Biochemical and molecular characterization of GES and NDM engineered variants: interactions with β-lactams and inhibitors

Antibiotic resistance occurs when bacteria causing an infection survive after being exposed to a drug that, under normal conditions, would kill it or inhibit its growth. As a result, these surviving strains multiply and spread due to the lack of competition from other strains sensitive to the same drug. Carbapenems and 3rd generation cephalosporins resistant Enterobacteriaceae represent one of the most critical group against which there is an urgent need to develop new antibiotics. These bacteria are common pathogens causing severe infections such as bloodstream infections, pneumonia, complicated urinary tract infections and complicated intra-abdominal infections. According to the Centers for Disease Control and Prevention (CDC) definition, CRE are defined as any Enterobacteriaceae which are resistant to any carbapenem or are documented to produce a carbapenemase. There are three major mechanisms by which Enterobacteriaceae become resistant to carbapenems: enzyme production, efflux pumps and porin mutations. Of these, enzyme production is the main resistance mechanism. Gram-negative bacteria generally develop resistances through the production of β-lactam-hydrolyzing enzymes, called β-lactamases. In the present study, two classes of these enzymes were analyzed and characterized also in association with a new molecule with inhibitory activity. In the first part of this thesis, the role of the Ω-loop (159-182 residues) in GES class A β-lactamase is well investigated. Recent studies report that, in these enzymes, carbapenemase activity is attributed to a single amino acid substitution at position 170. In our study, we decided to investigate other positions of the Ω-loop, in particular residue 174, occupied by a proline residue. This residue is well conserved in class A β-lactamases. Our kinetic and computational data have demonstrated that not only 170 residue is important in the carbapenemase activity, but also residue 174. Being Ω-loop a mobile structure of class A β-lactamases, we suppose that any mutation in this region could modify the substrate hydrolysis of the enzyme, not only toward carbapenems. The second part of the thesis is addressed to the involvement of some luecines located in the Loop 10 of NDM-1 and the effect of a new boronic acid molecule (taniborbactam) as inhibitors of metallo-β-lactamases. We have pointed the attention on residues L218, L221 and L269 that give hydrophobicity to the enzyme. Our results have demonstrated that the replacement of these leucines reduces the enzyme catalytic activity toward β-lactams. In addition, we studied the effect of Y229W substitution in our laboratory variant (L209F) with a drastic reduction in β-lactamase activity. This substitution is able to restore the catalytic activity in the L209F mutant enzyme. Actually, one of the most problem is related to the absence of inhibitors for metallo-β-lactamases in clinical therapy. For this reason, in collaboration with VenatoRx Pharmaceuticals (Malvern, PA, USA), we tested a new boronic acid, VNRX-5133, combinated with cefepime (taniborbactam), in NDM-1 and some engineered NDM-1 mutants able to hydrolyze β-lactams very efficiently. Taniborbactam was also tested in NDM-1- producing Enterobacteriaceae clinical strains. Our results revealed that the addition of VNRX-5133 restored cefepime activity in both NDM-1 producing recombinant strains of E. coli and clinical isolates of Enterobacteriaceae. The development of new non-β-lactam inhibitors would provide a new opportunity for the treatment of bacterial infections with existing antibiotics.