Identifying Small Molecules which Inhibit Autophagy: a Phenotypic Screen Using Image-Based High-Content Cell Analysis.

Autophagy plays an important role in cancer and it has been suggested that it functions not only as a tumor suppressor pathway to prevent tumor initiation, but also as a pro-survival pathway that helps tumor cells endure metabolic stress and resist death triggered by chemotherapeutic agents, including acquired resistance. We aimed to identify small-molecule autophagy inhibitors using a HTS/HCA approach through a phenotypic, cell image-based assay, in order to screen multiple biological targets simultaneously and to screen compounds in a physiologically relevant environment. LC3 is a component of the autophagosome, which undergoes a cytoplasmic redistribution from diffuse to punctate dots during autophagy. We employed HeLa cells stably expressing EGFP-LC3 in a primary phenotypic screen. As a first step, a "Validation Library" of about 8,000 pre-selected compounds, about 25% of which had known biological activity and the others representing a range of chemical structures, was run in duplicate both to assess screening suitability and likely hit rate, and to give a valuable preview of possible active structures or biological targets. The primary screen of about 0.25 million compounds yielded around 10,500 positive compounds. These were tested in a suite of further cellular assays designed to eliminate unwanted positives, together with the application of chemi- and bioinformatics to pick out compounds with known biological activity. These processes enabled the selection of compounds that were the most promisingly active and specific. The screening "tree" identified, amongst others with as yet unidentified targets, chemical series active against autophagy-relevant biological targets ULK or Vsp34, validating the phenotypic screening methods selected. Finally, about 400 compounds were fully qualified after following this triage. The development of the assays, compound screening process and the compound triage is described.

Activation of cellular responses to interleukin 6 is blocked by staurosporine.

Interleukin 6 (IL-6) acts on a wide spectrum of cells and can regulate differentiation or growth in these different cells. The effects of the microbial alkaloid staurosporine (SS) on IL-6 signaling through gp130, and also on the internalization of the IL-6 receptor complex, were studied using HepG2 cells which are well-characterized in their ability to respond to IL-6 by upregulating acute-phase protein production. SS was found effective in the blockade of the signaling cascade of IL-6: phosphorylation of both gp130 and Stat3 was eliminated by SS treatment and the production of IL-6 stimulated haptoglobin by the cells was abolished. In addition, SS reduced the internalization rate of 125I-IL-6 by 50%, resulting in a retention of 125I-IL-6 on the cell surface and a corresponding decrease in degraded 125I-IL-6 in the extracellular medium. SS is commonly employed as an apoptosis inducing agent but the mechanism of its action is not clear. The ability of SS to void the capacity of IL-6, and IL-6-related cytokines such as Oncostatin M, to deliver growth and differentiation signals may be one process by which this agent could promote apoptosis in a variety of cell types.

Effect of the selective and non-selective cysteine protease inhibitors on the intracellular processing of interleukin 6 by HEPG2 cells.

The effects were measured and compared of three nonselective cysteine cathepsin inhibitors (leupeptin, trans-Epoxy-succinyl-L-Leucylamido(4-guanidino)-butane (E-64), and Z-Phe-Ala-CH2F) and a selective cathepsin B inhibitor, CA074Me, on the intracellular processing of 125I-labeled human recombinant Interleukin 6 (IL-6) by HepG2 cells. The uptake and processing of 125I-IL-6 by cells treated with inhibitors was followed over a 7-h period. All inhibitors caused an increased residence time of IL-6 inside the cell and a corresponding decrease in the output of non-trichloroacetic acid-precipitable fragments of radiolabeled protein. Maximal effect was achieved with leupeptin at 200 microM, with which the rate of IL-6 digestion was reduced to 50% that of control cells. The specific inhibitor CA074Me was the least effective in slowing the intracellular processing of IL-6. The effects of all of the inhibitors on the production of haptoglobin, either stimulated by IL-6 or basal, was negligible over a similar time period, indicating continued cell viability. The data from this model suggest that cathepsin inhibitors would not interfere with lysosomal processing to an extent which would prohibit the development of selective and potent cathepsin inhibitors for the treatment of diseases in which individual cysteine cathepsins play clearly pathophysiological roles.

Characterization of an interleukin 6 cytokine family antagonist protein from a marine sponge, Callyspongia sp.

An inhibitor of IL-6 binding to the human hepatoma line HepG2 and myeloma cell line U266 was identified in a saline extract of the marine sponge, Callyspongia sp. Functional activity, measured through the increase in haptoglobin production by HepG2 cells stimulated with IL-6, could be strongly inhibited by the extract. Similarly, IL-6-induced production of IgM by the B cell line SKW6.4 was substantially reduced. In neither cell line was there evidence of toxicity produced by the extract. Other sponges of the Callyspongia species were found to contain analogous activity. The activity was destroyed by trypsin treatment or boiling of the extract, suggesting that the inhibition is due to a protein. When the binding of IL-6 to its receptor complex was dissected in vitro, inhibition of binding of IL-6 to soluble receptor by the extract was not detected, but binding of the IL-6-sIL-6R complex to soluble gp130 was inhibited in a dose-dependent fashion. This was borne out in cellular assays since the extract inhibited activation of HepG2 cells stimulated with oncostatin M or leukemia inhibitory factor, cytokines which also use gp130 for signal transduction. These results suggest that the Callyspongia extract contains a protein which blocks the interaction of the IL-6 family of cytokines with their signal transduction moiety, gp130. Elucidation of the structure and mode of action of such a protein would be helpful in designing gp130 antagonists to inhibit the functions of this cytokine family, overproduction of which has been associated with cancer and pathologies of autoimmune disease and AIDS.

The heterologous expression and characterization of human prostaglandin G/H synthase-2 (COX-2).

The open reading frame of human cyclooxygenase-2 was cloned by pcr amplification of IL-1 beta stimulated human dermal fibroblast cDNA. The coding region was used to construct a recombinant baculovirus which when used to infect Sf9 cells directed the expression of recombinant human cyclooxygenase-2. The heterologously expressed enzyme was characterized and found to display all salient features of cyclooxygenase. Large-scale microsomal preparations of infected cells yielded more than 20 units of enzyme with a specific activity of 240 nmoles prostaglandin product/mg protein.

Kinetics of a cellular response to interleukin 6.

The stimulation of the production of haptoglobin from the human hepatoma HepG2 was used as a model to examine the kinetics of a cellular response to interleukin 6 (IL-6). It was demonstrated that IL-6 upregulated the production of haptoglobin in a time-dependent manner: using a sensitive radioimmunoassay for haptoglobin, increases were already detectable 2 hr after IL-6 treatment began. The haptoglobin level continued to rise in a linear fashion to at least 16 hr. The stimulation of haptoglobin by IL-6 was abolished in the presence of 5 micrograms/ml actinomycin D and was thus likely pretranslational. It was further demonstrated that the upregulation of haptoglobin continued well after the removal of IL-6 from the system. An 8-hr "pulse" of IL-6 gave rise to haptoglobin secretion which was above control levels for the following 4 days. The effects of IL-6 in vivo can therefore be predicted to be long-lived despite its own short half-life, especially since the products of IL-6 stimulation, e.g., immunoglobulin and acute-phase proteins, are themselves long-lived in the circulation.

Pharmacologic profile of CGS 24128, a potent, long-acting inhibitor of neutral endopeptidase 24.11.

We compared the pharmacologic profiles of thiorphan, a neutral endopeptidase (NEP) inhibitor which is cleared rapidly from the circulation, and CGS 24128, an inhibitor with a much longer half-life (t1/2). Thiorphan and CGS 24128 inhibited NEP in vitro with IC50 values of 5.0 +/- 0.2 and 4.3 +/- 0.2 nM, respectively. After administration at 10 mg/kg intravenously (i.v.), the concentrations of CGS 24128 in the plasma were > 500 nM for 4 h but plasma thiorphan was detectable for only 60 min. Thiorphan 3 mg/kg administered intraarterially (i.a.) increased plasma atrial natriuretic peptide immunoreactivity (ANPir) levels by 58 +/- 12% in rats administered exogenous ANP(99-126). This response lasted < 60 min, whereas the same dose of CGS 24128 produced an average increase of 191 +/- 19% in ANPir concentrations that persisted for 4 h. ANP-induced (1 microgram/kg i.v.) natriuresis was significantly potentiated in anesthetized rats pretreated (60 min) with a bolus of CGS 24128 10 mg/kg i.v. The change in urinary sodium excretion (UNaV) produced by ANP was 28.8 +/- 4.0 and 15.8 +/- 1.8 muEq/kg/min in CGS 24128- and vehicle-treated rats, respectively. ANP-induced natriuresis was also greater during continuous infusion of thiorphan (5 mg/kg bolus + 0.1 mg/kg/min i.v.; delta UNaV = 29.2 +/- 5.8 and 13.8 +/- 3.2 muEq/kg/min in drug- and vehicle-treated rats, respectively) but not when thiorphan was administered as a bolus (10 mg/kg i.v.) 60 min before the ANP challenge.(ABSTRACT TRUNCATED AT 250 WORDS)

Design and synthesis of an orally active macrocyclic neutral endopeptidase 24.11 inhibitor.

A potent macrocyclic inhibitor of neutral endopeptidase (NEP) 24.11 was designed using a computer model of the active site of thermolysin. This 10-membered ring lactam represents a general mimic for any hydrophobic dipeptide in which the two amino acid side chains bind to an enzyme in a contiguous orientation. The parent 10-membered ring lactam was synthesized and exhibited excellent potency as an NEP 24.11 inhibitor (IC50 = 3 nM). In order to improve oral bioavailability, various functionality was attached to the macrocycle. These modifications lead to CGS 25155, an orally active NEP 24.11 inhibitor that slows down the degradation of the cardiac hormone atrial natriuretic factor, producing a lowering of blood pressure in the DOCA-salt rat model of hypertension.

Development of tolerance to the antihypertensive effects of highly selective adenosine A2a agonists upon chronic administration.

Three highly A2a-selective adenosine agonists were examined for their effects on blood pressure during chronic administration in conscious spontaneously hypertensive rats. Sodium 4-[2-[[6-amino-9-(N-ethyl-beta-D-ribofuranuronamidosyl)-9H-purin-2 -yl] amino]ethyl]benzenepropionate (CGS 21680C) 2-[(2-cyclohexyl-ethyl)amino]adenosine (CGS 22492) and 2-[[2-(1-cyclohexen-1-yl)ethyl]amino]adenosine (CGS 22989) were administered at a rate of 0.25 and 0.5 micrograms/kg/min i.v. for 2 weeks using osmotic minipumps. Significant systolic blood pressure reductions were seen in the A2a agonist-treated groups compared to vehicle-treated (50% dimethyl sulfoxide) animals. Maximum effects occurred on days 1 and 2 in the treated animals. However, the antihypertensive effect diminished with time such that no differences between treatments were seen at 2 weeks. In contrast, a sustained antihypertensive effect was evident with benazeprilat (an angiotensin converting enzyme inhibitor). Tolerance was associated with a decrease in Bmax values (375 +/- 22, 410 +/- 18 and 548 +/- 17 fmol/mg of protein in the CGS 21680C, CGS 22989- and vehicle-treated spontaneously hypertensive rats, respectively) without affecting the Kd value. In addition to a reduction in A2 receptor number, increased heart rates were seen on day 1 and 2 in both the CGS 21680C- and CGS 22989-treated animals and a mild stimulation of the renin angiotensin system occurred with CGS 21680C. In separate acute experiments using identical infusion rates, plasma concentrations of CGS 21680C were 157 +/- 41 ng/ml compared to 30.4 +/- 8.8 ng/ml after chronic administration.(ABSTRACT TRUNCATED AT 250 WORDS)

Feeding neonatal rats with IgG antibodies leads to humoral hyporesponsiveness in the adult.

Feeding monoclonal IgG2a or IgG1 anti-horseradish peroxidase (HRP) antibodies to 12-16-day-old neonatal rats caused a profound suppression of the humoral anti-HRP response in these rats as adults. The hyporesponsiveness to HRP was specific and long-lasting (up to 5 months). It was shown to be dose dependent, requiring relatively large doses of IgG (100-600 micrograms) for maximum effect. Secondary IgG (IgG1, IgG2a and IgG2b) responses were most depressed. The effect could be reproduced by i.p. injection of antibody. Hyporesponsiveness was not attributable to circulating antiidiotype antibodies directed against the monoclonal IgG, nor to the continued presence of the monoclonal anti-HRP since rats receiving antibody at or some weeks after the time of weaning and gut 'closure' responded well to subsequent HRP challenge. The effect was thus dependent on IgG administered over the identical period during which the neonatal circulation is rich in maternal IgG supplied via the milk. A direct function for maternal IgG in moulding the immune repertoire of the offspring, as well as providing passive protection, is suggested by these results.

A simple and rapid radioimmunoassay for human interleukin-6.

Studies were undertaken to develop a sensitive radioimmunoassay for human IL-6 using commercially available reagents. The assay utilized a polyclonal rabbit anti-IL-6 binding to iodinated IL-6; the reaction was carried out in solution and the immune complexes were precipitated using anti-rabbit IgG coupled to magnetic particles. Using a format where sample IL-6 was added in advance of the radiolabelled IL-6, the working range of the assay was found to fall between 0.1 and 10 ng/ml (IC50 around 1 ng/ml) for a 50 microliters sample volume, and was run overnight. However, the assay could be completed in 2 h, using a direct competitive format when less sensitivity is required (working range 1.5-25 ng/ml, IC50 12 ng/ml). The RIA correlated directly with a standard functional assay for IL-6 (proliferation of the mouse hybridoma B9.9) for both recombinant IL-6 and natural IL-6 from human monocytes. The total assay variance was less than 12% and no reactivity with interleukins-1-5 was found. Using the RIA, IL-6 produced in culture by human monocytes in response to various stimuli (LPS, IL-1, dibutyryl cAMP) was measured.

Transforming growth factor-beta enhances secretory component and major histocompatibility complex class I antigen expression on rat IEC-6 intestinal epithelial cells.

Transforming growth factor-beta (TGF-beta) has been implicated as having a role in inflammatory responses by inducing cellular infiltration and the release of inflammatory cytokines. In this study, the IEC-6 rat intestinal epithelial cell line was used as a model to assess the effect of TGF-beta 1 on the expression of various plasma membrane determinants. TGF-beta 1 induced a dose-dependent increase in the percentage of cells expressing surface secretory component (SC) and class I major histocompatibility (MHC) antigens. However, the expression of class II MHC was unaffected. In contrast, epidermal growth factor had no effect on any of the surface proteins studied. The TGF-beta 1-enhanced expression of SC was accompanied by an enhanced binding of polymeric, but not monomeric, immunoglobulin A (IgA). Preincubation of the TGF-beta 1-treated cells with an anti-human beta-galactosyltransferase (beta-GT) antiserum did not block the binding of the anti-SC antibody, indicating that the TGF-beta-induced increase in SC staining was due to SC expression and not the polymeric immunoglobulin-binding enzyme, beta-GT. These results indicate that TGF-beta 1 may be important in immune functions involving intestinal epithelial cells by enhancing the expression of surface class I MHC antigens and SC, a protein responsible for the transport of polymeric IgA into the intestinal lumen.

A comparison of lymphocyte subset distribution in rat lacrimal glands with cells from tissues of mucosal and non-mucosal origin.

The subset distribution of lymphocyte populations isolated from rat lacrimal glands (LG) was compared with those from tissues of both mucosal and non-mucosal origin. In spleen (SPL), mesenteric (MLN) and cervical lymph node (CLN) populations the percentages of cells bearing W3/13 (pan T) and OX19 (pan T) were greater than the percentages obtained for cells bearing the OX7 (Thy-1) marker. In contrast, for lacrimal (LG), salivary (SG) and mammary gland (MG) populations, cells bearing OX7 predominated over those bearing the W3/13 and OX19 markers, with the exception of day 1 post-partum MG tissue which displayed equal numbers of OX7 and OX19 cells. Except for MG, in which OX8 (T non-helper) predominated over W3/25 (T helper) populations, the proportions of these two subsets in the other tissues were generally similar. Analysis of SPL and LG cells for coexpression of OX7 with OX19 or L chain indicated that significant percentages of OX7 bearing cells also expressed T or B cell markers. However, the higher values noted for the OX7 population in LG were not attributable to increased numbers of cells coexpressing pan T or B cell markers. These findings show that lymphocyte subset distribution in LG and other glandular mucosal tissues is distinct from that of non-mucosal tissues, in that mucosal tissues contain a predominance of cells bearing the Thy-1 (OX7) phenotype.

Role of carbohydrate in binding of IgG to the Fc receptor of neonatal rat enterocytes.

Monoclonal IgG1, IgG2a, IgG2b and IgG2c were prepared from rat hybridoma cells treated with tunicamycin in order to inhibit N-linked glycosylation. The IgG produced by these cells was about 70% lower in carbohydrate content compared to IgG from equivalent untreated cells, but was similar to the corresponding normal IgG in terms of antigen binding. However, the ability of carbohydrate deficient (CHO-) IgG to bind in vitro to Fc receptor extracted from jejunum of neonatal rats was impaired in most cases and, in all but one case, the amount of CHO- IgG transported from gut lumen to blood in vivo was markedly reduced. No reduction in binding of normal IgG to extracted receptor was observed in the presence of various sugars. It is postulated that N-linked carbohydrate acts to stabilize the structure within the IgG molecule which is responsible for binding to this Fc receptor, possibly in the CH2 domain.

Antibody production in rats following ocular-topical or gastrointestinal immunization: kinetics of local and systemic antibody production.

The kinetics of the anti-DNP antibody response to DNP-pneumococcus appearing in tears and bile (IgA) and serum (IgM and IgG) was examined in rats after the application of antigen either via the ocular-topical (OT) or gastrointestinal (GI) routes. It was found that IgA responses were obtained each time in tears after either OT or GI antigen doses given monthly for three months, but that the OT route gave rise to consistently higher tear antibody titres than the GI route of immunization. Comparable IgA responses were found in bile using either route. In serum a small primary IgM response was consistently obtained but the main antibody found was IgG, the timing and degree of response being about the same for both routes. When the adjuvants Avridine in liposomes or MTP-PE were added along with the antigen it was found that with either immunization route, the tear IgA response was much reduced compared to when no adjuvant was used; the serum IgG response was marginally increased when adjuvant was added. The effects of binding anti-DNP monoclonal IgA or IgG1 to antigen before immunizing via the OT route was also studied. It was found that the presence of immunoglobulin of either isotype in the complex caused an increase in the serum IgG response, but that the tear IgA response was diminished in rats receiving IgA/antigen complexes compared with those receiving IgG/antigen or antigen alone.

Tolerogenic activity of polymerized type II collagen in preventing collagen-induced arthritis in rats.

Rats were exposed parenterally or pergastrically to polymerized type II collagen (POLCII) and became resistant to the subsequent induction of disease with arthritogenic type II collagen (CII) administered intradermally in Freund's incomplete adjuvant (FIA). POLCII was prepared by cross-linking native soluble arthritogenic CII, from bovine nasal septal cartilage, with glutaraldehyde. POLCII injected intradermally in FIA did not induce arthritis. Animals treated in this manner were resistant for a period of at least 100 days to induced disease. The change in the properties of the CII from an arthritogen to a tolerogen was related to the amount of glutaraldehyde (used to polymerize the CII) which was assumed to control the extent of cross-linking of the CII. Highly cross-linked POLCII administered pergastrically, like soluble CII, was not arthritogenic but was tolerogenic, inducing a state of unresponsiveness to a challenge with arthritogenic CII. In general serum anti-CII antibody levels were higher in arthritic than in tolerized non-arthritic rats. It is concluded that the breaking of self-tolerance to CII depends upon its physical state. When polymerized and insoluble, a form analogous to that in which it exists naturally, it is tolerogenic.

Studies on the origin and composition of IgA in rat tears.

Experiments were carried out to quantify the proportion of IgA in rat tears that arrives from the circulating IgA pool in the blood, as opposed to that supplied by local synthesis in the lacrymal gland. Rats were injected subcutaneously with an IgA-secreting rat hybridoma cell line (91c), which was allowed to grow in vivo, in order to raise the levels of plasma IgA of known antibody specificity. In rats where serum 91c IgA concentrations had built up to 0.3 mg/ml or more, unstimulated tears contained 91c antibody activity of a molecular weight range that corresponded to that of the polymeric sizes of IgA, and was associated with secretory component. The concentrations of IgA in tear samples were about 0.3% of those in matched serum samples. Thus, a plasma contribution is made to the IgA in tears, but greater than 99% of the tear IgA is synthesized locally in the lachrymal gland.