Stephen Felder

EGF induces tyrosine phosphorylation of phospholipase C-II: A potential mechanism for EGF receptor signaling

Binding of EGF to cells expressing human EGF receptor stimulated rapid tyrosine phosphorylation of phospholipase C-II (PLC-II), as revealed by immunoblotting analysis with phosphotyrosine-specific antibodies. Tyrosine phosphorylation of PLC-II was stimulated by low physiological concentrations of EGF (1 nM), was quantitative, and was already maximal after a 30 sec incubation with 50 nM EGF at 37 degrees C. Interestingly, antibodies specific for PLC-II were able to coimmunoprecipitate the EGF receptor and antibodies against EGF receptor also coimmunoprecipitated PLC-II. According to this analysis, approximately 1% of EGF receptor molecules were associated with PLC-II molecules. The protein tyrosine kinase inhibitor tyrphostin RG50864, which blocks EGF-dependent cell proliferation, blocked EGF-induced tyrosine phosphorylation of PLC-II, its association with EGF receptor, and EGF-induced Ca2+ release. Hence, EGF-induced tyrosine phosphorylation of PLC-II may be a regulatory event linking the tyrosine kinase activity of EGF receptor to the PIP2 hydrolysis signaling pathway.

Kinase activity controls the sorting of the epidermal growth factor receptor within the multivesicular body

We compared the internalization and intracellular sorting of epidermal growth factor receptor (EGF-R) and point mutant kinase-negative EGF-R separately expressed in NIH 3T3 cells lacking endogenous receptor. Both EGF-Rs internalized rapidly, but kinase-negative receptor was surface down-regulated only with monensin or at 20 degrees C. Furthermore, EGF internalized by mutant receptor alone was, in significant proportion, returned to the cell surface undegraded. Hence unlike wild-type receptor, kinase-negative EGF-R recycles. By electron microscopy the early pathways of endocytosis for the two receptors were identical; however, after 10-20 min the pathways diverged at the multivesicular body (MVB). Wild-type EGF-R, destined for degradation, localized to internal vesicles, while kinase-negative EGF-R, destined for recycling, localized to surface membranes of the MVBs and moved to small tubulovesicles. We conclude that sorting of internalized receptor for degradation or recycling can occur through spatial segregation within the MVB, and sorting of EGF-R is controlled by tyrosine kinase activity.

Automated solid-phase organic synthesis in micro-plate wells. Synthesis of N-(alkoxy-acyl)amino alcohols

Abstract The technique of concurrent solid-phase organic synthesis in 96-deep-well micro-titer plates is outlined and documented with the synthesis of N-(alkoxy-acyl)amino alcohols. Typically resin beads are separated from solvent using filtration approach that complicates automation. Instead we describe a method employing settling of resin, followed by aspiration of supernatant.

High-Throughput, High-Sensitivity Analysis of Gene Expression in Arabidopsis

High-throughput gene expression analysis of genes expressed during salt stress was performed using a novel multiplexed quantitative nuclease protection assay that involves customized DNA microarrays printed within the individual wells of 96-well plates. The levels of expression of the transcripts from 16 different genes were quantified within crude homogenates prepared from Arabidopsis (Arabidopsis thaliana) plants also grown in a 96-well plate format. Examples are provided of the high degree of reproducibility of quantitative dose-response data and of the sensitivity of detection of changes in gene expression within limiting amounts of tissue. The lack of requirement for RNA purification renders the assay particularly suited for high-throughput gene expression analysis and for the discovery of novel chemical compounds that specifically modulate the expression of endogenous target genes.

One bead, one chemical compound: Use of the selectide process for anticancer drug discovery

A technology for chemical synthesis and testing of libraries of millions of chemical entities has been developed for rapid molecular and cellular screening for drug leads. Each individual compound in the library is on a separate resin bead. Screening for binding activity can be conducted directly on the beads. Biological activity is assessed in solution phase assay by cleaving a portion of the compound from each bead. The molecular structure of the compound of interest is obtained by automated peptide sequencing from the bead of origin. We have applied this technology to anticancer drug discovery as well as to other pharmaceutical targets. For anticancer drug development, current molecular targets include B-cell lymphoma, the EGF receptor, and the HER2-neu receptor. Solution phase screening with dual cleavable libraries is being used for growth inhibition of human tumor cell lines. Initial in vitro leads have been identified in each of these areas of anticancer drug discovery.

Discovery of Potent 17β-Hydroxywithanolides for Castration-Resistant Prostate Cancer by High-Throughput Screening of a Natural Products Library for Androgen-Induced Gene Expression Inhibitors

Prostate cancer (PC) is the second most prevalent cancer among men in Western societies, and those who develop metastatic castration-resistant PC (CRPC) invariably succumb to the disease. The need for effective treatments for CRPC is a pressing concern, especially due to limited durable responses with currently employed therapies. Here, we demonstrate the successful application of a high-throughput gene-expression profiling assay directly targeting genes of the androgen receptor pathway to screen a natural products library leading to the identification of 17β-hydroxywithanolides 1-5, of which physachenolide D (5) exhibited potent and selective in vitro activity against two PC cell lines, LNCaP and PC-3. Epoxidation of 5 afforded physachenolide C (6) with higher potency and stability. Structure-activity relationships for withanolides as potential anti-PC agents are presented together with in vivo efficacy studies on compound 6, suggesting that 17β-hydroxywithanolides are promising candidates for further development as CRPC therapeutics.

Use of Large Combinatorial Chemical Libraries for Anticancer Drug Discovery

AbstractA new technology for drug discovery (Selectide technology) has been developed which greatly extends the capability for rapid molecular and cellular screening to large libraries containing millions of chemically synthesized compounds. Using a split synthesis approach to condensation chemistry, we recognized that each individual compound of potential interest is located on a different solid phase resin bead. Screening for binding activity on the bead surface: In order to screen for biological activity, we used several cleavable linkers which could release a portion of the compound from each bead into the solution phase in each of two sequential steps. For libraries comprised of peptides, there is sufficient residual peptide on each bead to permit sequence determination via Edman degradation. For non-peptide libraries, a system of peptide encoding is used on each bead so that the structure of the non-peptide compound can be determined by decoding the information conveyed by the peptide (which can be ...

Cloning, expression of the human substance K receptor, and analysis of its role in mitogenesis.

The primary strudure of the human substance K receptor was established from the sequences of complementary DNA clones isolated from a human jejunal complementary DNA library. It consists of 398 amino acids, including seven putative transmembrane regions. The gene for the human substance K receptor was localized to chromosome region 10p13-10q23, a region with frequent chromosomal abnormalities. The human substance K receptor was expressed in transfeded NIH-3T3 cells lacking endogenous substance K receptors, and Scatchard analysis of 125I labeled substance K binding indicates approximately 100,000 receptors/cell with a single dissociation constant of 12 nM. Covalent cross-linking experiments utilizing 125I-substanceK and three different chemical cross-linking reagents (disuccinimidyl suberate, disuccinimidyl tartrate, or 1-ethyl-3-(3dimethylaminopropyl)carbodiimide-HCI) demonstrate an apparent molecular weight of 45,000, consistent with little or no N-linked glycosylation. The binding of substance K to its receptor on transfeded cells led to a rapid increase in the produdion of total inositol phosphates and the release of Ca2� from internal stores. Growth of the cells transfeded with the human substance K receptor is stimulated by the addition of substance K to the medium to a level similar to 10% serum. Therefore, the human substance K receptor can function as a growth fador receptor when expressed in mouse 3T3 cells.