Patricia A. Pilling

Structure of the insulin receptor ectodomain reveals a folded-over conformation

The insulin receptor is a phylogenetically ancient tyrosine kinase receptor found in organisms as primitive as cnidarians and insects. In higher organisms it is essential for glucose homeostasis, whereas the closely related insulin-like growth factor receptor (IGF-1R) is involved in normal growth and development. The insulin receptor is expressed in two isoforms, IR-A and IR-B; the former also functions as a high-affinity receptor for IGF-II and is implicated, along with IGF-1R, in malignant transformation. Here we present the crystal structure at 3.8 Å resolution of the IR-A ectodomain dimer, complexed with four Fabs from the monoclonal antibodies 83-7 and 83-14 (ref. 4), grown in the presence of a fragment of an insulin mimetic peptide. The structure reveals the domain arrangement in the disulphide-linked ectodomain dimer, showing that the insulin receptor adopts a folded-over conformation that places the ligand-binding regions in juxtaposition. This arrangement is very different from previous models. It shows that the two L1 domains are on opposite sides of the dimer, too far apart to allow insulin to bind both L1 domains simultaneously as previously proposed. Instead, the structure implicates the carboxy-terminal surface of the first fibronectin type III domain as the second binding site involved in high-affinity binding.

Expression, purification and preliminary X-ray crystallographic analysis of PsaA, a putative metal-transporter protein of Streptococcus pneumoniae.

The putative metal-transporter protein PsaA of Streptococcus pneumoniae is of potential interest both as a vaccine and also as a drug target. The overexpression of the protein in E. coli, and its subsequent purification and crystallization are described. The crystals are rectangular rods and diffract to beyond 2.7 A resolution. The crystal space group is P212121 with unit-cell dimensions a = 59.9, b = 66.5 and c = 69.9 A.

Characterization of the Novel Broad-Spectrum Kinase Inhibitor CTx-0294885 As an Affinity Reagent for Mass Spectrometry-Based Kinome Profiling

Kinase enrichment utilizing broad-spectrum kinase inhibitors enables the identification of large proportions of the expressed kinome by mass spectrometry. However, the existing inhibitors are still inadequate in covering the entire kinome. Here, we identified a novel bisanilino pyrimidine, CTx-0294885, exhibiting inhibitory activity against a broad range of kinases in vitro, and further developed it into a Sepharose-supported kinase capture reagent. Use of a quantitative proteomics approach confirmed the selectivity of CTx-0294885-bound beads for kinase enrichment. Large-scale CTx-0294885-based affinity purification followed by LC-MS/MS led to the identification of 235 protein kinases from MDA-MB-231 cells, including all members of the AKT family that had not been previously detected by other broad-spectrum kinase inhibitors. Addition of CTx-0294885 to a mixture of three kinase inhibitors commonly used for kinase-enrichment increased the number of kinase identifications to 261, representing the largest kinome coverage from a single cell line reported to date. Coupling phosphopeptide enrichment with affinity purification using the four inhibitors enabled the identification of 799 high-confidence phosphosites on 183 kinases, ∼10% of which were localized to the activation loop, and included previously unreported phosphosites on BMP2K, MELK, HIPK2, and PRKDC. Therefore, CTx-0294885 represents a powerful new reagent for analysis of kinome signaling networks that may facilitate development of targeted therapeutic strategies. Proteomics data have been deposited to the ProteomeXchange Consortium ( http://proteomecentral.proteomexchange.org ) via the PRIDE partner repository with the data set identifier PXD000239.

A Human Monoclonal Antibody against Insulin-Like Growth Factor-II Blocks the Growth of Human Hepatocellular Carcinoma Cell Lines In vitro and In vivo

Elevated expression of insulin-like growth factor-II (IGF-II) is frequently observed in a variety of human malignancies, including breast, colon, and liver cancer. As IGF-II can deliver a mitogenic signal through both IGF-IR and an alternately spliced form of the insulin receptor (IR-A), neutralizing the biological activity of this growth factor directly is a potential alternative option to IGF-IR–directed agents. Using a Fab-displaying phage library and a biotinylated precursor form of IGF-II (1–104 amino acids) as a target, we isolated Fabs specific for the E-domain COOH-terminal extension form of IGF-II and for mature IGF-II. One of these Fabs that bound to both forms of IGF-II was reformatted into a full-length IgG, expressed, purified, and subjected to further analysis. This antibody (DX-2647) displayed a very high affinity for IGF-II/IGF-IIE (KD value of 49 and 10 pmol/L, respectively) compared with IGF-I (∼10 nmol/L) and blocked binding of IGF-II to IGF-IR, IR-A, a panel of insulin-like growth factor–binding proteins, and the mannose-6-phosphate receptor. A crystal complex of the parental Fab of DX-2647 bound to IGF-II was resolved to 2.2 Å. DX-2647 inhibited IGF-II and, to a lesser extent, IGF-I–induced receptor tyrosine phosphorylation, cellular proliferation, and both anchorage-dependent and anchorage-independent colony formation in various cell lines. In addition, DX-2647 slowed tumor progression in the Hep3B xenograft model, causing decreased tumoral CD31 staining as well as reduced IGF-IIE and IGF-IR phosphorylation levels. Therefore, DX-2647 offers an alternative approach to targeting IGF-IR, blocking IGF-II signaling through both IGF-IR and IR-A. Mol Cancer Ther; 9(6); 1809–19. ©2010 AACR.

c-Abl phosphorylates E6AP and regulates its E3 ubiquitin ligase activity.

In human papillomavirus (HPV)-infected cells, the p53 tumor suppressor is tightly regulated by the HPV-E6-E6AP complex, which promotes it for proteasomal degradation. We previously demonstrated that c-Abl tyrosine kinase protects p53 from HPV-E6-E6AP complex-mediated ubiquitination and degradation under stress conditions. However, the underlying mechanism was not defined. In this study, we explored the possibility that c-Abl targets E6AP and thereby protects p53. We demonstrated that c-Abl interacts with and phosphorylates E6AP. We determined that the E3 ligase activity of E6AP is impaired in response to phosphorylation by c-Abl. We mapped the phosphorylation site to tyrosine 636 within the HECT catalytic domain of E6AP, and using substitution mutants, we showed that this residue dictates the E3 ligase activity of E6AP, in a substrate-specific manner. On the basis of the crystal structure of the HECT domain of E6AP, we propose a model in which tyrosine 636 plays a regulatory role in the oligomerization of E6AP, which is a process implicated in its E3 ubiquitin ligase activity. Our results suggest that c-Abl protects p53 from HPV-E6-E6AP complex-mediated degradation by phosphorylating E6AP and impairing its E3 ligase activity, thus providing a molecular explanation for the stress-induced protection of p53 in HPV-infected cells.

Chapter Four – Structure and Function of Ecdysone Receptors—Interactions with Ecdysteroids and Synthetic Agonists

Abstract The binding of ecdysteroids and the bisacylhydrazine insecticide, tebufenozide, to recombinant ecdysone receptor ligand-binding domains from pest insects points to conserved and variable features of the receptors ligand-binding pocket. Fluorophores conjugated to the terminus of the ecdysteroid alkyl chain have surprisingly little effect on receptor binding, permitting the development of a fluorescence polarization chemical library screen that has led to the discovery of a new class of ecdysone receptor ligands, the methylene lactams. X-ray structures of ecdysone receptor ligand-binding domains have allowed identification of the conserved and variable features within the binding pocket. The structures offer explanations for the lepidopteran selectivity of the bisacylhydrazines, the effect of amino acid replacements on the binding of ecdysteroids and other chemistries, and the preference of a phytophagous pentatomomorphan for makisterone A; indeed, they speak to the control spectra of future ecdysone receptor-targeting insecticides. Possible ligands for nematode ecdysone receptor orthologs are also considered.

Abstract 5371: PRMT5 inhibitors as novel treatment for cancers

Increased expression or dysregulation of protein arginine methyltransferase 5 (PRMT5) activity is associated with poor prognosis in many cancers. Through increased methylation of epigenetic and non-epigenetic targets, the aberrant activity of PRMT5 has been associated with many pro-tumourigenic cellular changes such as, increased levels of protein synthesis, dysregulation of cell cycle, cellular adaptation to hypoxic conditions, and suppression of normal cell death pathways. Genetic studies suggest that suppression of PRMT5 activity can reverse many of these pro-tumourigenic effects making PRMT5 an attractive drug discovery target. We screened a library of 350,000 lead-like compounds with a biochemical assay measuring the methylation of a histone H4 peptide by the recombinant human PRMT5/MEP50 complex. Biochemical and biophysical profiling of the inhibitory compounds indicated that several distinct binding modes were exhibited by the different chemical scaffolds. Inhibitors displayed competitive, noncompetitive or uncompetitive interactions with respect to S-adenosyl methionine and the peptide substrate. Medicinal chemistry developed several classes of potent, highly selective inhibitors of PRMT5 methyltransferase activity from the hit set. The optimised tool compound, CTx-034, is a potent inhibitor of PRMT5 methyl transferase activity (KD = 2 nM), which is highly selective (>100-fold) versus a panel of 18 methyltransferases (including 6 PRMT family members), 11 lysine demethylases, and 15 safety related targets (GPCRs, ion channels, enzymes). Treatment of cancer cell lines with CTx-034 reduces cellular levels of symmetrically dimethylated H4 Arginine 3 (H4R3me2s), in a dose dependent manner (IC50 = 4 nM) to levels undetectable by Western blot. Furthermore, within this chemical series the ability of compounds to reduce cellular levels of H4R3me2s closely correlates with PRMT5 inhibitory activity supporting PRMT5 as the cellular target of these compounds, and suggesting that PRMT5 is the major writer of this histone mark in many cancer cell lines. CTx-034 also inhibits the symmetric dimethylation of arginine on other histone and non-histone cellular substrates of PRMT5, including H3R2me2s and SmD1. Conversely, CTx-034 treatment does not reduce levels of H4R3 asymmetric dimethylation, a histone mark catalysed by PRMT1. Finally, CTx-034 has good oral bioavailability and pharmacokinetic properties in rodents and twice-daily dosing (10 - 100 mg/kg) over 10-14 days produces a dose dependent reduction of the H4R3me2s mark in bone marrow cells and peripheral white blood cells. This treatment is well tolerated by the mice, with no significant reduction in body weight or changes in haematological parameters observed. CTx-034 provides an excellent tool compound for cellular and in vivo proof of concept studies. Citation Format: Hendrik Falk, Richard C. Foitzik, Elizabeth Allan, Melanie deSilva, Hong Yang, Ylva E. Bozikis, Marica Nikac, Scott R. Walker, Michelle A. Camerino, Ben J. Morrow, Alexandra E. Stupple, Rachel Lagiakos, Jo-Anne Pinson, Romina Lessene, Wilhelmus JA Kersten, Danny G. Ganame, Ian P. Holmes, Gill E. Lunniss, Matthew Chung, Stefan J. Hermans, Michael W. Parker, Alison Thistlethwaite, Karen White, Susan A. Charman, Brendon J. Monahan, Patricia Pilling, Julian Grusovin, Thomas S. Peat, Stefan Sonderegger, Emma Toulmin, Stephen M. Jane, David J. Curtis, Paul A. Stupple, Ian P. Street. PRMT5 inhibitors as novel treatment for cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5371. doi:10.1158/1538-7445.AM2015-5371