Abstract Description

Fungal pathogens such as Cryptococcus neoformans and Aspergillus fumigatus pose a significant threat to public health due to the rising incidence of infections. Cryptococcus neoformans is an opportunistic pathogen that causes cryptococcal meningitis, affecting the central nervous system. Aspergillus fumigatus is a filamentous environmental mould that causes pulmonary diseases, including invasive aspergillosis. Infectious fungal disease has a high mortality rate as a consequence of resistance against current antifungals and the delayed development of alternative treatment options. Ergosterol, the counterpart of cholesterol in mammals, is a component of the fungal cell membrane and is essential for the survival and proliferation of fungi. Thus, enzymes involved in ergosterol biosynthesis are possible drug targets. However, many enzymes in the ergosterol biosynthesis pathway share significant homology with those in cholesterol biosynthesis; thus, antifungal agents targeting these enzymes may pose a risk of toxicity to humans. To overcome this, enzymes that are unique to fungi, such as sterol C-24 methyltransferases (SMTs), can be explored. SMTs catalyse the methyl transfer from S-adenosylmethionine to the C-24 position of the sterol intermediates zymosterol or lanosterol, yielding the 24-alkylated sterols fecosterol or eburicol, and S-adenosylhomocysteine. In this study, we aim to establish the tools required to seed a target-based Medicinal Chemistry campaign against SMTs from C. neoformans and A. fumigatus. Here, we present the protocols for protein production, purification, biochemical assays, and initial crystallisation trials. Truncation libraries of the optimised SMT genes were created in the pNIC-NHSTIIT-GG expression vector, which encodes an N-terminal His-tag and a TEV protease cleavage site. The proteins were produced in Escherichia coli BL21-Gold(DE3) and purified using immobilised metal affinity chromatography (IMAC), followed by TEV digestion, reverse IMAC, and size-exclusion chromatography. Seven variants of A. fumigatus and six variants of C. neoformans SMT were successfully produced and purified. SDS-PAGE analysis indicated that unwanted truncation was present in some of the variants; however, three variants from A. fumigatus and one from C. neoformans SMT did not show truncation. The apoproteins of these variants were used to set up vapour-diffusion crystallisation trials, which are ongoing. In addition, S-adenosylhomocysteine nucleosidase and adenine deaminase from Escherichia coli were heterologously produced and purified to establish a coupled spectrophotometric SMT assay. In future, solving the 3D structures of SMTs may enable the development of novel antifungal therapies by structure-based drug discovery techniques such as X-ray crystallographic fragments screening.