Subject(s)
Cyclooxygenase Inhibitors/pharmacology , Isoenzymes/antagonists & inhibitors , Isoenzymes/pharmacology , Isoxazoles/pharmacology , Prostaglandin-Endoperoxide Synthases/pharmacology , Acylation , Animals , Cyclooxygenase 2 , Cyclooxygenase 2 Inhibitors , Cyclooxygenase Inhibitors/administration & dosage , Dogs , Injections , Isoxazoles/administration & dosage , Macaca fascicularis , Male , RatsABSTRACT
Monoclonal antibodies (mAb) made to the superpotent guanidino sweet tasting ligand, N-(p-cyanophenyl)-N'-(diphenylmethyl)-guanidineacetic acid were examined for their molecular recognition specificities using 14 different sweetener analogues in a competitive radioimmunoassay. The effects of variations in pH on ligand binding was also examined by radioimmunoassay. Photoaffinity labelling of the binding site was accomplished using a radiolabelled azido-derivative of the parent ligand, and L-chain or H-chain labelling was easily identified in several different mAb. For two of the mAb examined in this study (NC6.8 and NC10.14), the analogue binding studies are in agreement with the known Fab-ligand crystal structures. Monoclonal antibodies to this family of sweet tasting compounds may be useful probes for the study of sweet taste chemistry and identification of novel sweet taste ligands from combinatorial chemical libraries.
Subject(s)
Acetates/immunology , Guanidines/immunology , Sweetening Agents , Antibodies, Monoclonal/immunology , Hydrogen-Ion Concentration , Ligands , Molecular StructureABSTRACT
Azido-functionalized analogs of potently sweet guanidinoacetic acids have been synthesized for use as sweetener receptor photoaffinity labeling reagents. These compounds have been synthesized using readily available starting materials. One of the azido-labeled guanidinoacetic acids has been evaluated in an electrophysiological model in the Rhesus monkey. We found that the photoaffinity-labeling reagent caused irreversible inhibition in electrophysiological response to sweeteners upon exposure of the monkey tongue to a combination of the reagent and UV light.
Subject(s)
Affinity Labels/chemical synthesis , Glycine/analogs & derivatives , Taste/physiology , Affinity Labels/chemistry , Animals , Aspartame/pharmacology , Electrophysiology , Glycine/chemical synthesis , Glycine/chemistry , Macaca mulatta , Photolysis , Sweetening Agents/pharmacology , Taste/drug effects , Thiazines/pharmacology , Tongue/drug effects , Tongue/physiology , Ultraviolet RaysABSTRACT
The interactive residues for mouse mAb NC10.8, which binds a superpotent guanidinium sweetener N-(p-cyanophenyl)-N'-(diphenylmethyl)guanidinoacetic acid with high affinity (Kd = 5 nM), were examined by using radioligand competitive binding, photoaffinity labeling, absorption and fluorescence spectroscopy, computer-aided molecular modeling, and site-directed mutagenesis. Competitive ligand analogue binding data revealed important structural features and a pH sensitivity for ligand binding. Spectroscopy of the sweetener-mAb complex revealed ligand-induced fluorescence quenching and the presence of a charge-transfer band. Site-directed mutagenesis of L:96W abolished the ligand-induced fluorescence quenching and reduced Ab affinity. The apparent Kd increased from 5 nM to more than 200 nM after such modification. A theoretical model of the Fv region was generated with use of a knowledge-based algorithm, and this model was used to identify the locations of key residues in the complementarity determining regions. These experimental and theoretical studies support the prediction that the sweetener ligand coordinates with the following residues: L:34H contacts the cyanophenyl ring, L:27DR forms a salt bridge with the acetic acid moiety, L:96W forms a pi-pi interaction with the cyanophenyl ring, and H:95E contacts the positively charged aryl nitrogen. These studies are important to our understanding of Ab-ligand specificity and may also shed light on the important chemical motifs responsible for elevated levels of sweetness potency in organic compounds.
Subject(s)
Acetates/immunology , Antibodies, Monoclonal/immunology , Binding Sites, Antibody , Guanidines/immunology , Sweetening Agents , Amino Acid Sequence , Antibodies, Monoclonal/chemistry , Base Sequence , Cloning, Molecular , Computer Simulation , DNA Primers/chemistry , Ligands , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Structure, Tertiary , Spectrometry, Fluorescence , Structure-Activity RelationshipABSTRACT
N-(4-Cyanophenyl)-N'-(2-carboxyethyl)urea (2), an analogue of suosan [1,N-(4-nitrophenyl)-N'-(2-carboxyethyl)urea], is a known high-potency sweetener derived from beta-alanine. Sulfonic and phosphonic acid analogues of 2 were prepared to develop structure-activity relationships through modification of the carboxylic acid region of this family of sweeteners. Neither of the carboxylic acid replacements resulted in sweet analogues. However, we found that N-(4-cyanophenyl)-N'-[(sodiosulfo)methyl]urea (7) is an antagonist of the sweet taste response. The bitter taste response to caffeine, quinine, and naringin was also antagonized. Antagonist 7 was found to inhibit the sweet taste perception of a variety of sweeteners. Antagonist 7 had no effect on the sour or salty taste response.
Subject(s)
Aspartic Acid/analogs & derivatives , Carboxylic Acids/chemistry , Flavanones , Phenylurea Compounds/pharmacology , Sweetening Agents , Taste/drug effects , beta-Alanine/analogs & derivatives , beta-Alanine/pharmacology , Aspartic Acid/chemistry , Aspartic Acid/pharmacology , Caffeine/antagonists & inhibitors , Flavonoids/antagonists & inhibitors , Humans , Phenylurea Compounds/chemistry , Quinine/antagonists & inhibitors , Structure-Activity Relationship , beta-Alanine/chemistryABSTRACT
Sulbactam (1) is a beta-lactamase inhibitor with limited oral bioavailability. Lipophilic double-ester prodrug sulbactam pivoxil (2) significantly improves the oral absorption of sulbactam, as does the mutual prodrug double ester sultamicillin (3). We have found that double-ester prodrugs of sulbactam terminating in a carboxyl group (8) also were effective oral-delivery vehicles in rats. Carboxyl-terminated double esters have several potential advantages over their nonionizable lipophilic counterparts, including water solubility, crystallinity, choice of salts for dosage forms, and formation of innocuous byproducts on hydrolysis.
