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Updated: Jul 20, 2025
High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits
Published on: September 20, 2016
A Fluorogenic Disaccharide Substrate for α-Mannosidases Enables High-Throughput Screening and Identification of an
Sandeep Bhosale1, Matthew C Deen1, Cameron Proceviat1
1Department of Chemistry, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada.
Abstract:
Trimming of host glycans is a mechanism that is broadly employed by both commensal and pathogenic microflora to enable colonization. Host glycan trimming by the opportunistic Gram-positive bacterium Streptococcus pneumoniae has been demonstrated to be an important 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-needed new treatments of S. pneumoniae infection. Here we report the synthesis of 4-methylumbelliferyl α-d-mannopyranosyl-(1→2)-β-d-mannopyranoside (Manα1,2Manβ-4MU) as a fluorogenic disaccharide substrate and development of an assay for SpGH92 that overcomes its requirement for +1 binding site occupancy. We miniaturize our in vitro assay and apply it to a high-throughput screen of >65 000 compounds, identifying a single inhibitory chemotype, LIPS-343. We further show that Manα1,2Manβ-4MU is also a substrate of the human Golgi-localized α-mannosidase MAN1A1, suggesting that this substrate should be useful for assessing the activity of this and other mammalian α-mannosidases.
Insights
Researchers developed a new assay to screen for inhibitors of Streptococcus pneumoniae
Area of Science:
- Microbiology
- Biochemistry
- Enzymology
Background:
- Host glycan trimming is crucial for microbial colonization and virulence.
- Streptococcus pneumoniae utilizes multiple glycan-processing enzymes, with exo-mannosidase SpGH92 identified as a key virulence factor.
- SpGH92 is a potential therapeutic target for S. pneumoniae infections.
Purpose of the Study:
- To develop a novel fluorogenic substrate and assay for SpGH92 activity.
- To identify inhibitors of SpGH92 through high-throughput screening.
- To evaluate the substrate's utility for human alpha-mannosidases.
Main Methods:
- Synthesis of the fluorogenic disaccharide substrate Manα1,2Manβ-4MU.
- Development of an SpGH92 assay overcoming +1 binding site occupancy requirements.
- Miniaturization and high-throughput screening of over 65,000 compounds.
Main Results:
- Identification of a novel inhibitory chemotype, LIPS-343.
- Demonstration that Manα1,2Manβ-4MU is also a substrate for human MAN1A1.
- Successful development of a functional high-throughput screening assay for SpGH92.
Conclusions:
- The developed assay and substrate are effective for SpGH92 inhibition screening.
- LIPS-343 represents a potential starting point for novel S. pneumoniae therapeutics.
- The substrate Manα1,2Manβ-4MU can be used to assess mammalian alpha-mannosidase activity.
