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Fragment-based drug design of antivirals targeting the polyomavirus capsid and flaviviral NS3-helicase
Book - Dissertation
BK and JC viruses are closely related, non-enveloped, pathogenic DNA viruses of the polyomavirus family for which there are no approved treatments. These viruses cause latent infection in humans and have an extremely high prevalence worldwide (70-90% of the population is seropositive). However, clinical manifestation is seen only in immunocompromised patients which can lead to severe and often fatal complications. BK virus for instance, is a kidney and urogenital tropic virus which causes viral nephritis or cystitis and ultimately allograft failure in organ transplant recipients. At least 30-40% of all kidney transplant patients develop viruria, a quarter of which develop nephropathy and graft loss. In comparison, JC virus reactivation in the central nervous system (CNS) leads to the development of progressive multifocal leukoencephalopathy (PML), a devastating disease characterized by high mortality. PML is increasingly seen in patients with autoimmune diseases (Multiple Sclerosis, Rheumatoid Arthritis etc.) that are taking immune-suppressants (Natalizumab, Rituximab etc.). Currently, the only way to prevent the progression of viral disease is discontinuation of immunosuppressant therapy, which is not a viable therapeutic strategy as it either leads to aggravation of the underlying autoimmune disease or graft rejection by the host immune response. The small size of the virus genome provides limited prospects for the targeted design of antiviral drugs against viral proteins. The Large T-Antigen of polyomaviruses, which has a helicase function and VP1 capsid protein, are the only two prominent and indispensable viral proteins that can serve as drug-targets. The VP1 protein is an indispensable building block of the viral capsid, which exists in a pentameric form; oligomerization of 72 pentamers leads to the assembly of virus-like particles. Due to its central role in virus capsid assembly, viral entry and other host-interactions, we decided to take a mechanism agnostic approach to identifying drug-pockets and drug-ligands towards this protein. Fragment-based screening using two complementary biophysical techniques (i.e. a thermal shift assay and X-ray crystallography) enabled us to identify multiple novel drug-binding sites on the protein as well as fragment drug "hits" suitable for rational, structure-based optimization. One fragment-binding site, dubbed the F50 pocket, appears central to pentamer-pentamer interactions and thus virus assembly. Further work has led to the identification of multiple scaffolds that bind to this pocket providing a compelling pharmacophore that can be leveraged for the design of higher affinity compounds in the future. Multiple biological and mechanistic assays have also been developed for the testing of such drug compounds. Structure-based optimization has been initiated as part of this thesis work.