Design and synthesis of potent, selective, and orally bioavailable tetrasubstituted imidazole inhibitors of p38 mitogen-activated protein kinase.

Novel potent and selective diarylimidazole inhibitors of p38 MAP (mitogen-activated protein) kinase are described which have activity in both cell-based assays of tumor necrosis factor-alpha (TNF-alpha) release and an animal model of rheumatoid arthritis. The SAR leading to the development of selectivity against c-Raf and JNK2alpha1 kinases is presented, with key features being substitution of the 4-aryl ring with m-trifluoromethyl and substitution of the 5-heteroaryl ring with a 2-amino substituent. Cell-based activity was significantly enhanced by incorporation of a 4-piperidinyl moiety at the 2-position of the imidazole which also enhanced aqueous solubility. In general, oral bioavailability of this class of compounds was found to be poor unless the imidazole was methylated on nitrogen. This work led to identification of 48, a potent (p38 MAP kinase inhibition IC50 0.24 nM) and selective p38 MAP kinase inhibitor which inhibits lipopolysaccharide-stimulated release of TNF-alpha from human blood with an IC50 2.2 nM, shows good oral bioavailability in rat and rhesus monkey, and demonstrates significant improvement in measures of disease progression in a rat adjuvant-induced arthritis model.

The structures of the neurotrophin 4 homodimer and the brain-derived neurotrophic factor/neurotrophin 4 heterodimer reveal a common Trk-binding site.

The neurotrophins are growth factors that are involved in the development and survival of neurons. Neurotrophin release by a target tissue results in neuron growth along the neurotrophin concentration gradient, culminating in the eventual innervation of the target tissue. These activities are mediated through trk cell surface receptors. We have determined the structures of the heterodimer formed between brain-derived neurotrophic factor (BDNF) and neurotrophin 4 (NT4), as well as the structure of homodimer of NT4. We also present the structure of the Neurotrophin 3 homodimer, which is refined to higher resolution than previously published. These structures provide the first views of the architecture of the NT4 protomer. Comparison of the surface of a model of the BDNF homodimer with the structures of the neurotrophin homodimers reveals common features that may be important in the binding between the neurotrophins and their receptors. In particular, there exists an analogous region on the surface of each neurotrophin that is likely to be involved in trk receptor binding. Variations in sequence on the periphery of this common region serve to confer trk receptor specificity.

Pyrroles and other heterocycles as inhibitors of p38 kinase.

Investigation of furans, pyrroles and pyrazolones identified 3-pyridyl-2,5-diaryl-pyrroles as potent, orally bioavailable inhibitors of p38 kinase. 3-(4-pyridyl-2-(4-fluoro-phenyl)-5-(4-methylsulfinylphenyl)-pyrrol e (L-167307) reduces secondary paw swelling in the rat adjuvant arthritis model: ID50 = 7.4 mg/kg/b.i.d.

The interleukin-1 beta-converting enzyme (ICE) is localized on the external cell surface membranes and in the cytoplasmic ground substance of human monocytes by immuno-electron microscopy.

Interleukin-1 beta (IL-1 beta)-converting enzyme (ICE) is a novel cysteine protease that cleaves the 31-kD inactive cytoplasmic IL-1 beta precursor into active extracellular 17-kD IL-1 beta. The ICE gene product is a 45-kD proenzyme that requires proteolytic processing to activate ICE. Active ICE is a heterodimer consisting of equal amounts of p20 and p10 subunits. Generation of active ICE is affected by the removal of an 11-kD NH2-terminal precursor domain (p11) and an internal 19-amino acid sequence that separates the 20- and 10-kD subunits. Immuno-electron microscopy was performed on human monocytes with immunoglobulins recognizing the active (p20) or precursor (p11) domains of ICE. Elutriated monocytes were stimulated with 50 pM lipopolysaccharide followed by heat-killed Staphylococcus aureus under conditions that induce maximal rates of IL-1 beta secretion. Ultrathin cryosections were cut from fixed frozen pellets of these monocytes and were immunogold labeled with either antibody. Active and precursor domain ICE epitopes were localized in the cytoplasmic ground substance, but they were not detected within the endoplasmic reticulum, the Golgi apparatus, and secretory granules of activated or inactive monocytes. Importantly, numerous ICE p20 epitopes were also observed on the extracellular surfaces of the cell membrane, and were concentrated on the microvilli. Very similar patterns of ICE localization were obtained with unstimulated blood monocytes. In contrast, ICE p11 epitopes were not detected on the surfaces of these monocytes. Likewise, labeling of fixed ultrathin cryosections of monocytes with a biotinylated irreversible ICE inhibitor [Ac-Tyr-Val-Lys(biotin)-Asp-(acyloxy)-methyl-ketone] showed that the compound localized on the outer cell surface as well, and to a lesser extent, within the cytoplasmic ground substance. Furthermore, antipeptide antibodies specific for either the mature or precursor domains of IL-1 beta were both localized upon the cell membrane after stimulation of IL-1 beta secretion. Lipopolysaccaride-primed monocytes that synthesized, but did not secrete IL-1 beta, exhibited only cytoplasmic staining. The data suggests that mature IL-1 beta is generated via cleavage of the 31-kD inactive cytoplasmic IL-1 beta precursor by ICE after association with the plasma membrane during secretion.

IL-1 beta-converting enzyme is present in monocytic cells as an inactive 45-kDa precursor.

The major form of IL-1 beta-converting enzyme (ICE) identified in THP.1 monocytic cells and human monocytes is the 45-kDa precursor protein (p45), which is found in the cytoplasm. Cytoplasmic extracts of these cells show no pIL-1 beta cleavage activity, indicating that the p45 has no detectable catalytic activity. pIL-1 beta cleavage activity can only be observed after incubation in vitro when p45 breaks down to the active p20 form of the enzyme. LPS stimulation of human monocytes or THP.1 monocytic cells results in no change in the amount of p45 or its activity and no detectable appearance of p20 ICE. Immunoprecipitation of [35S]Met-labeled LPS-stimulated monocyte extracts revealed only p45 with no other co-precipitating protein. The inability to identify active ICE in stimulated monocytic cells was probably a reflection of the very low levels of active ICE present.

Dissociation of IL-1 beta synthesis and secretion in human blood monocytes stimulated with bacterial cell wall products.

Human blood monocytes synthesize but do not secrete IL-1 beta in response to low doses of bacterial cell-wall products. With this observation, we have developed a two-step staged release assay that separates IL-1 beta synthesis from secretion. This assay can be used to identify secretagogues or inhibitors of IL-1 beta secretion as well as the biochemical events leading to IL-1 beta release. Human blood monocytes are first treated with low (< 50 pg/ml) doses of LPS, which causes the synthesis of intracellular proIL-1 beta. Release of intracellular IL-1 beta can be induced by further treatment with 100 ng/ml of LPS or 1 x 10(6) CFU/ml of heat-killed Staphylococcus aureus. The amount and the efficiency of IL-1 beta secretion in the staged release assay was comparable with that of a standard method of treating blood monocytes with a single dose of 100 ng/ml LPS. Ongoing protein synthesis was not required for IL-1 beta secretion because mature IL-1 beta release occurred in the presence of the protein synthesis inhibitor cycloheximide. We have compared the effects of four different inhibitors of cytokine synthesis on IL-1 beta production in the standard and staged release assays. We find that dexamethasone or IL-10, when added together with 100 ng/ml LPS, inhibits IL-1 beta production with IC50 levels of 0.2 microM and 2.0 ng/ml, respectively. The IC50 levels increase greater than 50-fold when tested against monocytes pretreated with 50 pg/ml of LPS. These data suggest that dexamethasone and IL-10 have little effect on IL-1 beta secretion. Conversely, the IL-1 beta converting enzyme inhibitor, Ac-Tyr-Val-Ala-Asp-CHO (L-709,049) and the anti-inflammatory agent [5-(4-pyridyl)6(4-fluorophenyl)-2,3-dihydroimidazo(2,1-b)thiazole] (SK&F 86002) inhibited IL-1 beta release in both the standard and staged release assays with IC50 of 1 microM. The data suggest that L-709,049 and SK&F 86002 interfere with steps involved in IL-1 beta secretion, as opposed to synthesis. The results also document a novel method for delineating separate events in the pathway required for IL-1 beta biosynthesis and further distinguish two classes of compounds capable of modulating IL-1 beta secretion.

A novel heterodimeric cysteine protease is required for interleukin-1 beta processing in monocytes.

Interleukin-1 beta (IL-1 beta)-converting enzyme cleaves the IL-1 beta precursor to mature IL-1 beta, an important mediator of inflammation. The identification of the enzyme as a unique cysteine protease and the design of potent peptide aldehyde inhibitors are described. Purification and cloning of the complementary DNA indicates that IL-1 beta-converting enzyme is composed of two nonidentical subunits that are derived from a single proenzyme, possibly by autoproteolysis. Selective inhibition of the enzyme in human blood monocytes blocks production of mature IL-1 beta, indicating that it is a potential therapeutic target.

IL-1-converting enzyme requires aspartic acid residues for processing of the IL-1 beta precursor at two distinct sites and does not cleave 31-kDa IL-1 alpha.

IL-1 converting enzyme (ICE) specifically cleaves the human IL-1 beta precursor at two sequence-related sites: Asp27-Gly28 (site 1) and Asp116-Ala117 (site 2). Cleavage at Asp116-Ala117 results in the generation of mature, biologically active IL-1 beta. ICE is unusual in that preferred cleavage at Asp-X bonds (where X is a small hydrophobic residue), has not been described for any other eukaryotic protease. To further examine the substrate specificity of ICE, proteins that contain Asp-X linkages including transferrin, actin, complement factor 9, the murine IL-1 beta precursor, and human and murine IL-1 alpha precursors, were assayed for cleavage by 500-fold purified ICE. The human and murine IL-1 beta precursors were the only proteins cleaved by ICE, demonstrating that ICE is an IL-1 beta convertase. Analysis of human IL-1 beta precursor mutants containing amino acid substitutions or deletions within each processing site demonstrated that omission or replacement of Asp at site 1 or site 2 prevented cleavage by ICE. To quantitatively assess the substrate requirements of ICE, a peptide-based cleavage assay was established using a 14-mer spanning site 2. Cleavage between Asp [P1] and Ala [P1']2 was demonstrated. Replacement of Asp with Ala, Glu, or Asn resulted in a greater than 100-fold reduction in cleavage activity. The rank order in position P1' was Gly greater than Ala much greater than Leu greater than Lys greater than Glu. Substitutions at P2'-P4' and P6' had relatively little effect on cleavage activity. These results show that ICE is a highly specific IL-1 beta convertase with absolute requirements for Asp in P1 and a small hydrophobic amino acid in P1'.

Probing the role of interleukin-1 beta convertase in interleukin-1 beta secretion.

Interleukin-1 beta must be processed from its precursor form of 31.5 kDa to its mature form of 17 kDa in order to elaborate its wide array of bioactivities. The recent identification of a monocyte-specific endoprotease, termed interleukin-1 beta-converting enzyme (ICE), capable of generating authentic, bioactive 17 kDa IL-1 beta suggests that this protease may serve a specific role in the processing and subsequent secretion of IL-1 beta. To test this hypothesis, we describe initial attempts to establish a monocytic cell-based system to test if mutant preIL-1 beta molecules which are poor substrates for ICE in vitro will be processed and secreted by monocytic cells.

Interaction of adenovirus VA RNAl with the protein kinase DAI: nonequivalence of binding and function.

Adenovirus VA RNAL maintains protein synthesis by preventing activation of the double-stranded RNA (dsRNA)-dependent protein kinase DAI. There appears to be a single binding site for dsRNA on DAI, and this site is blocked by VA RNAl. VA RNAl binds to purified DAI and can be cross-linked to the enzyme by UV irradiation. To determine the relationship between DAI binding and VA RNAl structure and function, we examined the binding abilities of wild-type and mutant VA RNAs. In several cases, the ability to bind DAI efficiently in vitro did not correlate with function in vivo. Secondary structure analysis suggested that efficient binding requires an apical stem-loop structure, whereas inhibition of DAI activation requires the central domain of the VA RNA molecule. We propose that the duplex stem permits VA RNA to interact with the dsRNA binding site on DAI and inhibits activation by juxtaposing the central domain of the RNA with the enzyme's active site.

Identification of a 90-kDa polypeptide which associates with adenovirus VA RNAI and is phosphorylated by the double-stranded RNA-dependent protein kinase.

Interferon treatment of mammalian cells induces a double-stranded (ds) RNA-dependent protein kinase known as DAI. When activated, DAI phosphorylates the alpha-subunit of eukaryotic initiation factor eIF-2, impairing its ability to be recycled and leading to the inhibition of protein synthesis. We have identified a novel DAI substrate in the ribosomal salt wash of rabbit reticulocyte lysates. This substrate is a 90-kDa polypeptide which has been purified to apparent homogeneity. It can be cross-linked by ultraviolet irradiation to adenovirus VA RNAI, a small RNA polymerase III transcript RNA which acts as an inhibitor of DAI. As assayed by a nitrocellulose filter binding assay, the 90-kDa polypeptide is also able to associate with authentic double-stranded RNA, but not single-stranded RNA, made in vitro. Thus, this newly identified substrate of DAI appears to have affinity for dsRNA structures and may be involved in dsRNA-regulated processes in the reticulocyte. Polyclonal and monoclonal antibodies directed against the 90-kDa polypeptide co-precipitate DAI, suggesting that these two proteins may exist as a complex.

Identification of a monocyte specific pre-interleukin 1 beta convertase activity.

Interleukin 1 (IL-1) is a lymphokine secreted by monocytes in response to a variety of inflammatory stimuli. IL-1 beta, the predominant form of IL-1 produced by human monocytes, is synthesized as an inactive precursor of 31 kDa and is cleaved at Asp116-Ala117 to yield a 17.5-kDa extracellular form. The exact cellular site of cleavage and mechanism of secretion is at present unknown. We have prepared cell-free postnuclear extracts from freshly isolated human monocytes as well as THP.1 cells, a human monocyte-like cell line, and various blood lymphocytes and fibroblast cell lines. Using pre-IL-1 beta synthesized by in vitro transcription and translation, we have shown that only extracts derived from human monocytes and THP.1 cells were capable of cleaving precursor IL-1 beta to authentic mature IL-1 beta. Subcellular fractionation of the extracts suggested that the processing activity is found in the cytosol of monocytes or monocyte-like cell lines. The cleavage product of this protease is identical to authentic IL-1 beta as shown by mobility on SDS/PAGE and amino acid sequence analysis of the [3H]leucine-labeled product. The cleavage product is also capable of binding to the IL-1 receptor found on fibroblast membranes. Finally, mutation of Asp116----Ala116 rendered the IL-1 beta precursor resistant to cleavage by the processing activity. We conclude that a protease activity found only in monocytes will specifically process IL-1 beta to an active form.

Purification and activation of the double-stranded RNA-dependent eIF-2 kinase DAI.

The double-stranded RNA (dsRNA)-dependent protein kinase DAI (also termed dsI and P1) possesses two kinase activities; one is an autophosphorylation activity, and the other phosphorylates initiation factor eIF-2. We purified the enzyme, in a latent form, to near homogeneity from interferon-treated human 293 cells. The purified enzyme consisted of a single polypeptide subunit of approximately 70,000 daltons, retained its dependence on dsRNA for activation, and was sensitive to inhibition by adenovirus VA RNAI. Autophosphorylation required a suitable concentration of dsRNA and was second order with respect to DAI concentration, which suggests an intermolecular mechanism in which one DAI molecule phosphorylates a neighboring molecule. Once autophosphorylated, the enzyme could phosphorylate eIF-2 but seemed unable to phosphorylate other DAI molecules, which implies a change in substrate specificity upon activation. VA RNAI blocked autophosphorylation and activation but permitted the activated enzyme to phosphorylate eIF-2. VA RNAI also blocked the binding of dsRNA to the enzyme. The data are consistent with a model in which activation requires the interaction of two molecules of DAI with dsRNA, followed by intermolecular autophosphorylation of the latent enzyme. VA RNAI would block activation by preventing the interaction between DAI and dsRNA.

The cell-cycle regulated proliferating cell nuclear antigen is required for SV40 DNA replication in vitro.

Cell-free extracts prepared from human 293 cells, supplemented with purified SV40 large-T antigen, support replication of plasmids containing the SV40 origin of DNA replication. A cellular protein (Mr approximately 36,000) that is required for efficient SV40 DNA synthesis in vitro has been purified from these extracts. This protein is recognized by human autoantibodies and is identified as the cell-cycle regulated protein known as proliferating cell nuclear antigen (PCNA) or cyclin.

Functional identity of proliferating cell nuclear antigen and a DNA polymerase-delta auxiliary protein.

The mechanism of replication of the simian virus 40 (SV40) genome closely resembles that of cellular chromosomes, thereby providing an excellent model system for examining the enzymatic requirements for DNA replication. Only one viral gene product, the large tumour antigen (large-T antigen), is required for viral replication, so the majority of replication enzymes must be cellular. Indeed, a number of enzymatic activities associated with replication and the S phase of the cell cycle are induced upon SV40 infection. Cell-free extracts derived from human cells, when supplemented with immunopurified SV40 large-T antigen support efficient replication of plasmids that contain the SV40 origin of DNA replication. Using this system, a cellular protein of relative molecular mass 36,000 (Mr = 36K) that is required for the elongation stage of SV40 DNA replication in vitro has been purified and identified as a known cell-cycle regulated protein, alternatively called the proliferating cell nuclear antigen (PCNA) or cyclin. It was noticed that, in its physical characteristics, PCNA closely resembles a protein that regulates the activity of calf thymus DNA polymerase-delta. Here we show that PCNA and the polymerase-delta auxiliary protein have similar electrophoretic behaviour and are both recognized by anti-PCNA human autoantibodies. More importantly, both proteins are functionally equivalent; they stimulate SV40 DNA replication in vitro and increase the processivity of calf thymus DNA polymerase-delta. These results implicate a novel animal cell DNA polymerase, DNA polymerase-delta, in the elongation stage of replicative DNA synthesis in vitro.

Treatment of Chinese hamster ovary cells with the transcriptional inhibitor actinomycin D inhibits binding of messenger RNA to ribosomes.

Inhibitors of RNA synthesis such as actinomycin D, MPB, and cordycepin progressively inhibit the initiation of protein synthesis in intact, nucleated mammalian cells. This inhibition is not dependent on the levels of mRNA, ribosomes, or tRNA. Lysates prepared from CHO cells treated with actinomycin D do not incorporate labeled globin mRNA or ovalbumin mRNA into 80S initiation complexes at the rates of untreated control extract. The ability of the extracts to produce and accumulate 48S preinitiation complexes was assessed using the 60S subunit joining inhibitors edeine and 5'-guanylyl imidodiphosphate. Control extracts were able to accumulate both the 48S preinitiation complexes and the migration-related intermediates in the presence of both inhibitors. However, lysates derived from CHO cells treated with actinomycin D were unable to produce these complexes. This was also true at low temperature, a condition that does not inhibit mRNA binding but prevents migration of the 43S complex along the mRNA. Mixing experiments with extracts from untreated control or AMD-treated CHO cells provided no evidence for a translational inhibitor. Thus, our data are consistent with the hypothesis that treatment of whole cells with actinomycin D inhibits protein synthesis initiation at the level of mRNA binding and not at migration or 60S subunit joining.

Inhibition of protein synthesis in CHO cells by actinomycin D: lesion occurs after 40S initiation complex formation.

Inhibitors of RNA synthesis such as actinomycin D, 2-mercapto-1-(beta-4-pyridylethyl)benzimidazole, and cordycepin progressively inhibit the initiation of protein synthesis in intact nucleated mammalian cells independent of their effect on mRNA synthesis. The mechanism of this effect is unknown. The activity of cell-free lysates is not directly affected by these inhibitors, suggesting that their effect is indirect and requires an intact cell. However, lysates prepared from L-cells or CHO cells treated with the inhibitors do exhibit a decrease in initiation activity corresponding in magnitude to the effect seen in intact cells. Mixing experiments with lysates isolated from untreated or treated cells provide no evidence for a translational inhibitor. However, experiments analyzing the incorporation of [35S]methionine and [35S]Met-tRNAf into initiation complexes showed that while the level of labeled 40S initiation complex in lysates from treated cells was the same or higher than in control lysates, the rate or efficiency of formation of the 80S initiation complex was inhibited. These results imply that the transcriptional inhibitors do not affect the level or charging of the initiation tRNAMet, the activity of the eIF-2 initiation factor needed for ternary complex formation, and the availability of active 40S ribosomal subunits. Thus, this site of action is different from that observed in other translational control systems such as the hemin response in reticulocytes and the interferon-induced translation inhibition in virally infected cells. This effect may reflect the cell's coordination of nuclear transcription and cytoplasmic translation.