Protein Scaffolds to Enable Structure-Based Drug Discovery

Abstract

Structure-based drug discovery (SBDD) uses 3D structural information about disease targets to guide the design of effective drug compounds. A technical caveat to obtaining structural information is that some targets are not amenable to current structural techniques such as X-ray crystallography and cryo-electron microscopy (cryo-EM). This work aims to engineer protein scaffolds to display prospective drug targets (termed “cargo” molecules), adding the bulk, rigidity, and regularity required to enable structural elucidation of these targets using cryo-EM. Prospective scaffolds were designed based on a dodecameric acetohydroxy acid isomeroreducase (AHIR) and a Designed Ankyrin Repeat Protein (DARPin). Initial investigation and cryo-EM imaging of these scaffold approaches revealed that although the DARPin was not successfully resolved, AHIR could be readily reconstructed to resolutions beyond 2.5 Å. This demonstrated that AHIR could be imaged to high resolution by cryo-EM and was therefore deemed a promising scaffold for cryo-EM studies. Various approaches to display a range of small (<52 kDa) exemplar cargos on the AHIR scaffold were explored. These included a two-point binding interaction between cargo and scaffold, a rigid helical fusion of cargo to the AHIR C terminus and fusion of a cargo within a surface exposed loop on AHIR. Of these approaches, the rigid helical fusion and the binding interaction produced successful cryo-EM reconstructions reaching 3 Å and 5 Å resolution respectively. In parallel, cryo-EM experiments also informed development of a crystallographic Crysalin scaffold, composed of AHIR and streptavidin. Imaging of strategic AHIR mutants provided insights into generating order within the Crysalin lattice. In summary, results have indicated that AHIR is a versatile scaffold protein showing promise in both cryo-EM and crystallographic applications, with the ability to display cargo proteins effectively. Future work will focus on structural determination of novel targets using the AHIR scaffold, thereby expediting SBDD against important disease targets.