From a drug repositioning to a structure-based drug design approach to tackle acute lymphoblastic leukemia.

Cancer cells utilize the main de novo pathway and the alternative salvage pathway for deoxyribonucleotide biosynthesis to achieve adequate nucleotide pools. Deoxycytidine kinase is the rate-limiting enzyme of the salvage pathway and it has recently emerged as a target for anti-proliferative therapies for cancers where it is essential. Here, we present the development of a potent inhibitor applying an iterative multidisciplinary approach, which relies on computational design coupled with experimental evaluations. This strategy allows an acceleration of the hit-to-lead process by gradually implementing key chemical modifications to increase affinity and activity. Our lead compound, OR0642, is more than 1000 times more potent than its initial parent compound, masitinib, previously identified from a drug repositioning approach. OR0642 in combination with a physiological inhibitor of the de novo pathway doubled the survival rate in a human T-cell acute lymphoblastic leukemia patient-derived xenograft mouse model, demonstrating the proof-of-concept of this drug design strategy.

Discovery of Small-Molecule Inhibitors of the PTK7/ß-Catenin Interaction Targeting the Wnt Signaling Pathway in Colorectal Cancer.

Colorectal cancer (CRC), the second cause of death due to cancer worldwide, is a major public health issue. The discovery of new therapeutic targets is thus essential. Pseudokinase PTK7 intervenes in the regulation of the Wnt/ß-catenin pathway signaling, in part, through a kinase domain-dependent interaction with the ß-catenin protein. PTK7 is overexpressed in CRC, an event associated with metastatic development and reduced survival of nonmetastatic patients. In addition, numerous alterations have been identified in CRC inducing constitutive activation of the Wnt/ß-catenin pathway signaling through ß-catenin accumulation. Thus, targeting the PTK7/ß-catenin interaction could be of interest for future drug development. We have developed a NanoBRET screening assay recapitulating the interaction between PTK7 and ß-catenin to identify compounds able to disrupt this protein-protein interaction. A high-throughput screening allowed us to identify small-molecule inhibitors targeting the Wnt pathway signaling and inducing antiproliferative and antitumor effects in vitro in CRC cells harboring ß-catenin or adenomatous polyposis coli (APC) mutations. Thus, inhibition of the PTK7/ß-catenin interaction could represent a new therapeutic strategy to inhibit cell growth dependent on the Wnt signaling pathway. Moreover, despite a lack of enzymatic activity of its tyrosine kinase domain, targeting the PTK7 kinase domain-dependent functions appears to be of interest for further therapeutic development.

Fragment-based drug design targeting syntenin PDZ2 domain involved in exosomal release and tumour spread.

Syntenin stimulates exosome production and its expression is upregulated in many cancers and implicated in the spread of metastatic tumor. These effects are supported by syntenin PDZ domains interacting with syndecans. We therefore aimed to develop, through a fragment-based drug design approach, novel inhibitors targeting syntenin-syndecan interactions. We describe here the optimization of a fragment, 'hit' C58, identified by in vitro screening of a PDZ-focused fragment library, which binds specifically to the syntenin-PDZ2 domain at the same binding site as the syndecan-2 peptide. X-ray crystallographic structures and computational docking were used to guide our optimization process and lead to compounds 45 and 57 (IC50 = 33 µM and 47 µM; respectively), two representatives of syntenin-syndecan interactions inhibitors, that selectively affect the syntenin-exosome release. These findings demonstrate that it is possible to identify small molecules inhibiting syntenin-syndecan interaction and exosome release that may be useful for cancer therapy.

Gain of affinity for VEGF165 binding within the VEGFR2/NRP1 cellular complex detected by an HTRF-based binding assay.

Neuroplin 1 (NRP1), a transmembrane protein interacting with Vascular Endothelial Growth Factor VEGF-A165 (called here VEGF165) and the tyrosine kinase Receptor 2 (VEGFR2) promote angiogenesis and vascular homeostasis. In a pathophysiological context, several studies suggested that VEGFR2 and NRP1 mediate tumor development and progression. Given the involvement of the VEGF165 network in promoting tumor angiogenesis, NRP1, VEGFR2 and VEGF165 have been identified as targets for anti-angiogenic therapy. No binding assay exists to monitor specifically the binding of VEGF165 to the VEGFR2/NRP1 complex in intact cells. We established a binding assay based on the homogenous time-resolved fluorescence (HTRF®) technology. This unique binding assay enables to assess the interaction of VEGF165 with VEGFR2 or NRP1 within the VEGFR2/NRP1 complex. Ligand binding saturation experiments revealed that VEGF165 binds the VEGFR2/NRP1 complex at the cell surface with a ten to twenty-fold higher affinity compared to SNAP-VEGFR2 or SNAP-NRP1 receptors alone not engaged in the heteromeric complex. The assay allows characterizing the impact of NRP1 ligands on VEGF165 to the complex. It shows high specificity, reproducibility and robustness, making it compatible with high throughput screening (HTS) applications for identifying new VEGF165 antagonists selective for NRP1 or the VEGFR2/NRP1 complex.

Design and validation of a homogeneous time-resolved fluorescence-based leptin receptor binding assay.

The pleiotropic cytokine hormone leptin, by activating its receptor OB-R, plays a major role in many biological processes, including energy homeostasis, immune function, and cell survival and proliferation. Abnormal leptin action is associated with obesity, autoimmune diseases, and cancer. The pharmacological characterization of OB-R and the development of synthetic OB-R ligands are still in their infancy because currently available binding assays are not compatible with ligand saturation binding experiments and high-throughput screening (HTS) approaches. We have developed here a novel homogeneous time-resolved fluorescence-based binding assay that overcomes these limitations. In this assay, fluorescently labeled leptin or leptin antagonist binds to the SNAP-tagged OB-R covalently labeled with terbium cryptate (Tb). Successful binding is monitored by measuring the energy transfer between the Tb energy donor and the fluorescently labeled leptin energy acceptor. Ligand binding saturation experiments revealed high-affinity dissociation constants in the subnanomolar range with an excellent signal-to-noise ratio. The assay performed in a 384-well format shows high specificity and reproducibility, making it perfectly compatible with HTS applications to identify new OB-R agonists or antagonists. In addition, fluorescently labeled leptin and SNAP-tagged OB-R will be valuable tools for monitoring leptin and OB-R trafficking in cells and tissues.