ABSTRACT
OBJECTIVE AND DESIGN: We have explored the in vitro immunomodulatory effects of pure ruthenium red and a series of pyridine and imidazole substituted ruthenium complexes (RCs). MATERIAL: Human peripheral blood lymphocytes and purified T cells were used in these studies along with various cell lines. METHODS: Cells were treated with dilutions of RCs and assessed in various assays of immune function, cytotoxicity and cell cycle progression. RESULTS: RCs efficiently blocked T cell receptor (TCR)-mediated stimulation (IC(50)'s in the low nM range) of human peripheral blood lymphocytes (hPBL) by various agents, including tetanus toxoid, alloantigens, superantigens, and receptor-specific antibodies. RCs are not cytotoxic to T cells. Antiproliferative activity was also observed for B cells. Some non-lymphoid cell lines or primary cultures showed sensitivity to the RCs, but only at higher concentrations. The inhibitory effect on human T cells was assessed and demonstrated at the level of proliferation (DNA synthesis), IL-2 secretion, and IL-2 receptor (CD25) upregulation. RCs also inhibited IL-2-mediated proliferation of antigen-induced T-cell blasts and the IL-2-dependent T cell line Kit-225. Cell cycle analysis indicates that RCs inhibit the progression of activated T cells from G(0)/G(1) to S phase. CONCLUSIONS: Since the mechanism of T cell inhibition by RCs appears to be different than that of rapamycin (RAP) or cyclosporin A (CsA), they may provide a new tool to investigate intracellular signaling in T cells, and may present novel opportunities for immunosuppression
Subject(s)
Immunosuppressive Agents/pharmacology , Ruthenium Compounds/pharmacology , Animals , B-Lymphocytes/drug effects , Cell Division/drug effects , Cell Line , Cyclosporine/pharmacology , DNA/biosynthesis , Dogs , Fluorescent Antibody Technique , G1 Phase/drug effects , Humans , Imidazoles/chemical synthesis , Imidazoles/pharmacology , Immunity, Cellular/drug effects , In Vitro Techniques , Interleukin-2/biosynthesis , Lymphocyte Culture Test, Mixed , Pyridines/chemical synthesis , Pyridines/pharmacology , Receptors, Interleukin-2/biosynthesis , Resting Phase, Cell Cycle/drug effects , Ruthenium Red/pharmacology , S Phase/drug effects , Sirolimus/pharmacology , Superantigens/immunology , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Tetanus Toxoid/pharmacologyABSTRACT
The syntheses and immunosuppressive activity of ruthenium complexes are described. One of the complexes (1a) was shown to be a potent inhibitor of human T-lymphocyte proliferation with an IC50 of 5 nM. The activity of these complexes compares favorably to the well known immunosuppressants Cyclosporin A and Rapamycin.
Subject(s)
Immunosuppressive Agents/chemical synthesis , Ruthenium/chemistry , Cell Division/drug effects , Cells, Cultured , Humans , Immunosuppressive Agents/chemistry , Immunosuppressive Agents/pharmacology , T-Lymphocytes/cytology , T-Lymphocytes/drug effectsABSTRACT
T-cell receptors (TCRs) are created by a stochastic gene rearrangement process during thymocyte development, generating thymocytes bearing useful, as well as unwanted, specificities. Within the latter group, autoreactive thymocytes arise which are subsequently eliminated via a thymocyte-specific apoptotic mechanism, termed negative selection. The molecular basis of this deletion is unknown. Here, we show that TCR triggering by peptide/MHC ligands activates a caspase in double-positive (DP) CD4+ CD8+ thymocytes, resulting in their death. Inhibition of this enzymatic activity prevents antigen-induced death of DP thymocytes in fetal thymic organ culture (FTOC) from TCR transgenic mice as well as apoptosis induced by anti-CD3epsilon monoclonal antibody and corticosteroids in FTOC of normal C57BL/6 mice. Hence, a common caspase mediates immature thymocyte susceptibility to cell death.
Subject(s)
Apoptosis , Caspases , Cysteine Endopeptidases/metabolism , Major Histocompatibility Complex , Peptides/metabolism , Receptors, Antigen, T-Cell/metabolism , T-Lymphocytes/enzymology , Amino Acid Chloromethyl Ketones/pharmacology , Animals , Antibodies, Monoclonal , Caspase 3 , Caspase 6 , Caspase 7 , Cysteine Proteinase Inhibitors/pharmacology , Enzyme Activation , Enzyme Precursors/metabolism , Female , Glucocorticoids/pharmacology , Ligands , Male , Mice , Mice, Inbred C57BL , Receptors, Antigen, T-Cell/immunology , Signal Transduction , T-Lymphocytes/cytology , Thymus Gland/cytology , Thymus Gland/embryology , Thymus Gland/enzymologySubject(s)
Graft Rejection/prevention & control , Immunosuppressive Agents/pharmacology , Lymphocyte Activation/drug effects , Organometallic Compounds/pharmacology , Pyrroles/pharmacology , Skin Transplantation/immunology , T-Lymphocytes/immunology , Animals , Antibodies/pharmacology , CD2 Antigens/immunology , Cells, Cultured , Cyclosporine/pharmacology , Female , Graft Rejection/immunology , Humans , Interleukin-2/pharmacology , Mice , Mice, Inbred A , Mice, Inbred BALB C , Mice, Inbred C57BL , Organometallic Compounds/chemistry , Pyrroles/chemistry , T-Lymphocytes/drug effects , Time Factors , Transplantation, HomologousABSTRACT
A triene-modified analog of the potent immunosuppressive agent rapamycin was found to be a potent inhibitor of the peptidyl prolyl cis-trans isomerase activity of human FKBP (Ki = 12.5 nM). This analog was not immunosuppressive in a thymocyte proliferation assay itself, but was able to antagonize the effect of rapamycin. This new analog should be useful as a mechanistic probe for macrocyclic immunosuppressants.
Subject(s)
Amino Acid Isomerases/antagonists & inhibitors , Carrier Proteins/antagonists & inhibitors , Immunosuppressive Agents/pharmacology , Piperidines/pharmacology , Pyridazines/pharmacology , Animals , Cells, Cultured , Cyclosporins/metabolism , Humans , Kinetics , Lymphocyte Activation/drug effects , Mice , Mice, Inbred C3H , Molecular Structure , Peptidylprolyl Isomerase , Polyenes/pharmacology , Recombinant Proteins/antagonists & inhibitors , Sirolimus , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , Tacrolimus/metabolism , Tacrolimus Binding ProteinsABSTRACT
Modified heterocyclic phenylalanine analogues designed as replacements for the P3-P4 region were synthesized and incorporated into renin inhibitors. These inhibitors were found to have significant activity versus human recombinant renin, as well as in vivo activity. The compounds proved to be very resistant to chymotrypsin degradation, as exemplified by compound 8, which remained greater than 60% intact after a 24-h exposure to chymotrypsin. In contrast, the Boc-Phe analogue was nearly completely degraded after 1 h. Compound 6 proved to be the most potent renin inhibitor with an IC50 = 8.9 nM. These stable cyclized phenylalanines should prove to be generally useful as a substitute for Boc-Phe in protease inhibitors.
Subject(s)
Heterocyclic Compounds/chemical synthesis , Indoles/chemical synthesis , Phenylalanine/analogs & derivatives , Renin/antagonists & inhibitors , Angiotensin I/metabolism , Animals , Blood Pressure/drug effects , Chymotrypsin/metabolism , Drug Stability , Female , Heterocyclic Compounds/metabolism , Heterocyclic Compounds/pharmacology , Humans , Indoles/pharmacology , Macaca mulatta , Male , Molecular Structure , Renin/blood , Structure-Activity Relationship , X-Ray DiffractionABSTRACT
The design and synthesis of 3-amino-2-oxo-4-phenylbutanoic acid amides (alpha-keto amides), a new class of aminopeptidase inhibitor, are described. These compounds, illustrated by the Phe-Leu analogue 2, are effective inhibitors of arginyl aminopeptidase (Ki = 1.5 microM), cytosol aminopeptidase (Ki = 1.0 microM), and microsomal aminopeptidase (Ki = 2.5 microM). The ketone carbonyl of the alpha-keto amide was found to hydrate readily in an aqueous DMSO solution, due to the electron-withdrawing effect of the neighboring amide group. A mechanism of inhibition is proposed for the alpha-keto amides that is similar to that proposed for the structurally related aminopeptidase inhibitor bestatin and its analogues, wherein the inhibitor may interact with the S1'-S2' subsite of the enzyme rather than the S1-S1' subsite. Like bestatin, the alpha-keto amides are slow-binding inhibitors of all three enzymes.
Subject(s)
Aminopeptidases/antagonists & inhibitors , Amides/chemical synthesis , Amides/pharmacology , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Leucine/analogs & derivatives , Leucine/pharmacology , Magnetic Resonance Spectroscopy , Structure-Activity RelationshipABSTRACT
Sulfur-containing amino acid and peptide analogues of bestatin [((2S,3R)-3-amino-2-hydroxy-4-phenyl-butanoyl)-L-leucine] (1) have been synthesized and evaluated as inhibitors of aminopeptidase M (AP-M), leucine aminopeptidase (LAP), and aminopeptidase B (AP-B). The 2-thiolbestatin analogue (6) was found to be a potent inhibitor of all three aminopeptidases (AP-M, Ki = 4.4 microM; LAP, Ki = 0.55 microM; AP-B, Ki = 4.6 nM) but only a slightly better inhibitor of these aminopeptidases than the parent hydroxy-containing compound 1. Synthetic analogues of L-leucinethiol(4), a strong inhibitor of aminopeptidases, were prepared in which the carbon alpha to the thiol groups was substituted with methyl, methyl carboxylate, and carboxamide derivatives and found to be much weaker inhibitors of all aminopeptidases. A thioamide analogue of bestatin (49) is a modest inhibitor of AP-M (Ki = 40 microM), LAP (Ki = 0.33 microM), and AP-B (Ki = 2.4 microM). These results suggest that the sulfur atoms in 2-thiolbestatin and bestatin thioamide do not interact strongly with the active-site zinc atom of these aminopeptidases when the inhibitors are bound to the enzyme. These results are not consistent with proposed models for the inhibition of aminopeptidases by bestatin and related analogues.
Subject(s)
Aminopeptidases/antagonists & inhibitors , Leucine/analogs & derivatives , Sulfur/analysis , Amino Acids/analysis , Binding Sites , Chromatography, Thin Layer , Kinetics , Leucine/chemical synthesis , Leucine/pharmacology , Oxidation-Reduction , Structure-Activity Relationship , Sulfhydryl Compounds/analysisABSTRACT
L-Lysinethiol was found to be an extremely potent inhibitor of aminopeptidase B (AP-B) with a Ki = 9.1 X 10(-10) M. L-leucinethiol was also a potent inhibitor of AP-B (kj = 1.3 X 10(-7) M), while the D-isomer was much less effective (Ki = 9.8 X 10(-5) M). A thiol-zinc interaction at the active site is postulated for AP-B.
Subject(s)
Aminopeptidases/antagonists & inhibitors , Lysine/analogs & derivatives , Sulfhydryl Compounds/pharmacology , Amino Acids/pharmacology , Aminopeptidases/isolation & purification , Animals , Cytosol/enzymology , Kidney/enzymology , Leucyl Aminopeptidase/isolation & purification , Lysine/pharmacology , Microsomes/enzymology , SwineABSTRACT
Several amino acid derived azolides (I) have been synthesized and investigated for their inhibitory activity toward human leukocyte elastase and porcine pancreatic elastase. The inhibitory activity was found to be dependent on the nature of the precursor amino acid ester. Thus, compounds derived from L-valine methyl ester 3, L-norvaline methyl ester 5, DL-norleucine methyl ester 9, and L-methionine methyl ester 10 were found to inhibit irreversibly both enzymes. Compound 10 was found to be a specific and selective inhibitor of human leukocyte elastase. In contrast to these, inhibitors derived from glycine methyl ester 1, D-valine methyl ester 4, and D-norvaline methyl ester 6 were found to be inactive. The results of the present study show that latent isocyanates derived from appropriate amino acids can serve as selective inhibitors of serine proteases and are of potential pharmacological value.