Modulation of New Delhi Metallo-β-Lactamase-1 activity using Affimers

Metallo-β-lactamases (MBLs) are a class of Zn2+ containing enzymes that hydrolyse the β-lactam bond in β-lactam antibiotics and render them inactive. New Delhi metallo-β-lactamase 1 (NDM-1) is particularly troubling as the gene blaNDM-1 is plasmid borne resulting in rapid widespread dissemination. With few effective antibiotics against NDM-1 expressing bacteria, and resistance developing to those which remain, there is an urgent need to find new MBL inhibitors. Combining β-lactam antibiotics with β-lactamase inhibitors is considered amongst the most reliable of strategies for tackling resistant pathogens. However, the development of effective β-lactamase inhibitors has proved challenging. MBLs share a conserved active site with a number of human proteins and therefore orthosteric inhibitors often result in adverse side effects. Affimers may represent an alternative for the selection of novel MBL inhibitors. An Affimer that binds NDM-1 and inhibits it in a dose dependent manner was isolated, as confirmed in β-lactamase activity assays and cell based assays in clinical strains. Initial results indicated that Affimer 21 is an allosteric inhibitor, suggesting that it may be highly specific for NDM-1, reducing the possibility of off-target effects. To elucidate the binding interaction, attempts were made at obtaining a co-crystal structure and alanine scanning mutagenesis of the two variable regions was performed. The functional effects of the alanine substitutions on efficacy of NDM-1 inhibition were measured and the data used to guide the generation of sub-libraries to select for improved inhibitors. However, despite stringent screening approaches, the original Affimer has so far proven to be the the most effective inhibitor, with an estimated IC50 of 0.37 μM. Further investigation into the use of Affimers as a platform for identifying novel inhibitors effective against metallo-β-lactamases could represent a useful tool in the ongoing battle against antibiotic resistant pathogens