An ELISA for discovering protein-protein interaction inhibitors: Blocking lysinoalanine crosslinking between subunits of the spirochete flagellar hook as a test case

The inhibition of a specific protein-protein interaction is often difficult to achieve in targeted drug design. We report the development and optimization of a general-purpose, readily implemented enzyme-linked immunosorbent assay (ELISA) for high-throughput screening to identify small-molecule inhibitors of protein interactions. This ELISA does not involve the use of any capture antibodies, probes, or compounds coated on the plate and represents a general strategy to identify inhibitors of a given protein-protein interaction. We demonstrate its utility in blocking lysinoalanine crosslinking between subunits of the spirochete flagellar hook by targeting the native form of the FlgE protein, which differs from the strategies used in previous assays. The flagellar hook protein FlgE self-catalyzes the formation of a lysinoalanine (Lal) inter-subunit crosslink that is essential for the motility, and thus, infectivity of spirochetes. Prevention of Lal crosslinking through inhibition with small molecules thus represents an avenue for therapeutic development against spirochete-related diseases, such as Lyme and syphilis. Screening a library of ~700 compounds with the ELISA confirmed that hexachlorophene, currently the only known inhibitor of Lal crosslinking in FlgE, effectively inhibits the crosslinking reaction. In addition, the assay identified two new potential inhibitors, honokiol and zafirlukast, and several activators which belong to well-known classes of antibiotics.

• Publication year

  2025

• Venue

  SLAS discovery : advancing life sciences R & D

• Publication date

  2025-11-01

• Fields of study

  Biology, Medicine, Chemistry

• Identifiers
  DOI 10.1016/j.slasd.2025.100292PMID 41308746PMCID 12906330
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

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