Identification of lamins as the molecular targets of LBL1 using a clickable photoaffinity probe

Xiangshu Xiao, Bingbing X. Li

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Phenotypic screening is a powerful approach to discover small molecules targeting pathways or disease biology with complex genetic causes. Following the initial discovery of these small molecules is their target identification, which is at the cornerstone in addressing their biological and clinical utility. Yet, finding the needle in the haystack remains a challenge. Nuclear lamins are type V intermediate filament proteins that form a filamentous structure underneath the inner nuclear envelope to support the mechanical stability of the mammalian cell nucleus. They also participate a myriad of other cellular signaling processes with incompletely understood molecular mechanisms. Small molecules that can directly bind to nuclear lamins will be incredible tools to address lamins' roles in different aspects of biology. However, these small molecules did not exist until recently. We previously discovered an acylpyrroloquinazoline called LBL1 that selectively killed breast cancer cells without harming normal human cells. To help understand the mechanism of action of LBL1, we recently took an unbiased chemical proteomics approach to identify its direct binding targets from the entire human cellular proteome. In this chapter, we describe our detailed methods to identify and validate lamins as the direct targets of LBL1. In this approach, we developed a clickable photoaffinity probe called LBL1-P that contains acylpyrroloquinazoline, trifluoromethyldiazirine and alkyne groups. Furthermore, we described a fluorescence microscopic method to validate that LBL1 directly targets lamin A in living cells. When properly designed, this approach should be broadly applicable to other bioactive small molecules.

Original languageEnglish (US)
Title of host publicationMethods in Enzymology
PublisherAcademic Press Inc.
DOIs
StatePublished - Jan 1 2019

Publication series

NameMethods in Enzymology
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Keywords

  • Chemical proteomics
  • Click chemistry
  • Intermediate filament
  • Lamins
  • LBL1
  • LBL1-P
  • Photocrosslinking
  • Small molecule
  • Target identification

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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