Understanding pre-mRNA splicing regulation using molecular simulation and ligand design

Abstract

Splicing of RNA is a process where freshly transcribed pre-mRNA turns into mature messenger RNA ready for translation within eukaryotic species. This process is made possible by the spliceosome, a highly complex and dynamic molecular machine. Pre-mRNA splicing is an essential and conserved process and aberrant splicing has been linked to many genetic diseases and some cancers. Creating new drugs for these diseases and understanding how to regulate splicing is important but only possible with greater understanding of this complex molecular machine. Small molecule inhibitors are currently being developed to be able to modulate splicing and also to further understand its regulation. Snu114, a key GTPase protein involved in regulating splicing, is a spliceosomal protein that can be targeted for inhibiting ligand design. In silico methods were employed in this study first for structural analysis with results seemingly suggesting the Snu114 retains a rigid structure through all stages of the splicing cycle which is unlike the similar GTPase EF-G. Docking into the proposed binding site of Snu114 was done, including fusidic acid, an inhibitor of EF-G. Rapid overlay of chemical structures (ROCS) virtual screening was done using two steroids with differing conformations in their ring fusion system, fusidic acid and lithocholic acid. A range of compounds were discovered from this, some with the same steroidal core and others with significantly different structures. The top compounds from ROCS were docked into Snu114. The idea is proposed that ligand-protein binding can be achieved with ligand structures that deviate from the steroidal ring core. Molecular dynamics simulations were performed, and probe ligands were later included in the solvent to elucidate any hidden binding sites. The results yield a novel postulate for the role of Snu114: a structural scaffold. Finally, an alternative binding site is proposed rather than the GTP-binding area that was initially assumed.

Date of Award	6 Feb 2023
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Raymond O'Keefe (Supervisor), Richard Bryce (Supervisor) & Roger Whitehead (Supervisor)