Nadia Bouchemal

Structure–function analysis of the antiangiogenic ATWLPPR peptide inhibiting VEGF165 binding to neuropilin-1 and molecular dynamics simulations of the ATWLPPR/neuropilin-1 complex

Heptapeptide ATWLPPR (A7R), identified in our laboratory by screening a mutated phage library, was shown to bind specifically to neuropilin-1 (NRP-1) and then to selectively inhibit VEGF(165) binding to this receptor. In vivo, treatment with A7R resulted in decreasing breast cancer angiogenesis and growth. The present work is focused on structural characterization of A7R. Analogs of the peptide, obtained by substitution of each amino acid with alanine (alanine-scanning) or by amino acid deletion, have been systematically assayed to determine the relative importance of the side chains of each residue with respect to the inhibitory effect of A7R on VEGF(165) binding to NRP-1. We show here the importance of the C-terminal sequence LPPR and particularly the key role of C-terminal arginine. In solution, A7R displays significant secondary structure of the backbone adopting an extended conformation. However, the functional groups of arginine are very flexible in the absence of NRP-1 pointing to an induced fit upon binding to the receptor. A MD trajectory of the A7R/NRP-1 complex in explicit water, based on the recent tuftsin/NRP-1 crystal structure, has revealed the hydrogen-bonding network that contributes to A7Rs binding activity.

Identification of Serum Proton NMR Metabolomic Fingerprints Associated with Hepatocellular Carcinoma in Patients with Alcoholic Cirrhosis

Purpose: Metabolomics depicts metabolic changes in biologic systems using a multiparametric analysis technique. This study assessed the metabolomic profiles of serum, obtained by proton nuclear magnetic resonance (NMR) spectroscopy, from cirrhotic patients with and without hepatocellular carcinoma (HCC). Experimental Design: The study included 154 consecutive patients with compensated biopsy-proven alcoholic cirrhosis. Among these, 93 had cirrhosis without HCC, 28 had biopsy-proven HCC within the Milan criteria and were eligible for curative treatment (small HCC), and 33 had HCC outside the Milan criteria (large HCC). Proton spectra were acquired at 500 MHz. An orthogonal partial latent structure [orthogonal projection to latent structure (OPLS)] analysis model was built to discriminate large HCC spectra from cirrhotic spectra. Small HCC spectra were secondarily projected using previously built OPLS discriminant components. Results: The OPLS model showed discrimination between cirrhotic and large HCC spectra. Metabolites that significantly increased with large HCC were glutamate, acetate, and N-acetyl glycoproteins, whereas metabolites that correlated with cirrhosis were lipids and glutamine. Projection of small HCC samples into the OPLS model showed a heterogeneous distribution between large HCC and cirrhotic samples. Small HCC patients with metabolomic profile similar to those of large HCC group had higher incidences of recurrence or death during follow-up. Conclusions: Serum NMR-based metabolomics identified metabolic fingerprints that could be specific to large HCC in cirrhotic livers. From a metabolomic standpoint, some patients with small HCC, who are eligible for curative treatments, seem to behave as patients with advanced cancerous disease. It would be useful to further prospectively investigate these patients to define a subgroup with a worse prognosis. Clin Cancer Res; 18(24); 6714–22. ©2012 AACR.

Metabolomic approach by 1H NMR spectroscopy of serum for the assessment of chronic liver failure in patients with cirrhosis.

Assessment of chronic liver failure (CLF) in cirrhotic patients is needed to make therapeutic decisions. A biological score is usually performed, using the Model for End-Stage Liver Disease (MELD), to evaluate CLF. Nevertheless, MELD does not take into account metabolic perturbations produced by liver-function impairment. In contrast, metabolomics can investigate many metabolic perturbations within biological systems. The purpose of this study was to assess whether metabolomic profiles of serum, obtained by proton NMR spectroscopy from cirrhotic patients, are affected by the severity of CLF. An orthogonal projection to latent-structure analysis was performed to compare MELD scores and NMR spectra of 124 patients with cirrhosis. The statistical model obtained showed a good explained variance (R(2)X = 0.87 and R(2)Y = 0.86) and a good predictability (Q(2)Y = 0.64). Metabolomic profiles showed significant differences regarding various metabolites depending of severity of CLF: levels of high-density lipoprotein and phosphocholine resonances were significantly higher in patients with mild CLF compared to severe CLF. Other metabolites such as lactate, pyruvate, glucose, amino acids, and creatinine were significantly higher in patients with severe CLF than mild CLF. Our conclusion is that metabolomic NMR analysis provides new insights into metabolic processes related to the severity of hepatic function impairment in cirrhosis.

Serum 1H-NMR Metabolomic Fingerprints of Acute-On-Chronic Liver Failure in Intensive Care Unit Patients with Alcoholic Cirrhosis

Introduction Acute-on-chronic liver failure is characterized by acute deterioration of liver function in patients with compensated or decompensated, but stable, cirrhosis. However, there is no accurate definition of acute-on-chronic liver failure and physicians often use this term to describe different clinical entities. Metabolomics investigates metabolic changes in biological systems and identifies the biomarkers or metabolic profiles. Our study assessed the metabolomic profile of serum using proton nuclear magnetic resonance (1H-NMR) spectroscopy to identify metabolic changes related to acute-on-chronic liver failure. Patients Ninety-three patients with compensated or decompensated cirrhosis (CLF group) but stable liver function and 30 patients with cirrhosis and hospitalized for the management of an acute event who may be responsible of acute-on-chronic liver failure (ACLF group), were fully analyzed. Blood samples were drawn at admission, and sera were separated and stored at –80°C until 1H-NMR spectral analysis. Using orthogonal projection to latent-structure discriminant analyses, various metabolites contribute to the complete separation between these both groups. Results The predictability of the model was 0.73 (Q2 Y) and the explained variance was 0.63 (R2 Y). The main metabolites that had increased signals related to acute-on-chronic liver failure were lactate, pyruvate, ketone bodies, glutamine, phenylalanine, tyrosine, and creatinine. High-density lipids were lower in the ALCF group than in CLF group. Conclusion A serum metabolite fingerprint for acute-on-chronic liver failure, obtained with 1H-NMR, was identified. Metabolomic profiling may aid clinical evaluation of patients with cirrhosis admitted into intensive care units with acute-on-chronic liver failure, and provide new insights into the metabolic processes involved in acute impairment of hepatic function.

Metabolomic profiling with NMR discriminates between biphosphonate and doxorubicin effects on B16 melanoma cells.

The metabolomic profiles of B16 melanoma cells were investigated in vitro with high resolution‐magic angle spinning proton magnetic resonance spectroscopy and OPLS multivariate statistical analyse. We compared the profiles for untreated melanoma B16‐F10 cells and Ca2+ chelating EGTA, doxorubicin or BP7033 bisphosphonate treated cells. The two last molecules are known to induce anti‐proliferative effects by different mechanisms of action in cells. Untreated and EGTA treated cells had similar profiles and were considered together as control cells. Several spectral regions could discriminate control from doxorubicin as well as BP7033 treated cells. Doxorubicin and BP7033 displayed distinct metabolic profiles. Important changes in neutral lipids and inositol were related to doxorubicin activity whereas BP7033 affected essentially phospholipids and alanine/lactate metabolism. These results provide new putative targets for both drugs. Metabolomics by NMR is shown here to be a good tool for the investigation of the mechanisms of action of drugs in pre‐clinical studies. Copyright

NMR-based prediction of cardiovascular risk in diabetes

To the editor: It has been proposed that nuclear magnetic resonance (NMR) plasma analysis can improve lipoprotein subclass discrimination and predict coronary artery disease (CAD)1. In the recent paper by Kirschenlohr et al.2, it was concluded that proton NMR spectroscopy shows only weak discrimination for detecting angiographydefined CAD. An even more important question is whether proton NMR spectroscopy is able to predict cardiovascular events. Indeed, the mortality and morbidity for CAD in type 2 diabetes patients is two to four times higher than in nondiabetic subjects3, making cardiovascular disease the first cause of death and disability amongst this population. Moreover, CAD is often undetected in diabetic patients. The traditional risk factors do not fully explain the level of the cardiovascular risk in these patients, and new tools are being developed to better identify high-risk patients, who need invasive coronary evaluation and/or aggressive treatments. Indeed, the strategy must have a high degree of accuracy for detecting CAD, owing to the potential morbidity associated with angiographic procedures. To assess whether NMR spectroscopy can predict cardiovascular disease, we performed a case-control study nested in the placebo arm of a larger clinical trial, DIABHYCAR (ref. 4). DIABHYCAR, a randomized clinical trial, involved high-risk type 2 diabetic patients who were followed for 4 years and who, during that period, had fatal or nonfatal acute myocardial infarction. The study protocol was approved by the ethical committee of Angers University, France. All participants provided written informed consent. This nested case-control study included 190 patients (150 men and 40 women). We acquired proton NMR spectra of samples collected from patients upon their inclusion in DIABHYCAR. Patients of the ‘case’ group were defined as those who presented either a fatal or nonfatal acute myocardial infarction or who underwent sudden death during the 4-year follow-up. Control patients were defined as patients not presenting any cardiovascular events. Control patients were matched to the case patients for age ± 2 years, sex and geographical origin, and their followup lasted for at least as long as that of the case patients (Table 1). We obtained blood samples after an overnight fast, using tubes without anticoagulant. We stored sera at –80 °C before NMR analysis. We acquired spectra at 500 MHz (Varian Inova) with a 1.5-s presaturation of the water signal5. The acquisition sequence consisted of a 4-s-long relaxation delay (including water suppression delay) and a 90° excitation pulse. The signal was acquired on 16,384 data points. Fourier transform was applied without zero filling, and with a 0.1 Hz line-broadening factor. The chemical shift scale was calibrated on the fumaric acid signal at 6.53 p.p.m. We used two distinct methods to analyze the spectrum: determination of lipid subclasses by deconvolution in the frequency domain (method 1) and whole-spectra analysis (method 2). More specifically, for method 1, lipoprotein deconvolution was performed with WINNMR software as described previously5. Briefly, the region between 0.5 and 1.1 p.p.m. was deconvoluted into 11 Lorentzians whose areas were used for multivariate analysis. In method 2, the spectra were divided into bins of width 0.04 p.p.m., defined from 10 to 0.4 p.p.m., excluding the water signal between 4.9 to 4.5 p.p.m. This data reduction resulted in 207 elements for each spectrum1. To the results of each method, we then applied orthogonal signal correction (OSC) followed by partial least-squares discriminant analysis (PLS-DA), a similar approach to the one used previously1. This statistical analysis was performed twice: first on the complete set of 190 patients of the nested case-control study and then to evaluate the model predictability on an independent population, on a randomly chosen training group (70% of the subjects) and validation group (30%). To classify the patients of this validation group, we applied the model obtained from the OSC/PLS-DA

Synthesis and inclusion ability of anthracene appended β-cyclodextrins: unexpected effect of triazole linker

A new fluorescent β-cyclodextrin has been synthesized by coupling an anthracene moiety to the cyclic oligosaccharide via click chemistry. The influence of the triazole spacer was compared to the simple amino and amido linkers. While a sensing ability toward adamantan-1-ol was observed with the latter two spacers, the absence of inclusion capacity prevents the triazole modified cyclodextrin from showing any fluorescence variations. The difference in the binding behaviors studied by Isothermal Titration Calorimetry, UV-vis and fluorescence spectroscopies, was highlighted by the NOESY NMR spectra of the modified cyclodextrins: whereas a free cavity was observed for the amino and amido linkers, an important obstruction was obtained in the case of the triazole.

Tunable Design of Gold(III)–Doxorubicin Complex–PEGylated Nanocarrier. The Golden Doxorubicin for Oncological Applications

To date, the translation of Au (III) complexes into chemotherapeutic agents has been hindered by their low stability under physiological conditions, a crucial parameter in drug development. In this study, we report an innovative four-step synthesis of a stable Au (III)-doxorubicin (DOX) complex, acting as a key constitutive component of doxorubicin-loaded PEG-coated nanoparticles (DOX IN-PEG-AuNPs). For therapeutic purposes, such AuNPs were then functionalized with the anti-Kv11.1 polyclonal antibody (pAb), which specifically recognizes the hERG1 channel that is overexpressed on the membrane of human pancreatic cancer cells. The nature of the interactions between DOX and Au (III) ions was probed by various analytical techniques (Raman spectroscopy, UV-vis, and (1)H NMR), which enabled studying the Au (III)-DOX interactions during AuNPs formation. The theoretical characterization of the vibrational bands and the electronic transitions of the Au (III)-DOX complex calculated through computational studies showed significant qualitative agreement with the experimental observations on AuNPs samples. Stability in physiological conditions and efficient drug loading (up to to 85 w/w %) were achieved, while drug release was strongly dependent on the structure of DOX IN-PEG-AuNPs and on the pH. Furthermore, the interactions among DOX, PEG, and Au (III) ions in DOX IN-PEG-AuNPs differed significantly from those found in polymer-modified AuNPs loaded with DOX by covalent linkage, referred to as DOX ON-PEG-AuNPs. In vitro experiments indeed demonstrated that such differences strongly influenced the therapeutic potential of AuNPs in pancreatic cancer treatment, with a significant increase of the DOX therapeutic index when complexed to Au (III) ions. Collectively, our study demonstrated that Au (III)-DOX complexes as building blocks of PEGylated AuNPs constitutes a promising approach to transform promising Au (III) complexes into real chemotherapeutic drugs for the treatment of pancreatic cancer.

Energetics of endurance exercise in young horses determined by nuclear magnetic resonance metabolomics.

Long-term endurance exercise severely affects metabolism in both human and animal athletes resulting in serious risk of metabolic disorders during or after competition. Young horses (up to 6 years old) can compete in races up to 90 km despite limited scientific knowledge of energetic metabolism responses to long distance exercise in these animals. The hypothesis of this study was that there would be a strong effect of endurance exercise on the metabolomic profiles of young horses and that the energetic metabolism response in young horses would be different from that of more experienced horses. Metabolomic profiling is a powerful method that combines Nuclear Magnetic Resonance (NMR) spectrometry with supervised Orthogonal Projection on Latent Structure (OPLS) statistical analysis. 1H-NMR spectra were obtained from plasma samples drawn from young horses (before and after competition). The spectra obtained before and after the race from the same horse (92 samples) were compared using OPLS. The statistical parameters showed the robustness of the model (R2Y = 0.947, Q2Y = 0.856 and cros-validated ANOVA p < 0.001). For confirmation of the predictive value of the model, a test set of 104 sample spectra were projected by the model, which provided perfect predictions as the area under the receiving-operator curve was 1. The metabolomic profile determined with the OPLS model showed that glycemia after the race was lower than glycemia before the race, despite the involvement of lipid and protein catabolism. An OPLS model was calculated to compare spectra obtained on plasma taken after the race from 6-year-old horses and from experienced horses (cross-validated ANOVA p < 0.001). The comparison of metabolomic profiles in young horses to those from experienced horses showed that experienced horses maintained their glycemia with higher levels of lactate and a decrease of plasma lipids after the race.

Accumulation of Eu3+ chelates in cells expressing or not P-glycoprotein: Implications for blood–brain barrier crossing

Alzheimers disease (AD) is the most commonly form of dementia in the elderly. The development of molecules able to detect biomarkers characteristic of AD is critical to its understanding and treatment. However, such molecules must be able to pass blood-brain barrier (BBB) which is a major impediment to the entry of many therapeutic drugs into the brain. Such a limitation applies to the development of magnetic resonance imaging molecular neuroimaging agents using biomarkers of AD-like beta-amyloid deposits, as the common extracellular contrast agents (CAs) are not able to cross an intact BBB. In this work, we have studied the ability of a series of simple Eu(3+) complexes to enter cells overexpressing or not the ABCB1 (P-gp or P-glycoprotein) protein, which is expressed at the BBB and in human embryonic astrocytes. The intracellular uptake of the Eu(3+) complexes of linear and macrocyclic polyaminocarboxylate ligands with different charges and lipophilicities was followed by atomic absorption spectrometry. Based on biochemical argument, we propose that lipophilic contrast agents can be efficiently taken up by cells and accumulate inside mitochondria when they are positively charged. The important point is that they are not P-gp substrates, which is one of the major obstacles for them to cross the BBB.