Développement d'inhibiteurs allostériques de l'ecto-5'-nucléotidase (CD73) : application aux traitements anticancéreux

Le cancer représente l'un des problèmes majeurs en santé publique. Jusqu'à présent, en parallèle de l'intervention chirurgical, plusieurs traitements ont été mis au point et largement utilisés en thérapie clinique telles que les chimiothérapies. Cependant, leur efficacité est parfois limitée et couplée à des effets secondaires très néfastes, laissant les patients dans une impasse thérapeutique. Par conséquent, de nouvelles approches thérapeutiques doivent être développées sur de nouvelles cibles avérées en oncologie afin d'apporter des soins personnalisés aux patients. La première partie de mon travail de thèse a été dédiée à la compréhension des mécanismes moléculaires de la nucléotidase cytosolique de type II (cN-II), une enzyme du métabolisme des purines dont l'implication dans des phénomènes de résistance à des traitements anticancéreux a pu être démontrée. Aussi, une étude sur la cinétique enzymatique à l'état pré-stationnaire et stationnaire a été entreprise sur la forme sauvage et une forme mutée de l'enzyme lui conférant une activité accrue fortement impliquée dans les cas de résistance. Par cette approche, il a été possible de décortiquer le mécanisme cinétique, de définir l'étape cinétiquement limitant afin d'identifier les intermédiaires prépondérants de la réaction pouvant être ciblés pour le développement de nouveaux inhibiteurs. Cette étude cinétique est présentée dans ce premier volet de la thèse. En second lieu, mon travail s'est focalisée sur un second membre de cette famille d'enzyme qui est l'ecto-5'-nucléotidase (CD73). Cette enzyme exprimée sous forme dimérique à la surface extracellulaire régule la concentration en adénosine extracellulaire (par hydrolyse de l'adénosine monophosphate), ce dernier étant un puissant immunosuppresseur de la réponse immune anticancéreuse. L'objectif de mon travail de thèse a été de développer de nouveaux inhibiteurs de type allostérique en utilisant une approche basée sur la structure tridimensionnelle et la dynamique moléculaire. Une des étapes clés a été tout d'abord de mettre au point un système expression hétérologue afin d'obtenir l'enzyme recombinante en quantité suffisante pour les études enzymatiques ultérieures. Différents systèmes d'expression ont été testés et seul le système en cellules d'insecte infectées par le baculovirus a permis d'obtenir l'enzyme active en grande quantité. En parallèle, une étude in silico a permis de reproduire la dynamique fonctionnelle de l'enzyme requise pour sa fonction. A partir de ses données, un criblage virtuel d'une chimiothèque de 324 000 molécules a été réalisé sur le site de dimérisation et a permis d'identifier 33 composés chefs de files. Parmi, ces composés, dix molécules se sont avérés être de puissants inhibiteurs de CD73 (Ki < 1 µM) avec un mécanisme d'inhibition de type allostérique ou non-compétitif. La cytotoxicité des composés a été évaluée sur des lignées cellulaires transformées ou tumorales montrant un effet uniquement à des concentrations très élevées (supérieures à 100 µM). L'étude des relations structure-fonction devrait permettre à présent de proposer de voies d'optimisation afin d'améliorer l'efficacité des composés les plus actifs afin d'aboutir à de nouveaux candidats médicaments.Cancer burden still remains a major worldwide health problem. To date, several types of conventional anticancer treatments are widely used in clinical. However, the alternative effects of these treatments often leave patients impaired. Therefore, it is required to understand the unique medical needs of individual patients and to conduct effective, high–quality research focusing on the not yet identified oncotargets.The first part of my thesis is dedicated to decipher molecular basis of cN-II reaction. This study characterizes the steady state and transient state kinetics of cN-II wild type and hyperactive mutant which involved in cancer treatment resistance. Furthermore, the characterization of the rate-limiting step and reaction intermediates gave insights into the binding mechanisms and the development of small molecules inhibitors of cN-II.In the second part of this work, we aimed to investigate allosteric inhibitors of CD73 using structure-based drug design approach. In this study the suitable protein expression system was established for the production of sufficient quantities of fully active CD73. This work followed by in silico studies, including molecular dynamics, virtual screening, and hits identification and in vitro hits validations and kinetics characterizations. The cytotoxicity of the most powerful inhibitors exhibited on different cell types was determined. SAR studies gave insights into the binding mode of best compounds and function

Drug discovery and optimization targeting CD73 to restore anticancer immune response

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Identification of allosteric inhibitors of the ecto-5'-nucleotidase (CD73) targeting the dimer interface.

The ecto-5'-nucleotidase CD73 plays an important role in the production of immune-suppressive adenosine in tumor micro-environment, and has become a validated drug target in oncology. Indeed, the anticancer immune response involves extracellular ATP to block cell proliferation through T-cell activation. However, in the tumor micro-environment, two extracellular membrane-bound enzymes (CD39 and CD73) are overexpressed and hydrolyze efficiently ATP into AMP then further into immune-suppressive adenosine. To circumvent the impact of CD73-generated adenosine, we applied an original bioinformatics approach to identify new allosteric inhibitors targeting the dimerization interface of CD73, which should impair the large dynamic motions required for its enzymatic function. Several hit compounds issued from virtual screening campaigns showed a potent inhibition of recombinant CD73 with inhibition constants in the low micromolar range and exhibited a non-competitive inhibition mode. The structure-activity relationships studies indicated that several amino acid residues (D366, H456, K471, Y484 and E543 for polar interactions and G453-454, I455, H456, L475, V542 and G544 for hydrophobic contacts) located at the dimerization interface are involved in the tight binding of hit compounds and likely contributed for their inhibitory activity. Overall, the gathered information will guide the upcoming lead optimization phase that may lead to potent and selective CD73 inhibitors, able to restore the anticancer immune response

Drug discovery and optimization targeting CD73 to restore anticancer immune response

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Hydrophobic contacts involved in the binding of RR compounds.

<p>Docking poses obtained for the two structurally-related compounds, (A) <b>RR4</b> (yellow) and (B) <b>RR6</b> (cyan) highlighting the binding differences (thick sticks correspond to residues that are inversely involved). Binding mode of compound having a stretched structure as for <b>RR11</b> (C) and for <b>RR20</b> (D) depicted as blue and orange sticks, respectively. Comparison of the binding mode for the inhibitory compound <b>RR3</b> (E) and the activator <b>RR28</b> (F) assuming a common binding site for both. Residues making halogen bonds are depicted in yellow sticks. All residues contributing to hydrophobic contacts (either with backbone or sidechain atoms) are depicted in solvent accessible surface and in thin sticks (all compounds are not oriented identically).</p

Structure‐based design, synthesis and biological evaluation of a NAD+ analogue targeting Pseudomonas aeruginosa NAD kinase

International audienceMulti-drug resistance is a major public health problem that requires the urgent development of new antibiotics and therefore the identification of novel bacterial targets. The activity of nicotinamide adenine dinucleotide kinase, NADK, is essential in all bacteria tested so far, including many human pathogens that display antibiotic resistance leading to failure of current treatments. Inhibiting NADK is therefore a promising and innovative antibacterial strategy since there is currently no drug on the market targeting this enzyme. Through a fragment-based drug design approach, we have recently developed a NAD+-competitive inhibitor of NADKs, which displayed in vivo activity against Staphylococcus aureus. Here we show that this compound, a di-adenosine derivative, is inactive against the NADK enzyme from the Gram-negative bacteria Pseudomonas aeruginosa (PaNADK). This lack of activity can be explained by the crystal structure of PaNADK, which was determined in complex with NADP+ in this study. Structural analysis led us to design and synthesize a benzamide adenine dinucleoside analogue, active against PaNADK. This novel compound efficiently inhibited PaNADK enzymatic activity in vitro with a Ki of 4.6 µM. Moreover, this compound reduced P. aeruginosa infection in vivo in a zebrafish model

Comparison of hit compounds by using conventional metrics used in drug design.

<p>Inhibition constants (<i>K</i>_i) are expressed as pK_i (A) and ligand (B), ligand-lipophilicity (C), binding and surface (D) efficiencies correspond to LE, LLE, BEI and SEI, respectively. Note that for compounds exhibiting a mixed inhibition mode, two inhibition constants (“a” and “b”) were determined as for <b>RR2</b> and <b>RR16</b>.</p

Detailed analysis of the binding mode for best-ranked hit compounds.

<p>(A) Overlay of the docking poses from all selected hits at the dimerization interface. Compounds are depicted in sticks and CD73 as solvent accessible surface (yellow and pink for differentiating the two monomers). (B) Overlay of the three most active compounds, <b>RR3</b> (green) <b>RR6</b> (cyan) and <b>RR16</b> (purple). Main polar interactions involved in the binding of <b>RR3</b> (C), <b>RR6</b> (D) and <b>RR16</b> (E) viewed in the same orientation. (F) Binding pose of hit compound <b>RR11</b> (blue) holding an extended and dimeric structure.</p

Enzymatic inhibition assay in the presence of RR compounds using the purified recombinant enzyme.

<p>Red bars indicate the most active <b>RR</b> compounds promoting an enzyme inhibition as efficiently as APCP (5 μM) used as a positive control (green bar). Values of inhibitions are means from three independent experiments ± SD and negative values reflect enzyme activation.</p

Structure-based drug design including cavity selection and dynamics of the enzyme target.

<p>(A) Flowchart illustrating the global strategy for developing allosteric CD73 inhibitors. (B) Five cavities detected using Fpocket on the closed dimeric form of CD73 (4H2G) and shown in colored mesh representations. (C) Top view of superimposed structures of CD73 during the TMD simulation highlighting the large rotating motion of N-domains (centers of mass depicted as spheres in arc shape). (D) Volumes changes and mean local hydrophobic densities observed during TMD for the blue cavity from panel “B” located at the dimerization interface. (E) Target cavity (mesh representation) outside the substrate binding site (AMP and Zn ions are depicted in cyan sticks). (F) Illustration of the target binding site in complex with one hit compound (green sticks) obtained by docking (Glu543 residues are depicted as spheres).</p