Characterization of peptidyl boronic acid inhibitors of mammalian 20 S and 26 S proteasomes and their inhibition of proteasomes in cultured cells.

Proteasomes are large multisubunit proteinases which have several distinct catalytic sites. In this study a series of di- and tri-peptidyl boronic acids have been tested on the chymotrypsin-like activity of purified mammalian 20 S and 26 S proteasomes assayed with succinyl-Leu-Leu-Val-Tyr-amidomethylcoumarin (suc-Leu-Leu-Val-Tyr-AMC) as substrate. The inhibition of 20 S proteasomes is competitive but only slowly reversible. The K(i) values for the best inhibitors were in the range 10-100 nM with suc-Leu-Leu-Val-Tyr-AMC as substrate, but the compounds tested were much less effective on other proteasome activities measured with other substrates. Free boronic acid inhibitors exhibited equivalent potency to their pinacol esters. Both benzoyl (Bz)-Phe-boroLeu and benzyloxycarbonyl (Cbz)-Leu-Leu-boroLeu pinacol ester inhibited 20 S and 26 S proteasomes with non-ideal behaviour, differences in inhibition of the two forms of proteasomes becoming apparent at high inhibitor concentrations (above 3xK(i)). Both of these compounds were also potent inhibitors of 20 S and 26 S proteasomes in cultured cells. However, gel filtration of cell extracts prepared from cells treated with radiolabelled phenacetyl-Leu-Leu-boroLeu showed that only 20 S proteasomes were strongly labelled, demonstrating differences in the characteristics of inhibition of 20 S and 26 S proteasomes. The usefulness of peptidyl boronic acid inhibitors for investigations of proteasome-mediated protein degradation was confirmed by the observation that Bz-Phe-boroLeu and Cbz-Leu-Leu-boroLeu pinacol ester inhibited NFkappaB activation with IC(50) values comparable to their K(i) values for purified proteasomes. The latter result supports the view that the chymotrypsin-like activity of proteasomes assayed with suc-Leu-Leu-Val-Tyr-AMC is a critical one for protein degradation in cells.

Creation of a ribonuclease abzyme through site-directed mutagenesis.

The development of abzymes (antibody/enzymes) is one method of creating reagents with novel catalytic activity. To date, most abzymes have been obtained by immunization with transition state analogs. We have chosen to start with an existing antibody and convert it into an enzyme by the addition of catalytic residues to the binding site. We have introduced a histidine residue into antibody Jel 103 and converted it into an abzyme that cleaves poly(rI) with a kinetic efficiency of about 100 M(-1) sec(-1).

The natural product hymenialdisine inhibits interleukin-8 production in U937 cells by inhibition of nuclear factor-kappaB.

The nuclear factor-kappaB (NF-kappaB) family of transcription factors have been implicated in the inducible expression of genes involved in inflammatory and immune responses. As such, a specific inhibitor of NF-kappaB would be a useful therapeutic agent in a variety of inflammatory disorders. The marine natural product hymenialdisine was evaluated as an inhibitor of NF-kappaB in U937 cells. U937 cells were transfected with either a luciferase reporter plasmid containing the human immunodeficiency virus long terminal repeat or the interleukin-8 (IL-8) core promoter, both of which are activated by NF-kappaB. Hymenialdisine caused a concentration-dependent decrease in luciferase production from both reporters when the cells were stimulated with tumor necrosis factor-alpha, lipopolysaccharide or phorbol myristate acetate. An electrophoretic mobility shift assay confirmed its activity by inhibiting DNA binding of NF-kappaB. Hymenialdisine was shown to be a selective inhibitor of NF-kappaB in that it had no effect on the binding of other transcription factors to their DNA concensus motifs; these included activator protein-1, CCAAT/enhancer binding protein and Sp1. Functional studies showed hymenialdisine to be an inhibitor of IL-8 production and IL-8 mRNA formation in the U937 cell. Investigation into the mechanism of action of hymenialdisine showed that it was not due to inhibition of protein kinase C because the selective protein kinase C inhibitor RO 32-0432 was inactive against tumor necrosis factor-alpha-stimulated luciferase and IL-8 production. The compound also had no effect on IkappaB alpha or IkappaB beta phosphorylation and degradation. Thus, hymenialdisine is a potent inhibitor of NF-kappaB and IL-8 production in U937 cells.

1-substituted 4-aryl-5-pyridinylimidazoles: a new class of cytokine suppressive drugs with low 5-lipoxygenase and cyclooxygenase inhibitory potency.

A series of 1-alkyl- or -aryl-4-aryl-5-pyridinylimidazoles (A) were prepared and tested for their ability to bind to a recently discovered protein kinase termed CSBP and to inhibit lipopolysaccharide (LPS)-stimulated TNF production in mice. The kinase, CSBP, appears to be involved in a signaling cascade initiated by a number of inflammatory stimuli and leading to the biosynthesis of the inflammatory cytokines IL-1 and TNF. Two related imidazole classes (B and C) had previously been reported to bind to CSBP and to inhibit LPS-stimulated human monocyte IL-1 and TNF production. The members of the earlier series exhibited varying degrees of potency as inhibitors of the enzymes of arachidonic acid metabolism, PGHS-1 and 5-LO. Several of the more potent CSBP ligands and TNF biosynthesis inhibitors among the present series of N-1-alkylated imidazoles (A) were tested as inhibitors of PGHS-1 and 5-LO and were found to be weak to inactive as inhibitors of these enzymes. One of the compounds, 9 (SB 210313) which lacked measureable activity as an inhibitor of the enzymes of arachidonate metabolism, and had good potency in the binding and in vivo TNF inhibition assays, was tested for antiarthritic activity in the AA rat model of arthritis. Compound 9 significantly reduced edema and increased bone mineral density in this model.

Dissociation kinetics of actinomycin D from individual GpC sites in DNA.

We have examined the kinetics of dissociation of actinomycin from GpC sites in several DNA fragments containing synthetic DNA inserts, by a variation of the footprinting technique. Complexes of the ligand with radiolabelled DNA fragments were dissociated by adding a large excess of unlabelled calf thymus DNA. Samples were removed from this mixture at subsequent time intervals and subjected to DNase I footprinting. The rate of disappearance of the footprints varied considerably between the GpC sites located in different sequence environments. Actinomycin dissociates more slowly from GpC sites flanked by (AT)n than An.Tn. Within regions of alternating AT, TGCA represents a better binding site than AGCT, and CGCA is a better binding site than GGCA. GpC sites flanked by (AC)n.(GT)n present good binding sites; in this context, dissociation from CGCG is faster than from TGCA.

Visualising the dissociation of sequence selective ligands from individual binding sites on DNA.

We have used a modification of the footprinting technique to measure the dissociation of mithramycin, echinomycin and nogalamycin from their binding sites in a natural DNA fragment. Complexes with radiolabelled DNA were dissociated by addition of unlabelled DNA. Samples were removed at various times and subjected to DNase I digestion, and the rate of dissociation from each site was estimated from the time-dependent disappearance of the footprints. For echinomycin the slowest rate of dissociation is from ACGT, while the slowest site for mithramycin contains four contiguous guanines. The dissociation of nogalamycin is extremely slow, even from its weaker sites; the slowest rate was from ACGTA, which took longer than 4 h, even at 37 degrees C.

Dissociation kinetics of echinomycin from CpG binding sites in different sequence environments.

We have examined the kinetics of dissociation of echinomycin from CpG sites in several DNA fragments containing synthetic DNA inserts by a variation of the footprinting technique. Complexes of the ligand with radiolabeled DNA fragments were dissociated by adding an excess of unlabeled calf thymus DNA. Samples were removed from this mixture at subsequent time intervals and subjected to DNase I footprinting. The rate of disappearance of the footprints varied considerably between the various CpG sites. At 20 degrees C, echinomycin dissociates more slowly from CpG sites flanked by (AT)n (t1/2 approximately 40 min) and (CA)n.(TG)n (t1/2 approximately 11 min) than by An.Tn (t1/2 < 3 min). In each sequence context the dissociation from ACGT is slower than that from TCGA. (TAA)4CG(TTA)4 also represents a very good binding site (t1/2 approximately 35 min), which is less sensitive to changes in temperature than most other sites. Within sequences (AT)10(G/C)4(AT)10, the dissociation from CGGC is slower than that from CCCG or CCGC.

Pharmacological characterization of SB 202235, a potent and selective 5-lipoxygenase inhibitor: effects in models of allergic asthma.

The peptidoleukotrienes and leukotriene B4, formed from arachidonic acid through the action of 5-lipoxygenase (5-LO), exert a spectrum of biological effects. It has been proposed that potent and selective 5-LO inhibitors will be effective therapy in diseases in which the peptidoleukotrienes and leukotriene B4 have been implicated, such as asthma and arthritis. The novel compound (S)-N-hydroxy-N-(2,3-dihydro-6-phenylmethoxy-3-benzyofuranyl )urea (SB 202235) was evaluated as a selective inhibitor of 5-LO in a cell-free system as well as in various cellular assays. In addition, the potential therapeutic value of SB 202235 was assessed in preclinical models of allergic asthma. The activity of the 5-LO enzyme isolated from rat basophilic leukemia-1 cells was inhibited by SB 202235 in a concentration-dependent manner with an IC50 value of 1.9 microM. Consistent with its ability to inhibit 5-LO, SB 202235 inhibited the production of leukotriene B4 by human monocytes and in human whole blood (IC50 values of 1.5 microM and 1.1 microM, respectively). The selectivity of SB 202235 was confirmed by its lack of effect against several other enzymes and receptors. SB 202235 potently and effectively inhibited the contraction produced by a single concentration of ovalbumin in guinea pig trachea (IC50 = 20 microM) and of anti-IgE in human bronchus (IC50 = 2 microM). SB 202235 (3-30 microM) also inhibited the contraction of guinea pig trachea in response to increasing concentration of ovalbumin. When administered orally (30 mg/kg) to conscious guinea pigs, SB 202235 attenuated antigen-induced broncho-constriction and the subsequent eosinophil influx.(ABSTRACT TRUNCATED AT 250 WORDS)

Dissociation of the AT-specific bifunctional intercalator [N-MeCys3,N-MeCys7]TANDEM from TpA sites in DNA.

We have examined the dissociation of [N-MeCys3,N-MeCys7]TANDEM, an AT-selective bifunctional intercalator, from TpA sites in mixed-sequence DNAs by a modification of the footprinting technique. Dissociation of complexes between the ligand and radiolabelled DNA fragments was initiated by adding a vast excess of unlabelled calf thymus DNA. Portions of this mixture were subjected to DNAse I footprinting at various times after adding the competitor DNA. Dissociation of the ligand from each site was seen by the time-dependent disappearance of the footprinting pattern. Within a natural DNA fragment (tyrT) the ligand dissociates from TTAT faster than from ATAT. We found that the stability of complexes with isolated TpA steps decreases in the order ATAT > TTAA > TATA. Dissociation from each of these sites is much faster than from longer regions of (AT)n. These results confirm the requirement for A and T base-pairs surrounding the TpA step and suggest that the interaction is strongest with regions of alternating AT, possibly as a result of its unusual structure. The ligand dissociates more slowly from the centre of (AT)n tracts than from the edges, suggesting that variations in dissociation rate arise from sequence-dependent variations in local DNA structure.

Complex effects of phenylarsine oxide in T cells. Induction of tyrosine phosphorylation and calcium mobilization independent of CD45 expression.

The effects of phenylarsine oxide, a phosphotyrosine phosphatase inhibitor, on early signal transduction events in human T cells were studied. Simultaneous stimulation of T cells with anti-CD3 monoclonal antibody and phenylarsine oxide prevented increased tyrosine phosphorylation of phospholipase C gamma 1. In contrast, treatment of resting T cells with phenylarsine oxide alone resulted in increased tyrosine phosphorylation of a number of other intracellular substrates. Further, phenylarsine oxide was able to cause an immediate disruption of signal transduction in T cells after anti-CD3 stimulation, as measured by a return of intracellular calcium concentration and inositol 1,4,5-trisphosphate production to base-line levels. Surprisingly, in view of the inhibitory effects of phenylarsine oxide on T cell receptor signal transduction, treatment of T cells with phenylarsine oxide alone caused a dose-dependent increase in intracellular-free calcium concentration that was not accompanied with detectable increases in inositol 1,4,5-trisphosphate production. The phenylarsine oxide-induced increase in free calcium had distinct kinetics from antigen receptor-activated calcium mobilization and was derived from both intracellular sources and increased plasma membrane calcium permeability. This effect was independent of the CD45 transmembrane tyrosine phosphatase. Phenylarsine oxide thus has complex effects on signal transduction in T cells that suggests multiple intracellular targets, and these should be considered in the interpretation of experiments using this agent to study cellular kinase and phosphatase interactions. Finally, the effects of phenylarsine oxide on cellular calcium homeostasis may provide a mechanism of action for the therapeutic and/or toxic effects of arsenicals used for various forms of chemotherapy.

Visualising the kinetics of dissociation of actinomycin from individual sites in mixed sequence DNA by DNase I footprinting.

We have investigated the kinetics of dissociation of actinomycin D from DNA by a variation of the footprinting technique. Complexes of actinomycin with a radiolabelled DNA fragment (tyrT) were dissociated by addition of a large excess of unlabelled calf thymus DNA and the mixture subjected to DNase I footprinting at subsequent intervals. The rates at which the footprints disappeared varied between the different binding sites. The dissociation was temperature dependent with average time constants of 30 s, 10 mins and 2 hours at temperatures of 37 degrees C, 20 degrees C and 4 degrees C respectively. The dissociation from a DNA fragment containing the synthetic insert T9GCA9 was significantly faster, with a half-life of about 1 min at 20 degrees C. In contrast, the dissociation of distamycin was too fast to measure (< 5 s) even at 4 degrees C.

Defective signal transduction by the CD2 molecule in immature T-cell receptor/CD3- thymocytes.

The CD2 accessory molecule mediates an activation pathway in mature T cells, transducing signals similar to those observed following stimulation of the T-cell receptor/CD3 (TCR/CD3) complex. CD2 is also one of the earliest cell surface markers to appear during thymic ontogeny and has been proposed to be a stimulatory pathway for immature thymocytes that have not yet expressed TCRs on their surface (TCR/CD3-). To examine this hypothesis highly purified TCR/CD3- human thymocytes were stimulated using mitogenic combinations of anti-CD2 monoclonal antibodies or individual biotinylated anti-CD2 monoclonal antibodies crosslinked with avidin. TCR/CD3+ thymocytes responded readily to either stimulus as determined by anti-phosphotyrosine immunoblotting, and the pattern of tyrosine phosphorylated substrates was similar to that of mature T cells. In contrast, TCR/CD3- thymocytes responded weakly and with a distinct substrate pattern. In addition, the altered signal transduced by CD2 in TCR/CD3- thymocytes did not lead to a rise in intracellular calcium, failed to induce interleukin 2 receptor expression, and did not serve as a comitogen with phorbol ester or interleukin 2, functions that were all intact in TCR/CD3+ thymocytes. Failure of TCR/CD3- thymocytes to respond to CD2 stimulation was not due to an intrinsic defect in these cells as they responded normally to phorbol ester plus calcium ionophore. In TCR/CD3- thymocytes, CD2 stimulation also failed to affect steady-state mRNA levels of the recombination-activating genes RAG1 and RAG2, whereas in TCR/CD3+ cells activation of the CD2 pathway terminated their expression. Together, these data support the concept that CD2 engagement does not deliver a stimulus to TCR/CD3- thymocytes and suggests that this molecule may not directly participate in the earliest stages of thymic development.

Antibody and B7/BB1-mediated ligation of the CD28 receptor induces tyrosine phosphorylation in human T cells.

CD28 is an adhesion receptor expressed as a 44-kD dimer on the surface of a major subset of human T cells. The CD28 receptor regulates the production of multiple lymphokines, including interleukin 2 (IL-2), by activation of a signal transduction pathway that is poorly understood. Here we show that ligation of CD28 by a monoclonal antibody (mAb) or by a natural ligand, B7/BB1, induces protein tyrosine phosphorylation that is distinct from T cell receptor (TCR)-induced tyrosine phosphorylation. CD28-induced protein tyrosine phosphorylation was greatly enhanced in cells that had been preactivated by ligation of the TCR, or by pretreatment with phorbol esters. Rapid and prolonged tyrosine phosphorylation of a single substrate, pp100, was induced in T cells after interaction with B7/BB1 presented on transfected Chinese hamster ovary (CHO) cells. Anti-B7 mAb inhibited B7/BB1 receptor-induced tyrosine phosphorylation, indicating that B7-CD28 interaction was required. CD28-induced tyrosine phosphorylation was independent of the TCR because it occurred in a variant of the Jurkat T cell line that does not express the TCR. Herbimycin A, a protein tyrosine kinase inhibitor, could prevent CD28-induced tyrosine phosphorylation and CD28-induced IL-2 production in normal T cells. The simultaneous crosslinking of CD28 and CD45, a tyrosine phosphatase, could prevent tyrosine phosphorylation of pp100. These results suggest that specific tyrosine phosphorylation, particularly of pp100, occurs directly as a result of CD28 ligand binding and is involved in transducing the signal delivered through CD28 by accessory cells that express the B7/BB1 receptor. Thus, this particular form of signal transduction may be relevant to lymphokine production and, potentially may provide a means to study the induction of self-tolerance, given the putative role of the costimulatory signal in the induction of T cell activation or anergy.

Inhibition of tyrosine phosphorylation prevents T-cell receptor-mediated signal transduction.

The binding of antigen to the multicomponent T-cell receptor (TCR) activates several signal transduction pathways via coupling mechanisms that are poorly understood. One event that follows antigen receptor engagement is the activation of inositol phospholipid-specific phospholipase C (PLC). TCR activation by antigen, lectins, or anti-TCR monoclonal antibody has also been shown to cause increases in tyrosine phosphorylation of TCR-zeta and other substrates, suggesting stimulation of protein tyrosine kinase (PTK) activity. A critical question is whether these two pathways, PLC and PTK, are independently activated or whether one initiates and/or regulates the other. In the former case, PLC activation could be coupled to the TCR via a GTP-binding protein (G protein). We have reported, however, that tyrosine phosphorylation of intracellular substrates precedes detection of PLC activation and intracellular calcium elevation, suggesting that inositol phospholipid turnover in T cells is initiated by a PTK pathway. In this study, we test this hypothesis by treating T cells with the drug herbimycin A. We demonstrate that this agent inhibits substrate tyrosine phosphorylation, TCR-mediated inositol phospholipid hydrolysis, and calcium elevation. In contrast, under these conditions G-protein-mediated PLC activity, as tested by addition of aluminum fluoride, remains intact. Furthermore, whereas herbimycin treatment prevents TCR-mediated interleukin 2 production and interleukin 2 receptor expression, phorbol ester-induced effects are substantially resistant to herbimycin. The drug thus appears to abrogate TCR-mediated signaling without affecting distal signaling mechanisms.

Activation of tyrosine phosphorylation in human T cells via the CD2 pathway. Regulation by the CD45 tyrosine phosphatase.

In this study we compare the effect of CD3 and CD2 ligation on tyrosine kinase activation in human peripheral blood T cells. Using antiphosphotyrosine antibody to detect tyrosine phosphorylation of cellular substrates, we demonstrate that mAb stimulation of either CD3 or CD2 results in tyrosine phosphorylation of the TCR-zeta chain and 135- and 100-kDa proteins. However, differences are observed between CD3 and CD2 ligation; only the former results in rapid tyrosine phosphorylation of 72-, 65-, and 40-kDa substrates. Co-aggregation of CD2 and CD45, a tyrosine phosphatase, results in inhibition of intracellular calcium elevation and T cell proliferation. We demonstrate in this study that this manipulation also inhibits polyphosphoinositide hydrolysis and tyrosine phosphorylation of the 100-kDa substrate. The failure of tyrosine phosphorylation of the 100-kDa substrate is specific in that phosphorylation of the 135-kDa protein is not inhibited. Similar results are observed when CD2 and CD45 are independently cross-linked rather than co-aggregated. The observation that CD45 cross-linking alters tyrosine phosphorylation of T cell substrates and effects polyphosphoinositide hydrolysis is further evidence that tyrosine phosphorylation regulates early events in T cell activation including, perhaps, phospholipase C activity.

Increases in tyrosine phosphorylation are detectable before phospholipase C activation after T cell receptor stimulation.

Antiphosphotyrosine immunoblots were used to characterize tyrosine phosphorylated proteins after stimulation of the human TCR. Increased tyrosine phosphorylation was evident on at least 12 substrates within 2 min after ligation of the TCR with mAb. Analysis of the time course for increased tyrosine phosphorylation revealed distinct patterns. Increased phosphorylation of 135-kDa and 100-kDa substrates was evident within 5 s, whereas increased phosphorylation of the TCR-zeta-chain required several minutes after treatment with anti-CD3 mAb. This rapid cellular tyrosine phosphorylation occurred independent of the cell cycle, as it occurred after stimulation of resting T cells, T cell blasts, and the Jurkat T cell leukemia line. When the TCR complex was cross-linked together with the CD4 receptor by heteroconjugate anti-CD3/CD4 mAb, an increased magnitude of tyrosine phosphorylation occurred, although no new substrates could be detected. The increased tyrosine phosphorylation of the 135-kDa and 100-kDa substrates was specific in that anti-HLA class I, anti-CD6, anti-CD7, and anti-CD28 antibodies did not cause increased tyrosine phosphorylation. Anti-CD4 stimulation of resting T cells did not cause increased tyrosine phosphorylation of pp100 and pp135, suggesting that the CD4-associated kinase, lck, does not account for the tyrosine phosphorylation observed after TCR stimulation. Similarly, pharmacologic treatment of cells with phorbol ester and calcium ionophore did not cause increased tyrosine phosphorylation of these substrates, indicating that activation of protein kinase C or phospholipase C does not account for these early increases in tyrosine phosphorylation. The time of onset of pp100 phosphorylation, and the magnitude of phosphorylation correlated with the magnitude of calcium mobilization when cells were stimulated with different forms of TCR stimulation. When cells were labeled with [3H]myoinositol and analyzed after stimulation by anti-CD3 mAb, increased tyrosine phosphorylation of the 135-kDa and 100-kDa substrates preceded the activation of phospholipase C, as measured by the appearance of inositol 1,4,5-trisphosphate. This occurred in both T cell blasts and in the Jurkat T cell line. Thus, these findings show that increased tyrosine phosphorylation is the earliest yet detected signal observed after ligation of the TCR complex, and furthermore suggest that tyrosine phosphorylation might link the TCR to the phosphatidylinositolbisphosphate hydrolysis signaling pathway.