NMR and Docking Calculations Reveal Novel Atomistic Selectivity of a Synthetic High-Affinity Free Fatty Acid vs. Free Fatty Acids in Sudlow's Drug Binding Sites in Human Serum Albumin.

Saturation transfer difference (STD), inter-ligand NOEs (INPHARMA NMR), and docking calculations are reported for investigating specific binding sites of the high-affinity synthetic 7-nitrobenz-2-oxa-1,3-diazoyl-4-C12 fatty acid (NBD-C12 FA) with non-labeled human serum albumin (HSA) and in competition with the drugs warfarin and ibuprofen. A limited number of negative interligand NOEs between NBD-C12 FA and warfarin were interpreted in terms of a short-range allosteric competitive binding in the wide Sudlow's binding site II (FA7) of NBD-C12 FA with Ser-202, Lys-199, and Trp-214 and warfarin with Arg-218 and Arg-222. In contrast, the significant number of interligand NOEs between NBD-C12 FA and ibuprofen were interpreted in terms of a competitive binding mode in Sudlow's binding site I (FA3 and FA4) with Ser-342, Arg-348, Arg-485, Arg-410, and Tyr-411. NBD-C12 FA has the unique structural properties, compared to short-, medium-, and long-chain saturated and unsaturated natural free fatty acids, of interacting with well-defined structures with amino acids of both the internal and external polar anchor sites in Sudlow's binding site I and with amino acids in both FA3 and FA4 in Sudlow's binding site II. The NBD-C12 FA, therefore, interacts with novel structural characteristics in the drug binding sites I and II and can be regarded as a prototype molecule for drug development.

Molecular Basis for the Selectivity of DHA and EPA in Sudlow's Drug Binding Sites in Human Serum Albumin with the Combined Use of NMR and Docking Calculations.

Medium- and long-chain saturated and unsaturated free fatty acids (FFAs) are known to bind to human serum albumin (HSA), the main plasma carrier protein. Atomic-level structural data regarding the binding mode in Sudlow's sites I (FA7) and II (FA4, FA3) of the polyunsaturated ω-3 fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), however, are largely unknown. Herein, we report the combined use of saturation transfer difference (STD) and Interligand NOEs for Pharmacophore Mapping (INPHARMA) NMR techniques and molecular docking calculations to investigate the binding mode of DHA and EPA in Sudlow's sites Ι and ΙΙ of HSA. The docking calculations and the significant number of interligand NOEs between DHA and EPA and the drugs warfarin and ibuprofen, which are stereotypical ligands for Sudlow's sites I and II, respectively, were interpreted in terms of competitive binding modes and the presence of two orientations of DHA and EPA at the binding sites FA7 and FA4. The exceptional flexibility of the long-chain DHA and EPA and the formation of strongly folded structural motives are the key properties of HSA-PUFA complexes.

Structural Basis of Artemisinin Binding Sites in Serum Albumin with the Combined Use of NMR and Docking Calculations.

Artemisinin is known to bind to the main plasma protein carrier serum albumin (SA); however, there are no atomic level structural data regarding its binding mode with serum albumin. Herein, we employed a combined strategy of saturation transfer difference (STD), transfer nuclear Overhauser effect spectroscopy (TR-NOESY), STD-total correlation spectroscopy (STD-TOCSY), and Interligand Noes for PHArmacophore Mapping (INPHARMA) NMR methods and molecular docking calculations to investigate the structural basis of the interaction of artemisinin with human and bovine serum albumin (HSA/BSA). A significant number of inter-ligand NOEs between artemisinin and the drugs warfarin and ibuprofen as well as docking calculations were interpreted in terms of competitive binding modes of artemisinin in the warfarin (FA7) and ibuprofen (FA4) binding sites. STD NMR experiments demonstrate that artemisinin is the main analyte for the interaction of the A. annua extract with BSA. The combined strategy of NMR and docking calculations of the present work could be of general interest in the identification of the molecular basis of the interactions of natural products with their receptors even within a complex crude extract.

NMR and computational studies reveal novel aspects in molecular recognition of unsaturated fatty acids with non-labelled serum albumin.

An approach based on the combined use of saturation transfer difference (STD), Tr-NOESY and Inter-ligand NOEs for PHArmacophore Mapping (INPHARMA) NMR techniques and docking calculations is reported, for the first time, for mapping interactions and specific binding sites of caproleic acid (10 : 1 cis-9), oleic acid (18 : 1 cis-9), linoleic acid (18 : 2 cis-9,12) and linolenic (18 : 3, cis-9,12,15) free fatty acids (FFAs) with non-labelled serum albumin (BSA/HSA). Significant negative inter-ligand NOEs between the FFAs and the drugs ibuprofen and warfarin, through competition experiments, were observed. The inter-ligand NOEs and docking calculations were interpreted in terms of competitive binding mode, the significant folding of the bis allylic region and the presence of two orientations of the FFAs in the warfarin binding site (FA7), due to two potential distinctive anchoring polar groups of amino acids. This conformational flexibility is the reason that, the location and conformational states of the FFAs in the binding site of warfarin could not be determined accurately, despite numerous available X-ray structural studies. α-Linolenic acid competes favourably with warfarin at the binding site FA7. Isothermal titration calorimetry experiments of the preformed HSA/α-linolenic acid complex upon titration with warfarin show a significant reduction in the binding constant of warfarin, in very good agreement with NMR and computational data. The combined use, therefore, of STD, Tr-NOESY and INPHARMA NMR, ITC and docking calculations may find promising applications in the field of protein-lipid recognition research.

NGIWY-Amide: A Bioinspired Ultrashort Self-Assembled Peptide Gelator for Local Drug Delivery Applications.

Fibrillar structures derived from plant or animal origin have long been a source of inspiration for the design of new biomaterials. The Asn-Gly-Ile-Trp-Tyr-NH2 (NGIWY-amide) pentapeptide, isolated from the sea cucumber Apostichopus japonicus, which spontaneously self-assembles in water to form hydrogel, pertains to this category. In this study, we evaluated this ultra-short cosmetic bioinspired peptide as vector for local drug delivery applications. Combining nuclear magnetic resonance, circular dichroism, infrared spectroscopy, X-ray diffraction, and rheological studies, the synthesized pentapeptide formed a stiff hydrogel with a high ß-sheet content. Molecular dynamic simulations aligned well with scanning electron and atomic-force microscopy studies, revealing a highly filamentous structure with the fibers adopting a helical-twisted morphology. Model dye localization within the supramolecular hydrogel provided insights on the preferential distribution of hydrophobic and hydrophilic compounds in the hydrogel network. That was further depicted in the diffusion kinetics of drugs differing in their aqueous solubility and molecular weight, namely, doxorubicin hydrochloride, curcumin, and octreotide acetate, highlighting its versatility as a delivery vector of both hydrophobic and hydrophilic compounds of different molecular weight. Along with the observed cytocompatibility of the hydrogel, the NGIWY-amide pentapeptide may offer new approaches for cell growth, drug delivery, and 3D bioprinting tissue-engineering applications.

Serum albumin as a primary non-covalent binding protein for nitro-oleic acid.

This work explores the interaction of 9/10-nitro-oleic acid (NO2-OA) with human serum albumin (HSA). The molecular mechanism of the biological action of NO2-OA is to our knowledge based on a reversible covalent reaction-Michael addition of nucleophilic amino acid residues of proteins. Since HSA is an important fatty acid transporter, a key question is whether NO2-OA can bind covalently or non-covalently to HSA, similarly to oleic acid (OA), which can interact with the FA1-FA7 binding sites of the HSA molecule. 1H NMR studies and competition analysis with OA and the drugs ibuprofen and warfarin were used to investigate a potential non-covalent binding mode. NO2-OA/HSA binding was confirmed to compete with warfarin for FA-7 with significantly higher affinity. NO2-OA competes with ibuprofen for FA-3 and FA-6, however, in contrast to the situation with warfarin, the binding affinities are not significantly different. The described interactions are based exclusively on non-covalent binding. No covalent binding of NO2-OA to HSA was detected by MS/MS. More detailed studies based on MALDI-TOF-MS and Ellman's assay indicated that HSA can be covalently modified in the presence of NO2-OA to a very limited extent. It was also shown that NO2-OA has a higher affinity to HSA than that of OA.

Characterization of Protocatechuate 4,5-Dioxygenase from Pseudarthrobacter phenanthrenivorans Sphe3 and In Situ Reaction Monitoring in the NMR Tube.

The current study aims at the functional and kinetic characterization of protocatechuate (PCA) 4,5-dioxygenase (PcaA) from Pseudarthrobacter phenanthrenivorans Sphe3. This is the first single subunit Type II dioxygenase characterized in Actinobacteria. RT-PCR analysis demonstrated that pcaA and the adjacent putative genes implicated in the PCA meta-cleavage pathway comprise a single transcriptional unit. The recombinant PcaA is highly specific for PCA and exhibits Michaelis-Menten kinetics with Km and Vmax values of 21 ± 1.6 µM and 44.8 ± 4.0 U × mg-1, respectively, in pH 9.5 and at 20 °C. PcaA also converted gallate from a broad range of substrates tested. The enzymatic reaction products were identified and characterized, for the first time, through in situ biotransformation monitoring inside an NMR tube. The PCA reaction product demonstrated a keto-enol tautomerization, whereas the gallate reaction product was present only in the keto form. Moreover, the transcriptional levels of pcaA and pcaR (gene encoding a LysR-type regulator of the pathway) were also determined, showing an induction when cells were grown on PCA and phenanthrene. Studying key enzymes in biodegradation pathways is significant for bioremediation and for efficient biocatalysts development.

DFT Calculations of 1H NMR Chemical Shifts of Geometric Isomers of Conjugated Linolenic Acids, Hexadecatrienyl Pheromones, and Model Triene-Containing Compounds: Structures in Solution and Revision of NMR Assignments.

A DFT study of the 1H NMR chemical shifts, δ(1H), of geometric isomers of 18:3 conjugated linolenic acids (CLnAs), hexadecatrienyl pheromones, and model triene-containing compounds is presented, using standard functionals (B3LYP and PBE0) as well as corrections for dispersion interactions (B3LYP-D3, APFD, M06-2X and ωB97XD). The results are compared with literature experimental δ(1H) data in solution. The closely spaced "inside" olefinic protons are significantly more deshielded due to short-range through-space H…H steric interactions and appear close to or even beyond δ-values of aromatic systems. Several regularities of the computational δ(1H) of the olefinic protons of the conjugated double bonds are reproduced very accurately for the lowest-energy DFT-optimized single conformer for all functionals used and are in very good agreement with experimental δ(1H) in solution. Examples are provided of literature studies in which experimental resonance assignments deviate significantly from DFT predictions and, thus, should be revised. We conclude that DFT calculations of 1H chemical shifts of trienyl compounds are powerful tools (i) for the accurate prediction of δ(1H) even with less demanding functionals and basis sets; (ii) for the unequivocal identification of geometric isomerism of conjugated trienyl systems that occur in nature; (iii) for tackling complex problems of experimental resonance assignments due to extensive signal overlap; and (iv) for structure elucidation in solution.

DFT Calculations of 1H- and 13C-NMR Chemical Shifts of Geometric Isomers of Conjugated Linoleic Acid (18:2 ω-7) and Model Compounds in Solution.

A density functional theory (DFT) study of the 1H- and 13C-NMR chemical shifts of the geometric isomers of 18:2 ω-7 conjugated linoleic acid (CLA) and nine model compounds is presented, using five functionals and two basis sets. The results are compared with available experimental data from solution high resolution nuclear magnetic resonance (NMR). The experimental 1H chemical shifts exhibit highly diagnostic resonances due to the olefinic protons of the conjugated double bonds. The "inside" olefinic protons of the conjugated double bonds are deshielded than those of the "outside" protons. Furthermore, in the cis/trans isomers, the signals of the cis bonds are more deshielded than those of the trans bonds. These regularities of the experimental 1H chemical shifts of the olefinic protons of the conjugated double bonds are reproduced very accurately for the lowest energy DFT optimized single conformer, for all functionals and basis sets used. The other low energy conformers have negligible effects on the computational 1H-NMR chemical shifts. We conclude that proton NMR chemical shifts are more discriminating than carbon, and DFT calculations can provide a valuable tool for (i) the accurate prediction of 1H-NMR chemical shifts even with less demanding functionals and basis sets; (ii) the unequivocal identification of geometric isomerism of CLAs that occur in nature, and (iii) to derive high resolution structures in solution.

Simultaneous determination of artemisinin and its analogs and flavonoids in Artemisia annua crude extracts with the use of NMR spectroscopy.

Artemisia annua is a promising and potent antimalarial herbal drug. This activity has been ascribed to its component artemisinin, a sesquiterpene lactone. The ability to determine artemisinin and its known analogs in plant extracts is an especially difficult task because the compounds are present in low concentrations, are thermolabile, and lack ultraviolet or fluorescent chromophores. We report herein a facile and rapid 1-D 1 H, 1-D total correlation spectroscopy, 2-D 1 H-13 C heteronuclear single quantum coherence, and 1 H-13 C heteronuclear multiple bond correlation nuclear magnetic resonance techniques for the simultaneous identification and quantification of artemisinin and five of its analogs along with five flavonoids, an aromatic ketone, and camphor (in total, 13 compounds) in crude diethyl ether A. annua extract without the need of laborious isolation of the individual analytes. The above method was validated in terms of precision, linearity, and limit of detection. The analytical results were found to be in excellent agreement with those obtained with the use of the time consuming high-performance liquid chromatography with diode-array detection and liquid chromatography with tandem mass spectrometry for the compounds that standards were available.

Valorization of Carob Fruit Residues for the Preparation of Novel Bi-Functional Polyphenolic Coating for Food Packaging Applications.

The food industry has become interested in the development of innovative biomaterials with antioxidant and antimicrobial properties. Although several biopolymers have been evaluated for food packaging, the use of polyphenolic coatings has been unexplored. The purpose of this work was to develop an antioxidant and antimicrobial coating for food packaging through the polymerization of carob phenolics. At first, the polyphenolic coatings were deposited in glass surfaces polymerizing different concentrations of carob extracts (2 and 4 mg mL-1) at three pH values (7, 8 and 9). Results demonstrated that the coating produced at pH 8 and at a concentration of 4 mg mL-1 had the most potent antioxidant and antimicrobial potential. Then, the coating was applied directly on the salmon fillet (coating) and on the plastic container (active packaging). Peroxide and thiobarbituric acid-reactive substances (TBARS) methods were used to measure the potency to inhibit lipid oxidation in salmon fillets. Furthermore, the anti-Listeria activity of coatings was also assessed. Results showed a significant decrease of lipid oxidation during cold storage of salmon fillets for both treatments; the superiority of applied coating directly on the salmon fillets was also highlighted. Regarding the antimicrobial potency, the polyphenolic coating depleted the growth of Listeria monocytogenes after 10 days storage; while the active packaging had no effect on Listeria monocytogenes. Overall, we describe the use of low-cost carob polyphenols as precursors for the formation of bifunctional coatings with promising applications in food packaging.

Protective Effects of the Complement Inhibitor Compstatin CP40 in Hemorrhagic Shock.

Trauma-induced hemorrhagic shock (HS) plays a decisive role in the development of immune, coagulation, and organ dysfunction often resulting in a poor clinical outcome. Imbalanced complement activation is intricately associated with the molecular danger response and organ damage after HS. Thus, inhibition of the central complement component C3 as turnstile of both inflammation and coagulation is hypothesized as a rational strategy to improve the clinical course after HS.Applying intensive care conditions, anaesthetized, monitored, and protectively ventilated nonhuman primates (NHP; cynomolgus monkeys) received a pressure-controlled severe HS (60 min at mean arterial pressure 30 mmHg) with subsequent volume resuscitation. Thirty minutes after HS, animals were randomly treated with either an analog of the C3 inhibitor compstatin (i.e., Cp40) in saline (n = 4) or with saline alone (n = 4). The observation period lasted 300 min after induction of HS.We observed improved kidney function in compstatin Cp40-treated animals after HS as determined by improved urine output, reduced damage markers and a tendency of less histopathological signs of acute kidney injury. Sham-treated animals revealed classical signs of mucosal edema, especially in the ileum and colon reflected by worsened microscopic intestinal injury scores. In contrast, Cp40-treated HS animals exhibited only minor signs of organ edema and significantly less intestinal damage. Furthermore, early systemic inflammation and coagulation dysfunction were both ameliorated by Cp40.The data suggest that therapeutic inhibition of C3 is capable to significantly improve immune, coagulation, and organ function and to preserve organ-barrier integrity early after traumatic HS. C3-targeted complement inhibition may therefore reflect a promising therapeutic strategy in fighting fatal consequences of HS.

Probing the interaction of a quercetin bioconjugate with Bcl-2 in living human cancer cells with in-cell NMR spectroscopy.

In-cell NMR spectroscopy has emerged as a powerful technique for monitoring biomolecular interactions at an atomic level inside intact cells. However, current methodologies are inadequate at charting intracellular interactions of nonlabeled proteins and require their prior isotopic labeling. Herein, we describe for the first time the monitoring of the quercetin-alanine bioconjugate interaction with the nonlabeled antiapoptotic protein Bcl-2 inside living human cancer cells. STD and Tr-NOESY in-cell NMR methodologies were successfully applied in the investigation of the binding, which was further validated in vitro. In-cell NMR proved a very promising strategy for the real-time probing of the interaction profile of potential drugs with their therapeutic targets in native cellular environments and could, thus, open a new avenue in drug discovery.

Method development and validation for the quantitation of the complement inhibitor Cp40 in human and cynomolgus monkey plasma by UPLC-ESI-MS.

Cp40 is a 14-amino acid cyclic analog of the peptidic complement inhibitor compstatin that binds with sub-nanomolar affinity to complement component C3 and has already shown promise in various models of complement-related diseases. The preclinical and clinical development of this compound requires a robust, accurate, and sensitive method for quantitatively monitoring Cp40 in biological samples. In this study, we describe the development and validation of an ultra-high performance liquid chromatography electrospray mass spectrometry method for the quantitation of Cp40 in human and non-human primate (NHP) plasma. Isotope-labeled Cp40 was used as an internal standard, allowing for the accurate and absolute quantitation of Cp40. Labeled and non-labeled Cp40 were extracted from plasma using reversed phase-solid phase extraction, with recovery rates exceeding 80%, indicating minor matrix effects. The triply charged states of Cp40 and isotope-labeled Cp40 were detected at m/z 596.60 and 600.34, respectively, via a Q-TOF mass spectrometer and were used for quantitation. The method was linear in the range of 0.18-3.58µg/mL (r2≥0.99), with precision values below 0.71% in NHP and 0.77% in human plasma. The accuracy of the method ranged from -2.17% to 17.99% in NHP and from -0.26% to 15.75% in human plasma. The method was successfully applied to the quantitation of Cp40 in cynomolgus monkey plasma after an initial intravenous bolus of 2mg/kg followed by repetitive subcutaneous administration at 1mg/kg. The high reproducibility, accuracy, and robustness of the method developed here render it suitable for drug monitoring of Cp40, and potentially other compstatin analogs, in both human and NHP plasma samples during pharmacokinetic and pharmacodynamic studies.

Complement C3 inhibitor Cp40 attenuates xenoreactions in pig hearts perfused with human blood.

BACKGROUND: The complement system plays a crucial role in acute xenogeneic reactions after cardiac transplantation. We used an ex vivo perfusion model to investigate the effect of Cp40, a compstatin analog and potent inhibitor of complement at the level of C3. METHODS: Fifteen wild-type pig hearts were explanted, cardiopleged, and reperfused ex vivo after 150 minutes of cold ischemia. Hearts were challenged in a biventricular working heart mode to evaluate cardiac perfusion and function. In the treatment group (n=5), the complement cascade was blocked at the level of C3 using Cp40, using diluted human blood. Untreated human and porcine blood was used for controls. RESULTS: Throughout the perfusion, C3 activation was inhibited when Cp40 was used (mean of all time points: 1.11 ± 0.34% vs 3.12 ± 0.48% control activation; P<.01). Compared to xenoperfused controls, the cardiac index improved significantly in the treated group (6.5 ± 4.2 vs 3.5 ± 4.8 mL/min/g; P=.03, 180 minutes perfusion), while the concentration of lactate dehydrogenase as a maker for cell degradation was reduced in the perfusate (583 ± 187 U/mL vs 2108 ± 1145 U/mL, P=.02). Histological examination revealed less hemorrhage and edema, and immunohistochemistry confirmed less complement fragment deposition than in untreated xenoperfused controls. CONCLUSIONS: Cp40 efficiently prevents C3 activation of the complement system, resulting in reduced cell damage and preserved function in wild-type porcine hearts xenoperfused ex vivo. We suggest that this compstatin analog, which blocks all main pathways of complement activation, could be a beneficial perioperative treatment in preclinical and in future clinical xenotransplantation.

Understanding zinc(II) chelation with quercetin and luteolin: a combined NMR and theoretical study.

The Zn(II) chelation with natural flavonoids, quercetin and luteolin, was investigated by the use of NMR spectroscopy and various levels of ab initio calculations. Very sharp phenolic OH (1)H resonances in DMSO-d6 were observed for both free and complexed quercetin which allowed (i) the unequivocal assignment with the combined use of (1)H-(13)C HSQC and HMBC experiments and (ii) the determination of complexation sites which were found to be the CO-4 carbonyl oxygen and the deprotonated C-5 OH group of quercetin and CO-4 carbonyl oxygen and the deprotonated C-5 OH group of luteolin. DOSY experiments allowed the determination of the effective molecular weight of the Zn-quercetin complex which was shown to be mainly 1:1. DFT calculations of the 1:1 complex in the gas phase demonstrated that the C-3 O(-) and CO-4 sites are favored for quercetin at both GGA and LDA approximations and the C-5 O(-) and CO-4 groups of luteolin at the LDA approximation. Quantum chemical calculations were also performed by means of the conductor polarizable model in DMSO by employing various functionals. The energetically favored Zn chelation sites of the 1:1 complex were found to be either the C-3 O(-) and CO-4 or C-5 O(-) and CO-4 sites, depending on the functional used, for quercetin and the C-5 O(-) and CO-4 sites for luteolin.

Direct binding of Bcl-2 family proteins by quercetin triggers its pro-apoptotic activity.

Bcl-2 family proteins are important regulators of apoptosis and its antiapoptotic members, which are overexpressed in many types of cancer, are of high prognostic significance, establishing them as attractive therapeutic targets. Quercetin, a natural flavonoid, has drawn much attention because it exerts anticancer effects, while sparing normal cells. A multidisciplinary approach has been employed herein, in an effort to reveal its mode of action including dose-response antiproliferative activity and induced apoptosis effect, biochemical and physicochemical assays, and computational calculations. It may be concluded that, quercetin binds directly to the BH3 domain of Bcl-2 and Bcl-xL proteins, thereby inhibiting their activity and promoting cancer cell apoptosis.

Hydrogen bonding probes of phenol -OH groups.

Correlations between hydrogen bonds and solvent effects on phenol -OH proton shieldings, temperature coefficients (Δδ/ΔT) and effects on OH diffusion coefficients for numerous phenolic acids, flavonols, flavones, and oleuropein derivatives of biological interest were investigated in several organic solvents and were shown to serve as reliable indicators of hydrogen bonding and solvation state of -OH groups. The temperature coefficients span a range of -0.5 to -12.3 ppb K(-1). Shielding differences of 2.0 to 2.9 ppm at 298 K were observed for solvent exposed OH groups between DMSO-d(6) and CD(3)CN which should be compared with a shielding range of ~7 ppm. This demonstrates that the solvation state of hydroxyl protons is a key factor in determining the value of the chemical shift. For -OH protons showing temperature gradients more positive than -2.5 ppb K(-1), shielding changes between DMSO-d(6) and CD(3)CN below 0.6 ppm, and diffusion coefficients significantly different from those of traces of H(2)O, there is an intramolecular hydrogen bond predictivity value of 100%. The C-3 OH protons of flavonols show very significant negative temperature coefficients and shielding changes between DMSO-d(6) and CD(3)CN of ~2.3 ppm, which indicate the absence of persistent intramolecular hydrogen bonds, contrary to numerous X-ray structures.

Unexpected enzyme-catalyzed regioselective acylation of flavonoid aglycones and rapid product screening.

Unprecedented regioselective acylation of flavonoid aglycones was achieved using Candida antarctica lipase B (CALB). The rapid screening of product formation was performed by the use of the high resolution phenol-type OH (1)H NMR spectral region recorded after the addition of picric acid.