ABSTRACT
The binding of tumor necrosis factor alpha (TNF-alpha) to the type-1 TNF receptor (TNFRc1) plays an important role in inflammation. Despite the clinical success of biologics (antibodies, soluble receptors) for treating TNF-based autoimmune conditions, no potent small molecule antagonists have been developed. Our screening of chemical libraries revealed that N-alkyl 5-arylidene-2-thioxo-1,3-thiazolidin-4-ones were antagonists of this protein-protein interaction. After chemical optimization, we discovered IW927, which potently disrupted the binding of TNF-alpha to TNFRc1 (IC(50) = 50 nM) and also blocked TNF-stimulated phosphorylation of Ikappa-B in Ramos cells (IC(50) = 600 nM). This compound did not bind detectably to the related cytokine receptors TNFRc2 or CD40, and did not display any cytotoxicity at concentrations as high as 100 microM. Detailed evaluation of this and related molecules revealed that compounds in this class are "photochemically enhanced" inhibitors, in that they bind reversibly to the TNFRc1 with weak affinity (ca. 40-100 microM) and then covalently modify the receptor via a photochemical reaction. We obtained a crystal structure of IV703 (a close analog of IW927) bound to the TNFRc1. This structure clearly revealed that one of the aromatic rings of the inhibitor was covalently linked to the receptor through the main-chain nitrogen of Ala-62, a residue that has already been implicated in the binding of TNF-alpha to the TNFRc1. When combined with the fact that our inhibitors are reversible binders in light-excluded conditions, the results of the crystallography provide the basis for the rational design of nonphotoreactive inhibitors of the TNF-alpha-TNFRc1 interaction.
Subject(s)
Morpholines/chemistry , Receptors, Tumor Necrosis Factor/antagonists & inhibitors , Tumor Necrosis Factor-alpha/metabolism , Antigens, CD/chemistry , Antigens, CD/metabolism , Crystallography, X-Ray , Humans , Models, Molecular , Molecular Structure , Photochemistry , Receptors, Tumor Necrosis Factor/chemistry , Receptors, Tumor Necrosis Factor/metabolism , Receptors, Tumor Necrosis Factor, Type I , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/chemistry , Recombinant Proteins/metabolismABSTRACT
Studies conducted over the past decade have demonstrated a central role for tumour necrosis factor alpha (TNFalpha) in inflammatory diseases. As a result of this work, a number of biological agents that neutralise the activity of this cytokine have entered the clinic. The recent clinical data obtained with etanercept and infliximab highlight the relevance of this strategy. TNFalpha converting enzyme (TACE) is the metalloproteinase that processes the 26 kDa membrane bound precursor of TNFalpha (proTNFalpha) to the 17 kDa soluble component. Although a number of proteases have been shown to process proTNFalpha, none do so with the efficiency of TACE. A series of orally bioavailable, selective, and potent TACE inhibitors are currently in clinical development. These inhibitors effectively block TACE mediated processing of proTNFalpha and can reduce TNF production by lipopolysaccharide stimulated whole blood by >95%. Through a series of studies it is shown here that >80% of the unprocessed proTNFalpha is degraded intracellularly. The remainder appears to be transiently expressed on the cell surface. Although, in vitro, TACE inhibition has also been implicated in shedding of p55 and p75 surface TNFalpha receptors, the in vivo data cast doubt on the consequences of this finding. In a mouse model of collagen-induced arthritis, the inhibitors are efficacious both prophylactically and therapeutically. The efficacy seen is equivalent to strategies that neutralise TNFalpha. In many studies greater efficacy is observed with the TACE inhibitors, presumably owing to greater penetration to the site of TNFalpha production.
Subject(s)
Arthritis, Experimental/drug therapy , Enzyme Inhibitors/therapeutic use , Immunoglobulin G/therapeutic use , Metalloendopeptidases/antagonists & inhibitors , Protein Precursors/metabolism , Receptors, Tumor Necrosis Factor/antagonists & inhibitors , Receptors, Tumor Necrosis Factor/therapeutic use , Tumor Necrosis Factor-alpha/metabolism , ADAM Proteins , ADAM17 Protein , Animals , Arthritis, Experimental/immunology , Cell Membrane/metabolism , Collagen , Cytokines/metabolism , Etanercept , Humans , Lipopolysaccharides , Lymphocytes/metabolism , Male , Mice , Mice, Inbred BALB C , Monocytes/metabolism , Random AllocationABSTRACT
Starting with lead compound 2, we sought to increase the selectivity for alpha(v)beta(3)-mediated cell adhesion by examining the effects of structural changes in both the guanidine mimetic and the substituent alpha to the carboxylate. To prepare some of the desired aminoimidazoles, a novel reductive amination utilizing a trityl-protected aminoimidazole was developed. It was found that guanidine mimetics with a wide range of pK(a)'s were potent antagonists of alpha(v)beta(3). In general, it appeared that an acylated 2-aminoimidazole guanidine mimetic imparted excellent selectivity for alpha(v)beta(3)-mediated adhesion versus alpha(IIb)beta(3)-mediated platelet aggregation, with selectivity of approximately 3 orders of magnitude observed for compounds 3g and 3h. It was also found in this series that the alpha-substituent was required for potent activity and that 2,6-disubstituted arylsulfonamides were optimal. In addition, the selective alpha(v)beta(3) antagonist 3h was found to be a potent inhibitor of alpha(v)beta(3)-mediated cell migration.
Subject(s)
Isoxazoles/chemical synthesis , Receptors, Vitronectin/antagonists & inhibitors , beta-Alanine/analogs & derivatives , Cell Adhesion/drug effects , Cell Movement/drug effects , Chemotaxis/drug effects , Guanidines/chemistry , Humans , Hyperplasia/metabolism , In Vitro Techniques , Isoxazoles/chemistry , Isoxazoles/pharmacology , Kidney/cytology , Kidney/drug effects , Kidney/metabolism , Platelet Aggregation/drug effects , Receptors, Vitronectin/biosynthesis , Stereoisomerism , Structure-Activity Relationship , Tumor Cells, Cultured , Vitronectin/pharmacology , beta-Alanine/chemical synthesis , beta-Alanine/chemistry , beta-Alanine/pharmacologyABSTRACT
TNF-alpha converting enzyme (TACE) is the protease responsible for processing proTNF from the 26-kDa membrane-anchored precursor to the secreted 17-kDa TNF-alpha. We show here that a deletion mutant of TACE (dTACE), lacking the pro and catalytic domains of the protease, acts as a dominant negative for proTNF processing in transfected HEK293 cells. We used the same system to test the effect of dTACE on TNFRII processing. Overexpression of dTACE with TNFRII resulted in >80% inhibition of TNFRII shedding. Although significant inhibition of TNF-alpha and TNFRII shedding was achieved with dTACE, we could not detect a cell surface accumulation of the noncleaved substrates above that observed in the absence of dTACE. Our results suggest that TNFRII is a substrate for TACE, and that dTACE is capable of interfering with the function of endogenous TACE, either by binding and sequestering TACE substrates via the disintegrin domain, transmembrane domain, or cytoplasmic tail, or by some other mechanism that has yet to be determined.
Subject(s)
Antigens, CD/metabolism , Metalloendopeptidases/genetics , Metalloendopeptidases/physiology , Protein Precursors/antagonists & inhibitors , Protein Precursors/metabolism , Receptors, Tumor Necrosis Factor/metabolism , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Tumor Necrosis Factor-alpha/metabolism , ADAM Proteins , ADAM10 Protein , ADAM17 Protein , Amyloid Precursor Protein Secretases , Antigens, CD/biosynthesis , Antigens, CD/genetics , Cell Line , Cytoplasm/metabolism , Cytoplasm/physiology , DNA, Complementary/genetics , Disintegrins/genetics , Disintegrins/physiology , Enzyme Activation/genetics , Flow Cytometry , Humans , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/biosynthesis , Metalloendopeptidases/antagonists & inhibitors , Metalloendopeptidases/biosynthesis , Metalloendopeptidases/chemistry , Peptide Fragments/genetics , Peptide Fragments/physiology , Protein Precursors/biosynthesis , Protein Precursors/genetics , Protein Processing, Post-Translational/genetics , Protein Processing, Post-Translational/immunology , Receptors, Tumor Necrosis Factor/biosynthesis , Receptors, Tumor Necrosis Factor/genetics , Receptors, Tumor Necrosis Factor, Type II , Solubility , Substrate Specificity/genetics , Transfection , Tumor Necrosis Factor-alpha/biosynthesis , Tumor Necrosis Factor-alpha/geneticsABSTRACT
Human monocytes rapidly produce TNF-alpha following activation by bacterial LPS. The message for TNF-alpha encodes a 26-kDa protein that is proteolytically processed to the secreted 17-kDa form. Sequencing of the N terminus of the protein secreted by monocytes shows processing of the 26-kDa pro-TNF-alpha to a mature form at the projected metalloprotease cleavage site to generate 17-kDa TNF-alpha with the N terminus VRSSSR-. The addition of hydroxamic acid-based metalloprotease inhibitors to the cell culture is capable of blocking >95% of the production of soluble TNF-alpha and leads to a transient, but reproducible, increase in cell surface TNF-alpha as measured by FACS analysis. The cell surface TNF-alpha was demonstrated to increase the cell's ability to kill L929 tumor targets and induce PG production from human gingival fibroblasts. The buildup of cell surface TNF-alpha is unable to account for the TNF-alpha that is not secreted when inhibitor is present. Pulse-chase analysis of the cells demonstrates rapid degradation of the pro-TNF-alpha that remains unprocessed in the monocytes. Through inhibition of processing and secretion by brefeldin A, processing was shown to occur at a postendoplasmic reticulum site and is closely associated with movement to the cell surface.
Subject(s)
Metalloendopeptidases/metabolism , Monocytes/immunology , Tumor Necrosis Factor-alpha/metabolism , Biological Transport/drug effects , Cell Membrane/metabolism , Cells, Cultured , Enzyme Inhibitors/pharmacology , Humans , Lipopolysaccharides/immunology , Lipopolysaccharides/pharmacology , Metalloendopeptidases/antagonists & inhibitors , Metalloendopeptidases/immunology , Monocytes/cytology , Monocytes/metabolism , Protein Precursors/immunology , Protein Precursors/metabolism , Tumor Necrosis Factor-alpha/immunologyABSTRACT
Flow cytometry provides a rapid qualitative method for analyzing the binding of a fluorescent probe to a cell. To quantitate the binding of a probe using flow cytometry, one must be able to first calibrate the fluorescent signal with some known standard. We have compared a new one-step method with a previous two-step method for determining the number of binding sites (receptors) on the surface of cells using immunofluorescent staining of the cells and analysis by flow cytometry. Experimentally, recombinant chinese hamster ovary cells, expressing cell surface glycoprotein receptors, IIb/IIIa or Mac-1, were assayed using specific mouse monoclonal antibodies against these receptors. The two methods yielded comparable results and, depending on the cell type, detected anywhere from 100,000 to 300,000 antibody-binding sites per cell, respectively.
Subject(s)
Flow Cytometry/methods , Receptors, Cell Surface/analysis , Animals , Antibodies, Monoclonal/immunology , CHO Cells , Cricetinae , Fluorescent Antibody Technique , Humans , Macrophage-1 Antigen/analysis , Macrophage-1 Antigen/genetics , Microspheres , Platelet Membrane Glycoproteins/analysis , Platelet Membrane Glycoproteins/genetics , Recombinant Proteins/analysis , Recombinant Proteins/geneticsABSTRACT
The transmembrane and anchor region of pseudorabies virus gIII is postulated to be in the 35 hydrophobic amino acids (residues 436 to 470) found near the carboxy terminus of the 479-amino-acid envelope protein. In this study, we used a genetic approach to localize the functional gIII membrane anchor between amino acids 443 and 466. Mutant gIII proteins lacking the membrane anchor were not associated with virus particles, indicating that membrane retention is a prerequisite for virion localization. Unexpectedly, the specific hydrophobic gIII sequence defined by these deletions was not required for membrane anchor function since the entire region could be replaced with leucine residues without affecting gIII membrane retention, export, or virion localization. The hydrophobic region appears to encode more than the membrane anchor domain since both efficiency of posttranslational processing and localization to virions are affected by mutations in this region. We speculate that the composition of the hydrophobic domain influences the overall conformation of gIII, which in turn effects the efficiency of gIII export and processing. The virion localization phenotype is probably indirect and reflects the efficiency of protein processing. This conclusion provides insight into the mechanism of glycoprotein incorporation into virions.
Subject(s)
DNA, Viral/genetics , Herpesvirus 1, Suid/genetics , Mutagenesis, Site-Directed , Protein Processing, Post-Translational , Viral Envelope Proteins/genetics , Amino Acid Sequence , Animals , Base Sequence , Cell Line , Cell Membrane/physiology , Chromosome Deletion , DNA, Viral/isolation & purification , Herpesvirus 1, Suid/metabolism , Kinetics , Molecular Sequence Data , Oligodeoxyribonucleotides , Protein Conformation , Restriction Mapping , Signal Transduction , Viral Envelope Proteins/metabolismABSTRACT
Glycoprotein gIII of pseudorabies virus is a member of a conserved gene family found in at least seven diverse herpesviruses. We report here that the putative cytoplasmic domain of gIII is not required for transport to the cell surface and, unlike the prototype domain from herpes simplex virus type 1 glycoprotein C, is not required for stable membrane anchoring. Furthermore, this domain does not appear to be essential for incorporation of the glycoprotein into virions.
Subject(s)
Herpesvirus 1, Suid/metabolism , Protein Processing, Post-Translational , Viral Envelope Proteins/ultrastructure , Amino Acid Sequence , Base Sequence , Biological Transport , Cloning, Molecular , Cytoplasm , DNA Mutational Analysis , Herpesvirus 1, Suid/genetics , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Membrane Glycoproteins/ultrastructure , Molecular Sequence Data , Multigene Family , Restriction Mapping , Viral Envelope Proteins/genetics , Viral Envelope Proteins/metabolismABSTRACT
We have constructed in-frame lacZ protein fusions to the first three genes of the Escherichia coli unc operon, which codes for the subunits of the proton-translocating ATPase. We have used these constructions to measure the relative in vivo expression of these genes. The second and third genes, uncB and uncE, which code for the a and c subunits of the F0 sector, were expressed at relative levels of approximately 1:10, although the measured expression of uncB depended upon how much of the gene was fused to lacZ. These rates compared favorably with the relative numbers of a and c subunits (a1:c10) in the purified F1F0 complex. The in vivo expression of uncI, the first gene of the operon, was very low, at best 10 to 20 times less than the expression of uncB.
Subject(s)
Escherichia coli , Operon , Proton-Translocating ATPases/genetics , Bacterial Proteins/genetics , Cloning, Molecular , DNA, Bacterial/genetics , Escherichia coli/genetics , Gene Expression Regulation , Plasmids , Promoter Regions, Genetic , Recombinant Fusion Proteins/genetics , beta-Galactosidase/geneticsABSTRACT
Plasmid pRPG54, which carries the genes for the eight subunits of the proton-translocating ATPase of Escherichia coli, has been found to carry a single base change of a G to an A in the ribosome-binding site for uncE, the gene which codes for the N,N'-dicyclohexylcarbodiimide-binding subunit c of the Fo. This noncoding region mutation both lowers expression of uncE by a factor of 2-3 and affects the function of the ATPase, specifically of the Fo sector. The presence of the mutation results in a decrease in the proton permeability of the Fo or of the entire F1Fo-ATPase complex when either is synthesized from genes on a multicopy plasmid. Expression of uncE from an F1Fo plasmid carrying the wild type ribosome binding site results in increased membrane proton permeability and decreased ability of the resultant ATPase to couple a transmembrane proton gradient to ATP synthesis both in vitro and in vivo. Also, although an Fo plasmid carrying the correct ribosome-binding site causes harmful, F1-dependent proton permeability in unc+ cells (Brusilow, W. S. S. (1987) J. Bacteriol. 169, 4984-4990), an identical plasmid carrying the mutation does not, even though it still codes for a functional reconstitutable Fo. The results show a relationship between the relative level of expression of uncE from a multicopy plasmid and the assembly pathway, proton permeability, and energy-coupling characteristics of the ATPase.