Song, Weixiao; (2022) Fragment Based Drug Discovery of Southampton 3C-like Protease and SARS-CoV-2 Main Protease. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Fragment-based drug design (FBDD) is applied to develop potent compounds from fragments and has become increasingly important in drug discovery. Proteins constitute a broad category of drug targets and are widely used in drug discovery, and protease is one of the most focusing classes of protein drug targets. Viral 3C or 3C-like protease (3Cpro or 3CLpro) is responsible for the cleavages of the virus corresponding viral polyprotein or intermediate virus proteins. It is encoded in various viruses, including human rhinovirus, enterovirus, poliovirus, foot-and-mouth disease virus, hepatitis A virus, noroviruses and human coronavirus. 3Cpro or 3CLpro is an attractive drug target for antiviral drugs because of its role in viral replication and transcription. Here we focus on developing antiviral drug leads for Southampton 3C-like protease (SV3CP) based on virtual screening, kinetic assay and X-ray crystallography. We screened 1083 compounds based on our own designed screening library and identified five ligands (S01-S05), and we determined the crystal structures of SV3CP in complex with S02 and S05. We identified these five ligands with Ki values ranging from 0.077 to 1.325 mM against SV3CP. Our results suggested the efficacy of our fragment based inhibitor development. The emerging of a new coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is responsible for the 2019 pandemic of COVID-19. The main protease (Mpro) in SARS-CoV-2 is one of the key enzymes of coronaviruses and plays a vital role in mediating viral replication and transcription. It is also a 3C-like cysteine protease (3CLpro) and is a potential drug target for SARS-CoV-2. There are no approved drugs and antiviral treatments for SARS-CoV-2 currently. SARS-CoV-2 Mpro shares similar active sites with SV3CP, which led us to repurpose our ligands to it. We developed a kinetic assay for SARS-CoV-2 Mpro showing Ki values of these five ligands range from 9.3 μM to 0.87 mM, and we determined crystal structures of SARS-CoV-2 Mpro in complex with ligand S04 and S05. These ligands show their potential as broad-spectrum drug leads due to their inhibition activity in different 3CL proteases. We used another crystallography method – lipid cubic phase (LCP) to define the structures of different states of CRP when it binds to the membrane. LCP is widely used in understanding the structure and function of membrane proteins, while it is novel for crystallising CRP. Pentameric CRP can dissociate into monomeric CRP, and isoforms of CRP have different functions. The inhibition of pCRP dissociation may provide a new strategy for immunomodulatory therapy. We identified a monomer structure of CRP using LCP and determined the optimised condition for crystallising CRP through LCP. We identified the difference between pCRP and mCRP when bound to the membrane, and understanding the dissociation allows the development of novel anti-inflammatory drugs in the future.

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