The immuno-pharmacological profile of a novel immunosuppressive agent, produced by a streptomycete, is presented here. Weproceeded to test the effect of the agent on various in vitro immunesystems. It showed that mixed lymphocyte reaction, cytotoxic T cell generation, the production of T cell-derived soluble mediators such as interleukin 2 (IL-2), interleukin 3 and gamma-interferon and the expression of the IL-2 receptor were suppressed by this agent. The IC50 values of FK-506 and ciclosporin (CS) in all tests were approximately 0.1 nM and 10 nM, respectively. Therefore, the novel agent, FK-506 suppressed in vitro immunesystems at about hundred times lower concentration than CS.

Proteasomes are eukaryotic ring-shaped or cylindrical particles with multicatalytic protease activities. To clarify the involvement of proteasomes in tumorigenesis of human blood cells, we compared their expression in human hematopoietic malignant tumor cells with that in normal peripheral blood mononuclear cells. Immunohistochemical staining showed considerably increased concentrations of proteasomes in leukemic cells from the bone marrow of patients with various types of leukemia and the predominant localization of these proteasomes in the nuclei. Moreover, enzyme immunoassay and Northern blot analysis indicated that the concentrations of proteasomes and their mRNA levels were consistently much higher in a variety of malignant human hematopoietic cell lines than in resting peripheral lymphocytes and monocytes from healthy adults. Proteasome expression was also greatly increased in normal blood mononuclear cells during blastogenic transformation induced by phytohemagglutinin; their expression increased in parallel with induction of DNA synthesis and returned to the basal level with progress of the cell cycle. Thus, abnormally high expression of proteasomes may play an important role in transformation and proliferation of blood cells and in specific functions of hematopoietic tumor cells.named C2 (12) and C3 (13), from rat liver proteasomes and one component of 35 kDa of Drosophila proteasomes (14) have been determined by cDNA cloning. The overall amino acid sequence of rat C2 closely resembles that of the Drosophila 35-kDa protein, suggesting that these two components function similarly in most eukaryotes and that they evolved from the same ancestral gene. In contrast, no significant homologies of these components with any other known proteins were found by computer analysis, indicating that proteasomes are a novel type of enzyme complex.Proteasomes are ubiquitous in cells of eukaryotes ranging from humans to yeast (1,15,16). Moreover, proteasomes or the related 20S particles have been found in both the cytoplasm and the nuclei of a variety of mammalian cells (8,17,18) and also various lower eukaryotes (14,(19)(20)(21)(22)(23)(24)(25), suggesting that their diverse roles depend on their differential localizations in the cells. However, the exact function of proteasomes is still unknown. One way to obtain information on their physiological role(s) is to study their expression in cells in abnormal states. In this work, we examined the expression of proteasomes in resting and growth-stimulated normal peripheral blood mononuclear cells and in a variety of human hematopoietic tumor cell lines.Proteasomes are multicatalytic proteinase complexes that are thought to be major intracellular proteolytic enzyme complexes responsible for certain types of nonlysosomal pathways of protein breakdown that requires metabolic energy (1). They were found to catalyze the degradation of various proteins in an ATP-dependent fashion (2-4). Moreover, 20S proteasomes were demonstrated to assemble into 26S proteolytic complexes t...

A novel class of potent, selective, and orally active non-peptide bradykinin (BK) B2 receptor antagonists were designed and synthesized starting from 8-benzyloxyimidazo[1,2-a]pyridine derivative 2. The unique screening lead (2) was discovered by a two-step intentional random screening process, involving recognition of the relationship between BK and angiotensin II (Ang II) and the common structural features. Systematic chemical modification of 2 elucidated the structural requirements essential for B2 binding affinity leading to the identification of 8-[[3-(N-acylglycyl-N-methylamino)-2,6-dichlorobenzyl]oxy]-3-halo- 2- methylimidazo[1,2-a]pyridine skeleton as the basic framework of this new series of B2 antagonists. A molecular modeling study suggested the key role of the N-methylanilide moiety at the 3-position of the 2,6-dichlorobenzene ring to allow these compounds to adopt the characteristic active conformation. The representative lead compounds inhibited the specific binding of [3H]BK to guinea pig ileum membrane preparations expressing B2 receptors, with nanomolar IC50S and also displayed in vivo functional antagonistic activities against BK-induced bronchoconstriction in guinea pigs at an oral dose of 1 mg/kg. Pharmacokinetic studies of compounds 47c and 50b in rats highlighted their excellent oral bioavailabilities, indicating that they represent the first orally active non-peptide B2 antagonists reported to date.

An orally active, nonpeptide bradykinin (BK) B2 receptor antagonist, FR173657 (E)‐3‐(6‐acetamido‐3‐pyridyl)‐N‐[N‐[2‐4‐dichloro‐3‐[(2‐methyl‐8‐quinolinyl) oxymethyl]phenyl]‐N‐methylaminocarbonyl‐methyl]acrylamide) has been identified. This compound displaced [3H]‐BK binding to B2 receptors present in guinea‐pig ileum membranes with an IC50 of 5.6 × 10−10 M and in rat uterus with an IC50 of 1.5 × 10−9 M. It did not inhibit different specific radio‐ligand binding to other receptor sites. In human lung fibroblast IMR‐90 cells, FR173657 displaced [3H]‐BK binding to B2 receptors with an IC50 of 2.9 × 10−9 M and a Ki of 3.6 × 10−10 M, but did not reduce [3H]‐des‐Arg10‐kallidin binding to B1, receptors. In guinea‐pig isolated preparations, FR173657 antagonized BK‐induced contractions with an IC50 of 7.9 × 10−9 M, but did not antagonize acetylcholine or histamine‐induced contractions even at a concentration of 10−6 M. FR173657 caused parallel rightward shifts of the concentration‐response curves to BK at concentrations of 10−9 M and 3.2 × 10−9 M, and a little depression of the maximal response in addition to the parallel rightward shift of the concentration‐response curve at a concentration of 10−8 M. Analysis of the data yield a pA2 of 9.2 ± 0.2 (n = 5) and a slope of 1.5 ± 0.2 (n = 5). In vivo, the oral administration of FR173657 inhibited BK‐induced bronchoconstriction dose‐dependently in guinea‐pigs with an ED50 of 0.075 mg kg−1, but did not inhibit histamine‐induced bronchoconstriction even at 1 mg kg−1. FR173657 also inhibited carrageenin‐induced paw oedema with an ED50 of 6.8 mg kg−1 2 h after the carrageenin injection in rats. These results show that FR173657 is a potent, selective, and orally active bradykinin B2 receptor antagonist.

In the course of our studies on non-peptide bradykinin (BK) B(2) receptor ligands, it was suggested that the 4-substituent of the quinoline ring may play a critical role in determining binding affinities for human and guinea pig B(2) receptors, as well as agonist/antagonist properties. We carried out an extensive investigation to elucidate the structure-activity relationships (SAR) for this key pharmacophore. Introduction of lower alkoxy groups to the 4-position of the quinoline ring of 3 led to the identification of 4-ethoxy derivative 22b as a unique partial agonist. This compound significantly stimulated inositol phosphates (IPs) formation in Chinese hamster ovary cells expressing the cloned human B(2) receptor at concentrations greater than 10 nM and displayed one-tenth of the intrinsic activity of BK. The agonist activity of 22b was selective for the B(2) receptor and was inhibited by selective peptide and non-peptide B(2) antagonists. On the other hand, 22b strongly suppressed BK-induced IPs formation through the cloned human B(2) receptor. Further studies on the key pharmacophore led to identification of a 2-picolyloxy moiety as a powerful agonist switch, leading to the discovery of a potent and efficacious non-peptide B(2) agonist, 19a. Successive optimization of the acyl side chain afforded 38, which exhibited full agonist activity on stimulation of IPs formation. Furthermore, this strategy could be applied successfully to the benzimidazole series. The representative 1-(2-picolyl)benzimidazole derivative 47c increased PGE(2) production at a 1 microM concentration to the same level as the maximum effect of BK. Thus, we have established the medicinal chemistry modifications required to convert our highly potent non-peptide B(2) antagonists to agonists with potent efficacy.