Jacqueline Norris-Drouin

UNC1062, a new and potent Mer inhibitor.

Abnormal activation of Mer kinase has been implicated in the oncogenesis of many human cancers including acute lymphoblastic and myeloid leukemia, non-small cell lung cancer, and glioblastoma. We have discovered a new family of small molecule Mer inhibitors, pyrazolopyrimidine sulfonamides, that potently inhibit the kinase activity of Mer. Importantly, these compounds do not demonstrate significant hERG activity in the PatchXpress assay. Through structure-activity relationship studies, 35 (UNC1062) was identified as a potent (IC50 = 1.1 nM) and selective Mer inhibitor. When applied to live tumor cells, UNC1062 inhibited Mer phosphorylation and colony formation in soft agar. Given the potential of Mer as a therapeutic target, UNC1062 is a promising candidate for further drug development.

Discovery of Mer Specific Tyrosine Kinase Inhibitors for the Treatment and Prevention of Thrombosis

The role of Mer kinase in regulating the second phase of platelet activation generates an opportunity to use Mer inhibitors for preventing thrombosis with diminished likelihood for bleeding as compared to current therapies. Toward this end, we have discovered a novel, Mer kinase specific substituted-pyrimidine scaffold using a structure-based drug design and a pseudo ring replacement strategy. The cocrystal structure of Mer with two compounds (7 and 22) possessing distinct activity have been determined. Subsequent SAR studies identified compound 23 (UNC2881) as a lead compound for in vivo evaluation. When applied to live cells, 23 inhibits steady-state Mer kinase phosphorylation with an IC50 value of 22 nM. Treatment with 23 is also sufficient to block EGF-mediated stimulation of a chimeric receptor containing the intracellular domain of Mer fused to the extracellular domain of EGFR. In addition, 23 potently inhibits collagen-induced platelet aggregation, suggesting that this class of inhibitors may have utility for prevention and/or treatment of pathologic thrombosis.

Pseudo-Cyclization through Intramolecular Hydrogen Bond Enables Discovery of Pyridine Substituted Pyrimidines as New Mer Kinase Inhibitors

Abnormal activation or overexpression of Mer receptor tyrosine kinase has been implicated in survival signaling and chemoresistance in many human cancers. Consequently, Mer is a promising novel cancer therapeutic target. A structure-based drug design approach using a pseudo-ring replacement strategy was developed and validated to discover a new family of pyridinepyrimidine analogues as potent Mer inhibitors. Through SAR studies, 10 (UNC2250) was identified as the lead compound for further investigation based on high selectivity against other kinases and good pharmacokinetic properties. When applied to live cells, 10 inhibited steady-state phosphorylation of endogenous Mer with an IC50 of 9.8 nM and blocked ligand-stimulated activation of a chimeric EGFR-Mer protein. Treatment with 10 also resulted in decreased colony-forming potential in rhabdoid and NSCLC tumor cells, thereby demonstrating functional antitumor activity. The results provide a rationale for further investigation of this compound for therapeutic application in patients with cancer.

UNC569, a Novel Small-Molecule Mer Inhibitor with Efficacy against Acute Lymphoblastic Leukemia In Vitro and In Vivo

Acute lymphoblastic leukemia (ALL) is the most common malignancy in children. Although survival rates have improved, patients with certain biologic subtypes still have suboptimal outcomes. Current chemotherapeutic regimens are associated with short- and long-term toxicities and novel, less toxic therapeutic strategies are needed. Mer receptor tyrosine kinase is ectopically expressed in ALL patient samples and cell lines. Inhibition of Mer expression reduces prosurvival signaling, increases chemosensitivity, and delays development of leukemia in vivo, suggesting that Mer tyrosine kinase inhibitors are excellent candidates for targeted therapies. Brain and spinal tumors are the second most common malignancies in childhood. Multiple chemotherapy approaches and radiotherapies have been attempted, yet overall survival remains dismal. Mer is also abnormally expressed in atypical teratoid/rhabdoid tumors (AT/RT), providing a rationale for targeting Mer as a therapeutic strategy. We have previously described UNC569, the first small-molecule Mer inhibitor. This article describes the biochemical and biologic effects of UNC569 in ALL and AT/RT. UNC569 inhibited Mer activation and downstream signaling through ERK1/2 and AKT, determined by Western blot analysis. Treatment with UNC569 reduced proliferation/survival in liquid culture, decreased colony formation in methylcellulose/soft agar, and increased sensitivity to cytotoxic chemotherapies. MYC transgenic zebrafish with T-ALL were treated with UNC569 (4 μmol/L for two weeks). Fluorescence was quantified as indicator of the distribution of lymphoblasts, which express Mer and enhanced GFP. UNC569 induced more than 50% reduction in tumor burden compared with vehicle- and mock-treated fish. These data support further development of Mer inhibitors as effective therapies in ALL and AT/RT. Mol Cancer Ther; 12(11); 2367–77. ©2013 AACR.

Discovery of Peptidomimetic Ligands of EED as Allosteric Inhibitors of PRC2

The function of EED within polycomb repressive complex 2 (PRC2) is mediated by a complex network of protein-protein interactions. Allosteric activation of PRC2 by binding of methylated proteins to the embryonic ectoderm development (EED) aromatic cage is essential for full catalytic activity, but details of this regulation are not fully understood. EEDs recognition of the product of PRC2 activity, histone H3 lysine 27 trimethylation (H3K27me3), stimulates PRC2 methyltransferase activity at adjacent nucleosomes leading to H3K27me3 propagation and, ultimately, gene repression. By coupling combinatorial chemistry and structure-based design, we optimized a low-affinity methylated jumonji, AT-rich interactive domain 2 (Jarid2) peptide to a smaller, more potent peptidomimetic ligand (Kd = 1.14 ± 0.14 μM) of the aromatic cage of EED. Our strategy illustrates the effectiveness of applying combinatorial chemistry to achieve both ligand potency and property optimization. Furthermore, the resulting ligands, UNC5114 and UNC5115, demonstrate that targeted disruption of EEDs reader function can lead to allosteric inhibition of PRC2 catalytic activity.

Discovery of Macrocyclic Pyrimidines as MerTK-Specific Inhibitors

Macrocycles have attracted significant attention in drug discovery recently. In fact, a few de novo designed macrocyclic kinase inhibitors are currently in clinical trials with good potency and selectivity for their intended target. In this study, we successfully engaged a structure‐based drug design approach to discover macrocyclic pyrimidines as potent Mer tyrosine kinase (MerTK)‐specific inhibitors. An enzyme‐linked immunosorbent assay (ELISA) in 384‐well format was employed to evaluate the inhibitory activity of macrocycles in a cell‐based assay assessing tyrosine phosphorylation of MerTK. Through structure–activity relationship (SAR) studies, analogue 11 [UNC2541; (S)‐7‐amino‐N‐(4‐fluorobenzyl)‐8‐oxo‐2,9,16‐triaza‐1(2,4)‐pyrimidinacyclohexadecaphane‐1‐carboxamide] was identified as a potent and MerTK‐specific inhibitor that exhibits sub‐micromolar inhibitory activity in the cell‐based ELISA. In addition, an X‐ray structure of MerTK protein in complex with 11 was resolved to show that these macrocycles bind in the MerTK ATP pocket.

Quantitative Characterization of Bivalent Probes for a Dual Bromodomain Protein, Transcription Initiation Factor TFIID Subunit 1

Multivalent binding is an efficient means to enhance the affinity and specificity of chemical probes targeting multidomain proteins in order to study their function and role in disease. While the theory of multivalent binding is straightforward, physical and structural characterization of bivalent binding encounters multiple technical difficulties. We present a case study where a combination of experimental techniques and computational simulations was used to comprehensively characterize the binding and structure-affinity relationships for a series of Bromosporine-based bivalent bromodomain ligands with a bivalent protein, Transcription Initiation Factor TFIID subunit 1 (TAF1). Experimental techniques-Isothermal Titration Calorimetry, X-ray Crystallography, Circular Dichroism, Size Exclusion Chromatography-Multi-Angle Light Scattering, and Surface Plasmon Resonance-were used to determine structures, binding affinities, and kinetics of monovalent ligands and bivalent ligands with varying linker lengths. The experimental data for monomeric ligands were fed into explicit computational simulations, in which both ligand and protein species were present in a broad range of concentrations, and in up to a 100 s time regime, to match experimental conditions. These simulations provided accurate estimates for apparent affinities (in good agreement with experimental data), individual dissociation microconstants and other microscopic details for each type of protein-ligand complex. We conclude that the expected efficiency of bivalent ligands in a cellular context is difficult to estimate by a single technique in vitro, due to higher order associations favored at the concentrations used, and other complicating processes. Rather, a combination of structural, biophysical, and computational approaches should be utilized to estimate and characterize multivalent interactions.

Abstract B206: Ror2 as a therapeutic target in renal cell carcinoma and other invasive cancers.

Protein kinases play key roles in defining the transformation of many solid tumors, including renal cell carcinoma (RCC), and have become attractive drug targets. The Ror-family receptor tyrosine kinases (RTKs) are transmembrane proteins with putative tyrosine kinase activities that play crucial roles during the development of various organs and tissues. One of these receptors, the RTK-like orphan receptor 2 (Ror2) is known as a developmentally regulated receptor that enhances tumor cell migration and tumor invasiveness. Recently, our lab reported on the expression of Ror2 in human RCC tumors and cell lines, and that its expression is correlated with invasive growth in culture. In mammals, Ror2 has been shown to act as a receptor or co-receptor for Wnt5a, a member of the Wnt family, inducing a noncanonical Wnt signaling cascade. We have found that Ror2 is expressed in various cell lines, including 786-O, HEK293, HeLa, SaOS2, and U2OS. Cell migration analysis using single cell tracking confirmed that Ror2 promotes cell migration, further enhanced by Wnt5a stimulation. Separately, we have shown that Ror2 expression correlates with enhanced canonical Wnt-signaling through an increased pool of downstream stable β-catenin in RCC and activation of canonical targets. However, the kinase activity of Ror2 has been controversial. Using 786-O Ror2 overexpressing cells (786-O/Ror2), we detected that Ror2 becomes phosphorylated upon Wnt5a treatment. Based on a report of antibody induced homodimerization of Ror2 necessary for stimulation, we treated 786-O/Ror2 cells with Ror2 antibody and verified a significantly enhanced phosphorylation. Based on these findings, we hypothesize that receptor dimerization via Wnt ligand engagement or antibody treatment is necessary for effective signal transduction. We have thus utilized the PathHunter Ror2 activity assay developed by DiscoveRX, to use blockade of dimerization as an assay for Ror2 targeted drug development. This system utilizes an EGFR/Ror2 chimera cell line that expresses the cytosolic portion of ROR2 containing the kinase domain tagged with a ProLink tag at the C-terminus and fused to the extracellular and transmembrane domains of EGFR. Receptor activation is mediated by EGF addition, which results in a dose-dependent increase in signal caused by complementation of the SH2 tagged with the complementary EA enzyme fragment binding to the phosphorylated receptor. Thus, activation reads out in dimerization and phosphorylation which in turn, results in enzyme fragment complementation in this assay. Our data using this EGFR/Ror2 chimera U2OS cell line show that stimulation of these cells with EGF induces phosphorylation to a great extent both by IP/western, and chimeric signal. We believe these tools are useful in screening compounds in search of Ror2 inhibitors for RCC or other cancer therapeutics. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B206. Citation Format: Zufan Debebe, Melissa Porter, Emily E. Hull-Ryde, Neal Rasmussen, Adam Sendor, Jacqueline Norris-Drouin, Keefe Chan, James E. Bear, William P. Janzen, Kimryn Rathmell. Ror2 as a therapeutic target in renal cell carcinoma and other invasive cancers. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B206.