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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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Blockade of probe–protein interactions in cells by nonclickablecompetitor analogues. (A) Structure of probes 24 and 31 and their nonclickable competitor agents 60 and 64, respectively. (B) SILAC plots for total proteinsidentified in experiments comparing PC3 cells treated with test probe 31 (10 μM) and either 2× competitor 64 (20 μM, heavy cells), or DMSO (light). Red dashed line marksa light:heavy ratio of 0.5; protein ratios at or below this line indicatesubstantial competition. See Figure S5 and TableS2 for competition data for additional probes. (C) RepresentativeMS1 peptide traces for protein targets of probes 24 (PEBP1)and 31 (CUTA) in competition experiments with 2×competitor (60 and 64, respectively). (D)Comparison of enrichment ratios for representative targets in testprobe-versus-3 and test probe-versus-competitor experiments.A good correlation is observed between the test probe showing thehighest target enrichment and the corresponding nonclickable analogueshowing the highest competition (depicted using the inverse of thecompetition ratio shown in panel B and FigureS5) of target–probe interactions.
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fig6: Blockade of probe–protein interactions in cells by nonclickablecompetitor analogues. (A) Structure of probes 24 and 31 and their nonclickable competitor agents 60 and 64, respectively. (B) SILAC plots for total proteinsidentified in experiments comparing PC3 cells treated with test probe 31 (10 μM) and either 2× competitor 64 (20 μM, heavy cells), or DMSO (light). Red dashed line marksa light:heavy ratio of 0.5; protein ratios at or below this line indicatesubstantial competition. See Figure S5 and TableS2 for competition data for additional probes. (C) RepresentativeMS1 peptide traces for protein targets of probes 24 (PEBP1)and 31 (CUTA) in competition experiments with 2×competitor (60 and 64, respectively). (D)Comparison of enrichment ratios for representative targets in testprobe-versus-3 and test probe-versus-competitor experiments.A good correlation is observed between the test probe showing thehighest target enrichment and the corresponding nonclickable analogueshowing the highest competition (depicted using the inverse of thecompetition ratio shown in panel B and FigureS5) of target–probe interactions.

Mentions: Based on their different interaction profiles withthe test probes (Figure 5A), each protein couldbe assigned a preferred probe (Table 1). Weconfirmed these preferential probe interactions for representativeprotein targets by recombinantly expressing these proteins in HEK293Tcells by transient transfection and testing the transfected cellsagainst the test probe set (Figure 5B). Wethen generated “non-clickable” alkane (or alkene) versionsof each probe for use in competitive profiling experiments to estimatetarget engagement in cells (Figure 6A and Figure S5). Initial competition experiments wereperformed by treating PC3 cells with probes (10 μM) and 2×competitors (20 μM) or DMSO for 30 min, followed by UV-lightexposure (10 min at 4 °C), cell lysis, coupling of probe-labeledproteins to Rh–N3 by CuAAC, and analysis by gel-basedprofiling. Several probe-labeled proteins showed reduced signals incompetitor-treated versus DMSO-treated cell preparations (Figure S5).


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)

Blockade of probe–protein interactions in cells by nonclickablecompetitor analogues. (A) Structure of probes 24 and 31 and their nonclickable competitor agents 60 and 64, respectively. (B) SILAC plots for total proteinsidentified in experiments comparing PC3 cells treated with test probe 31 (10 μM) and either 2× competitor 64 (20 μM, heavy cells), or DMSO (light). Red dashed line marksa light:heavy ratio of 0.5; protein ratios at or below this line indicatesubstantial competition. See Figure S5 and TableS2 for competition data for additional probes. (C) RepresentativeMS1 peptide traces for protein targets of probes 24 (PEBP1)and 31 (CUTA) in competition experiments with 2×competitor (60 and 64, respectively). (D)Comparison of enrichment ratios for representative targets in testprobe-versus-3 and test probe-versus-competitor experiments.A good correlation is observed between the test probe showing thehighest target enrichment and the corresponding nonclickable analogueshowing the highest competition (depicted using the inverse of thecompetition ratio shown in panel B and FigureS5) of target–probe interactions.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: Blockade of probe–protein interactions in cells by nonclickablecompetitor analogues. (A) Structure of probes 24 and 31 and their nonclickable competitor agents 60 and 64, respectively. (B) SILAC plots for total proteinsidentified in experiments comparing PC3 cells treated with test probe 31 (10 μM) and either 2× competitor 64 (20 μM, heavy cells), or DMSO (light). Red dashed line marksa light:heavy ratio of 0.5; protein ratios at or below this line indicatesubstantial competition. See Figure S5 and TableS2 for competition data for additional probes. (C) RepresentativeMS1 peptide traces for protein targets of probes 24 (PEBP1)and 31 (CUTA) in competition experiments with 2×competitor (60 and 64, respectively). (D)Comparison of enrichment ratios for representative targets in testprobe-versus-3 and test probe-versus-competitor experiments.A good correlation is observed between the test probe showing thehighest target enrichment and the corresponding nonclickable analogueshowing the highest competition (depicted using the inverse of thecompetition ratio shown in panel B and FigureS5) of target–probe interactions.
Mentions: Based on their different interaction profiles withthe test probes (Figure 5A), each protein couldbe assigned a preferred probe (Table 1). Weconfirmed these preferential probe interactions for representativeprotein targets by recombinantly expressing these proteins in HEK293Tcells by transient transfection and testing the transfected cellsagainst the test probe set (Figure 5B). Wethen generated “non-clickable” alkane (or alkene) versionsof each probe for use in competitive profiling experiments to estimatetarget engagement in cells (Figure 6A and Figure S5). Initial competition experiments wereperformed by treating PC3 cells with probes (10 μM) and 2×competitors (20 μM) or DMSO for 30 min, followed by UV-lightexposure (10 min at 4 °C), cell lysis, coupling of probe-labeledproteins to Rh–N3 by CuAAC, and analysis by gel-basedprofiling. Several probe-labeled proteins showed reduced signals incompetitor-treated versus DMSO-treated cell preparations (Figure S5).

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