Conformational dynamics associated with ligand binding to vertebrate hexa-coordinate hemoglobins

Neuroglobin (Ngb) and cytoglobin (Cygb) are two new additions to the globin family, exhibiting heme iron hexa-coordination, a disulfide bond and large internal cavities. These proteins are implicated in cytoprotection under hypoxic-ischemic conditions, but the molecular basis of their cytoprotective function is unclear. Herein, a photothermal and spectroscopic study of the interactions of diatomic ligands with Ngb, Cygb, myoglobin and hemoglobin is presented. The impact of the disulfide bond in Ngb and Cygb and role of conserved residues in Ngb His64, Val68, Cys55, Cys120 and Tyr44 on conformational dynamics associated with ligand binding/dissociation were investigated. Transient absorption and photoacoustic calorimetry studies indicate that CO photo-dissociation from Ngb leads to a volume expansion (13.4±0.9 mL mol-1), whereas a smaller volume change was determined for Ngb with reduced Cys (ΔV=4.6±0.3 mL mol-1). Furthermore, Val68 side chain regulates ligand migration between the distal pocket and internal hydrophobic cavities since Val68Phe geminate quantum yield is ∼2.7 times larger than that of WT Ngb. His64Gln and Tyr44Phe mutations alter the thermodynamic parameters associated with CO photo-release indicating that electrostatic/hydrogen binding network that includes heme propionate groups, Lys 67, His64, and Tyr 44 in Ngb modulates the energetics of CO photo-dissociation. In Cygb, CO escape from the protein matrix is fast (< 40 ns) with a ΔH of 18±2 kcal mol-1 in Cygbred, whereas disulfide bridge formation promotes a biphasic ligand escape associated with an overall enthalpy change of 9±4 kcal mol-1. Therefore, the disulfide bond modulates conformational dynamics in Ngb and Cygb. I propose that in Cygb with reduced Cys the photo-dissociated ligand escapes through the hydrophobic tunnel as occurs in Ngb, whereas the CO preferentially migrates through the His64 gate in Cygbox. To characterize Cygb surface 1,8-ANS interactions with Cygb were investigated employing fluorescence spectroscopy, ITC and docking simulations. Two 1,8-ANS binding sites were identified. One binding site is located close to the extended N-terminus of Cygb and was also identified as a binding site for oleate. Furthermore, guanidinium hydrochloride-induced unfolding studies of Cygb reveal that the disulfide bond does not impact Cygb stability, whereas binding of cyanide slightly increases the protein stability.

Effet du CP-3(iv), un ligand du récepteur CD36, sur le stress oxydatif suite à une ischémie cardiaque transitoire chez la souris

Le récepteur éboueur CD36 facilite l’internalisation des acides gras libres non estérifiés (AGNE) au niveau des tissus cardiaque et périphériques. Lors d’une ischémie-reperfusion du myocarde (MI/R), les dommages produits sont en partie liés à l’internalisation des AGNE et à la production d’espèces réactives de l’oxygène, contrairement à ce qui est observé chez des souris déficientes en CD36 (CD36-/-). Nous avons émis l’hypothèse selon laquelle le CP-3(iv), un ligand synthétique du récepteur CD36, exercerait un effet cardioprotecteur en réduisant la taille de la zone myocardique infarcie lors d’une ischémie transitoire du myocarde. Nos objectifs étaient 1) de déterminer l’effet cardioprotecteur du CP-3(iv) et 2) de définir son mécanisme. Pour cela, des études in vivo et ex vivo ont été faites. Des souris de type sauvage ont été traitées avec le CP-3(iv) (289 nmol/kg) par voie sous-cutanée pendant 14 jours avant d’être soumises à 30 minutes d’ischémie suivant la ligature de l’artère coronaire gauche descendante et de sa reperfusion pendant une période de 6 ou 48 heures. De plus, des coeurs isolés de souris ont été perfusés 30 minutes, suivi de 40 minutes à faible débit (10%) et de 30 minutes de reperfusion pendant laquelle le coeur est perfusé avec le CP-3(iv) à une concentration de 10-6 M. Nos travaux ont montré que l’effet cardioprotecteur d’un traitement préventif par le CP-3(iv) permet de diminuer la taille de l’infarctus et préserve l’hémodynamie cardiaque de façon dépendante du CD36 puisque cet effet est non visible chez les souris CD36-/-. De plus, le CP-3(iv) exerce non seulement un effet systémique, mais aussi un effet cardioprotecteur direct sur le coeur isolé.

Conception et synthèse d'un ligand de l'intégrine αVβ3 susceptible d'être greffé à un polymère, dans le but de cibler les processus d'angiogenèse dans des tissus cancéreux

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Development and Application of Covalent-Labeling Strategies for the Large-Scale Thermodynamic Analysis of Protein Folding and Ligand Binding

Thermodynamic stability measurements on proteins and protein-ligand complexes can offer insights not only into the fundamental properties of protein folding reactions and protein functions, but also into the development of protein-directed therapeutic agents to combat disease. Conventional calorimetric or spectroscopic approaches for measuring protein stability typically require large amounts of purified protein. This requirement has precluded their use in proteomic applications. Stability of Proteins from Rates of Oxidation (SPROX) is a recently developed mass spectrometry-based approach for proteome-wide thermodynamic stability analysis. Since the proteomic coverage of SPROX is fundamentally limited by the detection of methionine-containing peptides, the use of tryptophan-containing peptides was investigated in this dissertation. A new SPROX-like protocol was developed that measured protein folding free energies using the denaturant dependence of the rate at which globally protected tryptophan and methionine residues are modified with dimethyl (2-hydroxyl-5-nitrobenzyl) sulfonium bromide and hydrogen peroxide, respectively. This so-called Hybrid protocol was applied to proteins in yeast and MCF-7 cell lysates and achieved a ~50% increase in proteomic coverage compared to probing only methionine-containing peptides. Subsequently, the Hybrid protocol was successfully utilized to identify and quantify both known and novel protein-ligand interactions in cell lysates. The ligands under study included the well-known Hsp90 inhibitor geldanamycin and the less well-understood omeprazole sulfide that inhibits liver-stage malaria. In addition to protein-small molecule interactions, protein-protein interactions involving Puf6 were investigated using the SPROX technique in comparative thermodynamic analyses performed on wild-type and Puf6-deletion yeast strains. A total of 39 proteins were detected as Puf6 targets and 36 of these targets were previously unknown to interact with Puf6. Finally, to facilitate the SPROX/Hybrid data analysis process and minimize human errors, a Bayesian algorithm was developed for transition midpoint assignment. In summary, the work in this dissertation expanded the scope of SPROX and evaluated the use of SPROX/Hybrid protocols for characterizing protein-ligand interactions in complex biological mixtures.

Conception et synthèse d'un ligand de l'intégrine αVβ3 susceptible d'être greffé à un polymère, dans le but de cibler les processus d'angiogenèse dans des tissus cancéreux

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Changes of Soluble CD40 Ligand in the Progression of Acute Myocardial Infarction Associate to Endothelial Nitric Oxide Synthase Polymorphisms and Vascular Endothelial Growth Factor But Not to Platelet CD62P Expression

Reported in vitro data implicated soluble CD40 ligand (sCD40L) in endothelial dysfunction and angiogenesis. However, whether sCD40L could exert that influence in endothelial dysfunction and angiogenesis after injury in acute myocardial infarction (AMI) patients remains unclear. In the present study, we evaluated the association of sCD40L with markers of platelet activation, endothelial, and vascular function during a recovery period early after AMI. To achieve this goal, the time changes of soluble, platelet-bound, and microparticle-bound CD40L levels over 1 month were assessed in AMI patients and correlated with endothelial nitric oxide synthase (eNOS) polymorphisms, vascular endothelial growth factor (VEGF) concentrations, and platelet expression of P-selectin (CD62P). The association of soluble form, platelet-bound, and microparticle-bound CD40L with CD62P expression on platelets, a marker of platelet activation, was also assessed to evaluate the role of CD40L in the thrombosis, whereas the association with eNOS and VEGF was to evaluate the role of CD40L in vascular dysfunction. This work shows for the first time that time changes of sCD40L over 1 month after myocardial infarct onset were associated with G894T eNOS polymorphism and with the VEGF concentrations, but not to the platelet CD62P expression. These results indicate that, in terms of AMI pathophysiology, the sCD40L cannot be consider just as being involved in thrombosis and inflammation but also as having a relevant role in vascular and endothelial dysfunction.

Towards NIR emission: 2-(1H-tetrazol-1-yl)pyridine as versatile ligand for the synthesis of two new families of cationic and neutral Ir(III) complexes.

In the last decades, cyclometalated Ir(III) complexes have drawn a large interest for their unique properties: they are excellent triplet state emitters, thus the emission is phosphorescent in nature; typically high quantum yields and good stability make them good candidates for luminescent materials. Moreover, through an opportune choice of the ligands, it is possible to tune the emission along the whole visible spectra. Thanks to these interesting features, Ir(III) complexes have found different applications in several areas of applied science, from OLEDs to bioimaging. In particular, regarding the second application, a remarkable red-shift in the emission is required, in order to minimize the problem of the tissue penetration and the possible damages for the organisms. With the aim of synthesizing a new family of NIR emitting Ir(III) complexes, we envisaged the possibility to use for the first time 2-(1H-tetrazol-1-yl)pyridine as bidentate ligand able to provide the required red-shift of the emission of the final complexes. Exploiting the versatility of the ligand, I prepared two different families of heteroleptic Ir(III) complexes. In detail, in the first case the 2-(1H-tetrazol-1-yl)pyridine was used as bis-chelating N^N ligand, leading to cationic complexes, while in the second case it was used as cyclometalating C^N ligand, giving neutral complexes. The structures of the prepared molecules have been characterised by NMR spectroscopy and mass spectrometry. Moreover, the neutral complexes’ emissive properties have been measured: emission spectra have been recorded in solution at both room temperature and 77K, as well as in PMMA matrix. DFT calculation has then been performed and the obtained results have been compared to experimental ones.

Recombinant expression of Aryl Hydrocarbon Receptor for quantitative ligand-binding analysis

Recombinant expression of the Aryl Hydrocarbon Receptor (AhR) yields small amounts of ligand- binding competent AhR. Therefore, Spodoptera frugiperda (Sf9) cells and baculovirus have been evaluated for high level and functional expression of AhR. Rat and human AhR were expressed as soluble protein in significant amounts. Expression of ligand-binding competent AhR was sensitive to the protein concentration of Sf9 extract, and co-expression of the chaperone p23 failed to affect the yield of functional ligand-binding AhR. The expression system yielded high levels of functional protein, with the ligand-binding capacity (Bmax) typically 20- fold higher than that obtained with rat liver cytosol. Quantitative estimates of the ligand-binding affinity of human and rat AhR were obtained; the Kd for recombinant rat AhR was indistinguishable from that of native rat AhR, thereby validating the expression system as a faithful model for native AhR. The human AhR bound TCDD with significantly lower affinity than the rat AhR. These findings demonstrate high-level expression of ligand-binding competent AhR, and sufficient AhR for quantitative analysis of ligand-binding.

2-(4-Amino-3-methylphenyl)-5-fluorobenzothiazole is a ligand and shows species-specific partial agonism of the Aryl Hydrocarbon Receptor

2-(4-Amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203) and related compounds are a series of anti-cancer candidate pharmaceuticals (Table 1.), that have been shown to activate the AhR. We show that these compounds are high affinity ligands for the rat AhR, but a quantitative assay for their ability to induce CYP1A1 RNA in H4IIEC3 cells, a measure of activation of the AhR, showed a poor relationship between affinity for the AhR and ability to induce CYP1A1 RNA. 5F 203, an agonist with low potency, was able to antagonise the induction of CYP1A1 RNA by TCDD, while IH 445, a potent agonist, did not antagonise the induction of CYP1A1 RNA by TCDD, and Schild analysis confirmed 5F 203 to be a potent antagonist of the induction of CYP1A1 RNA by TCDD in H4IIEC3 cells. In contrast, several benzothiazoles show potent induction of CYP1A1 RNA in human MCF-7 cells, and 5F 203 is unable to detectably antagonise the induction of CYP1A1 RNA in MCF-7 cells, showing a species difference in antagonism. Evaluation of the antiproliferative activity of benzothiazoles showed that the ability to agonise the AhR correlated with growth inhibition both in H4IIEC3 cells for a variety of benzothiazoles, and between H4IIEC3 and MCF-7 cells for 5F 203, suggesting an important role of agonism of the AhR in the anti-proliferative activity of benzothiazoles.

Ligand effects on the structure and vibrational properties of the thiolated Au18 cluster

Most of the studies devoted to thiolated gold clusters suppose that their core and Au-S framework do not suffer from distortion independently of the protecting ligands (-SR) and it is assumed as correct to simplify the ligand as SCH3. In this work is delivered a systematic study of the structure and vibrational properties (IR and Raman) of the Au18(SR)14 cluster. The pursued goal is to understand the dependency of the displayed vibrational properties of the thiolated Au18 cluster with the ligands type. A set of six ligands was considered during calculations of the vibrational properties based on density functional theory (DFT) and in its dispersioncorrected approach (DFT-D)

Ligand effects on the optical and chiroptical properties of the thiolated Au18cluster

The effect of chiral and achiral ligands protecting the inner Au9 core of the Au18(SR)14 cluster is studied based on density functional theory (DFT) and its corrected long-range interaction (DFT-D) approach. It was found that the electronic properties (energy levels) depend on the specific ligands, which induce distinct distortions on the Au–S framework. However, the substitution of S-c-C6H11 as SCH3 ligands may be considered to be correct given the obtained resemblance to the displayed bonding, optical and chiroptical properties. A further comparison of the CD and UV spectra displayed by the Au18 cluster protected by chiral and achiral ligands attests that more intense profiles are featured by ligands including phenyl rings and/or oxygen atoms such that the Au18 cluster protected by either achiral metamercaptobenzoic acid (m-MBA) or achiral SPh ligands displays more intense UV and CD signals. These results provide new insight into the effect of ligands on thiolated gold clusters

Ligand-Free Palladium-Catalyzed Oxyarylation of Dihydronaphthal­enes and Chromenequinone with o-Iodophenols and 3-Iodolawsone in PEG-400: An Efficient Synthesis of 5-Carbapterocarpans and Pterocarpanquinones

Dihydronaphthalenes were oxyarylated with o-iodophenols, in PEG-400 at 140 or 170 °C, leading regio- and stereoselectively to 5-carbapterocarpans. By using Pd(OAc)2 (5–10 mol%) as precatalyst and Ag2CO3 (1.1 equiv) as base (conditions A), products were obtained in good to excellent chemical yields, in 5–30 minutes, irrespective of the pattern of substitution the starting materials. Alternatively, when p-hydroxyacetophenone oxime derived palladacycle (1 mol%) was used as precatalyst, and dicyclohexylamine (2 equiv) was used as base (silver-free, conditions B), the corresponding adducts were obtained in moderate to good yields, in 0.5 to 4 hours. Finally, the oxyarylation of dihydronaphthalenes­ and chromenquinone with o-iodophenols and 3-iodolawsone in PEG-400 under conditions A led regio- and stereoselectively to the formation of carbapterocarpanquinones and pterocarpanquinones in moderate yield.

A new post-metallocene-ti catalyst with maltolate bidentade ligand: an investigation in heterogeneous polymerization reactions in different mesoporous supports

A new titanium catalyst easily synthesized from ethylmaltol bidentate chelator ligand was studied in homogeneous and heterogeneous ethylene polymerization. The dichlorobis(3-hydroxy-2-ethyl-4-pyrone)titanium(IV) complex was characterized by 1H and 13C NMR (nuclear magnetic resonance), UV-Vis and elemental analysis. Theoretical study by density functional theory (DFT) showed that the complex chlorines exhibit cis configuration, which is important for the activity in olefin polymerization. The complex was supported by two methods, direct impregnation or methylaluminoxane (MAO) pre-treatment, in five mesoporous supports: MCM-41 (micro and nano), SBA-15 and also the corresponding modified Al species. All the catalytic systems were active in ethylene polymerization and the catalytic activity was strongly influenced by the method of immobilization of the catalyst and the type of support.