Investigating the role of cryo-EM as a tool for structure-based drug design

Structure based drug design (SBDD) programs have traditionally relied upon structural information derived from X-ray crystallography. However, despite the high-resolution structures, the information obtained is a static snapshot of flexible biological systems. As the proteins are not constrained by a crystal lattice, EM can provide unique insights into the dynamics and conformational states of the system. Moreover, the structures of biological systems which were previously intractable to X-ray crystallography projects, such as membrane proteins, have now been determined providing a wealth of structural information to be utilised in SBDD programs. This thesis presents two examples of cryo-EM being used to aid SBDD for different membrane proteins. One example is Cytochrome bc1, which is a validated anti-parasitic drug target. Cryo-EM has been used in a proof of principle approach to determine four structures of bovine bc1 in the absence and presence of four different inhibitors to ~3.3-4.6 Å resolution. The resolutions attained have enabled the inhibitors to be unambiguously positioned in the density, thereby Chapter 3 provides one of the first examples of inhibitor density being visualised using cryo-EM. The second system studied is the Vacuolar ATPase (V-ATPase) which is at an early-stage of the drug discovery pipeline. By using a virtual screening approach, several hit compounds have been identified to act upon V-ATPase. Derivatives of the hit compound have been synthesised, resulting in three compounds which have <100uM IC50 values. Moreover, the cryo-EM structure of V-ATPase in the apo and inhibitor bound form were determined. Although resolutions were not sufficient to visualise inhibitor binding, a novel state of V-ATPase has been identified which shows the complex without the H-subunit present. Overall the work in this thesis shows that cryo-EM can play an important role in SBDD programs.