ß-Alanine and N-terminal cationic substituents affect polyamide-DNA binding.

Minor-groove binding hairpin polyamides (PAs) bind specific DNA sequences. Synthetic modifications can improve PA-DNA binding affinity and include flexible modules, such as ß-alanine (ß) motifs to replace pyrroles (Py), and increasing compound charge using N-terminal cationic substituents. To better understand the variations in kinetics and affinities caused by these modifications on PA-DNA interactions, a comprehensive set of PAs with different numbers and positions of ß and different types of N-cationic groups was systematically designed and synthesized to bind their cognate sequence, the λB motif. The λB motif is also a strong binding promoter site of the major groove targeting transcription factor PU.1. The PA binding affinities and kinetics were evaluated using a spectrum of powerful biophysical methods: thermal melting, biosensor surface plasmon resonance and circular dichroism. The results show that ß inserts affect PA-DNA interactions in a number and position dependent manner. Specifically, a ß replacement between two imidazole heterocycles (ImßIm) generally strengthens binding. In addition, N-terminal cationic groups can accelerate the association between PA and DNA, but the bulky size of TMG can cause steric hindrance and unfavourable repulsive electrostatic interactions in some PAs. The future design of stronger binding PA requires careful combination of ßs and cationic substituents.

Evaluation of the effects of passion fruit peel flour (Passiflora edulis fo. flavicarpa) on metabolic changes in HIV patients with lipodystrophy syndrome secondary to antiretroviral therapy

ABSTRACT This study evaluated the effects of using passion fruit peel flour together with diet therapy and counseling in 36 patients with HIV lipodystrophy who were in an ambulatory clinic in a university hospital. The patients were divided into two groups. One received 30 g of passion fruit peel flour daily for 90 days and diet therapy counseling. The other group received only diet therapy counseling. The metabolic changes were analyzed before and after the intervention, with a significance level predetermined at p ≤ 0.05. The use of passion fruit peel flour was effective in reducing total cholesterol and triacylglycerides after 30 days. The concentrations of LDL-C decreased, while HDL-C increased in the blood of lipodystrophy patients after 90 days passion fruit peel flour treatment. No significant differences in food consumption were seen between groups. The use of 30 g of passion fruit peel flour for 90 days together with diet therapy counseling was effective in improving plasma concentrations of total cholesterol, LDL-C, HDL-C and triacylglycerides.

Phytochemicals and botanical extracts regulate NF-κB and Nrf2/ARE reporter activities in DI TNC1 astrocytes.

The increase in oxidative stress and inflammatory responses associated with neurodegenerative diseases has drawn considerable attention towards understanding the transcriptional signaling pathways involving NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) and Nrf2 (Nuclear Factor Erythroid 2-like 2). Our recent studies with immortalized murine microglial cells (BV-2) demonstrated effects of botanical polyphenols to inhibit lipopolysaccharide (LPS)-induced nitric oxide (NO) and enhance Nrf2-mediated antioxidant responses (Sun et al., 2015). In this study, an immortalized rat astrocyte (DI TNC1) cell line expressing a luciferase reporter driven by the NF-κB or the Nrf2/Antioxidant Response Element (ARE) promoter was used to assess regulation of these two pathways by phytochemicals such as quercetin, rutin, cyanidin, cyanidin-3-O-glucoside, as well as botanical extracts from Withania somnifera (Ashwagandha), Sutherlandia frutescens (Sutherlandia) and Euterpe oleracea (Açaí). Quercetin effectively inhibited LPS-induced NF-κB reporter activity and stimulated Nrf2/ARE reporter activity in DI TNC1 astrocytes. Cyanidin and the glycosides showed similar effects but only at much higher concentrations. All three botanical extracts effectively inhibited LPS-induced NF-κB reporter activity. These extracts were capable of enhancing ARE activity by themselves and further enhanced ARE activity in the presence of LPS. Quercetin and botanical extracts induced Nrf2 and HO-1 protein expression. Interestingly, Ashwagandha extract was more active in inducing Nrf2 and HO-1 expression in DI TNC1 astrocytes as compared to Sutherlandia and Açaí extracts. In summary, this study demonstrated NF-kB and Nrf2/ARE promoter activities in DI TNC1 astrocytes, and further showed differences in ability for specific botanical polyphenols and extracts to down-regulate LPS-induced NF-kB and up-regulate the NRF2/ARE activities in these cells.

UPLC–QTOF–MS and NMR analyses of graviola (Annona muricata) leaves

ABSTRACT Graviola leaves (Annona muricata L., Annonaceae) are used by some people to try to treat or even cure cancer, even though over-consumption of the fruit, which contains the neurotoxins annonacin and squamocin has caused an atypical form of Parkinson's disease. In previous analyses, the fruits were extracted with methanol under ambient conditions before analyses. In the present study, UPLC–QTOF–MS and NMR were used to analyze freeze-dried graviola leaves that were extracted using dry methanol and ethanol at 100 ºC and 10 MPa (100 atm) pressure in a sealed container. Methanol solubilized 33% of the metabolites in the lyophilized leaves. Ethanol solubilized 41% of metabolites in the lyophilized leaves. The concentrations of total phenolic compounds were 100.3 ± 2.8 and 93.2 ± 2.0 mg gallic acid equivalents per g of sample, for the methanolic and ethanolic extracts, respectively. Moreover, the toxicophore (unsaturated γ-lactone) that is present in neurotoxic acetogenins was found in the lipophilic portion of this extract. The concentrations of the neurotoxins annonacin and squamocin were found by UPLC–QTOF–MS to be 305.6 ± 28.3 and 17.4 ± 0.89 µg/g-dw, respectively, in the dried leaves. Pressurized methanol solubilized more annonacin and squamocin than ethanol. On the other hand, a hot, aqueous infusion solubilized only 0.213% of the annonacin and too little of the squamocin to be detected. So, graviola leaves contain significant amounts of the neurotoxins annonacin and squamocin, as well as some potentially healthy phenolic compounds. Finally, the potential neurotoxicity of whole leaves in dietary supplements could be much higher than that of a tea (hot aqueous infusion) that is made from them.

Using NMR to Develop New Allosteric and Allo-Network Drugs.

NMR is becoming an important tool for developing new allosteric and allo-network drugs that bind to allosteric sites on enzymes, partially inhibiting them and causing fewer side effects than drugs already developed that target active sites. This is based on systems thinking, in which active enzymes and other proteins are known to be flexible and interact with each other. In other words, proteins can exist in an ensemble of different conformations whose populations are tunable. NMR is being used to find the pathways through which the effects of binding of an allosteric ligand propagate. There are NMR screening assays for studying ligand binding. This includes determining the changes in the spin lattice relaxation due to changes in the mobility of atoms involved in the binding, measuring magnetization transfer from the protein to the ligand by saturation difference transfer NMR (STD-NMR) and the transfer of bulk magnetization to the ligand by water-Ligand Observed via Gradient Spectroscopy, or waterLOGSY. The chemical shifts of (1)H and (15)N of some of the atoms in amino acids change when an allosteric ligand binds to a protein. So, (1)H-(15)N heteronuclear single quantum coherence (HSQC) spectra can be used to identify key amino acids and ligand binding sites. The NMR chemical shifts of amino acids affected by ligand binding form a network that can be characterized. Allosteric networks can be identified by chemical shift covariance analysis (CHESCA). This approach has been used recently to study the binding of new molecular entities (NMEs) to potentially therapeutic drug targets.

Neurotoxicity of Dietary Supplements from Annonaceae Species.

Dietary supplements containing plant materials of Annonaceae species (Annona muricata L., A. squamosa L., A. mucosa JACQ., A. squamosa × cherimola Mabb.) were extracted by hot, pressurized ethyl acetate and analyzed for their effect in vitro on Lund human mesencephalic neurons. Cell viability was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and cell death was determined by lactate dehydrogenase levels. Three supplements strongly decreased the cell viability at extract concentrations of 1 µg/mL, of which 1 decreased cell viability at 0.1 µg/µL. Also, strong neuronal toxicities of these supplements were found. Cell death was observed at concentrations of 10 µg/mL. The degree of toxicity was comparable to the ones found in Annonaceous fruit extracts. Two fruit pulps of Annonaceae (A. muricata and A. squamosa) showed a reduction in cell viability at lower concentrations. The fruit pulp extract of A. muricata revealed the strongest neurotoxic effect, with 67% cell death at a concentration of 1 µg/mL. A high reduction in cell viability coupled with pronounced cell death was found at 0.1 µg/mL for an Annonaceous seed extract. These results demonstrate that the intake of dietary supplements containing plant material from Annonaceae may be hazardous to health in terms of neurotoxicity.

Determination of Neurotoxic Acetogenins in Pawpaw (Asimina triloba) Fruit by LC-HRMS.

The concentrations of the neurotoxins, annonacin and squamocin, were determined in a lyophilized sample of the fruit pulp of the North American pawpaw (Asimina triloba) by LC coupled to high resolution mass spectrometry or LC-HRMS. The sample was extracted using dry methanol at 100 °C and 10 MPa pressure in a sealed container. The extraction of annonacin and squamocin was optimal at 100 °C with 7.72 and 0.162 mg/g, respectively, being found. Also, several isomers of annonacin and squamocin were separated and detected but not quantified.

Prediction of solubility of drugs and other compounds in organic solvents.

We have set out a procedure for the prediction of solubilities of drugs and other compounds in a wide range of solvents, based on the Abraham solvation equations. The method requires a knowledge of solubilities of a given compound in a few solvents, as shown by our own experimental data on apocynin, diapocynin, dehydrodivanillin, and dehydrodi(methyl vanillate). The procedure is especially useful for very hydrophobic compounds such as cholesteryl acetate and cholesterol that we give as examples. Other examples include vanillin and 3,4-dichlorobenzoic acid. If the solubility in water is available, then this alone is sufficient to predict solubilities in organic solvents, provided that the Abraham descriptors are available for the compound. Predictions can be made for solubilities in some 85 solvents.

Dose formulation and analysis of diapocynin.

Procedures based on high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection and liquid chromatography-mass spectrometry (LC-MS) are described for analyzing diapocynin. Diapocynin was synthesized by oxidative coupling of two apocynin monomers, through the in situ generation of sulfate radicals. It was purified by washing 3 times each with boiling water, followed by boiling methanol. HPLC was used to determine the concentration of unreacted apocynin and other impurities and the purity of the diapocynin that had been synthesized. Negative-ion, atmospheric pressure chemical ionization (APCI) LC-MS was used to determine the molecular weights of impurities. The method using HPLC with UV detection provided a calibration curve that was linear from 0.16 to 24 microg/mL. The LC-MS method was linear from 0.005 to 2 microg/mL. It was found that diapocynin has low solubility in deionized water and corn oil but is soluble in dimethylsulfoxide (DMSO) and alkaline aqueous solutions. Also, diapocynin is 13 times more lipophilic than apocynin, even though both compounds have the same p K a of 7.4. The log of the octanol/water partition coefficient (log P) was 1.01 for apocynin and 1.82 for diapocynin. A solution of 5.5 mg/mL (16.7 mM) diapocynin in DMSO was found to be stable for at least 30 days when stored at room temperature.

Bioavailability of apocynin through its conversion to glycoconjugate but not to diapocynin.

Apocynin (4-hydroxy-3-methoxyacetophenone) is a major active ingredient from the rhizomes of Picrorhiza kurroa, a botanical plant used as an herbal medicine for treatment of a number of inflammatory diseases. Recently, apocynin is regarded as a specific inhibitor for NADPH oxidase in cell and animal models. In vitro studies indicated conversion of apocynin to diapocynin in the presence of peroxidases, e.g., myloperoxidase, posing the possibility that diapocynin also contributes to the anti-oxidative action of apocynin. The objectives of this study are to examine the bioavailability of apocynin to plasma, liver and brain tissue after intraperitoneal (i.p.) injection, and to examine whether apocynin is converted to diapocynin in vivo. Diapocynin was chemically synthetized and characterized by NMR and IR. Apocynin (5mg/kg body wt) was injected i.p. to adult male Sprague-Dawley rats and plasma, liver and brain were collected at different times (30min, 1 and 2h) after injection. Samples were treated with beta-glucuronidase to hydrolyze the glycosyl linkage and analyzed by HPLC/MS. At 30min and 1h after injection, approximately 50% of apocynin was converted to its glycosyl derivative and was distributed in plasma, liver and brain. No diapocynin was detected in any samples. These results indicate rapid glycosylation of apocynin and its transport to blood and other organs but no apparent conversion to diapocynin in vivo.

Dual fluorescence from an isonido ReIII rhenacarborane phosphine complex, [7,10-mu-H-7-CO-7,7-(PPh3)2-isonido-7,8,9-ReC2B7H9].

The complex [7,10-mu-H-7-CO-7,7-(PPh3)2-isonido-7,8,9-ReC2B7H9] has been synthesized by treatment of the complex salt [NHMe3][3,3-Cl2-3,3-(CO)2-closo-3,1,2-ReC2B9H11] with PPh3 in refluxing THF (tetrahydrofuran) and isolated as intensely colored orange-red microcrystals. Spectroscopic NMR and IR data have suggested that the product has a highly asymmetric structure with two inequivalent PPh3 ligands and a single CO ligand. Measurement of 11B NMR spectra in particular have indicated seven distinct boron vertexes, although the resulting cage degradation by removal of two BH vertexes was confirmed only following X-ray crystallographic analysis, which revealed the pentadecahedral isonido-7,8,9-ReC2B7 architecture. The 11B NMR resonances span an enormous chemical shift range (Deltadelta = 113), and this appears to be a direct consequence of the deshielding of the boron vertex directly opposite the quadrilateral |ReCCB| aperture. The new complex has been shown by electrochemical measurements to undergo a reversible one-electron oxidation. Digitally simulated cyclic voltammograms support a proposed square scheme (E(1/2) = 0.58, 0.69 V vs ferrocene) involving a reversible isonido-closo transition of the metallacarborane cage. Most unusually for a metallacarborane complex, ambient temperature solutions in CH2Cl2 and DMF have been shown to be intensely turquoise-blue fluorescent (lambda(em) = 442 nm, Phi = 0.012). Fluorescence spectroscopy measurements in MeTHF (2-methyltetrahydrofuran) glass at 77 K have indicated that the likely cause of such a broad emission is dual fluorescence (lambda(em) = 404, 505 nm), with both emissions displaying vibronic structure. Following excited-state lifetime decay analysis, the emissive behavior has been accredited to metal-perturbed 1IL states, with the lower energy emission arising from a slight geometric distortion of the initially excited complex.

Three-dimensional 13C-detected CH3-TOCSY using selectively protonated proteins: facile methyl resonance assignment and protein structure determination.

Recent advances in instrumentation and isotope labeling methodology allow proteins up to 100 kDa in size to be studied in detail using NMR spectroscopy. Using 2H/13C/15N enrichment and selective methyl protonation, we show that newly developed 13C direct detection methods can be used to rapidly yield proton and carbon resonance assignments for the methyl groups of Val, Leu, and Ile residues. We present a highly sensitive 13C-detected CH3-TOCSY experiment that, in combination with standard 1H-detected backbone experiments, allows the full assignment of side chain resonances in methyl-protonated residues. Selective methyl protonation, originally developed by Kay and co-workers (Rosen, M. K.; Gardner, K. H.; Willis, R. C.; Parris, W. E.; Pawson, T.; Kay, L. E. J. Mol. Biol. 1996, 263, 627-636; Gardner, K. G.; Kay, L. E. Annu. Rev. Biophys. Biomol. Struct. 1998, 27, 357-406; Goto, N. K.; Kay, L. E. Curr. Opin. Struct. Biol. 2000, 10, 585-592), improves the nuclear relaxation behavior of larger proteins compared to their fully protonated counterparts, allows significant simplification of spectra, and facilitates NOE assignments. Here, we demonstrate the usefulness of the 13C-detected CH3-TOCSY experiment through studies of (i) a medium-sized protein (CbpA-R1; 14 kDa) with a repetitive primary sequence that yields highly degenerate NMR spectra, and (ii) a larger, bimolecular protein complex (p21-KID/Cdk2; 45 kDa) at low concentration in a high ionic strength solution. Through the analysis of NOEs involving amide and Ile, Leu, and Val methyl protons, we determined the global fold of CbpA-R1, a bacterial protein that mediates the pathogenic effects of Streptococcus pneumoniae, demonstrating that this approach can significantly reduce the time required to determine protein structures by NMR.

Solution NMR studies of an intrinsically unstructured protein within a dilute, 75 kDa eukaryotic protein assembly; probing the practical limits for efficiently assigning polypeptide backbone resonances.

This paper describes an efficient NMR strategy for assigning the backbone resonances of an intrinsically unstructured protein (IUP), p21-KID, bound to its biological target, Cdk2/cyclin A. In order to overcome the challenges associated with the high molecular weight (75 kDa) and low solubility of the ternary complex (0.2 mM), we used perdeuteration, TROSY, and high-sensitivity cryogenic NMR probes at high magnetic-field strengths (i.e. 16.4, 18.8 and 21.1 Tesla). p21-KID was also prepared by using specific amino acid isotope labels. Most importantly, we studied binary, subcomplexes that allowed resonance assignments to be made in stages. We show that subdomains of p21-KID folded within binary complexes into the same conformations as observed in the ternary, Cdk2/cyclin A complex. This is a general feature of IUPs, which often adopt highly extended conformations when bound to other proteins. This strategy is suitable for studies of IUPs within considerably larger biomolecular assemblies as long as the IUP can be uniformly and selectively isotope labeled.

Solution structure of choline binding protein A, the major adhesin of Streptococcus pneumoniae.

Streptococcus pneumoniae (pneumococcus) remains a significant health threat worldwide, especially to the young and old. While some of the biomolecules involved in pneumococcal pathogenesis are known and understood in mechanistic terms, little is known about the molecular details of bacterium/host interactions. We report here the solution structure of the 'repeated' adhesion domains (domains R1 and R2) of the principal pneumococcal adhesin, choline binding protein A (CbpA). Further, we provide insights into the mechanism by which CbpA binds its human receptor, polymeric immunoglobulin receptor (pIgR). The R domains, comprised of 12 imperfect copies of the leucine zipper heptad motif, adopt a unique 3-alpha-helix, raft-like structure. Each pair of alpha-helices is antiparallel and conserved residues in the loop between Helices 1 and 2 exhibit a novel 'tyrosine fork' structure that is involved in binding pIgR. This and other structural features that we show are conserved in most pneumococcal strains appear to generally play an important role in bacterial adhesion to pIgR. Interestingly, pneumococcus is the only bacterium known to adhere to and invade human cells by binding to pIgR.