Glucocorticoids (GCs) are commonly used to treat inflammatory disease; unfortunately, the long-term use of these steroids leads to a large number of debilitating side effects. The antiinflammatory effects of GCs are a result of GC receptor (GR)-mediated inhibition of expression of proinflammatory genes as well as GR-mediated activation of antiinflammatory genes. Similarly, side effects are most likely due to both activated and repressed GR target genes in affected tissues. An as yet unachieved pharmaceutical goal is the development of a compound capable of separating detrimental side effects from antiinflammatory activity. We describe the discovery and characterization of AL-438, a GR ligand that exhibits an altered gene regulation profile, able to repress and activate only a subset of the genes normally regulated by GCs. When tested in vivo, AL-438 retains full antiinflammatory efficacy and potency comparable to steroids but its negative effects on bone metabolism and glucose control are reduced at equivalently antiinflammatory doses. The mechanism underlying this selective in vitro and in vivo activity may be the result of differential cofactor recruitment in response to ligand. AL-438 reduces the interaction between GR and peroxisomal proliferator-activated receptor gamma coactivator-1, a cofactor critical for steroid-mediated glucose up-regulation, while maintaining normal interactions with GR-interacting protein 1. This compound serves as a prototype for a unique, nonsteroidal alternative to conventional GCs in treating inflammatory disease.

A general approach is illustrated for providing detailed structural information on large enzyme/inhibitor complexes using NMR spectroscopy. The method involves the use of isotopically labeled ligands to simplify two-dimensional NOE spectra of large molecular complexes by isotope-editing techniques. With this approach, the backbone and side-chain conformations (at the P2 and P3 sites) of a tightly bound inhibitor of porcine pepsin have been determined. In addition, structural information on the active site of pepsin has been obtained. Due to the sequence homology between porcine pepsin and human renin, this structural information may prove useful for modeling renin/inhibitor complexes with the ultimate goal of designing more effective renin inhibitors. Moreover, this general approach can be applied to study other biological systems of interest such as other enzyme/inhibitor complexes, ligands bound to soluble receptors, and enzyme/substrate interactions.

Engagement of the T cell antigen receptor (TcR)1 with the antigen-major histocompatibility complex on antigen-presenting cells triggers a complex TcR signaling cascade that leads to T cell activation and cytokine secretion (1). During this process, T cells express the autocrine growth factor interleukin 2 (IL-2), which promotes T cell proliferation by interacting with the IL-2 receptor, which is also up-regulated on activated T cells. The transcriptional regulation of the IL-2 gene has been extensively analyzed at the IL-2 promoter, a 275-bp region located upstream of the transcriptional start site of the gene (2, 3). Several transcription factors have been identified to bind elements within this regulatory region, including AP-1, NF-B, and the nuclear factor of activated T cells (NFAT) (2).The transcription factor NFAT plays an essential role in IL-2 expression. Binding sites for NFATs have also been found within the promoter regions of several other cytokine genes, including IL-3, IL-4, IL-5, IL-8, IL-13, tumor necrosis factor ␣, granulocyte-macrophage colony-stimulating factor, and ␥-IFN (4, 5). NFAT is a complex composed of a cytoplasmic subunit and an inducible nuclear component comprised of AP-1 (Fos/ Jun) family members. At least four structurally related NFAT cytoplasmic subunit members, NFATp/NFAT1, NFATc/ NFAT2, NFAT3, and NFATX/NFATc3/NFAT4, have been identified (5). NFAT proteins share a conserved domain located toward the C terminus (6) that binds DNA and also participates in cooperative protein-protein interactions with AP-1 transcription factors (7,8). Immediately N-terminal to the DNA-binding domain is a second conserved module of ϳ300 residues known as the NFAT homology (NFAT-h) region. The N terminus of NFAT, including the NFAT-h region, regulates nuclear/cytoplasm trafficking in response to changes in intracellular Ca 2ϩ concentrations. In resting T cells, the protein is retained in the cytoplasm and its NFAT-h domain is heavily phosphorylated. Engagement of the TcR or treatment of cells with the Ca 2ϩ ionophore activates the Ca 2ϩ /calmodulin-dependent Ser/Thr phosphatase, calcineurin. CaN dephosphorylates the NFAT-h domain, resulting in translocation of NFAT to the nucleus (9).

A novel class of functional ligands for the human glucocorticoid receptor is described. Substituents in the C-10 position of the tetracyclic core are essential for glucocorticoid receptor (GR) selectivity versus other steroid receptors. The C-5 position is derivatized with meta-substituted aromatic groups, resulting in analogues with a high affinity for GR (K(i) = 2.4-9.3 nM) and functional activity comparable to prednisolone in reporter gene assays of glucocorticoid-mediated gene transcription. The biological activity of these novel quinolines was also prednisolone-equivalent in whole cell assays of glucocorticoid function, and compound 13 was similar to prednisolone (po ED(50) = 2.8 mpk for 13 vs ED(50) = 1.2 mpk for prednisolone) in a rodent model of asthma (sephadex-induced eosinophil influx).