Encoded chemical synthesis coupled to screening: "Pot Assay".

A variety of screening methodologies is available to identify lead compounds. Screening methods that would permit the direct use of libraries made via the Radiofrequency Encoded Combinatorial chemistry paradigm (each individual small molecule in the library is presented separately on an individual encoded support) have the potential to diminish burdensome steps in this process. Here we report on our studies leading to such a direct method, which we have termed a Pot Assay.

An immunoassay for assessment of receptor tyrosine kinase autophosphorylation.

We have developed an immunoassay that can be used to assess autophosphorylation activity of receptor tyrosine kinases, yet does not require the use of synthetic peptide substrates or anti-receptor antibodies. In the assay described here, receptor autophosphorylation and detection take place entirely within the wells of 96 well microplates coated with Protein G and an anti-phosphotyrosine antibody. As the kinase reaction takes place, the antibodies capture the phosphorylated products in situ.

A fluorometric assay for the measurement of endothelial cell density in vitro.

A fluorometric assay for determining endothelial cell numbers based on the endogenous enzyme acid phosphatase is described. In preliminary studies, three substrates--p-nitrophenyl phosphate, 4-methylumbelliferyl phosphate, and 2'-[2-benzthiazoyl]-6'-hydroxy-benthiazole phosphate (AttoPhos)--were compared with respect to their kinetic, optimum assay conditions, sensitivity, and detection limits. Only AttoPhos was found to have a high degree of sensitivity, reliability, and reproducibility for measuring both high and low cell numbers in the same plate.

Vascular smooth muscle cell cytotoxicity and sustained inhibition of neointimal formation by fibroblast growth factor 2-saporin fusion protein.

Basic fibroblast growth factor (FGF2) is an important mediator of smooth muscle cell (SMC) proliferation following arterial injury that results in neointimal growth. The present study was designed to explore the effects of recombinant FGF2 linked to the ribosome-inactivating protein saporin-6 (rFGF2-SAP) on vascular SMC cytotoxicity and neointimal formation following arterial injury. Cultured rat aortic SMCs were exposed to various concentrations of rFGF2-SAP, FGF2, and saporin-6 (SAP).

Large-scale purification and characterization of recombinant fibroblast growth factor-saporin mitotoxin.

In order to produce sufficient quantities of fibroblast growth factor-saporin (rFGF-2-SAP) mitotoxin for preclinical evaluation in models of diseases such as cancer and restenosis, we have undertaken the large-scale expression, purification, and characterization of the recombinant molecule. The fusion gene encoding rFGF-2-SAP was cloned into the inducible pET 11a expression vector and transformed into Escherichia coli strain BL21 (DE3). The transformants were grown using a fed-batch fermentation until the A600 reached 85.

Antiproliferative effect of basic fibroblast growth factor-saporin mitotoxin on keratocytes in culture.

We evaluated the effect of the conjugate of basic fibroblast growth factor (FGF2) and saporin (FGF2-SAP) on proliferation of cultured keratocytes. Cultured rabbit and human keratocytes were incubated in medium containing 0.01 to 100 nM of chemical conjugate of EGF2 conjugated by disulfide bond to saporin (CCFS1), FGF2 genetically fused to saporin (rFGF2-SAP), FGF2, or saporin for three hours or four days and cell proliferation was quantified four days after the drug treatment.

Synthesis and characterization of a homogeneous chemical conjugate between basic fibroblast growth factor and saporin.

Basic fibroblast growth factor (FGF-2) and saporin were chemically conjugated using the crosslinker, N-succinimidyl-3(2-pyridyldithio)-propionate. When purified, the conjugate was found to be heterogeneous as analyzed by SDS/PAGE, size-exclusion HPLC and reverse-phase HPLC. Therefore, we sought to synthesize a molecule that would be homogeneous and thus easier to characterize and evaluate its efficacy and toxicity for pharmaceutical drug development.

Progesterone and oestrogen receptors in the decidualized mouse uterus and effects of different types of anti-progesterone treatment.

Pseudopregnant mice were treated systemically with monoclonal anti-progesterone antibody (DB3) (model 1), or progesterone receptor antagonists RU486 or ZK98,299 (ZK299) (model 2) on day 3 post coitum. On day 4, sesame oil was administered intraluminally into one uterine horn to induce decidualization. On day 7, the average mass of the oil-injected horn was 335.

Maturational differences in acetazolamide-altered pH and HCO3 of choroid plexus, cerebrospinal fluid, and brain.

The carbonic anhydrase inhibitor acetazolamide is useful for analyzing ion transport, pH regulation, and fluid formation in developing central nervous system. We used the 14C-labeled dimethadione technique to measure alterations in steady-state pH, and to estimate the HCO3 concentration [HCO3], in choroid plexus (CP), cerebrospinal fluid (CSF), and cerebral cortex of 1- and 3-wk-old Sprague-Dawley rats treated with acetazolamide or probenecid. These drugs can suppress transport of HCO3 and other anions in some cells, consequently altering intracellular pH.

Interaction of glucocorticoid analogues with the human glucocorticoid receptor.

Transient co-transfection of receptor cDNA and suitable reporter genes was used to study human glucocorticoid receptor (hGR) function in a neutral mammalian cell background. A variety of natural and synthetic steroids were analyzed for their ability to activate gene expression through the hGR and to bind to extracts of cells expressing the hGR cDNA. There was very good correlation between these two in vitro parameters for these compounds.

An adenoviral vector system for functional identification of nuclear receptor ligands.

A recombinant adenovirus system has been designed that confers glucocorticoid responsiveness upon infected cells in culture. Two mutually dependent viruses are required: a trans-activator virus containing the human glucocorticoid receptor transcription unit and a second receptor virus harboring a glucocorticoid response element linked to the firefly luciferase gene. Another reciprocal pair of viruses has been generated; one member expresses the rat thyroid hormone receptor alpha, while the other contains the luciferase gene regulated by a thyroid hormone-responsive DNA element.

Changes in diacylglycerol and membrane associated protein kinase C activity reflect the growth status of xenografted human mammary carcinoma treated with 8-Cl-cAMP.

The intracellular accumulation of cAMP inhibits the growth of transformed cells in vitro and in vivo, and exposure to various cAMP analogs produces similar results. The influence of such analogs on the growth of neoplastic cells in vivo is less well defined, and the relevance of these analogs for the phosphoinositide pathway has not been established. The present report details the inhibition of tumor growth that occurred when human mammary xenografts were treated with 8-Cl-cAMP, the subsequent rebound in tumor growth that occurred when treatment ceased, and the levels of diacylglycerol and membrane-associated protein kinase C activity that characterized tumors in different growth states.

Changes in diacylglycerol and cyclic GMP during the differentiation of human myeloid leukemia K562 cells.

When the human myeloid leukemia cell line, K562, was induced to differentiate along the erythroid lineage by a 4 day treatment with 10 microM tiazofurin, the cellular content of diacylglycerol decreased to 35% of the value in untreated control cells. Under the same conditions the content of cGMP decreased to 61% of the control value. Tiazofurin inhibits guanine nucleotide biosynthesis and lowers cellular GTP.

Tiazofurin and selenazofurin induce depression of cGMP and phosphatidylinositol pathway in L1210 leukemia cells.

The synthetic nucleoside tiazofurin(2-beta-ribofuranosylthiazole-4-carboxyamide) and its selenium analog selenazofurin inhibited the growth of L1210 leukemia cell culture in a dose dependent manner with IC50 value of 2.0 and 0.2 Um respectively.

Stimulation of phosphoinositide signaling pathway in murine B lymphocytes by a novel guanosine analog, 7-thia-8-oxoguanosine.

A novel guanosine analog, 7-thia-8-oxoguanosine, is shown to induce proliferation in nude mouse splenic lymphocytes in vitro as measured by increased DNA synthesis. Determination of the levels of sn-1,2-diacylglycerol and inositol 1,4,5-trisphosphate in the 7-thia-8-oxoguanosine-treated B lymphocyte cultures revealed an enhanced formation of these second messengers leading to the activation and possible de novo synthesis of protein kinase C. The relevance of these results are discussed in regard to the transduction mechanism linking these guanosine analog generated signals with subsequent B lymphocyte activation events.

Nucleoside and nucleotide modulation of genetic expression--a new approach to chemotherapy.

Unlike conventional enzymes, receptors that activate G proteins do not catalyze the direct formation or cleavage of covalent bonds but act instead as a catalyst for the exchange of GTP vs GDP, which results in major conformational changes in the alpha subunit of G proteins and dissociation and selective binding of the alpha subunit which provokes direct enzyme activation eventually resulting in stimulation of protein kinase A, B or C. Each of these kinases can phosphorylate specific DNA binding proteins which allow new portions of DNA to be read and expressed. Such a series of events can act as switches to control cellular genetic expression resulting in cellular proliferation, differentiation or hormonal secretion of growth factors (Scheme I).

Synergistic inhibition of growth of breast and colon human cancer cell lines by site-selective cyclic AMP analogues.

Our past studies on the mechanism of cyclic AMP (cAMP)-mediated control of tumor growth, using the experimental rat mammary tumor models as well as human breast cancer cell lines, indicated that the action of cAMP is mediated by the RII cAMP receptor protein, the regulatory subunit of cAMP-dependent protein kinase type II (Y. S. Cho-Chung, J.

Cytochrome P-450 isozymes 2 and 5 in rabbit lung and liver. Comparisons of structure and inducibility.

Rabbit cytochrome P-450 isozymes 2 and 5 were purified from pulmonary and hepatic microsomal preparations. Purification of isozyme 5 was monitored by immunochemical methods so that contamination by isozymes 2, 4, and 6 could be avoided. Partial proteolysis of hepatic and pulmonary isozyme 5 showed minor differences in peptide formation when analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and visualized by the silver staining method.

Unique properties of NADPH- and NADH-dependent metabolism of p-nitroanisole catalyzed by cytochrome P-450 isozyme 2 in pulmonary and hepatic microsomal preparations from rabbits.

Approximately 90% of the NADPH- and NADH-dependent O-demethylation of p-nitroanisole (PNA) in the hepatic microsomal fraction from phenobarbital (PB)-treated rabbits and in the pulmonary microsomal fraction from untreated rabbits is catalyzed by the same isozyme of cytochrome P-450. This isozyme of cytochrome P-450 catalyzes less than 60% of this reaction in the hepatic microsomal fraction from untreated rabbits. Antibodies to NADPH-cytochrome P-450 reductase inhibit NADPH-dependent metabolism of p-nitroanisole by about 90% but have no effect on NADH-dependent metabolism.

Cl-HCO3 exchange in choroid plexus: analysis by the DMO method for cell pH.

[14C]DMO distribution was used to measure steady-state intracellular pH (pHi) and [HCO3]i in adult rat choroid plexus (CP) incubated in synthetic cerebrospinal fluid (CSF) for 30 min. In controls at 37 degrees C, mean pHi (6.95 at PCO2 = 30 mmHg) was close to corresponding in vivo values; and [HCO3]i/[HCO3]csf, i.

The influence of in vitro procedures which diminish NADPH cytochrome c reductase activity on oxidative drug metabolism in lung and liver microsomes.

Rabbit lung and liver microsomes were subjected to three procedures which decreased NADPH cytochrome c reductase activity; flavoprotein antibody, trypsin and subtilisin digestion. The effects on benzphetamine and p-nitroanisole demethylation and amine metabolic-intermediate complex formation were investigated. In general, the proteolytic digestion had a greater inhibitory effect on oxidation reactions for a given loss of NADPH cytochrome c reductase activity than did flavoprotein antibody; and of the two proteases, subtilisin, which also diminishes the cytochrome b5 reduction pathway, had a greater inhibitory effect than trypsin.

Developmental changes in rabbit pulmonary cytochrome P-450 subpopulations.

Of the two characterized cytochrome P-450 subpopulations present in adult lung microsomes, only one (P-450II) appears to be present in the neonate. Both this subpopulation and a second subpopulation (P-450I) gradually increase over a period of several months, and account for most of the increase in lung cytochrome P-450 concentration during maturation. A third fraction of the cytochrome P-450, which is incapable of forming metabolic-intermediate complexes remains constant in concentration during maturation, thus decreasing from 60% of the total in the neonate to 20% in the adult.

Ontogeny of blood-brain barrier permeability to, and cerebrospinal fluid sink action on, [14C]urea.

To ascertain the ontogeny of CSF sink action on a small hydrophilic solute, we investigated the rate and extent of uptake of [14C]urea by lateral ventricle choroid plexus (CP), cisternal cerebrospinal fluid (CSF), cerebral cortex, and cerebellum in 0.5- to 4-wk-old etherized nephrectomized rats. Five hours after ip injection, radiourea penetrated the entire H2O of tissue and CSF in 1-wk-old brain; moreover, for animals 1-4 wk old there was a marked inverse relationship between age and magnitude of steady-state [14C]urea space in all regions.