The discovery of antibiotics has revolutionized medicine practice in many respects, from the prevention to the treatment of
infections. The proliferation of multi drug resistant pathogens has compromised these achievements, and the hard-won victories
against infectious diseases of the last fifty years are jeopardized. β-Lactamases, in particular metallo-β-lactamases (MBLs), represent
the most worrisome resistance mechanism since they can hydrolyze almost all β-lactam antibiotics including the so-called “last
resort” carbapenems, and no inhibitors are currently available on the market for MBLs (in the pipeline only taniborbactam is
currently in clinical phase III).
This project (Fighting CArbapenemase RESistance by kinetic target guided Synthesis, CARESS) aims to use an innovative approach,
Kinetic-Guided Target Synthesis (KTGS), to discover novel inhibitors of MBLs, with a particular focus on NDM-1, to tackle
antimicrobial resistance. KTGS is an innovative strategy for drug-discovery where the biological target is employed to catalyse the
synthesis of its own inhibitors from a library of reagents. This platform has enabled the development of several inhibitors throughout
a rapid and efficient screening of several small-molecules. Most of experiments reported for KTGS focus on the formation of triazoles
starting from alkynes and azides and the inner biocompatibility of these starting materials, combined to their easy availability, has
favoured the development of “in situ click chemistry”, where the biological target itself, lowering the activation energy of the
reaction, promotes the assembly of triazole. The tremendous potential of KTGS, and more specifically of in situ click chemistry, has
not been exploited for the development of novel β-lactamases inhibitors yet. In CARESS we propose four different azide-bearing
warheads, specifically designed to bind the catalytic Zinc ions of NDM-1, and several reasoned sets of diverse alkynes, to perform
KTGS by in situ click chemistry. The compounds formed into the catalytic pocket will be detected by High Resolution Mass
Spectrometry (HRMS) and synthesized on mgs scale to perform kinetic analysis against NDM-1 and other MBLs variants such as
VIM-1, IMP-1 and their natural variants of clinical interest. The active hit compounds will be tested with a collection of isogenic laboratory strains of E. coli, K. pneumoniae and P. aeruginosa producing NDM-1. For the best performing inhibitors crystal structures
of the complex enzyme-inhibitor will be obtained, and few analogs will be synthesized and evaluated, aiming to improve affinity and
bioavailability. Advanced microbiological profiling of selected inhibitor-β-lactam combinations will then be carried out with a
collection of clinical strains of Enterobacterales and P. aeruginosa expressing different patterns of β-lactamases and other β-lactam
resistance mechanisms.