to almost 25Å of the centre of geometry (centroid) of the protein ligands. Of note, the two most tested substitutions (resistance substitution T790M and activating substitution L858R) showed very high coefficients of difference but different locations with respect to the binding pocket (0.80 and 9.77Å, and 1.01 and 16.60Å, respectively). These two substituted residues are in the binding pocket of EGFR and correspond, respectively, to the gatekeeper residue and the back cleft. In contrast, HIV-1 RNaseH-RT harboured 31 single substitutions, of which 64.52% had an Epstein coefficient of difference lower than 0.4 (Figure ). Of note, these substitutions were concentrated around the non-nucleoside reverse transcriptase inhibitor (NNRTI) binding site, with distances to the ligand centroid mostly below 15Å. The only E. coli proteins with structural data, acetylglucosamine deacetylase LPXC (P0A725), and dihydrofolate reductase DYR (P0ABQ4), showed six substitutions affecting either size or polarity (Figure ), and were located around 15Å of the ligand centroid. The type of amino acid substitution, as well as the distance from the substituted residue to the ligand binding site, could affect the bioactivity of certain small molecules towards different variants. From a biological point of view, enriched human variants are likely to be disease-related whereas variants in pathogenic organisms are more likely linked to drug resistance. The extent of such an effect and its potential relevance in bioactivity modelling was analysed in the following sections.