Developing Spindlin1 small-molecule inhibitors by using protein microarrays

Abstract

The discovery of inhibitors of methyl- and acetyl-binding domains has provided evidence for the “druggability” of epigenetic effector molecules. The small molecule probe UNC1215 prevents methyl-dependent protein-protein interactions by engaging the aromatic cage of MBT domains, and with lower affinity, Tudor domains. Using a library of tagged UNC1215 analogs we screened a protein domain microarray of human methyl-lysine effector molecules to rapidly detect compounds with novel binding profiles - either improved or loosened specificity. Using this approach, we identified a compound (EML405) that acquired a novel interaction with the Tudor domain-containing protein Spindlin1 (SPIN1). Structural studies facilitated the rational synthesis of more selective SPIN1 inhibitors (EML631–633), which engage SPIN1 in cells, block its ability to “read” H3K4me3 marks, and inhibit its transcriptional coactivator activity. Protein microarrays can thus be used as a platform to “target hop” and identify small molecules that bind and compete with domain–motif interactions.

Kinetic and high-throughput profiling of epigenetic interactions by 3D-carbene chip-based surface plasmon resonance imaging technology

“…S1B). The ITC curve of histone H3 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15) un, H3K4 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15) me3, H3.1 (28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41) K36me3, and H3.3 (28)(29)(30)(31)(32)(33)(34)(35)(36)…”

“…Verified interactions can then be subjected to in-depth mechanistic and functional studies, such as cocrystal structural analysis. Besides the 3D-carbene SPRi platform, other fluorescencebased peptide or reader microarray technologies have also been successfully applied for profiling epigenetic targets (29,30). Recently, DNA-barcoded designer nucleosome libraries (DNLs) have emerged as a unique technology that analyzes histone recognition and signaling, especially combinatorial histone modification readout at the nucleosome level (31).…”

A transcriptional coregulator, SPIN·DOC, attenuates the coactivator activity of Spindlin1

“…Because of the emerging role of SPIN1 in transformation and cancer, there is a growing interest in developing small-molecule inhibitors that target this effector molecule. Indeed, we and others have identified compounds that block the SPIN1-H3K4me3 interaction and repress the transcriptional coactivator function of SPIN1 (14,15).…”

Driving Integrative Structural Modeling with Serial Capture Affinity Purification

“…The structure of SPIN1 has been solved by X-ray crystallography [31][32][33] . As a result, multiple researchers are pursuing the development of a SPIN1 inhibitor 10,16,[40][41][42][43] . C11orf84, which was recently renamed SPINDOC by Bae and colleagues 9 , is a SPIN1-interacting protein that is less well understood, and the complex containing these two proteins remains poorly characterized.…”

“…We demonstrate the striking capabilities of this technology using a pair of proteins, Spindlin1 (SPIN1) and SPINDOC (c11orf84), which have previously been proposed to directly interact in biochemical 9 and computational 3 studies. SPIN1 is a well characterized histone methylation reader [9][10][11][12][13][14][15][16][17][18][19][20][21] , while SPINDOC has only been defined by its ability to bind SPIN1 9 . In this study, we first characterize the direct interaction and co-diffusion of SPIN1 and SPINDOC in live cells using imaging methods.…”