Kurt R. Auger

Oncogenes and signal transduction

The purpose of this review is to incorporate recent discoveries into a general biochemical pathway by wich the protein products of the oncogenes send signals from the cell surface to the nucleus .The protein-tyrosine kinase oncogenes will be the primary focus of the review .However, biochemical connections between the protein tyrosine kinases and oncoproteins of the Ras,Raf,Fos,Jun,and Rel families as well as the protein kinase C family are also discussed .

Discovery of GSK2126458, a Highly Potent Inhibitor of PI3K and the Mammalian Target of Rapamycin.

Phosphoinositide 3-kinase α (PI3Kα) is a critical regulator of cell growth and transformation, and its signaling pathway is the most commonly mutated pathway in human cancers. The mammalian target of rapamycin (mTOR), a class IV PI3K protein kinase, is also a central regulator of cell growth, and mTOR inhibitors are believed to augment the antiproliferative efficacy of PI3K/AKT pathway inhibition. 2,4-Difluoro-N-{2-(methyloxy)-5-[4-(4-pyridazinyl)-6-quinolinyl]-3-pyridinyl}benzenesulfonamide (GSK2126458, 1) has been identified as a highly potent, orally bioavailable inhibitor of PI3Kα and mTOR with in vivo activity in both pharmacodynamic and tumor growth efficacy models. Compound 1 is currently being evaluated in human clinical trials for the treatment of cancer.

Adult human endothelial cell coverage of small-caliber dacron and polytetrafluoroethylene vascular prostheses in vitro

Culture of endothelial cells on synthetic vascular grafts has heretofore met with limited success. We report here a technique which allows attachment and subsequent growth of adult human vascular endothelial cells on the synthetic materials polytetrafluoroethylene (PTFE) and Dacron which are currently used for vascular reconstructive surgery. Studies were conducted on both untreated materials and those pretreated with the extracellular matrix proteins collagen and fibronectin. Collagen was applied to the graft materials with positive pressure and then allowed to gel in the interstices. Fibronectin was added to the collagen-lined lumen followed by a cell suspension. Cell coverage on the grafts was assessed by scanning electron microscopy after various lengths of time. Cells adhered poorly to and did not grow on untreated Dacron and PTFE. Protein-treated materials did allow cell attachment and growth but with distinct differences. On PTFE (n = 30), cells could form a confluent monolayer within 9 days while cell coverage was generally incomplete at this time on the more irregular surface of Dacron (n = 5). Thus, adult human endothelial cells can grow on collagen- and fibronectin-coated prosthetic materials. This approach to lining graft materials in vitro may be useful in improving the performance of small-caliber vascular grafts.

Discovery of a Potent Class of PI3Kα Inhibitors with Unique Binding Mode via Encoded Library Technology (ELT)

In the search of PI3K p110α wild type and H1047R mutant selective small molecule leads, an encoded library technology (ELT) campaign against the desired target proteins was performed which led to the discovery of a selective chemotype for PI3K isoforms from a three-cycle DNA encoded library. An X-ray crystal structure of a representative inhibitor from this chemotype demonstrated a unique binding mode in the p110α protein.

Discovery of the First Potent and Selective Inhibitor of Centromere-Associated Protein E: GSK923295.

Inhibition of mitotic kinesins represents a novel approach for the discovery of a new generation of anti-mitotic cancer chemotherapeutics. We report here the discovery of the first potent and selective inhibitor of centromere-associated protein E (CENP-E) 3-chloro-N-{(1S)-2-[(N,N-dimethylglycyl)amino]-1-[(4-{8-[(1S)-1-hydroxyethyl]imidazo[1,2-a]pyridin-2-yl}phenyl)methyl]ethyl}-4-[(1-methylethyl)oxy]benzamide (GSK923295; 1), starting from a high-throughput screening hit, 3-chloro-4-isopropoxybenzoic acid 2. Compound 1 has demonstrated broad antitumor activity in vivo and is currently in human clinical trials.

Quantitative Assays of Mdm2 Ubiquitin Ligase Activity and Other Ubiquitin‐Utilizing Enzymes for Inhibitor Discovery

Mdm2 is a negative regulator of p53 activity and functions as an E3 ubiquitin ligase of p53. Inhibition of mdm2 E3 ligase activity will block ubiquitination and subsequent proteasome-mediated degradation of p53, resulting in the stabilization of p53 protein that could lead to the restoration of its tumor-suppressor activity. This chapter describes quantitative biochemical assays for mdm2 E3 activity that can be applied to other ubiquitin-utilizing enzyme systems. Our unique assay format relies on the generation of labeled Ub-E2 conjugate that functions as a substrate for the E3 ligase enzyme. Reducing the E1-E2-E3 ubiquitin cascade to a single enzyme (E3) and bisubstrate (Ub-E2 and target protein) reaction makes it possible to carry out detailed biochemical characterization of the reaction mechanism, high-throughput screening to identify inhibitors of specific E3 ligases, and detailed characterization of the mode of inhibitor interactions with the target enzyme. In addition, preforming the Ub-E2 conjugate as an enzyme substrate for inhibitor screening minimizes interference from thiol-modifying compounds and from nucleotide analogs and other ATP-interfering compounds that might affect the E1 reaction. Using this type of format, we were able to identify small molecule inhibitors of mdm2 E3 ligase activity that are selective against E1 and other E3 ligases, including mdm2s own autoubiquitination activity. Detailed protocols on the labeling of Ub, the generation of Ub-E2, and the use of Ub-E2 in the E3 ligase reaction for inhibitor discovery and characterization are provided.

Baculovirus production of fully-active phosphoinositide 3-kinase alpha as a p85α–p110α fusion for X-ray crystallographic analysis with ATP competitive enzyme inhibitors☆

Phosphoinositide 3-kinases have been targeted for therapeutic research because they are key components of a cell signaling cascade controlling proliferation, growth, and survival. Direct activation of the PI3Kalpha pathway contributes to the development and progression of solid tumors in breast, endometrial, colon, ovarian, and gastric cancers. In the context of a drug discovery effort, the availability of a robust crystallographic system is a means to understand the subtle differences between ATP competitive inhibitor interactions with the active site and their selectivity against other PI3Kinase enzymes. To generate a suitable recombinant design for this purpose, a p85alpha-p110alpha fusion system was developed which enabled the expression and purification of a stoichiometrically homogeneous, constitutively active enzyme for structure determination with potent ATP competitive inhibitors (Raha et al., in preparation) [56]. This approach has yielded preparations with activity and inhibition characteristics comparable to those of the full-length PI3Kalpha from which X-ray diffracting crystals were grown with inhibitors bound in the active site.

Phosphatidylinositol 3-kinase: A Novel Signal Transduction Pathway?

Publisher Summary This chapter discusses the existence of newly described polyphosphoinositides that are responsive to mitogen stimulation or oncogenic transformation in cultured mammalian cells. The time course of production of novel polyphosphoinositides suggests that they are important signaling molecules in the signal transduction pathway for mitogenic protein tyrosine kinases. The structures of polyphosphoinositides, PtdIns-3,4-P2 and PtdIns-3,4,5-P3, provide evidence that they are not direct precursors for the well-characterized second messenger Ins-1,4,5-P3. Because the purified Ptdlns 3-kinase can utilize Ptdlns, PtdIns-4-P, and PtdIns-4,5-P2 as substrates in vitro, it seems likely that activation of Ptdlns 3-kinase may result in the generation of multiple signals that branch off from the conventional polyphosphoinositide pathway. Polyphosphoinositides have been extensively studied because of their involvement in intracellular signaling. Phosphatidylinositol 4,5-bisphosphate and phosphatidyHnositol 4-phosphate are precursors of the second messenger pathway involving inositol 1,4,5-trisphosphate and diacylglycerol.