Fragment growing exploiting dynamic combinatorial chemistry of inhibitors of the aspartic protease endothiapepsin

Fragment-based drug design (FBDD) has emerged as an efficient hit-identification and/or -optimization strategy with a higher hit rate than high-throughput screening (HTS). Whereas fragment linking is more challenging, fragment growing has become the preferred fragment-optimization strategy, requiring synthesis of derivatives and validation of their binding mode at each step of the optimization cycle. Dynamic combinatorial chemistry (DCC) is a powerful and efficient strategy to identify ligands for biological targets. Here, we have demonstrated that the novel combination of fragment-growing and DCC is a highly powerful strategy to grow a fragment into a more potent, non-covalent inhibitor of the aspartic protease endothiapepsin. We have designed a library of acylhydrazones using fragment growing starting from a known fragment in complex with endothiapepsin. We have used DCC and a fluorescence-based enzymatic assay to identify the best hit(s) from the dynamic combinatorial libraries, displaying double-digit micromolar inhibition of endothiapepsin. In addition, each DCC experiment requires only very small amounts of protein compared with established methods of analysis and the protein needs to be in the assay mixture only for a short period of time, making this protocol ideal for precious and unstable proteins. These results constitute a proof of concept that the combination of fragment growing and DCC is a powerful and efficient strategy to convert a fragment into a hit.