Acetylenic TACE inhibitors. Part 1. SAR of the acyclic sulfonamide hydroxamates.

The SAR of a series of potent sulfonamide hydroxamate TACE inhibitors, all bearing a butynyloxy P1' group, was explored. In particular, compound 5j has excellent in vitro potency against isolated TACE enzyme and in cells, good selectivity over MMP-1 and MMP-9, and oral activity in an in vivo model of TNF-alpha production and a collagen-induced arthritis model.

Synthesis and SAR of bicyclic heteroaryl hydroxamic acid MMP and TACE inhibitors.

Potent and selective bicyclic heteroaryl hydroxamic acid MMP and TACE inhibitors were synthesized by a novel convergent route. Selectivity and efficacy versus MMPs and TACE could be controlled by appropriate substitution on the scaffolds and by variation of the P1' group. Select compounds were found to be effective in in vivo models of arthritis.

Anthranilate sulfonamide hydroxamate TACE inhibitors. Part 2: SAR of the acetylenic P1' group.

The SAR of a series of potent sulfonamide hydroxamate TACE inhibitors bearing novel acetylenic P1' groups was explored. In particular, compound 4t bearing a butynyloxy P1' moiety has excellent in vitro potency against isolated TACE enzyme and in cells, good selectivity over MMP-1 and oral activity in an in vivo model of TNF-alpha production.

The discovery of anthranilic acid-based MMP inhibitors. Part 3: incorporation of basic amines.

Anthranilic acid derivatives bearing basic amines were prepared and evaluated in vitro and in vivo as inhibitors of MMP-1, MMP-9, MMP-13, and TACE. Piperazine 4u has been identified as a potent, selective, orally active inhibitor of MMP-9 and MMP-13.

The discovery of anthranilic acid-based MMP inhibitors. Part 2: SAR of the 5-position and P1(1) groups.

A novel series of anthranilic acid-based inhibitors of MMP-1, MMP-9, MMP-13, and TACE was prepared and evaluated. Selective inhibitors of MMP-9, MMP-13, and TACE were identified, including the potent, orally active MMP-13 inhibitor 4p.

Expression of serum- and glucocorticoid-regulated kinase (sgk) mRNA is up-regulated by GM-CSF and other proinflammatory mediators in human granulocytes.

Stimulation of human peripheral blood granulocytes with the proinflammatory cytokine, granulocyte-macrophage colony-stimulating factor (GM-CSF), increases incorporation of [3H]uridine into RNA. We investigated the nature of the RNA synthesized under these conditions. Using transcription inhibitors, gel electrophoresis, and high-salt precipitation, it was concluded that as much as 90% of this radiolabeled RNA represents polymerase II transcripts. Differential display reverse transcription-polymerase chain reaction was used to identify and clone GM-CSF-responsive mRNAs. Serum- and glucocorticoid-regulated kinase (sgk) mRNA was identified that could be up-regulated 10- to 20-fold by > or =0. 1 ng/mL recombinant human GM-CSF. The 2.6-kb sgk mRNA was induced rapidly (within 30 min) by GM-CSF and remained at high levels for at least 12 h. Up-regulation was blocked completely by the transcription inhibitor, actinomycin D, but not by the translation inhibitor, cycloheximide, nor by the tyrosine kinase inhibitor, genistein. Up-regulation did not appear to be caused by enhanced mRNA stability. Other inflammatory mediators could also increase sgk mRNA levels (GM-CSF > > lipopolysaccharide > fMLP = tumor necrosis factor alpha). The function of sgk in granulocytes remains unknown.

Characterization of a polyclonal antibody to human thymidylate synthase suitable for the study of colorectal cancer specimens.

Measurement of thymidylate synthase (hTS) using immunohistochemical techniques has been reported in several clinical studies. However, its value as a prognostic indicator is still not clear. To pursue this, we have developed a new rabbit polyclonal antibody, hTS7.4. The antigen was recombinant hTS containing an N-terminal His(6)-tag. Antiserum hTS7.4 detected recombinant hTS by ELISA at a titer of 1:100,000. Western blot analysis of several human cell lines showed a single band of the expected 36-kD molecular size. HeLa cells treated with the TS inhibitor 5-FUdR showed the expected additional band corresponding to the ternary complex of hTS-dFUMP-reduced folate. hTS7.4 detected TS in bacterial, rat, mouse, and monkey cell extracts, and hTS8.3 (a closely related antiserum) immunoprecipitated a 36-kD [(35)S]-methionine-labeled protein from HeLa extracts. TS was detectable by indirect immunofluorescence in HeLa cells. Proliferating normal human fibroblasts in culture showed staining, but nonproliferating cells did not. Lymphocytes in the germinal center of human tonsil tissue, which are known to be proliferating, stained with hTS7.4 and also with monoclonal antibody TS106. TS may therefore be useful as an immunohistochemical marker of cell proliferation. Normal colon mucosa showed weak staining, whereas some colorectal cancer specimens stained very strongly with hTS7.4. A clinical study of colorectal cancer using this antibody is in progress. (J Histochem Cytochem 47:1563-1573, 1999)

Tumor necrosis factor-alpha upregulates angiotensin II type 1 receptors on cardiac fibroblasts.

Angiotensin II (Ang II) plays an important role in post-myocardial infarction (MI) remodeling. Most Ang II effects related to remodeling involve activation of the type 1 receptor (AT(1)). Although the AT(1) receptor is upregulated on cardiac fibroblasts post-MI, little is known about the mechanisms involved in the process. Consequently, we tested whether growth factors known to be present in the remodeling heart increased AT(1) mRNA levels. Using quantitative competitive reverse transcription-polymerase chain reaction, we found that norepinephrine, endothelin, atrial natriuretic peptide, and bradykinin had no significant effect on AT(1) mRNA levels. Ang II, transforming growth factor-beta(1), and basic fibroblast growth factor reduced AT(1) mRNA levels (P<0.02). Tumor necrosis factor-alpha (TNF-alpha), however, produced a marked increase in AT(1) mRNA. After 24 hours of TNF-alpha incubation, AT(1) mRNA increased by 5-fold above control levels (P<0.01). The EC(50) for the TNF-alpha effect was 4.6 ng/mL (0.2 nmol/L). Interleukin (IL)-1beta caused a 2.4-fold increase, whereas IL-2 and IL-6 had no significant effect. Studies of TNF-alpha enhancement of AT(1) mRNA levels demonstrate that the increase was not due to a change in transcript stability. TNF-alpha treatment for 48 hours also resulted in a 3-fold increase in AT(1) surface receptor and a 2-fold increase in Ang II-induced production of inositol phosphates. The present findings provide evidence for TNF-alpha regulation of AT(1) receptor density on cardiac fibroblasts. Because TNF-alpha concentration and AT(1) receptor density increase in the myocardium after MI, these results raise the possibility that TNF-alpha modulates post-MI remodeling by enhancing Ang II effects on cardiac fibroblasts.

The interleukin-7 receptor alpha chain transmits distinct signals for proliferation and differentiation during B lymphopoiesis.

The interleukin 7 receptor (IL7R), which contains a unique alpha chain and a gamma chain shared by other cytokine receptors, is indispensable for normal lymphocyte development. The basis for this role is poorly understood. Here we show that the IL7R alpha chain not only causes progenitors to proliferate, but also has a distinct activity in inducing differentiation. First, we identify a single cytoplasmic tyrosine residue in the IL7R alpha chain that is essential for cell cycle entry and proliferation dependent on phosphatidylinositol 3-kinase. We use a mutant alpha chain in which this residue has been altered to reconstitute B lymphopoiesis by retrovirus-mediated gene transfer in cultures of bone marrow from mice deficient in IL7R alpha chain. The mutation abrogates the proliferation of B-lymphocyte progenitors, but reveals a novel function of the alpha chain in promoting immunoglobulin heavy chain gene rearrangement leading to B-cell differentiation. This function is lost (but proliferation sustained) when the cytoplasmic domain of IL7R alpha is replaced by corresponding sequences from the IL2R, despite the similarity on their signalling mechanisms. Thus, the signals which mediate a differentiative function of the IL7R in B lymphopoiesis are specific and distinct from those causing proliferation.

X-ray spectroscopy of the iron site in soybean lipoxygenase-1: changes in coordination upon oxidation or addition of methanol.

Iron K-edge X-ray spectroscopy (XANES and EXAFS) was used to study iron coordination in frozen solutions of soybean lipoxygenase-1 (SLO). The intensity of the 1s-->3d pre-edge transition of native iron(II) lipoxygenase is greater than what was found for six-coordinate high-spin iron(II) model complexes, but comparable to that of a five-coordinate model. This and a relatively short average bond length determined by EXAFS (2.13 A) indicate that the native lipoxygenase in our frozen samples is five-coordinate, excluding possible bonds longer than 2.5 A. The coordination of the iron(II) in native lipoxygenase changes when methanol (as low as 0.1%) or glycerol (20%) is added to the buffer prior to freezing. The addition of methanol diminishes the pre-edge transition and increases EXAFS-derived bond lengths by 0.04 A, indicating a change to six-coordination. The small pre-edge feature in active iron(III) lipoxygenase suggests six-coordination. EXAFS indicates a short, 1.88 A Fe-O bond, which, given other spectroscopic and crystallographic evidence, is assigned to coordinated hydroxide. The average of the remaining bond lengths is 2.11 A. The iron coordination in iron(III) lipoxygenase is less affected by the presence of alcohols than is the site in the iron(II) enzyme. Bond valence sums indicate that the bond lengths for lipoxygenase derived from our EXAFS analyses are comparable to those of crystallographically characterized model complexes. The flexibility of the coordination number in SLON (native SLO) and the presence of an [FeIIIOH]2+ unit in SLOA (active SLO) are of possible mechanistic importance.

Incorporation of [32P]orthophosphate into inorganic polyphosphates by human granulocytes and other human cell types.

When human peripheral blood granulocytes were metabolically labeled at 37 degrees C with [32P]orthophosphate, inorganic polyphosphates became preferentially radiolabeled. Incorporation of radiolabel into the polymer appeared to be ATP-independent. [32P]Polyphosphate was identified by its (i) characteristic lability to acid hydrolysis, (ii) insolubility in barium acetate (pH 4.5), (iii) conversion to [32P]trimetaphosphate, (iv) hydrolysis to [32P]orthophosphate by an exopolyphosphate (Saccharomyces cerevisiae scPPX1), and (v) conversion to a "phosphate ladder" which co-migrated on a polyacrylamide gel with a synthetic phosphate ladder. Also, indirect evidence suggested that the [32P]polyphosphate was strongly, noncovalently associated with another unknown molecule. Particulate fractions (13,000 x g) from lysates of human granulocytes, skin fibroblasts, HL-60 and SK-N-SH cells, all demonstrated radiolabeling of polyphosphate when incubated at 37 degrees C with [32P]orthophosphate.

Interleukin 7 receptor functions by recruiting the tyrosine kinase p59fyn through a segment of its cytoplasmic tail.

Engagement of the cell surface receptor for interleukin 7 (IL-7R) provokes protein tyrosine phosphorylation, although the receptor lacks a kinase catalytic domain in its cytoplasmic tail. The molecular basis of this response is not known. Here we report that the IL-7R functions by recruiting p59fyn, an intracellular tyrosine kinase of the src family. Treatment of pre-B cells with IL-7 causes an enhancement of the catalytic activity of p59fyn, but not of the related kinase p62yes. IL-7-dependent stimulation of the enzyme phosphatidylinositol 3-kinase, a tyrosine kinase substrate, provides further evidence suggestive of p59fyn activation. We demonstrate that p59fyn forms part of a protein complex with the IL-7R. A chimeric receptor comprising the CD8 extracellular domain and the IL-7R cytoplasmic tail (CD8/IL-7R) recruits tyrosine kinase activity in transfected myeloma cells, and p59fyn can be detected in association with it by immunoprecipitation and immunoblotting. Conversely, p59fyn immunoprecipitates contain the phosphorylated CD8/IL-7R. We have identified a segment of the IL-7R cytoplasmic tail which mediates p59fyn recruitment: a truncated CD8/IL-7R containing only this segment recruits tyrosine kinase activity, associates with p59fyn, and activates phosphatidylinositol 3-kinase. Interestingly, this segment contains no tyrosine residues, although it is the phosphotyrosine-binding src homology domains of p59fyn and phosphatidylinositol 3-kinase which mediate their association with many growth factor receptors. Thus our results suggest that an unusual interaction links IL-7R to these two important signaling pathways.