ABSTRACT
The first nonpeptidic, noncovalent inhibitors of the cysteine protease cathepsin S (CatS) are described. Electronic database searching using the program DOCK generated a screening set of potential CatS inhibitors from which two lead structures were identified as promising starting points for a drug discovery effort. Lead optimization afforded potent (IC(50) < 50 nM) and selective inhibitors of CatS demonstrating cellular activity and reversibility of enzyme inhibition.
Subject(s)
Cathepsins/antagonists & inhibitors , Cysteine Proteinase Inhibitors/chemistry , Cysteine Proteinase Inhibitors/pharmacology , B-Lymphocytes/drug effects , B-Lymphocytes/metabolism , Cathepsins/chemistry , Cells, Cultured , Drug Evaluation, Preclinical/methods , Histocompatibility Antigens Class II/drug effects , Histocompatibility Antigens Class II/metabolism , Humans , Immunoglobulin Constant Regions/drug effects , Immunoglobulin Constant Regions/metabolism , Inhibitory Concentration 50 , Peptides/chemistry , Peptides/pharmacology , Protein Conformation , Structure-Activity RelationshipABSTRACT
IL-4 and CD40 ligand stimulate transcription of CD23 (Fc epsilonRII) in B cells and are necessary for the expression of germline epsilon mRNA and production of IgE. Because in vivo studies have shown that the Fc epsilonRII is involved in the regulation of IgE, a study was initiated to compare how IL-4 and engagement of CD40 up-regulate the Fc epsilonRII and epsilon genes. Herein, we describe the preparation of a series of linker-scanning mutants that cover the IL-4 response region in the murine Fc epsilonRII promoter, and their function when transfected into M12.4.5 and M12.4.1 B lymphoma cell lines. Several discrete elements were found to be necessary for IL-4 induction of the Fc epsilonRII gene, some of which have homology with the binding sites of known transcription factors, including NF-IL-4 and NF-kappaB. In contrast, the response element for anti-CD40 (plus IL-4) mapped to a single discrete sequence, a NF-kappaB-like site. Aligning the Fc epsilonRII and germline epsilon promoters in the region that is highly conserved between the human and mouse homologues of both genes reveals a high degree of identity, particularly within discrete clusters. Comparing the function of linker-scanning mutants of the Fc epsilonRII promoter with a similar report for germline epsilon shows that both genes require at least two homologous and similarly located DNA elements in their promoters for a full IL-4 induction. Moreover, the similar response of Fc epsilonRII and epsilon promoter-driven chloramphenicol acetyl transferase plasmids to several cytokines and other agents suggests that the two proximal promoter regions are activated by a similar cassette of factors.
Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/pharmacology , CD40 Antigens/immunology , Embryonic and Fetal Development/immunology , Interleukin-4/pharmacology , Promoter Regions, Genetic/genetics , Receptors, IgE/drug effects , Receptors, IgE/genetics , Transcription, Genetic/drug effects , Transcription, Genetic/genetics , Animals , Base Sequence , Genes, Immunoglobulin/drug effects , Genes, Immunoglobulin/genetics , Histocompatibility Antigens Class II/drug effects , Histocompatibility Antigens Class II/genetics , Humans , Immunoglobulin Constant Regions/drug effects , Immunoglobulin Constant Regions/genetics , Immunoglobulin E/genetics , Lymphoma , Mice , Molecular Sequence Data , Mutation/genetics , Receptors, IgE/biosynthesis , Sequence Homology, Nucleic Acid , Tumor Cells, CulturedABSTRACT
Trifluoroethanol (TFE) is known to stabilize the alpha-helical structure in proteins and their fragments. However, the relationship between the TFE-induced structures and the native structure is not clear. Here we show that beta-lactoglobulin, which consists predominantly of beta-sheets, exhibited a markedly high propensity to form an alpha-helical structure in the presence of TFE, as measured by far-UV circular dichroism. A cooperative transformation from the beta-sheet structure to an alpha-helical structure occurred at a TFE concentration between 10% and 20%. These results were in contrast to a gradual beta-sheet to alpha-helix transition of the constant fragment of the immunoglobulin light chain, which is also a beta-sheet protein. To understand the significance of the high helical propensity of beta-lactoglobulin, we measured the TFE-induced conformational transition of more than 20 proteins of various secondary structural types. Whereas the alpha-helical proteins showed a propensity to form an extensive helical structure in TFE, the helical propensity of proteins with a low helical content in the native state varied. The helical content in TFE was correlated more with the helical content predicted by a secondary structure prediction than with the helical content of the native structure, suggesting that the stability of the helical structure in TFE is determined by local interactions between nearby amino acid residues. Our results suggest that an alpha-helical intermediate can accumulate during the refolding process of beta-lactoglobulin and that a hierarchical model of protein folding is not necessarily true for some beta-sheet proteins including beta-lactoglobulin.
