Inhibitors of NF-kappaB and AP-1 gene expression: SAR studies on the pyrimidine portion of 2-chloro-4-trifluoromethylpyrimidine-5-[N-(3', 5'-bis(trifluoromethyl)phenyl)carboxamide].

We investigated the structure-activity relationship studies of N-[3, 5-bis(trifluoromethyl)phenyl][2-chloro-4-(trifluoromethyl)pyrimidin-5 -yl]carboxamide (1), an inhibitor of transcription mediated by both NF-kappaB and AP-1 transcription factors, with the goal of improving its potential oral bioavailability. Compounds were examined for cell-based activity, were fit to Lipinski's rule of 5, and were examined for potential gastrointestinal permeability using the intestinal epithelial cell line, Caco-2. Selected groups were substituted at the 2-, 4-, and 5-positions of the pyrimidine ring using solution-phase combinatorial methodology. The introduction of a fluorine in the place of 2-chlorine of 1 resulted in a compound with comparable activity. However, other substitutions at the 2-position resulted in a loss of activity. The trifluoromethyl group at the 4-position could be replaced with a methyl, ethyl, chlorine, or phenyl without a substantial loss of activity. The carboxamide group at the 5-position is critical for activity. If it was moved to the 6-position, the activity was lost. The 2-methyl analogue of 1 (81) showed comparable in vitro activity and improved Caco-2 permeability compared to 1.

Novel inhibitors of AP-1 and NF-kappaB mediated gene expression: structure-activity relationship studies of ethyl 4-[(3-methyl-2,5-dioxo(3-pyrrolinyl))amino]-2-(trifluoromethyl)++ +pyrimidi ne-5-carboxylate.

In an effort to identify novel inhibitors of AP-1 and NF-kappaB mediated transcriptional activation, several analogues of ethyl 4-[(3-methyl-2,5-dioxo(3-pyrrolinyl))amino]-2-(trifluoromethyl)pyr imidine-5-carboxylate (1) were synthesized and tested in two in vitro assays. The 2-(2'-thienyl) substituted compound (11) was identified as the most potent in this series.

The effect of a T cell-specific NF-kappa B inhibitor on in vitro cytokine production and collagen-induced arthritis.

NF-kappa B plays a key role in the production of cytokines in inflammatory diseases. The effects of a novel T cell-specific NF-kappa B inhibitor, SP100030, were evaluated in cultured Jurkat cells and in murine collagen-induced arthritis (CIA). Chemical libraries were screened for NF-kappa B-inhibitory activity. SP100030, a compound identified in this process, inhibited NF-kappa B activation in PMA/PHA-activated Jurkat cells by EMSA at a concentration of 1 microM. Jurkat cells and the monocytic cell line THP-1 were transfected with an NF-kappa B promotor/luciferase construct and activated. SP100030 inhibited luciferase production in the Jurkat cells (IC50 = 30 nM). ELISA and RT-PCR confirmed that IL-2, IL-8, and TNF-alpha production by activated Jurkat and other T cell lines were inhibited by SP100030. However, cytokine expression was not blocked by the compound in THP-1 cells, fibroblasts, endothelial cells, or epithelial cells. Subsequently, DBA/1J mice were immunized with type II collagen. Treatment with SP100030 (10 mg/kg/day i.p. beginning on day 21) significantly decreased arthritis severity from onset of clinical signs to the end of the study on day 34 (arthritis score, 5.6 +/- 1.7 for SP100030 and 9.8 +/- 1.5 for control; p < 0.001). Histologic evaluation demonstrated a trend toward improvement in SP100030-treated animals. EMSA of arthritic mouse ankles in CIA showed that synovial NF-kappa B binding was suppressed in the SP100030-treated mice. SP100030 inhibits NF-kappa B activation in T cells, resulting in reduced NF-kappa B-regulated gene expression and decreased CIA. Its selectivity for T cells could provide potent immunosuppression with less toxicity than other NF-kappa B inhibitors.

2-Chloro-4-(trifluoromethyl)pyrimidine-5-N-(3',5'- bis(trifluoromethyl)phenyl)-carboxamide: a potent inhibitor of NF-kappa B- and AP-1-mediated gene expression identified using solution-phase combinatorial chemistry.

Described is the identification of a novel series of compounds that blocks the activation of two key transcription factors, AP-1 and NF-kappa B. These transcription factors regulate the expression of several critical proinflammatory proteins and cytokines and represent attractive targets for drug discovery. Through the use of high throughput screening and solution-phase parallel synthesis, inhibitors of both NF-kappa B and AP-1 were identified. In subsequent testing, these compounds were also shown to block both IL-2 and IL-8 levels in the same cells. One of the most potent compounds in this series, 28, was active in several animal models of inflammation and immunosuppression, thus validating the importance of AP-1 and NF-kappa B as potential therapeutic targets. The synthesis and preliminary structure-activity relationships of these compounds is addressed.

Association between proto-oncoprotein Rel and TATA-binding protein mediates transcriptional activation by NF-kappa B.

The c-Rel protein is able to associate in vitro and in vivo with the TATA-binding protein (TBP) of the TFIID complex. Coexpression of TBP with c-Rel augments transactivation from the kappa B site in Drosophila Schneider cells. DNA-binding mutants of TBP not only fail to cooperate, but they repress transactivation by c-Rel. There may be a direct communication between kappa B enhancer binding proteins and basal transcription factors which leads to enhanced transcription.

The bZIP domains of Fos and Jun mediate a physical association with the TATA box-binding protein.

Fos and Jun oncoproteins form a complex that regulates transcription from promoters containing AP-1 binding sites. These two proteins, like other transcriptional activators, are likely to stimulate transcription through direct and/or indirect interactions with members of the basal transcriptional machinery. The ability of c-Fos and c-Jun proteins to interact directly with the TATA box-binding protein (TBP), the general transcription factor required for initiating the assembly of transcription complexes, was investigated. Using co-immunoprecipitation and protein-protein association assays, we show that both c-Fos and c-Jun bind specifically and stably to TBP. Mutational analysis demonstrates that both the basic region and leucine zipper domains of c-Fos and c-Jun are necessary and sufficient for stable association with TBP. A 51-residue region from the conserved C-terminal region of TBP, previously shown to be the binding site for the viral activator protein E1A, interacts with c-Fos and c-Jun proteins. We propose that c-Fos and c-Jun proteins function as transcriptional activators, in part by recruiting TBP to form complexes to initiate RNA synthesis.

Transformation by Jun: requirement for leucine zipper, basic region and transactivation domain and enhancement by Fos.

Mutants in the leucine zipper and basic regions of mouse c-jun were tested for transformation in chicken embryo fibroblast cultures. Reduction or elimination of the ability of Jun to dimerize or to bind to DNA severely decreased transformation. A chicken v-jun gene from which the major transactivation domain was deleted also failed to transform. We conclude that an intact leucine zipper, basic region and transactivation domain are required for Jun-induced oncogenic transformation. Coexpression of chicken c-Fos increased formation of transformed foci by Jun proteins of moderate to low oncogenic potency but had no effect on highly transforming Jun. Chicken c-Fos could also transform chicken embryo fibroblasts on its own, albeit after prolonged culture and at a low efficiency.

Functional antagonism between c-Jun and MyoD proteins: a direct physical association.

The product of the proto-oncogene Jun inhibits myogenesis. Constitutive expression of Jun in myoblasts interferes with the expression and the function of MyoD protein. In transient transfection assays Jun inhibits transactivation of the MyoD promoter, the muscle creatine kinase enhancer, and a reporter gene linked to MyoD DNA-binding sites. Conversely, MyoD suppresses the transactivation by Jun of genes linked to an AP-1 site. We demonstrate that both in vivo and in vitro MyoD and Jun proteins physically interact. Mutational analysis suggests that this interaction occurs via the leucine zipper domain of Jun and the helix-loop-helix region of MyoD.

Retinoic acid is a negative regulator of AP-1-responsive genes.

We present evidence that retinoic acid can down-regulate transcriptional activation by the nuclear protooncogene c-jun. All three members of the retinoic acid receptor (RAR) subfamily (RAR alpha, RAR beta, and RAR gamma) can repress transcriptional induction of the human collagenase gene or a heterologous promoter that contains the collagenase promoter AP-1-binding site. In contrast, the retinoid X receptor fails to repress Jun/AP-1 activity, demonstrating a significant difference between the two regulatory systems through which retinoids exert their transcriptional control. Analysis of RAR alpha mutants in transfection studies reveals that the DNA-binding domain is important for the inhibition of Jun/AP-1 activity, even though the RAR does not bind the collagenase AP-1 site. Rather, gel-retardation assays reveal that bacterially expressed full-length RAR alpha inhibits binding of Jun protein to target DNA. These data suggest that the RAR alpha may form a nonproductive complex with c-Jun and provides a simple mechanisms by which retinoic acid may limit cell growth and possibly malignant progression.

c-rel activates but v-rel suppresses transcription from kappa B sites.

We show that the product of the protooncogene c-rel is a constituent of an NF-kappa B-like complex that binds to the kappa B site originally identified in the enhancer of immunoglobulin kappa light chain gene. c-rel protein synthesized in bacteria binds to the kappa B site in a sequence-specific manner. The rel-kappa B complex can be disrupted by incubation with anti-rel antibodies. The rel protein can form oligomers. The c-rel protein can activate transcription from promoters containing kappa B sites; v-rel, on the other hand, suppresses the transcription of genes linked to kappa B sites. Thus, v-rel may interfere with the normal transcriptional machinery of the cell by acting as a dominant negative mutant.

Domain swapping reveals the modular nature of Fos, Jun, and CREB proteins.

The products of the Jun and Fos proto-oncogenes form a heterodimer that binds to and activates transcription from 12-O-tetradecanoylphorbol-13-acetate-responsive promoter elements (TGACTCA) and AP-1-binding sites (TGACATCA). These two proteins belong to a family of related transcription factors which contain similar domains required for protein dimerization and DNA binding but display different protein and DNA binding specificities. The basic region, required for DNA binding, is followed by a leucine zipper structure, a domain that mediates protein-protein interactions. To assess the role of these two domains in three related proteins, Fos, Jun, and CREB, we carried out extensive domain-swapping analysis. We found that (i) dimers formed by two Jun leucine zipper-containing proteins were unable to bind DNA as efficiently as a Fos-Jun combination, regardless of the source of the basic region; (ii) the Fos leucine zipper was unable to form either homo- or heterodimers with a chimeric protein containing a Fos leucine zipper; (iii) the Fos basic region was capable of binding to an AP-1 site; (iv) replacement of the Jun amino terminus with that of CREB had little effect on dimerization, whereas replacement with the amino terminus of Fos disrupted both protein-protein and protein-DNA interactions; (v) changes in relative affinities of the Fos and Jun basic regions for the AP-1 element were dependent on the secondary contributions of amino-terminal residues; and (vi) the Fos-Jun chimeric constructs cooperated in transcriptional transactivation of the Jun promoter in NIH 3T3 cells.

Functional antagonism between oncoprotein c-Jun and the glucocorticoid receptor.

We present evidence that the glucocorticoid receptor (GR) and transcription factor Jun/AP-1 can reciprocally repress one another's transcriptional activation by a novel mechanism that is independent of DNA binding. Overexpression of c-Jun prevents the glucocorticoid-induced activation of genes carrying a functional glucocorticoid response element (GRE). Conversely, GR is able to repress AP-1-mediated transcriptional activation. Mutant analysis reveals that the ligand binding and DNA binding domains of GR and the region including the leucine zipper of c-Jun are required for repression. Gel retardation analysis demonstrates that bacterially expressed c-Jun disrupts GR-GRE complexes. These data indicate that members of two distinct classes of transcription factors can oppose one another's activity through a mechanism likely involving protein-protein interactions.

Trans-dominant negative mutants of Fos and Jun.

Jun and Fos nuclear oncoproteins form a complex that regulates transcription from promoters containing activator protein AP-1 binding sites. The leucine-zipper and basic-region domains of both Fos and Jun are necessary for formation of the heterodimer that binds to DNA. Reciprocal mutations in the basic region of Fos or Jun can influence the binding of the heterodimer to DNA, implying a symmetrical binding site. DNA-binding mutants of Jun exhibit increased affinity for Fos and are capable of suppressing wild-type Fos-Jun DNA-binding activity. In contrast, mutations in the basic domain of Fos, which prevent binding to DNA in association with Jun, do not significantly diminish the ability of the wild-type heterodimer to bind to DNA. These dominant negative mutants are functional in vivo and can be exploited to study the role of Fos and Jun in normal and transformed cells.

Cross-talk in signal transduction: TPA-inducible factor jun/AP-1 activates cAMP-responsive enhancer elements.

The product of the jun proto-oncogene has been identified as one form of the transcription factor AP-1. The p55fos protein associates with jun/AP-1 by means of a heterodimer which requires intact 'leucine zipper' domains of both proteins. The fos/jun heterodimer binds to and activates transcription from TPA-responsive promoter elements (TGACTCA), which represent one final target of the protein kinase C pathway. The other main signal transduction pathway, initiated by the activation of the adenylate cyclase, involves the transcription factor CREB. The promoter element recognized by CREB, a cyclic AMP responsive element (CRE), consist of a palyndromic sequence similar to a TRE (TGACGTCA). We show that jun efficiently trans-activates CRE sequences and that fos and jun efficiently bind and cooperate in activating CRE promoter elements. The similarity between TRE and CRE sequences may involve an interplay in transcriptional regulation and 'cross-talk' between components of the two major signal transduction pathways.

fos-jun conspiracy: implications for the cell.

Two nuclear oncoproteins, fos and jun (AP-1), cooperate in forming a very stable heterodimeric complex that binds to the AP-1 site on DNA with high affinity. The 'leucine zipper' domain of both fos and jun is necessary for the formation of this heterodimer. Mutations of single residues within the leucine zipper domain have no effect on protein complex formation. However, results from mutagenesis of the first leucine of the heptad repeat in either fos or jun basic regions and alteration of the spacing between the basic and leucine zipper domains indicate that the basic region of fos plays a crucial role in determining the DNA binding affinity of the transcriptional complex. Mutations of the basic amino acids in fos protein prevent binding to the tumour promoter response element (TRE) in the presence of wild-type jun protein. Thus fos protein appears to be dominant in jun-fos binding to DNA, even though fos alone cannot bind to TRE. Mutants in the basic region of fos and jun can be exploited as dominant-negative mutants to ablate the normal fos cellular function.

Multiple isoelectric forms of poliovirus RNA-dependent RNA polymerase: evidence for phosphorylation.

Poliovirus-specific RNA-dependent RNA polymerase (3Dpol) was purified to apparent homogeneity. A single polypeptide of an apparent molecular weight of 63,000 catalyzes the synthesis of dimeric and monomeric RNA products in response to the poliovirion RNA template. Analysis of purified 3Dpol by two-dimensional electrophoresis showed multiple forms of 3Dpol, suggesting posttranslational modification of the protein in virus-infected cells. The two major forms of 3Dpol appear to have approximate pI values of 7.1 and 7.4. Incubation of purified 3Dpol with calf intestinal phosphatase resulted in almost complete disappearance of the pI 7.1 form and a concomitant increase in the intensity of the pI 7.4 form of 3Dpol. Addition of 32P-labeled Pi during infection of HeLa cells with poliovirus resulted in specific labeling of 3Dpol and 3CD, a viral protein which contains the entire 3Dpol sequence. Both 3Dpol and 3CD appear to be phosphorylated at serine residues. Ribosomal salt washes prepared from both mock- and poliovirus-infected cells contain phosphatases capable of dephosphorylating quantitatively the phosphorylated form (pI 7.1) of 3Dpol.