Drug repurposing : fast-tracking antifungal drug discovery for cryptococcal meningitis

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Cryptococcal meningitis is the most common form of meningitis in HIV‐infected persons. It is a life-threatening fungal infection of the brain that disproportionately affects the poorest and most resource‐limited regions of the world. Current antifungal therapies used to treat cryptococcal meningitis are critically limited, toxic, expensive and have issues with drug resistance, highlighting an urgent need for more effective and affordable drugs. However, developing a new drug can cost over $2 billion and take over a decade to succeed, causing many development pipelines to dry up. To address this issue, this thesis uses drug repurposing as an alternative approach to accelerate antifungal drug discovery efforts to treat cryptococcal meningitis. This involves investigating existing drugs, approved for other purposes, for candidates with antifungal activities that can be developed into new antifungal drugs. An untested library of drugs was screened for compounds that inhibited the growth of 𝘊𝘳𝘺𝘱𝘵𝘰𝘤𝘰𝘤𝘤𝘶𝘴 𝘥𝘦𝘶𝘵𝘦𝘳𝘰𝘨𝘢𝘵𝘵𝘪𝘪. This approach successfully identified multiple candidates that are not currently approved to treat fungal infections. The most potent candidate was flubendazole, a drug used to treat intestinal worms in veterinary and clinical settings. A diverse list of 𝘊𝘳𝘺𝘱𝘵𝘰𝘤𝘰𝘤𝘤𝘶𝘴 species were highly susceptible to low concentrations of flubendazole and this effect was fungicidal. This is important for efficient clearing of fungal burden, and may subsequently help improve clinical outcomes, prevent clinical relapse and deter the development of drug resistance. Flubendazole was also equally effective against fluconazole‐resistant 𝘊𝘳𝘺𝘱𝘵𝘰𝘤𝘰𝘤𝘤𝘶𝘴 strains, and was highly specific to Cryptococcus species and did not inhibit other clinically‐important, human fungal pathogens. To understand the antifungal mechanism of action of flubendazole, a combination of RNA‐Seq and microscopy approaches were used. Gene expression data and image analyses demonstrated that tubulin was inhibited and cell morphology was compromised in 𝘊𝘳𝘺𝘱𝘵𝘰𝘤𝘰𝘤𝘤𝘶𝘴 cells treated with flubendazole. This provides evidence to support the involvement of tubulin as a probable target of flubendazole in its antifungal mechanism of action. The RNA‐Seq data generated also represents a resource for future studies to comparatively investigate genes of interest for drug discovery, such as transcription factors and kinases. This thesis encompasses a body of work that exemplifies the benefits of combining drug screening and repurposing efforts in an attempt to address the unmet medical needs of cryptococcal meningitis. The discovery of flubendazole as a potential antifungal agent and preliminary characterisation of its antifungal action provides a platform that may inform and aid future developmental work including medicinal chemistry studies.
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