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NADPH-cytochrome P450 reductase is the electron transfer partner for the cytochromes P450, heme oxygenase, and squalene monooxygenase, and is a component of the nitric oxide synthases and methionine synthase reductase. P450 reductase shows very high selectivity for NADPH and uses NADH only poorly. Substitution of tryptophan 677 with alanine (W677A) has been shown by others to yield a 3-fold increase in turnover with NADH, but profound inhibition by NADP+ makes the enzyme unsuitable for in vivo applications. In the present study site-directed mutagenesis of amino acids in the 2'-phosphate-binding site of the NADPH domain, coupled with the W677A substitution, was used to generate a reductase that was able to use NADH efficiently in vivo without inhibition by NADP+. Of 11 single, double, and triple mutant proteins, two (R597M/W677A and R597M/K602W/W677A) showed up to a 500-fold increase in catalytic efficiency (kcat/Km) with NADH. Inhibition by NADP+ was reduced by up to four orders of magnitude relative to the W677A protein and was equal to or less than that of the wild-type reductase. Both proteins were 2- to 3-fold more active than wild-type reductase with NADH in reconstitution assays with cytochrome P450 1A2 and with squalene monooxygenase. In a recombinant cytochrome P450 2E1 Ames bacterial mutagenicity assay the R597M/W677A protein increased the sensitivity to dimethylnitrosamine by approximately 2-fold, suggesting that the ability to use NADH afforded a significant advantage in this in vivo assay. In addition to providing a valuable tool for understanding the determinants of nucleotide cofactor specificity in this and related enzymes, these mutants might also lend themselves to creation of bioremediation schemes with increased enzymatic activity and robustness in situ, as well as cost-effective reconstitution of enzyme systems in vitro that do not require the use of expensive reducing equivalents from NADPH.

Food protein-derived peptides have increasingly become important sources of ingredients for the formulation of therapeutic products. The main aim of this work was to study the in vitro and in vivo bioactive properties of structurally diverse group of peptides produced through enzymatic hydrolysis of flaxseed proteins (FP). Hydrolysis of FP with seven proteases followed by fractionation into low-molecular-weight (LMW) cationic fractions yielded multifunctional peptides that inhibited angiotensin converting enzyme (ACE) and renin activities, which are molecular targets for antihypertensive agents. The LMW peptides also exhibited antioxidant properties by scavenging free radicals and inhibiting amine oxidase activity. The peptide fractions showed inhibition of calmodulin-dependent phosphodiesterase, an enzyme that has been implicated in the pathogenesis of several chronic diseases. Moreover, FP hydrolysis with thermolysin and pronase followed by mixing with activated carbon yielded branched-chain amino acids (BCAA)-enriched multifunctional peptide mixture (Fischer ratio of 23.65) with antioxidant properties and in vitro ACE inhibition; Fischer ratio of 20.0 is considered minimum for therapeutic purposes. The BCAA-enriched peptide product can be used in clinical nutrition to treat muscle wasting symptoms associated with hepatic diseases. Furthermore, an arginine-rich peptide mixture (31% arginine versus 11% in the original flaxseed protein) was produced by hydrolysis of FP with trypsin and pronase followed by separation using electrodialysis-ultrafiltration. Arginine plays important physiological roles especially as precursor to vasodilator, nitric oxide. The arginine-rich peptide mixture exhibited in vitro ACE and renin inhibition and led to decreased systolic blood pressure (–17.9 and –11.7 mmHg, respectively at 2 and 4 h) after oral administration to spontaneously hypertensive rats. For the first time in the literature, we showed that arginine peptides have superior physiological effects when compared to the amino acid form of arginine. Lastly, quantitative structure-activity relationship studies using partial least squares (PLS) regression yielded two predictive models for renin-inhibiting dipeptides with z-scales amino acid descriptors. The PLS models indicated that hydrophobic and bulky side chain-containing amino acids contribute to renin inhibition if present at the amino- and carboxyl-terminal of dipeptides, respectively. Based on this study, Ile-Trp was discovered as potent renin-inhibiting dipeptide, and may serve as a useful template for the development of potent antihypertensive peptidomimetics.

Food protein-derived peptides have increasingly become important sources of ingredients for the formulation of therapeutic products. The main aim of this work was to study the in vitro and in vivo bioactive properties of structurally diverse group of peptides produced through enzymatic hydrolysis of flaxseed proteins (FP). Hydrolysis of FP with seven proteases followed by fractionation into low-molecular-weight (LMW) cationic fractions yielded multifunctional peptides that inhibited angiotensin converting enzyme (ACE) and renin activities, which are molecular targets for antihypertensive agents. The LMW peptides also exhibited antioxidant properties by scavenging free radicals and inhibiting amine oxidase activity. The peptide fractions showed inhibition of calmodulin-dependent phosphodiesterase, an enzyme that has been implicated in the pathogenesis of several chronic diseases. Moreover, FP hydrolysis with thermolysin and pronase followed by mixing with activated carbon yielded branched-chain amino acids (BCAA)-enriched multifunctional peptide mixture (Fischer ratio of 23.65) with antioxidant properties and in vitro ACE inhibition; Fischer ratio of 20.0 is considered minimum for therapeutic purposes. The BCAA-enriched peptide product can be used in clinical nutrition to treat muscle wasting symptoms associated with hepatic diseases. Furthermore, an arginine-rich peptide mixture (31% arginine versus 11% in the original flaxseed protein) was produced by hydrolysis of FP with trypsin and pronase followed by separation using electrodialysis-ultrafiltration. Arginine plays important physiological roles especially as precursor to vasodilator, nitric oxide. The arginine-rich peptide mixture exhibited in vitro ACE and renin inhibition and led to decreased systolic blood pressure (–17.9 and –11.7 mmHg, respectively at 2 and 4 h) after oral administration to spontaneously hypertensive rats. For the first time in the literature, we showed that arginine peptides have superior physiological effects when compared to the amino acid form of arginine. Lastly, quantitative structure-activity relationship studies using partial least squares (PLS) regression yielded two predictive models for renin-inhibiting dipeptides with z-scales amino acid descriptors. The PLS models indicated that hydrophobic and bulky side chain-containing amino acids contribute to renin inhibition if present at the amino- and carboxyl-terminal of dipeptides, respectively. Based on this study, Ile-Trp was discovered as potent renin-inhibiting dipeptide, and may serve as a useful template for the development of potent antihypertensive peptidomimetics.

Ces travaux de thèse s'articulent autour de deux projets : les études structure-fonction de l'aminopeptidase A, d'une part, et celles du récepteur de l'apéline, d'autre part. I/ L'aminopeptidase A (APA, EC 3.4.11.7) est une aminopeptidase monozinc membranaire qui, dans le cerveau, produit l'angiotensine (Ang) III à partir de l'Ang II. L'Ang III est l'un des principaux peptides effecteurs du système rénine-angiotensine cérébral qui exerce un effet stimulateur tonique sur le contrôle central de la pression artérielle chez le rat hypertendu. Ainsi le blocage de l'APA par un inhibiteur spécifique et sélectif, l'EC33 ou sa prodrogue, le RB150, normalise la pression artérielle dans deux modèles expérimentaux d'hypertension artérielle (HTA). L'APA constitue une cible thérapeutique potentielle pour le traitement de l'HTA qui justifie le développement de nouveaux inhibiteurs de cette enzyme plus puissants et plus sélectifs que l'EC33 et avec un profil pharmacodynamique et pharmacocinétique amélioré par rapport au RB150. Pour cela, nous avons construit un modèle tridimensionnel (3D) de l'APA sur la base de la structure cristallographique de l'APA humaine récemment publiée. Nous avons ensuite validé ce modèle par des études structure-fonction par modélisation moléculaire et mutagénèse dirigée en démontrant l'implication, d'un résidu du sous-site S1 dans la spécificité de substrat acide de l'APA et de deux résidus formant le sous-site S2' interagissant avec le résidu P2' acide d'inhibiteurs tripeptidiques précédemment développés dans le laboratoire.II/ L'apéline est le ligand naturel du récepteur orphelin humain APJ (ApélineR), un récepteur à sept domaines transmembranaires couplé aux protéines G. L'apéline et son récepteur sont impliqués dans le maintien de l'équilibre hydrique et des fonctions cardiovasculaires. L'ApélineR constitue une cible thérapeutique potentielle dans le traitement de l'insuffisance cardiaque et des rétentions hydriques. Etant donné que la demi-vie de l'apéline dans la circulation sanguine est de l'ordre de la minute, l'objectif est de développer des analogues de l'apéline métaboliquement stables. Pour développer de tels composés, nous avons entrepris de comprendre comment l'apéline se lie à son récepteur et comment elle l'active. Dans ce but, nous avons construit un modèle 3D de l'ApélineR basé sur la structure cristallographique du récepteur aux chimiokines, CXCR4. Nous avons validé ce modèle par des études structure-fonction par modélisation moléculaire et mutagénèse dirigée. Nous avons identifié à la surface du récepteur, les résidus acides des boucles extracellulaires qui interagissent avec les résidus basiques de l'apéline. Nous avons ensuite développé des analogues de l'apéline-17 (K17F) métaboliquement stables par deux stratégies différentes. Premièrement, nous avons substitué chacun des résidus de l'apéline par son énantiomère de la série D ou par un acide aminé synthétique. Deuxièmement, nous avons ajouté une chaîne fluoroalkyle à l'extrémité N-terminale de l'apéline. Ces deux stratégies ont permis d'obtenir plusieurs composés dont les plus actifs sont le P92 et le LIT01-196 qui conservent des propriétés pharmacologiques identiques à celles de K17F et qui présentent une demi-vie plasmatique largement supérieure à celle du peptide endogène. Ces deux analogues se sont révélés particulièrement actifs in vivo avec une capacité à diminuer la pression artérielle et à réduire la sécrétion de vasopressine dans le sang conduisant à une augmentation de la diurèse aqueuse. Les modèles 3D validés de l'APA et de l'ApélineR seront utilisés pour des campagnes de criblage in silico de chimiothèques virtuelles afin de découvrir de nouveaux inhibiteurs de l'APA et des agonistes de l'ApélineR qui pourraient conduire à terme à de nouveaux candidats-médicaments. Ces composés pourraient être utiles pour le traitement de l'HTA et de l'insuffisance cardiaque. / The doctoral work was divided in two parts, one on the structure-function studies of aminopeptidase A, and the second one, on those of the apelin receptor. I/ Aminopeptidase A (APA) is a membrane bound monozinc aminopeptidase which generates, in the brain, angiotensin (Ang) III from Ang II. Ang III is one of the main effector peptides of the brain renin-angiotensin system, which exerts a tonic stimulatory action on the control of blood pressure in hypertensive rats. Thus, the blockade of brain APA by a specific and selective inhibitor, EC33 or its prodrug, RB150, normalizes blood pressure in two animal models of arterial hypertension (HTA). APA constitutes a potential therapeutic target for the treatment of HTA that justifies the development of more potent and selective APA inhibitors than EC33, with enhanced pharmacodynamic and pharmacokinetic profiles when compared to RB150. With this aim, we built a three dimensional (3D) model of APA based on the recently published crystal structure of human APA. We validated this model by structure-function studies combining molecular modeling and site-directed mutagenesis demonstrating the crucial role of one residue in the S1 subsite responsible for substrate specificity of APA for N-terminal acidic amino-acid residues and two other residues constituting the S2' subsite of APA involved in the binding of the P2' acidic residue of tripeptidic inhibitors, previously developed in the laboratory. II/ Apelin is the endogenous ligand of the human orphan receptor named APJ (ApelinR), a G protein-coupled receptor. Apelin and ApelinR are involved in the control of body fluid homeostasis and cardiovascular functions. ApelinR constitutes a potential therapeutic target for the treatment of heart failure and water retentions. Given that apelin half-life in the blood circulation is in the minute range, we aimed to develop potent metabolically stable apelin analogs.. In this context, it is necessary to understand how apelin binds to ApelinR and how it is activated. To do so, we build a 3D model of ApelinR based on the crystal structure of the chemokine receptor, CXCR4. We validated this model by structure-function studies by molecular modeling and site-directed mutagenesis. We showed that apelin interacts with the receptor through interactions between the basic residues of the peptide and the acidic residues of the ApelinR, located in the extracellular loops. ,We then developed metabolically stable apelin-17 (K17F) analogs following two different strategies. First, we substituted each residue of K17F by its D-isomer or a synthetic amino-acid. Secondly, we added a fluoroalkyl chain at the N-terminal part of K17F. These two strategies allowed to significantly improve plasma half-life of the modified peptides for several hours without modifying their pharmacological properties as compared to K17F. Two apelin metabolically stable analogs, P92 and LIT01-196, were found to have significantly higher in vivo activity than K17F with a strong capacity to decrease blood pressure and to inhibit vasopressin release in the blood stream inducing an increased aqueous diuresis. These new validated 3D models will be now used to perform in silico screening of virtual chemical libraries to discover new APA inhibitors and ApelinR agonists that could ultimately lead to new drug candidates. These compounds could be useful for the treatment of HTA and heart failure.