Repositioning the Substrate Activity Screening (SAS) Approach as a Fragment‐Based Method for Identification of Weak Binders

Fragment‐based drug discovery (FBDD) has evolved into an established approach for “hit” identification. Typically, most applications of FBDD depend on specialised cost‐ and time‐intensive biophysical techniques. The substrate activity screening (SAS) approach has been proposed as a relatively cheap and straightforward alternative for identification of fragments for enzyme inhibitors. We have investigated SAS for the discovery of inhibitors of oncology target urokinase (uPA). Although our results support the key hypotheses of SAS, we also encountered a number of unreported limitations. In response, we propose an efficient modified methodology: “MSAS” (modified substrate activity screening). MSAS circumvents the limitations of SAS and broadens its scope by providing additional fragments and more coherent SAR data. As well as presenting and validating MSAS, this study expands existing SAR knowledge for the S1 pocket of uPA and reports new reversible and irreversible uPA inhibitor scaffolds.

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