Morling, Katherine Lisa; (2024) Design and characterisation of small molecules that enhance and restrict lentiviral infection. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Human immunodeficiency virus 1 (HIV-1) both utilises and evades host proteins to maximise replication and minimise innate immune activation. We have designed and characterised molecules that manipulate host-pathogen interactions to restrict and enhance HIV-1 infectivity. Combination therapy can rapidly suppress HIV-1 replication, but resistance and side eIects can occur, therefore there is an ongoing need for inhibitors with novel mechanisms. The HIV capsid is a molecular machine built of ~250 hexamers and 12 pentamers of capsid protein assembled in a conical lattice. Capsid has multiple essential functions during the HIV lifecycle, particularly in safeguarding reverse transcription, and is thus an attractive drug target. We have developed HIV-1 capsid inhibitors that bind a conserved pocket that sequentially interacts with host cofactors Sec24C, Nup153 and CPSF6. These molecules (allosteres) inhibit HIV-1 with nanomolar potency. Allosteres destabilise capsid cones, causing HIV-1 to activate innate immune sensing and inhibiting reverse transcription. We find the same resistance profiles between allosteres and capsid- targeting drug lenacapavir. Despite these escape mutants arising rapidly in vivo, we find that they trigger cGAS-dependent innate immune sensing more eIectively than wild-type virus, implying a potential barrier to transmission. HIV-derived viral vectors are used in hematopoietic stem cell (HSC) gene therapy to deliver therapeutic genes, providing lifesaving treatments for many genetic diseases. However, transduction eIiciency is limited by intrinsic expression of interferon-stimulated genes in HSCs, including restriction factor IFITM3 which inhibits viral entry. We have developed cyclosporine A (CsA) derivatives with reduced cyclophilin A binding that transiently induce IFITM3 degradation and enhance lentiviral vector transduction. However, the direct target of these molecules is unclear. We developed CsA-PROTAC probes and performed global proteomics to identify novel CsA targets to explain transduction enhancer mechanism. In both projects, our results inform cofactor and restriction factor mechanisms and are guiding design of improved inhibitors.

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