Dataset Information

Small molecule interactome mapping by photo-affinity labeling (SIM-PAL)

ABSTRACT: Non-steroidal anti-inflammatory drugs (NSAIDs) are one of the most common pharmaceutical agents in the world. However, despite well-studied interactions with cyclooxygenase-2, little is known about the broader proteomic interactions and additional mechanisms that the NSAIDs elicit. Here we present a global binding site identification platform for the direct in cellulo characterization of NSAID binding sites. The platform uses (1) photochemical conjugation of NSAID derivatives in the whole proteome, (2) enrichment of the binding sides, for (3) targeted mass spectrometry-based assignment. Using our approach, we identified the NSAID “interactome” consisting of nearly 200 binding sites in Jurkat and K562 cells. We observed a binding site hotspot on the nucleosome where three NSAIDs (but not fragment-based small molecules) interacted. These data suggest that the NSAIDs may behave as a novel type of epigenetic mark. Our approach is potentially amenable to production of precise, whole proteome binding site maps for virtually any molecule of interest.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): B Cell, T Cell, Cell Culture

DISEASE(S): Cardiovascular System Disease,Colon Cancer,Inflammation

SUBMITTER: Christina Woo

LAB HEAD: Christina May Woo

PROVIDER: PXD007094 | Pride | 2019-01-25

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	170207LMRSAM02539_JG_Jurkat_DMSO_trypsin.raw	Raw
	170207LMRSAM02540_JG_Jurkat_PHOTOA_trypsin.raw	Raw
	170207LMRSAM02541_JG_Jurkat_PHOTOB_trypsin.raw	Raw
	170207LMRSAM02542_JG_Jurkat_PHOTOC_trypsin.raw	Raw
	170207LMRSAM02543_JG_Jurkat_DMSO_cleaveage.raw	Raw

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Publications

Small Molecule Interactome Mapping by Photoaffinity Labeling Reveals Binding Site Hotspots for the NSAIDs.

Gao Jinxu J Mfuh Adelphe A Amako Yuka Y Woo Christina M CM

Journal of the American Chemical Society 20180315 12

Many therapeutics elicit cell-type specific polypharmacology that is executed by a network of molecular recognition events between a small molecule and the whole proteome. However, measurement of the structures that underpin the molecular associations between the proteome and even common therapeutics, such as the nonsteroidal anti-inflammatory drugs (NSAIDs), is limited by the inability to map the small molecule interactome. To address this gap, we developed a platform termed small molecule inte ...[more]

PMID: 29543447

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Project description:PRIDE > Archive > PXD013420 Project PXD013420 Download Project Files Summary Title A binding site hotspot map of the FKBP12–rapamycin–FRB ternary complex by photo-affinity labeling and mass spectrometry-based proteomics Description Structural characterization of small molecule binding site hotspots within the global proteome is uniquely enabled by photo-affinity labeling (PAL) coupled with chemical enrichment and unbiased analysis by mass spectrometry (MS). MS-based binding site hotspot maps provide structural resolution of interaction sites in conjunction with identification of target proteins. However, binding site hotspot mapping has been confined to relatively simple small molecules to date; extension to more complex compounds would enable the structural definition of new binding modes in the proteome. Here, we extend PAL and MS methods to derive a binding site hotspot map for the immunosuppressant rapamycin, a complex macrocyclic natural product that forms a ternary complex with the proteins FKBP12 and FRB. Photo-rapamycin was developed as a diazirine-based PAL probe for rapamycin, and the FKBP12–photo-rapamycin–FRB ternary complex formed readily in vitro. Photo-irradiation, digestion, and MS analysis of the ternary complex revealed a McLafferty rearrangement product of photo-rapamycin conjugated to specific surfaces on FKBP12 and FRB. Molecular modeling of the ternary complex based on the binding site map revealed a 5.0 Å minimum distance constraint between the conjugated residues and the diazirine carbon. Molecular dynamics further predicted a 9.0 Å labeling radius for the diazirine upon photo-activation that may be useful in the interpretation of binding site measurements from PAL more broadly. Thus, in characterizing the ternary complex of photo-rapamycin by MS, we applied binding site hotspot mapping to a macrocyclic natural product and extracted a precise structural measurement for interpretation of PAL products that may enable the discovery of new ligand space in the “undruggable” proteome.