Drug repurposing has become an important branch of drug discovery. Several computational approaches that help to uncover new repurposing opportunities and aid the discovery process have been put forward, or adapted from previous applications. A number of successful exam-ples are now available. Overall, future developments will greatly benefit from integration of different methods, approaches and disciplines. Steps forward in this direction are expected to help to clarify, and therefore to rationally predict, new drug-target, target-disease, and ulti-mately drug-disease associations.

Aim: The EGFR inhibitors represent the first-line treatment of non-small-cell lung cancer. However, the emergence of resistance urgently requires the development of new inhibitors targeting drug-resistant mutants. Methodology: A recently released structure of an EGFR kinase domain in complex with an allosteric inhibitor and a mutant protein model derived from it were used to set up a low-cost high-throughput docking protocol for the fast identification of EGFR allosteric inhibitors. Conclusion: The virtual screening of commercially available compounds led to the identification of interesting new hit compounds. The most promising hit was confirmed to be a new allosteric inhibitor of wild-type and T790M/L858R double mutant EGFR which was able to inhibit the growth of non-small-cell lung cancer cell lines. Graphical abstract

More than 141 000 inhibitors of human kinases and their activity data were assembled to perform an in-depth analysis of inhibitor promiscuity (single- versus multi-kinase activity) at varying activity data confidence levels. For ∼20% of these inhibitors, it was also possible to consider test frequency and inactivity information. Only small subsets of highly promiscuous inhibitors were identified. Nearly 95% of more than 45 000 inhibitors with high-confidence data were only active against one or at most two kinases. At decreasing data confidence levels, more than 92 000 kinase inhibitors were on average active against two kinases. When taking all activity information without any restrictions into account, the mean promiscuity degree of kinase inhibitors was less than four and notably biased by small numbers of highly promiscuous inhibitors. Even under these conditions, more than 70% of all inhibitors were active against a single kinase. There was only small-scale progression of inhibitor promiscuity when data confidence criteria were iteratively removed during the analysis. Furthermore, the majority of inhibitors that were tested against 10 to 20 different kinases were only active against a single kinase. The results of our activity data-driven analysis indicate that promiscuity of kinase inhibitors cannot generally be assumed. Many inhibitors retain single-kinase activity at decreasing data confidence criteria or increasing test frequency. Hence, on the basis of currently available data, many kinase inhibitors are selective, which is an important aspect for drug development.

The recent discovery of the fourth generation EAI045 allosteric inhibitor, which potently and selectively inhibits mutant EGFR, represents an important step forward for the treatment of non-small cell lung cancer. However, the structural determinants of EAI045 selectivity with respect to the wild type (wt) protein have not been fully investigated. To this aim, we performed a comparative analysis of long-scale molecular dynamics simulations and binding free energy calculations on wt and T790M EGFR in complexes with the EAI001 and EAI045 allosteric ligands. Unexpectedly, we found that the observed selectivity for T790M EGFR over wt is not due to more favorable interactions of the two ligands with the mutated gatekeeper residue, as previously suggested. Rather, the allosteric ligands were engaged in a direct hydrogen bond with the Asp855 residue of the DFG motif in mutant T790M but not in wt, in which the hydrogen bond was found to be water-mediated. Per-residue decomposition of binding free energies suggests that the loss of a direct interaction with Asp855 is the main cause of inhibitor selectivity. Moreover, the possibility that the allosteric ligands and adenosine triphosphate may have synergistic binding effects, as previously observed in MEK allosteric inhibitors, was investigated. Altogether, the results suggest that ligand selectivity arises from direct hydrogen bonds with the Asp855 side chain, and that the design of mutant-selective inhibitors should be focused on ligands that form direct hydrogen bonds with Asp855 in T790M EGFR but not in wt EGFR. These results may provide useful hints for future structural design of mutant-selective allosteric inhibitors that spare wt EGFR, which is a highly desirable goal.