Selectivity determining features in N-myristoyltransferases – a model system for drug targets with conserved binding sites
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Abstract
In rational drug design projects one major goal is to obtain high affinity ligands for a target, while maintaining selectivity over potential off-targets and thereby reducing unwanted side effects. However, this proves to be challenging when facing highly conserved binding sites. In this project, the focus was laid on a model system of two well investigated essential eukaryotic enzymes, which catalyze the transfer of myristate to a various number of substrates – the N-myristoyltransferases (NMT) of Leishmania major (LmNMT) and the human homologue HsNMT1. The enzymes share an overall sequence identity of over 40 % and an identical first protein-ligand interaction shell. Many non-selective NMT-inhibitors were found previously, but only a few selective ones are known. Further, the molecular basis for selective inhibition was unclear. A combination of molecular dynamic simulations (MDs), isothermal titration calorimetry (ITC), fluorescence-based enzyme inhibition assay and X-ray crystallography was used to analyze protein dynamics, water network formation and their changes upon ligand binding. Two different selectivity determining features were identified and validated by site-directed mutagenesis – the impairment of protein flexibility upon ligand binding close to the catalytically active C-terminus and a highly stable water molecule, only present in the binding site of HsNMT1. Based on these findings, a virtual screening was conducted and three novel and selective LmNMT inhibitors were revealed.