isopropanoyl-sulfonamide core that can have either H or benzyl group. The in vitro inhibition activity against recombinant protease showed a general beneficial effect of both carbamoyl moiety and the benzyl group, ranging the IC50 values between 11 and 0.6 nM. In particular, benzofuryl and indolyl derivatives showed IC50 values among the best for such structurally simple inhibitors. Docking analysis allowed to identify the favorable situation of such derivatives in terms of number of interactions in the active site, supporting the experimental results. The inhibition activity was also confirmed in HEK293 mammalian cells and was maintained against protease mutants. Furthermore, the metabolic stability was comparable with that of the commercially available inhibitors.

Prediction of self-assembly of adenosine analogues in solution: a computational approach validated by isothermal titration calorimetry.

The recent discovery of the role of adenosine-analogues as neuroprotectants and cognitive enhancers has sparked interest in these molecules as new therapeutic drugs. Understanding the behavior of these molecules in solution and predicting their ability to self-assemble will accelerate new discoveries. We propose a computational approach based on density functional theory, a polarizable continuum solvation description of the aqueous environment, and an efficient search procedure to probe the potential energy surface, to determine the structure and thermodynamic stability of molecular clusters of adenosine analogues in solution, using caffeine as a model. The method was validated as a tool for the prediction of the impact of small structural variations on self-assembly using paraxanthine. The computational results were supported by isothermal titration calorimetry experiments. The thermodynamic parameters enabled the quantification of the actual percentage of dimer present in solution as a function of concentration. The data suggest that both caffeine and paraxanthine are present at concentrations comparable to the ones found in biological samples.

Chlorogenic Compounds from Coffee Beans Exert Activity against Respiratory Viruses.

Chlorogenic acids are secondary metabolites in diverse plants. Some chlorogenic acids extracted from traditional medicinal plants are known for their healing properties, e.g., against viral infections. Also, green coffee beans are a rich source of chlorogenic acids, with 5-O-caffeoylquinic acid being the most abundant chlorogenic acid in coffee. We previously reported the synthesis of the regioisomers of lactones, bearing different substituents on the quinidic core. Here, 3,4-O-dicaffeoyl-1,5-γ-quinide and three dimethoxycinnamoyl-γ-quinides were investigated for in vitro antiviral activities against a panel of 14 human viruses. Whereas the dimethoxycinnamoyl-γ-quinides did not show any antiviral potency in cytopathogenic effect reduction assays, 3,4-O-dicaffeoyl-1,5-γ-quinide exerted mild antiviral activity against herpes simplex viruses, adenovirus, and influenza virus. Interestingly, when the compounds were evaluated against respiratory syncytial virus, a potent antiviral effect of 3,4-O-dicaffeoyl-1,5-γ-quinide was observed against both subtypes of respiratory syncytial virus, with EC50 values in the submicromolar range. Time-of-addition experiments revealed that this compound acts on an intracellular post-entry replication step. Our data show that 3,4-O-dicaffeoyl-1,5-γ-quinide is a relevant candidate for lead optimization and further mechanistic studies, and warrants clinical development as a potential anti-respiratory syncytial virus drug.

Synthesis of Chiral, Enantiopure Allylic Amines by the Julia Olefination of α-Amino Esters.

The four-step conversion of a series of N-Boc-protected l-amino acid methyl esters into enantiopure N-Boc allylamines by a modified Julia olefination is described. Key steps include the reaction of a lithiated phenylalkylsulfone with amino esters, giving chiral ß-ketosulfones, and the reductive elimination of related α-acetoxysulfones. The overall transformation takes place under mild conditions, with good yields, and without loss of stereochemical integrity, being in this respect superior to the conventional Julia reaction of α-amino aldehydes.

In Vitro and In Vivo Evaluation of the Effects of Drug 2c and Derivatives on Ovarian Cancer Cells.

BACKGROUND: The identification of novel therapeutic strategies for ovarian cancer (OC), the most lethal gynecological neoplasm, is of utmost urgency. Here, we have tested the effectiveness of the compound 2c (4-hydroxy-2,6-bis(4-nitrobenzylidene)cyclohexanone 2). 2c interferes with the cysteine-dependent deubiquitinating enzyme (DUB) UCHL5, thus affecting the ubiquitin-proteasome-dependent degradation of proteins. METHODS: 2c phenotypic/molecular effects were studied in two OC 2D/3D culture models and in a mouse xenograft model. Furthermore, we propose an in silico model of 2c interaction with DUB-UCHL5. Finally, we have tested the effect of 2c conjugated to several linkers to generate 2c/derivatives usable for improved drug delivery. RESULTS: 2c effectively impairs the OC cell line and primary tumor cell viability in both 2D and 3D conditions. The effectiveness is confirmed in a xenograft mouse model of OC. We show that 2c impairs proteasome activity and triggers apoptosis, most likely by interacting with DUB-UCHL5. We also propose a mechanism for the interaction with DUB-UCHL5 via an in silico evaluation of the enzyme-inhibitor complex. 2c also reduces cell growth by down-regulating the level of the transcription factor E2F1. Eventually, 2c activity is often retained after the conjugation with linkers. CONCLUSION: Our data strongly support the potential therapeutic value of 2c/derivatives in OC.

The crystal structure of sterol carrier protein 2 from Yarrowia lipolytica and the evolutionary conservation of a large, non-specific lipid-binding cavity.

Sterol carrier protein 2 (SCP2), a small intracellular domain present in all forms of life, binds with high affinity a broad spectrum of lipids. Due to its involvement in the metabolism of long-chain fatty acids and cholesterol uptake, it has been the focus of intense research in mammals and insects; much less characterized are SCP2 from other eukaryotic cells and microorganisms. We report here the X-ray structure of Yarrowia lipolytica SCP2 (YLSCP2) at 2.2 Å resolution in complex with palmitic acid. This is the first fungal SCP2 structure solved, and it consists of the canonical five-stranded ß-sheet covered on the internal face by a layer of five α-helices. The overall fold is conserved among the SCP2 family, however, YLSCP2 is most similar to the SCP2 domain of human MFE-2, a bifunctional enzyme acting on peroxisomal ß-oxidation. We have identified the common structural elements defining the shape and volume of the large binding cavity in all species characterized. Moreover, we found that the cavity of the SCP2 domains is distinctly formed by carbon atoms, containing neither organized water nor rigid polar interactions with the ligand. These features are in contrast with those of fatty acid binding proteins, whose internal cavities are more polar and contain bound water. The results will help to design experiments to unveil the SCP2 function in very different cellular contexts and metabolic conditions.

On the absolute configuration of chiral 1,4-dihydropyridazines synthesized by organocatalysed reactions.

A computational investigation of the specific optical rotation and of the electronic circular dichroism spectra of two chiral 1,4-dihydropyridazines was performed and compared with existing experimental data to verify a previous assignment of their absolute configuration based on a well-accepted mechanism of catalysis of the organocatalyst used in their synthesis. Both the optical rotation and circular dichroism calculations indicate that the absolute configuration is opposite to the one assigned on the basis of the mechanism originally assumed. An alternative reaction mechanism is therefore suggested.

Synthesis and biological evaluation of novel small non-peptidic HIV-1 PIs: the benzothiophene ring as an effective moiety.

Synthesis and biological evaluation of a new series of potential HIV-1 protease inhibitors incorporating different heterocycles are described. The variation of heteroatom in such molecules has displayed totally different biological activities and a benzothiophene containing inhibitor has shown high potency against wild type HIV-1 protease with IC(50)=60 nM, thanks to the lower desolvation penalty to be payed by such hydrophobic moiety.

Short peptides as biosensor transducers.

This review deals with short peptides (up to 50 amino acids) as biomimetic active recognition elements in sensing systems. Peptide-based sensors have been developed in recent years according to different strategies. Synthetic peptides have been designed on the basis of known interactions between single or a few amino acids and targets, with attention being paid to the presence of peptide motifs known to allow intermolecular self-organization of the sensing peptides over the sensor surface. Sensitive and sophisticated sensors have been obtained in this way, but the use of designed peptides is limited by severe difficulties in their in silico design. Short peptides from random phage display have been selected in a random way from large, unfocussed, and often preexisting and commercially available phage display libraries, with no design elements. Such peptides often perform better than antibodies, but they are difficult to select when the target is a small molecule because of the need to immobilize it with considerable modifications of its structure. Artificial, miniaturized receptors have been obtained from the reduction of the known sequence of a natural receptor down to a synthesizable and yet stable one. Alternatively, binding sites have been created over a designed, stable peptide scaffold. Short peptides have also been used as active elements for the detection of their own natural receptors: pathogenic bacteria have been detected with antimicrobial and cell-penetrating peptides, but key challenges such as detection of bacteria in real samples, improved sensitivity, and improved selectivity have to be faced. Peptide substrates have been conjugated to fluorescent quantum dots to obtain disposable sensors for protease activity with high sensitivity. Ferrocene-peptide conjugates have been used for electrochemical sensing of protease activity.

Novel synthesis of 1,2-diaza-1,3-dienes with potential biological activity from cinnamic acids and diazonium salts of anilines.

Cinnamic acids are an important class of phenolic compounds, which have many beneficial effects on human health but are also interesting synthetic intermediates thanks to the presence of several reactive sites. While studying the reactivity of cinnamic acids with diazonium salts from aromatic amines, an unexpected reactivity has been discovered, leading to the formation of 1,2-diaza-1,3-dienes instead of traditional diazo-coupling products. The new compounds have been fully characterized by mono and bidimensional NMR spectroscopy and mass spectrometry. Preliminary studies on the biological activity of the compounds have been carried out testing both their antibacterial and antitumor activity, leading to promising results.

Aldolase activity of serum albumins.

Bovine and human serum albumins catalyze the aldol reaction of aromatic aldehyedes and acetone, with saturation kinetics and moderate and opposite enantioselectivity. The reaction occurs at the binding site in domain IIa, and is inhibited by warfarin. Kinetic data are consistent with an enamine mechanism. The activity is conserved in a 103 aminoacid peptide derived from the albumin sequence.

Albumin-directed stereoselective reduction of 1,3-diketones and ß-hydroxyketones to anti diols.

The reduction of 1,3-diketones and ß-hydroxyketones with NaBH(4) in aqueous acetonitrile is highly stereoselective in the presence of stoichiometric amounts of bovine or human albumin, giving anti 1,3-diols with d.e. up to 96%. The same reaction, without albumin, gives syn and anti 1,3-diols in approximately 1:1 ratio. The presence of an aromatic carbonyl group is essential for diastereoselectivity in the NaBH(4)/albumin reduction of both 1,3-diketones and ß-hydroxyketones. Thus, 3-hydroxy-1-(p-tolyl)-1-butanone is stereoselectively reduced in the presence of albumin, while reduction of its isomer 4-(p-tolyl)-4-hydroxy-2-butanone is not stereoselective. The albumin-controlled reduction is not stereospecific as both enantiomers of 1-aryl-3-hydroxy-1-butanones are reduced to diols with identical stereoselectivities. Circular dichroism of the bound substrates confirms that aromatic ketones are recognized by the protein's IIA binding site. Binding studies also suggest that 1,3-diketones are recognized in their enol form. From the effect of pH on binding of a diketone it is concluded that, in the complex with the substrate, ionizable residues His242 and Lys199 are in the neutral and protonated forms, respectively. A homology model of BSA was obtained and docking of model substrates confirms the preference of the protein for aromatic ketones. Modelling of the complexes with the substrates also allows us to propose a mechanism for the reduction of 1,3-diketones in which the chemoselective reduction of the first (aliphatic) carbonyl is followed by the diastereoselective reduction of the second (aromatic) carbonyl. The role of albumin is thus a combination of chemo- and stereocontrol.