Sandrine Hilairet

Detection of beta 2-adrenergic receptor dimerization in living cells using bioluminescence resonance energy transfer (BRET).

Heptahelical receptors that interact with heterotrimeric G proteins represent the largest family of proteins involved in signal transduction across biological membranes. Although these receptors generally were believed to be monomeric entities, a growing body of evidence suggests that they may form functionally relevant dimers. However, a definitive demonstration of the existence of G protein-coupled receptor (GPCR) dimers at the surface of living cells is still lacking. Here, using bioluminescence resonance energy transfer (BRET), as a protein-protein interaction assay in whole cells, we unambiguously demonstrate that the human beta(2)-adrenergic receptor (beta(2)AR) forms constitutive homodimers when expressed in HEK-293 cells. Receptor stimulation with the hydrophilic agonist isoproterenol led to an increase in the transfer of energy between beta(2)AR molecules genetically fused to the BRET donor (Renilla luciferase) and acceptor (green fluorescent protein), respectively, indicating that the agonist interacts with receptor dimers at the cell surface. Inhibition of receptor internalization did not prevent agonist-promoted BRET, demonstrating that it did not result from clustering of receptors within endosomes. The notion that receptor dimers exist at the cell surface was confirmed further by the observation that BS3, a cell-impermeable cross-linking agent, increased BRET between beta(2)AR molecules. The selectivity of the constitutive interaction was documented by demonstrating that no BRET occurred between the beta(2)AR and two other unrelated GPCR. In contrast, the well characterized agonist-dependent interaction between the beta(2)AR and the regulatory protein beta-arrestin could be monitored by BRET. Taken together, the data demonstrate that GPCR exist as functional dimers in vivo and that BRET-based assays can be used to study both constitutive and hormone-promoted selective protein-protein interactions.

Hypersensitization of the Orexin 1 receptor by the CB1 receptor: evidence for cross-talk blocked by the specific CB1 antagonist, SR141716.

In the present study, we observed evidence of cross-talk between the cannabinoid receptor CB1 and the orexin 1 receptor (OX1R) using a heterologous system. When the two receptors are co-expressed, we observed a major CB1-dependent enhancement of the orexin A potency to activate the mitogen-activated protein kinase pathway; dose-responses curves indicated a 100-fold increase in the potency of orexin-mediated mitogen-activated protein kinase activation. This effect required a functional CB1 receptor as evidenced by the blockade of the orexin response by the specific CB1 antagonist, N-(piperidino-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-pyrazole-3-carboxamide (SR141716), but also by pertussis toxin, suggesting that this potentiation is Gi-mediated. In contrast to OX1R, the potency of direct activation of CB1 was not affected by co-expression with OX1R. In addition, electron microscopy experiments revealed that CB1 and OX1R are closely apposed at the plasma membrane level; they are close enough to form hetero-oligomers. Altogether, for the first time our data provide evidence that CB1 is able to potentiate an orexigenic receptor. Considering the antiobesity effect of SR141716, these results open new avenues to understand the mechanism by which the molecule may prevent weight gain through functional interaction between CB1 and other receptors involved in the control of appetite.

SAR103168: a tyrosine kinase inhibitor with therapeutic potential in myeloid leukemias

Abstract SAR103168, a tyrosine kinase inhibitor of the pyrido [2,3-d] pyridimidine subclass, inhibited the kinase activities of the entire Src kinase family, Abl kinase, angiogenic receptor kinases (vascular endothelial growth factor receptor [VEGFR] 1 and 2), Tie2, platelet derived growth factor (PDGF), fibroblast growth factor receptor (FGFR) 1 and 3, and epidermal growth factor receptor (EGFR). SAR103168 was a potent Src inhibitor, with 50% inhibitory concentration (IC50) = 0.65 ± 0.02 nM (at 100 μM ATP), targeting the auto-phosphorylation of the kinase domain (Src260-535) and activity of the phosphorylated kinase. Phosphorylation of Src, Lyn and Src downstream signaling pathways (PYK2, P-130CAS, FAK, JNK and MAPK) were inhibited in a dose-dependent manner. SAR103168 inhibited the phosphorylation of STAT5 in KG1 cells and fresh cells from patients with acute myeloid leukemia (AML). SAR103168 inhibited proliferation and induced apoptosis in acute and chronic myeloid leukemic cells at nanomolar IC50. SAR103168 induced anti-proliferation of leukemic progenitors (CFU-L) from 29 patients with AML, and > 85% of AML patient samples were sensitive to SAR103168. These antagonist activities of SAR103168 were independent of FLT3 expression. SAR103168 treatment was effective in 50% of high-risk patient samples carrying chromosome 7 abnormalities or complex rearrangement. SAR103168 administration (intravenous or oral) impaired tumor growth and induced tumor regression in animals bearing human AML leukemic cells, correlating with potent inhibition of Src downstream signaling pathways in AML tumors. SAR103168 showed potent anti-tumor activity in SCID (severe combined immunodeficiency) mice bearing AML (KG1, EOL-1, Kasumi-1, CTV1) and chronic myeloid leukemia (CML) (K562) tumors. The combination of cytarabine and SAR103168 showed synergistic activity in AML and CML tumor models. These results highlight the therapeutic potential of SAR103168 in myeloid leukemias and support the rationale for clinical investigations.

Development of an Equine Groove Model to Induce Metacarpophalangeal Osteoarthritis: A Pilot Study on 6 Horses

The aim of this work was to develop an equine metacarpophalangeal joint model that induces osteoarthritis that is not primarily mediated by instability or inflammation. The study involved six Standardbred horses. Standardized cartilage surface damage or “grooves” were created arthroscopically on the distal dorsal aspect of the lateral and medial metacarpal condyles of a randomly chosen limb. The contralateral limb was sham operated. After 2 weeks of stall rest, horses were trotted 30 minutes every other day for 8 weeks, then evaluated for lameness and radiographed. Synovial fluid was analyzed for cytology and biomarkers. At 10 weeks post-surgery, horses were euthanized for macroscopic and histologic joint evaluation. Arthroscopic grooving allowed precise and identical damage to the cartilage of all animals. Under the controlled exercise regime, this osteoarthritis groove model displayed significant radiographic, macroscopic, and microscopic degenerative and reactive changes. Histology demonstrated consistent surgically induced grooves limited to non-calcified cartilage and accompanied by secondary adjacent cartilage lesions, chondrocyte necrosis, chondrocyte clusters, cartilage matrix softening, fissuring, mild subchondral bone inflammation, edema, and osteoblastic margination. Synovial fluid biochemistry and cytology demonstrated significantly elevated total protein without an increase in prostaglandin E2, neutrophils, or chondrocytes. This equine metacarpophalangeal groove model demonstrated that standardized non-calcified cartilage damage accompanied by exercise triggered altered osteochondral morphology and cartilage degeneration with minimal or inefficient repair and little inflammatory response. This model, if validated, would allow for assessment of disease processes and the effects of therapy.

Abstract 2201: The molecular signature of tumor cells that are sensitives to a new tight binder NAMPT inhibitor.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC SAR154782 is a tight binder, low reversible NAMPT inhibitor that has been identified by a phenotypic screen and chemogenomic approaches. This class of NAMPT inhibitor induces a profound and time dependent modulation of tumor metabolism which in turn induces the reprogrammation of tumor cell transcriptome. The transcriptional profile revealed that different metabolic and oncogenic signaling pathways are modulated by SAR154782. In order to define the context of tumor cell sensitivity to NAMPT inhibition, the effects of this compound on tumor cell growth and viability have been assessed against a panel of about 240 cell lines from different tissue origin. Based on the difference of tumor cell lines sensitivity to NAMPT inhibitor, a panel of 60 genes that are differentially expressed in the sensitive versus non-sensitive cell lines has been identified. Interestingly, the low expression of NAMPT concomitant with the high expression of Sirtuin1 and PARP1 are the main outstanding features in the panel of genes identified for the most sensitive cell lines. This finding is in agreement with high NAD turnover. The relevance of the preceding biomarkers to determine the potential cancer target population that could benefit upon treatment with a NAMPT inhibitor has been investigated in in vivo settings by evaluating the antitumor activity of an advanced new class of NAMPT inhibitors in several mouse tumor xenografts and human primary tumor models. The results obtained from these studies highlighted the modulation upon NAMPT inhibition of important tumor growth pathways and revealed for the first time a potential molecular signature of tumor sensitivity and support further investigation into use of the signature for patient stratification in the clinical setting Citation Format: Sandrine Hilairet, Jerome Arigon, Frederico Nardi, Samir Jegham, Annick Peleraux, Sylvie Cosnier, Regine Floutard, Omar Jbilo, Paul August, Maysoun Shomali, Francois Autelitano, Rosalia Arrebola, Stephan Reiling, Jack Pollard, Francisco Adrian, Jason Sager, Christoph Lengauer, Marion Dorsh, Carlos Garcia-Echeverria, Monsif Bouaboula. The molecular signature of tumor cells that are sensitives to a new tight binder NAMPT inhibitor. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2201. doi:10.1158/1538-7445.AM2013-2201