ABSTRACT
The protein-protein interaction between leukocyte functional antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) is critical to lymphocyte and immune system function. Here, we report on the transfer of the contiguous, nonlinear epitope of ICAM-1, responsible for its association with LFA-1, to a small-molecule framework. These LFA-1 antagonists bound LFA-1, blocked binding of ICAM-1, and inhibited a mixed lymphocyte reaction (MLR) with potency significantly greater than that of cyclosporine A. Furthermore, in comparison to an antibody to LFA-1, they exhibited significant anti-inflammatory effects in vivo. These results demonstrate the utility of small-molecule mimics of nonlinear protein epitopes and the protein epitopes themselves as leads in the identification of novel pharmaceutical agents.
Subject(s)
Immunosuppressive Agents/pharmacology , Intercellular Adhesion Molecule-1/immunology , Intercellular Adhesion Molecule-1/metabolism , Lymphocyte Function-Associated Antigen-1/metabolism , Thiophenes/chemical synthesis , Thiophenes/pharmacology , beta-Alanine/chemical synthesis , beta-Alanine/pharmacology , Amino Acid Sequence , Animals , Anti-Inflammatory Agents, Non-Steroidal/chemical synthesis , Anti-Inflammatory Agents, Non-Steroidal/chemistry , Anti-Inflammatory Agents, Non-Steroidal/metabolism , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Cyclosporine/pharmacology , Dermatitis, Irritant/drug therapy , Dinitrofluorobenzene , Drug Design , Enzyme-Linked Immunosorbent Assay , Epitopes , Female , Humans , Immunoglobulin Fab Fragments/immunology , Immunoglobulin Fab Fragments/pharmacology , Immunosuppressive Agents/chemical synthesis , Immunosuppressive Agents/chemistry , Immunosuppressive Agents/metabolism , Intercellular Adhesion Molecule-1/chemistry , Lymphocyte Culture Test, Mixed , Lymphocyte Function-Associated Antigen-1/immunology , Mice , Mice, Inbred BALB C , Molecular Mimicry , Mutagenesis , Protein Structure, Secondary , Structure-Activity Relationship , Thiophenes/chemistry , Thiophenes/metabolism , beta-Alanine/analogs & derivatives , beta-Alanine/chemistry , beta-Alanine/metabolismABSTRACT
Strains of Aspergillus nidulans carrying the sltA1 mutation, conferring sensitivity to KCl and NaCl, also showed an arginine-sensitive phenotype whereby concentrations of the L-amino acid at or above 10 mM were toxic to growth. Sexual progeny of a cross between a sltA1 mutant and a wild-type strain showed a co-segregation of salt and arginine sensitivity. Similarly, revertants to salt tolerance showed a loss of arginine sensitivity as did sltA1 strains that were transformed with a cosmid carrying the putative sltA1+ wild-type allele. In addition, arginine sensitivity could be relieved by L-ornithine. It is suggested that sltA1 is a salt-sensitive allele of the arginase gene (agaA).
Subject(s)
Alleles , Arginase/genetics , Aspergillus nidulans/genetics , Chlorides/pharmacology , Genes, Fungal , Arginine/analogs & derivatives , Arginine/pharmacology , Aspergillus nidulans/drug effects , Aspergillus nidulans/enzymology , Aspergillus nidulans/growth & development , Crosses, Genetic , Drug Resistance, Microbial , Meiosis , Mutagenesis , Phenotype , Potassium Chloride/pharmacology , Protoplasts/drug effects , Sodium Chloride/pharmacology , Transformation, GeneticABSTRACT
Reexamination of the hexapeptide GH-releasing peptide (GHRP-6) structure/function has lead to the development of four novel classes of compound that stimulate GH release. Each class is represented as follows: a pentapeptide, G-7039; a tetrapeptide, G-7134; a pseudotripeptide, G-7502; and a rigid cyclic heptapeptide, G-7203. The EC50 values for these compounds, determined by GH dose-response curves using primary cultures of rat pituitary cells, were 0.18, 0.34, 10.6, and 0.43 nM, respectively. To demonstrate that these compounds were acting at the putative GHRP receptor, challenges were made using combinations that included GHRP-6 and GH-releasing hormone (GHRH). All four new classes further increased GH release in combination with GHRH, but not with GHRP-6. Homologous desensitization occurred after 45 min of exposure to the new compounds while the cells remained sensitive to GHRH. Somatostatin inhibited all of these compounds. Additionally, G-7039 elevated free calcium, as occurs with GHRP-6. All four classes elicited a robust GH release, a small increase in PRL, and no change in LH, FSH, ACTH, or TSH. We conclude that these novel compounds are potent and direct stimulators of pituitary GH release, with in vitro attributes that suggest mediation via a specific GHRP-like mechanism.
Subject(s)
Growth Hormone-Releasing Hormone/pharmacology , Animals , Calcium/metabolism , Dose-Response Relationship, Drug , Female , Growth Hormone/metabolism , In Vitro Techniques , Rats , Rats, Sprague-Dawley , Somatostatin/pharmacology , Structure-Activity RelationshipABSTRACT
Another class of growth hormone (GH) secretagogues has been discovered by altering the backbone structure of a flexible linear GH-releasing peptide (GHRP). In vitro and in vivo characterization confirms these GH secretagogues as the most potent and smallest (M(r) < 500) reported. Anabolic efficacy is demonstrated in rodents with intermittent delivery. A convergent model of the bioactive conformation of GHRPs is developed and is supported by the NMR structure of a highly potent cyclic analog of GHRP-2. The model and functional data provide a logical framework for the further design of low-molecular weight secretagogues and illustrate the utility of an interdisciplinary approach to elucidating potential bound-state conformations of flexible peptide ligands.
Subject(s)
Growth Hormone/metabolism , Hormones/chemistry , Oligopeptides/chemistry , Peptides, Cyclic/chemistry , Amino Acid Sequence , Animals , Consensus Sequence , Female , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Sequence Data , Pituitary Gland, Anterior/metabolism , Protein Structure, Secondary , Rats , Rats, Sprague-Dawley , Secretory Rate , Structure-Activity RelationshipABSTRACT
A structural survey of protein Zn2+ binding geometries was instigated based upon the functional requirement of Ras farnesyltransferase for Zn2+. The Cys-X-X-Cys motif found in Zn(2+)-binding proteins such as aspartate transcarbamylase was used as a template to devise a bidentate-coordination model for Cys-A1-A2-X peptide inhibitors. Accordingly, replacement of the central dipeptide with the hydrophobic scaffold 3-amino-1-carboxymethyl-2,3-dihydro-5- phenyl-1H-1,4-benzodiazepin-2-one (BZA) yielded a peptidomimetic inhibitor, Cys(BZA)Met, of moderate potency (IC50 = 400 nM). N-Methylation of the cysteine amide improved potency almost 100-fold (IC50 = 0.3-1 nM). The increased affinity presumably correlates with a preferred conformation of the inhibitor which maximizes a hydrophobic interaction between the scaffold and the enzyme, and the proper presentation of cysteine and methionine to allow bidentate coordination at Zn2+. These non-peptide inhibitors have been shown to block farnesylation of the Ras protein in intact cells and provide lead compounds for the development of new cancer therapeutic agents.
Subject(s)
Alkyl and Aryl Transferases , Benzodiazepines/pharmacology , Transferases/antagonists & inhibitors , Amino Acid Sequence , Benzodiazepines/chemical synthesis , Cell Membrane Permeability , Farnesyltranstransferase , Models, Molecular , Molecular Sequence Data , Oligopeptides/chemical synthesis , Oligopeptides/metabolism , Oligopeptides/pharmacology , Structure-Activity Relationship , Transferases/metabolismABSTRACT
Human papillomavirus infection of the uterine cervix is associated with a spectrum of benign, premalignant, and malignant epithelial lesions, a process that appears to require the coordinated effects of secondary cellular and environmental events. We have used flow cytometry and immunohistochemistry to examine the expression of the cellular markers for proliferation (interleukin-1, epidermal growth factor receptor, and transferrin receptor) and the markers of cellular differentiation (filaggrin and low-molecular-weight cytokeratin) in normal and human papillomavirus--infected human cervical tissues representing the natural range of human papillomavirus--induced disease. The results were correlated with the histologic grade of disease, human papillomavirus type, cellular deoxyribonucleic acid content, and cell cycle status. Interleukin-1 and transferrin receptor were slightly increased in high-grade dysplasias and in squamous cell carcinomas. Filaggrin expression was found to be inversely related and cytokeratin and epidermal growth factor receptor expression directly related to the degree of neoplasia. These findings indicate that cytokeratin and epidermal growth factor receptor are useful markers of cell proliferation in human papillomavirus--infected tissues and that their expression may directly increase as a result of infection.
Subject(s)
Cervix Uteri/microbiology , Cytokines/metabolism , Papillomaviridae/genetics , Receptors, Cell Surface/analysis , Tumor Virus Infections/metabolism , Blotting, Southern , Cell Differentiation , Cervix Uteri/pathology , DNA, Viral/analysis , ErbB Receptors/analysis , Female , Filaggrin Proteins , Humans , Interleukin-1/analysis , Intermediate Filament Proteins/analysis , Keratins/analysis , Ploidies , Receptors, Transferrin/analysisABSTRACT
In order to determine the conformational requirements for binding of L-glutamate to the proteins involved in the process of neurotransmission, rigid analogues containing an embedded glutamate moiety have been prepared. These "conformer mimics", the pyrrolidine-2,4-dicarboxylates 4, 7, 11, and 14, were synthesized from commercially available trans-4-hydroxy-L-proline and cis-4-hydroxy-D-proline, and then were tested for their ability to inhibit the high-affinity transport of [3H]-L-glutamate into synaptosomes and to block the binding of radioligands to the NMDA (N-methyl-D-aspartate), KA (kainate), and QA (quisqualate) glutamate neurotransmitter receptor sites. While none of the four analogues binds effectively to the excitatory receptors, the L-trans-isomer 7 is a potent and selective competitive inhibitor of L-glutamate transport. These results delineate a specific structural/conformational preference for binding to the uptake system that is distinct from that required for binding to the NMDA, KA, and QA receptors.