DiscussionDrug target validation is the process of identifying and validating a novel target prior to the development of small molecule inhibitors. Current methods for validation include RNA interference (RNAi) and development of small molecule probes. RNAi reduces levels of the protein so potentially affecting multiple signalling pathways, while small molecule probes can take a long time and are expensive to develop. Consequently, alternative methods for target validation that are quick, cheap and act in a similar manner to small molecules are highly sought.
Here we describe the development of a novel, artificial binding protein (Adhiron – commercially marketed as Affimers) from which a high quality phage-display library has been constructed. The Adhirons are based on a consensus sequence, have a high melting temperature and constrain two nine amino acid loops for molecular recognition. We initially screened the Adhiron library against yeast SUMO and within two weeks isolated reagents with high binding affinities and specificity. To further test the ability of the library to be used in target validation we screened against two SH2 domains from target proteins, p85 and Grb2, and tested the isolated Adhirons for specificity in ELISA and the ability to inhibit protein function in a range of in vitro and in vivo assays. Our work demonstrates the ability to use the Adhirons to identify reagents to study and inhibit protein function highlighting their potential as novel drug target validation tools.
Rebecca Ross1*, Matthew Balmford3 Christian Tiede2*, Anna Tang2, Rob Bedford2, Upasana Mandal2, Margaret Knowles1, Michael McPherson2,3, Darren Tomlinson2,3.
1 Section of Experimental Biology, Leeds Institute of Cancer and Pathology, University of Leeds, UK.
2BioScreening Technology Group, Institute of Molecular and Cellular Biology, University of Leeds, UK.
3Astbury Centre for Structural and Molecular Biology, University of Leeds, UK.
*contributed equally to this work