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The interleukin-2 (IL-2) promoter consists of several independent T cell receptor (TcR) responsive elements. The induction of promoters dependent on these elements is inhibitable by the immunosuppressants cyclosporin A (CsA) and tacrolimus . Calcineurin, a Ca2+/calmodulin-dependent protein phosphatase, is the FK-506-and CsA-sensitive enzyme required for TcR mediated activation of the LL-2 promoter. We report that a constitutively active form of calcineurin partially substitutes for the Ca2+ co-stimulus required to activate the IL-2 promoter elements IL-2A (which binds the factors OAP and Oct-i) and LL-2E (which binds NF-AT), and completely substitutes for the Ca2+ co-stimulus required to stimulate an NF-xB-dependent element. Calcineurin stimulates the NF-xB element by enhancing inactivation of IxB/MAD3, an inhibitor of NF-xB, thereby increasing the amount of nuclear NF-xB DNA binding activity. These data provide the first demonstration in vivo that activation of a protein phosphatase can inactivate IxB, and suggest one possible explanation for mechanism-based toxicities associated with FK-506 and CsA by demonstrating that these drugs can inhibit the calcineurin-dependent activation of a virtually ubiquitous transcription factor.

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.

Three critical enzymes, catechol oxygenase II (chlorocatechol dioxygenase)$ muconate cycloisomerase II, and diehelactone hydrolase, are involved in the degradation of chlorocatechols, which are obligatory intermediates in the catabolism of chlorinated aromatic compounds. The organization and complete nucleotide sequence of the genes for these enzymes have been determined on a 4.2-kilobase-pair (kbp) Bgl II fragment cloned from the plasmid pAC27, based on the agreement of open reading frame lengths with apparent mobilities of polypeptides expressed in Escherichia coli maxicells, predicted N-terminal amino acid sequences with those of the purified proteins, and predicted total amino acid compositions with those of the purified proteins. (4,5). While the plasmid pJP4 encodes the complete 2,4-dichlorophenoxyacetic acid degradative pathway, the plasmid pAC27 encodes only a chlorocatechol degradative pathway. The two enzymes that are involved in the conversion of 3-chlorobenzoate to 3-chlorocatechol are chromosomally encoded in Pseudomonas putida and are assumed to be the same needed for growth on benzoate. Thus, transfer of pAC27 to P. putida allows total degradation of 3-chlorobenzoate through both chromosomal and plasmid genes. Knackmuss and Reineke (6, 7) showed that there are three enzymes critical for chlorocatechol degradation: catechol oxygenase II (chlorocatechol dioxygenase, or pyrocatechase II), muconate cycloisomerase II, and dienelactone hydrolase (Fig. 1). Whereas the analogous chromosomally encoded enzymes involved in the oxidation of catechol (catechol oxygenase I, cycloisomerase I, and enol-lactone hydrolase) have high specificity for the nonchlorinated substrates with little activity toward chlorinated analogues, the plasmidencoded catechol oxygenase II and cycloisomerase II are highly active toward chlorinated substrates but retain diminished activity toward the nonchlorinated substrates. Dienelactone hydrolase is, however, specific for dienelactone and has no activity toward enol-lactones. The plasmid pAC27 was demonstrated to complement P. putida chromosomal Benmutant PRS2015 (8), which is deficient in cycloisomerase I activity, to Ben+ (allowing growth on benzoate as well as 3-chlorobenzoate) (9), suggesting that the plasmid-encoded enzyme can substitute for the defective chromosomal enzyme while retaining a different substrate specificity. These similarities and differences in substrate specificity raise the interesting question as to how genes encoding degradation of synthetic chlorinated compounds such as 3-chlorocatechol evolve in nature, how they are organized and regulated on a plasmid, and whether any clue to such a process can be obtained by comparing the nucleotide sequences of the newly evolved genes to the sequences for the corresponding chromosomal genes specifying degradation of nonchlorinated analogues. In this paper we report the organization and complete nucleotide sequence of the pAC27 gene cluster specifying 3-chlorocatechol degradation. We compare the promoter region o...

The serine/threonine kinase p38 is a ubiquitous, highly conserved, stress responsive, signal-transducing enzyme. It regulates the production of proinflammatory mediators and is the target of the cytokine synthesis inhibitory pyridinylimidazoles. We have expressed human p38 in Drosophila S2 cells and characterized preparations of mixed unphosphorylated/monophosphorylated (inactive) and homogeneously diphosphorylated (active) forms of the enzyme. We observed that only the active preparation of the enzyme has significant kinase activity when assayed using an ATF2-GST fusion protein as the substrate. We determined that the value of KM[ATP] in this reaction is 25 microM and that the pyridinylimidazole inhibitor of p38 kinase activity, SB203580, competes with ATP. We have found that a tritiated pyridinylimidazole, SB202190, has an equal affinity for both the active and inactive forms of the enzyme and that SB203580 competes with it equally well for binding to either form of the enzyme. However, ATP can compete with the tritiated inhibitor for binding to only the active form of the enzyme. Further, we demonstrate in vivo that at concentrations consistent with its IC50 as a cytokine inhibitor, SB203580 can inhibit stimulus-induced phosphorylation of p38 at the Thr-Gly-Tyr activation motif. Our observations suggest that pyridinylimidazoles may block the biological activity of p38 kinase by binding to the inactive form of p38 and reducing its rate of activation. Under these conditions, ATP would not effectively compete with the inhibitors in vivo.