A Fluorogenic Disaccharide Substrate for alpha;-Mannosidases Enables High-Throughput Screening and Identification of an Inhibitor of the GH92 Virulence Factor from Streptococcus pneumoniae

摘要

Trimming of host glycans is a mechanism that is broadlyemployedby both commensal and pathogenic microflora to enable colonization.Host glycan trimming by the opportunistic Gram-positive bacterium Streptococcus pneumoniae has been demonstrated to be animportant mechanism of virulence. While S. pneumoniae employs a multitude of glycan processing enzymes, the exo-mannosidase SpGH92 has been shown to be an important virulence factor.Accordingly, SpGH92 is hypothesized to be a target for much-needednew treatments of S. pneumoniae infection. Here wereport the synthesis of 4-methylumbelliferyl alpha;-d-mannopyranosyl-(1 RARR;2)- beta;-d-mannopyranoside (Man alpha;1,2Man beta;-4MU) as a fluorogenicdisaccharide substrate and development of an assay for SpGH92 thatovercomes its requirement for +1 binding site occupancy. We miniaturizeour in vitro assay and apply it to a high-throughputscreen of >65 000 compounds, identifying a single inhibitorychemotype, LIPS-343. We further show that Man alpha;1,2Man beta;-4MUis also a substrate of the human Golgi-localized alpha;-mannosidaseMAN1A1, suggesting that this substrate should be useful for assessingthe activity of this and other mammalian alpha;-mannosidases.