Synthesis and Spectroscopic Analysis of Piperine- and Piperlongumine-Inspired Natural Product Scaffolds and Their Molecular Docking with IL-1ß and NF-κB Proteins.

Inspired by the remarkable bioactivities exhibited by the natural products, piperine and piperlongumine, we synthesised eight natural product-inspired analogues to further investigate their structures. For the first time, we confirmed the structure of the key cyclised dihydropyrazolecarbothioamide piperine analogues including the use of two-dimensional (2D) 15N-based spectroscopy nuclear magnetic resonance (NMR) spectroscopy. Prior investigations demonstrated promising results from these scaffolds for the inhibition of inflammatory response via downregulation of the IL-1ß and NF-κB pathway. However, the molecular interaction of these molecules with their protein targets remains unknown. Ab initio calculations revealed the electronic density function map of the molecules, showing the effects of structural modification in the electronic structure. Finally, molecular interactions between the synthesized molecules and the proteins IL-1ß and NF-κB were achieved. Docking results showed that all the analogues interact in the DNA binding site of NF-κB with higher affinity compared to the natural products and, with the exception of 9a and 9b, have higher affinity than the natural products for the binding site of IL-1ß. Specificity for the molecular recognition of 3a, 3c and 9b with IL-1ß through cation-π interactions was determined. These results revealed 3a, 3c, 4a, 4c and 10 as the most promising molecules to be evaluated as IL-1ß and NF-κB inhibitors.

Nuclear magnetic resonance and small-angle X-ray scattering studies of mixed sodium dodecyl sulfate and N,N-dimethyldodecylamine N-oxide aqueous systems performed at low temperatures.

Surfactant crystallisation is important in many applications in the food, consumer product and medical sectors. However, these processes are not well understood. In particular, surfactant crystallisation can be detrimental to the stability of detergent formulations, such as dish liquid products, resulting in a turbid solution that fails appearance criteria. With the rising global demand for detergent products, understanding the factors that influence formulation stability is of increasing importance. To enable industry to build more robust formulations, it is important to understand the underlying chemistry of the crystallisation process. Here, a model system containing anionic (sodium dodecyl sulfate, SDS) and amphoteric (N,N-dimethyldodecylamine N-oxide, DDAO) surfactants, at concentrations typical of dish liquid products, is studied. Variable temperature 1H nuclear magnetic resonance (NMR) spectroscopy and small-angle X-ray scattering (SAXS) is used to probe the compositional and structural properties of this system, as a function of pH. On cooling, at pH 9, a mixture of hydrated crystals, predominately composed of SDS, and micelles containing both surfactants, have been observed prior to complete freezing. At pH 2, both surfactants appear to undergo a simultaneous phase transition, resulting in the removal of micelles and the formation of hydrated crystals of mixed composition.

Synthesis and Biological Evaluation of ( E)-4-Hydroxy-3-methylbut-2-enyl Phosphate (HMBP) Aryloxy Triester Phosphoramidate Prodrugs as Activators of Vγ9/Vδ2 T-Cell Immune Responses.

The aryloxy triester phosphoramidate prodrug approach has been used with success in drug discovery. Herein, we describe the first application of this prodrug technology to the monophosphate derivative of the phosphoantigen HMBPP and one of its analogues. Some of these prodrugs exhibited specific and potent activation of Vγ9/Vδ2 T-cells, which were then able to lyse bladder cancer cells in vitro. This work highlights the promise of this prodrug technology in the discovery of novel immunotherapeutics.

Role of the Adsorbed Oxygen Species in the Selective Electrochemical Reduction of CO2 to Alcohols and Carbonyls on Copper Electrodes.

The electrochemical reduction of CO2 into fuels has gained significant attention recently as source of renewable carbon-based fuels. The unique high selectivity of copper in the electrochemical reduction of CO2 to hydrocarbons has called much interest in discovering its mechanism. In order to provide significant information about the role of oxygen in the electrochemical reduction of CO2 on Cu electrodes, the conditions of the surface structure and the composition of the Cu single crystal electrodes were controlled over time. This was achieved using pulsed voltammetry, since the pulse sequence can be programmed to guarantee reproducible initial conditions for the reaction at every fraction of time and at a given frequency. In contrast to the selectivity of CO2 reduction using cyclic voltammetry and chronoamperometric methods, a large selection of oxygenated hydrocarbons was found under alternating voltage conditions. Product selectivity towards the formation of oxygenated hydrocarbon was associated to the coverage of oxygen species, which is surface-structure- and potential-dependent.

Kinetin Riboside and Its ProTides Activate the Parkinson's Disease Associated PTEN-Induced Putative Kinase 1 (PINK1) Independent of Mitochondrial Depolarization.

Since loss of function mutations of PINK1 lead to early onset Parkinson's disease, there has been growing interest in the discovery of small molecules that amplify the kinase activity of PINK1. We herein report the design, synthesis, serum stability, and hydrolysis of four kinetin riboside ProTides. These ProTides, along with kinetin riboside, activated PINK1 in cells independent of mitochondrial depolarization. This highlights the potential of modified nucleosides and their phosphate prodrugs as treatments for neurodegenerative diseases.

The Blood Flow Shutdown Induced by Combretastatin A4 Impairs Gemcitabine Delivery in a Mouse Hepatocarcinoma.

In recent clinical studies, vascular disrupting agents (VDAs) are mainly used in combination with chemotherapy. However, an often overlooked concern in treatment combination is the VDA-induced impairment of chemotherapy distribution in the tumor. The work presented here investigated the impact of blood flow shutdown induced by Combretastatin A4 (CA4) on gemcitabine uptake into mouse hepatocarcinoma. At 2 h after CA4 treatment, using DCE-MRI, a significant decrease in the perfusion-relevant parameters Ktrans and Vp were observed in treated group compared with the control group. The blood flow shutdown was indeed confirmed by a histology study. In a third experiment, the total gemcitabine uptake was found to be significantly lower in treated tumors, as assessed in a separate experiment using ex vivo fluorine nuclear magnetic resonance spectroscopy. The amount of active metabolite gemcitabine triphosphate was also lower in treated tumors. In conclusion, the blood flow shutdown induced by VDAs can impact negatively on the delivery of small cytotoxic agents in tumors. The present study outlines the importance of monitoring the tumor vascular function when designing drug combinations.

Induction of highly curved structures in relation to membrane permeabilization and budding by the triterpenoid saponins, α- and δ-Hederin.

The interactions of triterpenoid monodesmosidic saponins, α-hederin and δ-hederin, with lipid membranes are involved in their permeabilizing effect. Unfortunately, the interactions of these saponins with lipid membranes are largely unknown, as are the roles of cholesterol or the branched sugar moieties (two for α-hederin and one for δ-hederin) on the aglycone backbone, hederagenin. The differences in sugar moieties are responsible for differences in the molecular shape of the saponins and the effects on membrane curvature that should be the most positive for α-hederin in a transbilayer direction. In large unilamellar vesicles and monocyte cells, we showed that membrane permeabilization was dependent on the presence of membrane cholesterol and saponin sugar chains, being largest for α-hederin and smallest for hederagenin. In the presence of cholesterol, α-hederin induced the formation of nonbilayer phases with a higher rate of Brownian tumbling or lateral diffusion. A reduction of Laurdan's generalized polarization in relation to change in order of the polar heads of phospholipids was observed. Using giant unilamellar vesicles, we visualized the formation of wrinkled borders, the decrease in liposome size, budding, and the formation of macroscopic pores. All these processes are highly dependent on the sugars linked to the aglycone, with α-hederin showing a greater ability to induce pore formation and δ-hederin being more efficient in inducing budding. Hederagenin induced intravesicular budding but no pore formation. Based on these results, a curvature-driven permeabilization mechanism dependent on the interaction between saponin and sterols and on the molecular shape of the saponin and its ability to induce local spontaneous curvature is proposed.

"Clip" and "click" chemistries combination: toward easy PEGylation of degradable aliphatic polyesters.

α-Methoxy-ω-alkyne poly(ethylene glycol) (PEG) was tagged with pendent N-hydroxy-succinimidyl activated esters by photografting of a molecular clip. This easily synthesized heterofunctional PEG was found to be a versatile building block for (i) conjugation with an amino derivative and (ii) grafting to azido functional aliphatic polyesters backbone by Huisgen's 1,3-dipolar cycloaddition. This original combination of "clip" and "click" reactions provides a versatile and straightforward pathway for the synthesis of functional amphiphilic and degradable copolymers valuable for biomedical applications such as in drug-delivery.

Biochemical characterization of RAR1 cysteine- and histidine-rich domains (CHORDs): a novel class of zinc-dependent protein-protein interaction modules.

Disease resistance in plants requires the activation of defense signaling pathways to prevent the spread of infection. The protein Required for Mla12 Resistance (RAR1) is a component of such pathways, which contains cysteine- and histidine-rich domains (CHORDs) that bind zinc. CHORDs are 60 amino acid domains, usually arranged in tandem, found in almost all eukaryotes, where they are involved in processes ranging from pressure sensing in the heart to maintenance of diploidy in fungi, and exhibit distinct protein-protein interaction specificity. In the case of RAR1, CHORD-I is known to interact with heat-shock protein 90 (HSP90) and CHORD-II is known to interact with the Suppressor of the G2 allele of Skp1 (SGT1). The focus of this work on RAR1 from barley and Arabidopsis was to address the paucity of biochemical information on RAR1 and its constituent CHORDs, particularly the role of the metal ion. Sedimentation experiments indicated RAR1 to be an extended monomer in solution with few intramolecular interactions. This was reinforced by denaturation experiments, where little difference between the stability of the individual domains and intact RAR1 could be detected by intrinsic tryptophan fluorescence. Electrospray ionization-mass spectrometry and atomic absorption showed that, contrary to previous reports, RAR1 binds five zinc ions; each CHORD binds two, and the plant-specific, 20 amino acid cysteine- and histidine-containing motif (CCCH motif) located between the two CHORDs binds the fifth. Fluorescence, ultraviolet circular dichroism (UV CD), and nuclear magnetic resonance (NMR) spectroscopy further demonstrated that zinc ions are essential for maintaining CHORD structure. Finally, we used isothermal titratrion colarimetry to show that zinc is essential for the specific binding interactions of CHORD-II with SGT1. Our study provides the first biochemical and biophysical data on the zinc metalloprotein RAR1, defines its metal stoichiometry and that of its constituent CHORDs, and reveals that the metal ions are essential for structural integrity and specific protein-protein associations.

Characterisation of the conformational properties of urea-unfolded Im7: implications for the early stages of protein folding.

The colicin immunity protein Im7 folds from its unfolded state in 6 M urea to its native four-helix structure through an on-pathway intermediate that lacks one of the helices of the native structure (helix III). In order to further characterize the folding mechanism of Im7, we have studied the conformational properties of the protein unfolded in 6 M urea in detail using heteronuclear NMR. Triple-resonance experiments with 13C/15N-labelled Im7 in 6 M urea provided almost complete resonance assignments for the backbone nuclei, and measurement of backbone 15N relaxation parameters allowed dynamic ordering of the unfolded polypeptide chain to be investigated. Reduced spectral density mapping and fitting backbone R2 relaxation rates to a polymer dynamics model identified four clusters of interacting residues, each predicted by the average area buried upon folding for each residue. Chemical shift analyses and measurement of NOEs detected with a long mixing-time 1H-1H-15N NOESY-HSQC spectrum confirmed the formation of four clusters. Each cluster of interacting side-chains in urea-unfolded Im7 occurs in a region of the protein that forms a helix in the protein, with the largest clusters being associated with the three long helices that are formed in the on-pathway folding intermediate, whilst the smallest cluster forms a helix only in the native state. NMR studies of a Phe15Ala Im7 variant and a protein in which residues 51-56 are replaced by three glycine residues (H3G3 Im7*), indicated that the clusters do not interact with each other, possibly because they are solvated by urea, as indicated by analysis of NOEs between the protein and the solvent. Based on these data, we suggest that dilution of the chaotrope to initiate refolding will result in collapse of the clusters, leading to the formation of persistent helical structure and the generation of the three-helix folding intermediate.

Equilibrium hydrogen exchange reveals extensive hydrogen bonded secondary structure in the on-pathway intermediate of Im7.

The four-helical immunity protein Im7 folds through an on-pathway intermediate that has a specific, but partially misfolded, hydrophobic core. In order to gain further insight into the structure of this species, we have identified the backbone hydrogen bonds formed in the ensemble by measuring the amide exchange rates (under EX2 conditions) of the wild-type protein and a variant, I72V. In this mutant the intermediate is significantly destabilised relative to the unfolded state (deltadeltaG(ui) = 4.4 kJ/mol) but the native state is only slightly destabilised (deltadeltaG(nu) = 1.8 kJ/mol) at 10 degrees C in 2H2O, pH* 7.0 containing 0.4 M Na2SO4, consistent with the view that this residue forms significant non-native stabilising interactions in the intermediate state. Comparison of the hydrogen exchange rates of the two proteins, therefore, enables the state from which hydrogen exchange occurs to be identified. The data show that amides in helices I, II and IV in both proteins exchange slowly with a free energy similar to that associated with global unfolding, suggesting that these helices form highly protected hydrogen-bonded helical structure in the intermediate. By contrast, amides in helix III exchange rapidly in both proteins. Importantly, the rate of exchange of amides in helix III are slowed substantially in the Im7* variant, I72V, compared with the wild-type protein, whilst other amides exchange more rapidly in the mutant protein, in accord with the kinetics of folding/unfolding measured using chevron analysis. These data demonstrate, therefore, that local fluctuations do not dominate the exchange mechanism and confirm that helix III does not form stable secondary structure in the intermediate. By combining these results with previously obtained Phi-values, we show that the on-pathway folding intermediate of Im7 contains extensive, stable hydrogen-bonded structure in helices I, II and IV, and that this structure is stabilised by both native and non-native interactions involving amino acid side-chains in these helices.