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Mapping the protein interaction landscape for fully functionalized small-molecule probes in human cells.

Kambe T, Correia BE, Niphakis MJ, Cravatt BF - J. Am. Chem. Soc. (2014)

Bottom Line: Phenotypic screening provides a means to discover small molecules that perturb cell biological processes.In-depth mass spectrometry-based analysis revealed a diverse array of probe targets in human cells, including enzymes, channels, adaptor and scaffolding proteins, and proteins of uncharacterized function.For many of these proteins, ligands have not yet been described.

View Article: PubMed Central - PubMed

Affiliation: The Skaggs Institute for Chemical Biology and Department of Chemical Physiology, The Scripps Research Institute , 10550 North Torrey Pines Road, La Jolla, California 92037, United States.

ABSTRACT
Phenotypic screening provides a means to discover small molecules that perturb cell biological processes. Discerning the proteins and biochemical pathways targeted by screening hits, however, remains technically challenging. We recently described the use of small molecules bearing photoreactive groups and latent affinity handles as fully functionalized probes for integrated phenotypic screening and target identification. The general utility of such probes, or, for that matter, any small-molecule screening library, depends on the scope of their protein interactions in cells, a parameter that remains largely unexplored. Here, we describe the synthesis of an ~60-member fully functionalized probe library, prepared from Ugi-azide condensation reactions to impart structural diversity and introduce diazirine and alkyne functionalities for target capture and enrichment, respectively. In-depth mass spectrometry-based analysis revealed a diverse array of probe targets in human cells, including enzymes, channels, adaptor and scaffolding proteins, and proteins of uncharacterized function. For many of these proteins, ligands have not yet been described. Most of the probe-protein interactions showed well-defined structure-activity relationships across the probe library and were blocked by small-molecule competitors in cells. These findings indicate that fully functionalized small molecules canvas diverse segments of the human proteome and hold promise as pharmacological probes of cell biology.

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Quantitative proteomic data for representativetargets of the probelibrary. (A) SILAC plots for total proteins identified in experimentscomparing cells treated with test probe (8, 22, 24, 26, 31, and 55) versus 3 (10 μM probes, 30 min). Proteins withmedian SILAC ratios ≥3 (test probe/3) are designatedas preferred targets of the test probe (red dashed line marks 3-foldenrichment). Results are a combination of duplicate proteomic experimentsperformed in PC3 cells. See Table S1 forfull proteomic data sets. (B) Representative MS1 peptide traces forprotein targets of probes 24 (PEBP1) and 31 (CUTA) in experiments comparing the test probes to probe 3 (“probe vs 3”), to no-UV-light controls(“no UV”), and to themselves (“probe vs probe”).SILAC ratios ≥20 are reported as 20. SILAC ratios are shownas heavy:light.
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fig3: Quantitative proteomic data for representativetargets of the probelibrary. (A) SILAC plots for total proteins identified in experimentscomparing cells treated with test probe (8, 22, 24, 26, 31, and 55) versus 3 (10 μM probes, 30 min). Proteins withmedian SILAC ratios ≥3 (test probe/3) are designatedas preferred targets of the test probe (red dashed line marks 3-foldenrichment). Results are a combination of duplicate proteomic experimentsperformed in PC3 cells. See Table S1 forfull proteomic data sets. (B) Representative MS1 peptide traces forprotein targets of probes 24 (PEBP1) and 31 (CUTA) in experiments comparing the test probes to probe 3 (“probe vs 3”), to no-UV-light controls(“no UV”), and to themselves (“probe vs probe”).SILAC ratios ≥20 are reported as 20. SILAC ratios are shownas heavy:light.

Mentions: In our initial MS studies, we measured the relativeprotein enrichmentsfor each test probe versus a common comparison probe 3, which showed a protein-interaction profile that included most ofthe proteins that interacted broadly with the probe library, but limitedevidence of unique protein-labeling events. Quantification of relativeprotein enrichments was achieved by SILAC (Stable isotope labelingby amino acids in cell culture15) methods,following previously described protocols.7,16 Inbrief, we treated heavy-isotope-labeled (“heavy”) andlight-isotope-labeled (“light”) PC3 cells with the testprobe (8, 22, 24, 26, 31, or 55) and probe 3 (10μM of each probe), respectively, following the general protocoldescribed for gel-based profiling except that the heavy and lightproteomic samples were mixed prior to CuAAC conjugation to an azide-biotintag. Biotinylated proteins were enriched using streptavidin chromatography,digested on-bead with trypsin, and the resulting tryptic peptide mixtureanalyzed by liquid chromatography–mass spectrometry (LC-MS)using an LTQ-Orbitrap mass spectrometer. Proteins that exhibited heavy:lightSILAC ratios ≥3 were designated as preferred targets of thetest probe. These SILAC experiments identified a distinct set of preferredtargets for each test probe, with some probes showing a restrictedinteraction profile (e.g., 24, which selectively enricheda single protein PEBP1 relative to probe 3) and othersexhibiting a larger number of targets (e.g., 31, whichenriched nine protein targets relative to probe 3) (Figure 3A). Interestingly, many of the protein targets couldbe matched with confidence to the gel-based profiles on the basisof their predicted molecular weights and selective interactions acrossthe test probes (Figure S3). Control experimentswith representative probes confirmed that targets showed approximately1:1 ratios in comparisons of heavy and light cells treated with thesame probe and that target enrichment was, in general, dependent onUV-light exposure (Figure 3B and Table S1).


Mapping the protein interaction landscape for fully functionalized small-molecule probes in human cells.

Kambe T, Correia BE, Niphakis MJ, Cravatt BF - J. Am. Chem. Soc. (2014)

Quantitative proteomic data for representativetargets of the probelibrary. (A) SILAC plots for total proteins identified in experimentscomparing cells treated with test probe (8, 22, 24, 26, 31, and 55) versus 3 (10 μM probes, 30 min). Proteins withmedian SILAC ratios ≥3 (test probe/3) are designatedas preferred targets of the test probe (red dashed line marks 3-foldenrichment). Results are a combination of duplicate proteomic experimentsperformed in PC3 cells. See Table S1 forfull proteomic data sets. (B) Representative MS1 peptide traces forprotein targets of probes 24 (PEBP1) and 31 (CUTA) in experiments comparing the test probes to probe 3 (“probe vs 3”), to no-UV-light controls(“no UV”), and to themselves (“probe vs probe”).SILAC ratios ≥20 are reported as 20. SILAC ratios are shownas heavy:light.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4120992&req=5

fig3: Quantitative proteomic data for representativetargets of the probelibrary. (A) SILAC plots for total proteins identified in experimentscomparing cells treated with test probe (8, 22, 24, 26, 31, and 55) versus 3 (10 μM probes, 30 min). Proteins withmedian SILAC ratios ≥3 (test probe/3) are designatedas preferred targets of the test probe (red dashed line marks 3-foldenrichment). Results are a combination of duplicate proteomic experimentsperformed in PC3 cells. See Table S1 forfull proteomic data sets. (B) Representative MS1 peptide traces forprotein targets of probes 24 (PEBP1) and 31 (CUTA) in experiments comparing the test probes to probe 3 (“probe vs 3”), to no-UV-light controls(“no UV”), and to themselves (“probe vs probe”).SILAC ratios ≥20 are reported as 20. SILAC ratios are shownas heavy:light.
Mentions: In our initial MS studies, we measured the relativeprotein enrichmentsfor each test probe versus a common comparison probe 3, which showed a protein-interaction profile that included most ofthe proteins that interacted broadly with the probe library, but limitedevidence of unique protein-labeling events. Quantification of relativeprotein enrichments was achieved by SILAC (Stable isotope labelingby amino acids in cell culture15) methods,following previously described protocols.7,16 Inbrief, we treated heavy-isotope-labeled (“heavy”) andlight-isotope-labeled (“light”) PC3 cells with the testprobe (8, 22, 24, 26, 31, or 55) and probe 3 (10μM of each probe), respectively, following the general protocoldescribed for gel-based profiling except that the heavy and lightproteomic samples were mixed prior to CuAAC conjugation to an azide-biotintag. Biotinylated proteins were enriched using streptavidin chromatography,digested on-bead with trypsin, and the resulting tryptic peptide mixtureanalyzed by liquid chromatography–mass spectrometry (LC-MS)using an LTQ-Orbitrap mass spectrometer. Proteins that exhibited heavy:lightSILAC ratios ≥3 were designated as preferred targets of thetest probe. These SILAC experiments identified a distinct set of preferredtargets for each test probe, with some probes showing a restrictedinteraction profile (e.g., 24, which selectively enricheda single protein PEBP1 relative to probe 3) and othersexhibiting a larger number of targets (e.g., 31, whichenriched nine protein targets relative to probe 3) (Figure 3A). Interestingly, many of the protein targets couldbe matched with confidence to the gel-based profiles on the basisof their predicted molecular weights and selective interactions acrossthe test probes (Figure S3). Control experimentswith representative probes confirmed that targets showed approximately1:1 ratios in comparisons of heavy and light cells treated with thesame probe and that target enrichment was, in general, dependent onUV-light exposure (Figure 3B and Table S1).

Bottom Line: Phenotypic screening provides a means to discover small molecules that perturb cell biological processes.In-depth mass spectrometry-based analysis revealed a diverse array of probe targets in human cells, including enzymes, channels, adaptor and scaffolding proteins, and proteins of uncharacterized function.For many of these proteins, ligands have not yet been described.

View Article: PubMed Central - PubMed

Affiliation: The Skaggs Institute for Chemical Biology and Department of Chemical Physiology, The Scripps Research Institute , 10550 North Torrey Pines Road, La Jolla, California 92037, United States.

ABSTRACT
Phenotypic screening provides a means to discover small molecules that perturb cell biological processes. Discerning the proteins and biochemical pathways targeted by screening hits, however, remains technically challenging. We recently described the use of small molecules bearing photoreactive groups and latent affinity handles as fully functionalized probes for integrated phenotypic screening and target identification. The general utility of such probes, or, for that matter, any small-molecule screening library, depends on the scope of their protein interactions in cells, a parameter that remains largely unexplored. Here, we describe the synthesis of an ~60-member fully functionalized probe library, prepared from Ugi-azide condensation reactions to impart structural diversity and introduce diazirine and alkyne functionalities for target capture and enrichment, respectively. In-depth mass spectrometry-based analysis revealed a diverse array of probe targets in human cells, including enzymes, channels, adaptor and scaffolding proteins, and proteins of uncharacterized function. For many of these proteins, ligands have not yet been described. Most of the probe-protein interactions showed well-defined structure-activity relationships across the probe library and were blocked by small-molecule competitors in cells. These findings indicate that fully functionalized small molecules canvas diverse segments of the human proteome and hold promise as pharmacological probes of cell biology.

Show MeSH
Related in: MedlinePlus