The main mechanism of resistance to β-lactam antibiotics is represented by the appearance of β-lactamases. A representative example is the class A KPC-2 β-lactamase that in a few years became one of the most menacing β-lactamase currently spreading worldwide. We have developed a protocol using the PMX software that allows the evaluation of the energetic cost associated with a mutation in the active site of a β-lactamase, in the presence of the β-lactam substrate. The identification of the most favourable mutations will guide the development of new β-lactamase inhibitors, which will be active not only against the currently known mutants, but also against those that are likely to appear in the near future. This protocol include three steps: i) conformational space sampling; ii) in silico mutation (morphing) and iii) free energy estimation. The molecular dynamics simulations are carried out using Gromacs and the OPLS-AA force field. The force field parameters of ligands are taken from Ligandbook (https://ligandbook.org) and the experimental data from the Beta-Lactamase DataBase (BLDB, http://bldb.eu). This protocol was validated by comparison with various experimental data. The difficulties associated with the mutations involving a change in the overall charge will be discussed. A variant of this protocol involves mutations on a ligand in complex with the protein of interest. The use of this variant for our participation to the D3R Grand Challenge 2 will also be presented.

Towards in silico prediction of β-lactamase-mediated antibiotic resistance using free energy calculations

255th National Meeting (2018)

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