ABSTRACT
9G4H9, a catalytic antibody displaying ß-lactamase-like activity, has been developed by the anti-idiotypic approach using ß-lactamase as the first antigen. Thus 9G4H9 represents the 'internal image' of ß-lactamase. We selected a cyclic peptide anchored to a bacteriophage M13 library using 9G4H9 as the target. Pep90 is a cyclic heptapeptide enclosed between two cysteine residues. We showed that Pep90 could inhibit both TEM-1 ß-lactamase (K(i) = 333 µm) and several penicillin-binding proteins (IC50 values ranging from 6-62 µm). We determined that the tryptophan residue of Pep90 is of crucial importance for its inhibitory activity. Using Pep90 as a scaffold, we generated a new class of peptidomimetics that retained inhibitory activity towards TEM-1 ß-lactamase.
Subject(s)
Penicillin-Binding Proteins/antagonists & inhibitors , Peptides, Cyclic/chemistry , Peptides, Cyclic/pharmacology , beta-Lactamase Inhibitors , Antibodies, Catalytic/chemistry , Antibodies, Catalytic/genetics , Bacteriophage M13/genetics , Catalytic Domain , Kinetics , Models, Molecular , Mutagenesis , Peptide Library , Peptides, Cyclic/genetics , Peptidomimetics/chemistry , Peptidomimetics/pharmacology , Protein Engineering , Tryptophan/chemistry , beta-LactamasesABSTRACT
Data on catalytic antibodies (abzymes) having critical roles in pathologies, for example in some auto-immune diseases accumulate at overwhelming pace. Nevertheless, the misunderstanding of how antibodies can mimic a catalytic activity may hamper the development of therapeutic tools. We thus investigated the structure function roles of residues of a catalytic antibody endowed with a beta-lactamase activity. The catalytic antibody 9G4H9 was generated using the internal image properties of anti-idiotypic antibodies. The single-chain fragment was cloned and produced in Escherichia coli. Based on the structure function knowledge of beta-lactamases pattern and on the tridimensional model of the scFv, five residues were selected for mutagenic analysis to learn about the contribution of putative residues in catalysis. Light chain mutants R24A, S26A, S28A, and E98A lost catalytic activity significantly. Mutant K27A retained catalytic activity but the estimated K(M) was affected. Kinetic outcomes support the argument that S26 and S28 function as nucleophile and E98 as general acid/base catalyst. We have selected a peptide Pep90 able to inhibit 9G4H9 catalytic activity. We also demonstrate the tryptophan and proline residues of Pep90 (YHFLWGP) are responsible for binding and inhibitory properties of Pep90 on 9G4H9. A three-dimensional model docked with Pep90 is therefore built in which critical residues of Pep90 are buried at the interface of CDR-L1 and CDR-L3 loops whereas other are exposed to the solvent.
