Limits...
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.

Show MeSH

Related in: MedlinePlus

Frequency of detection of probe targetsin the CRAPome database(http://www.crapome.org/).31 Targets show a broad distribution of detection frequencies, indicatingthat they span a wide range of abundances in human cells.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4120992&req=5

fig4: Frequency of detection of probe targetsin the CRAPome database(http://www.crapome.org/).31 Targets show a broad distribution of detection frequencies, indicatingthat they span a wide range of abundances in human cells.

Mentions: Protein targets of the probe library not only exhibiteddiversityin their structure and function, but also in their relative abundancein cells, as estimated by searches of the public CRAPome (ContaminantRepository for Affinity Purification) database,31 which inventories the detection frequency of proteins across∼400 negative-control affinity chromatography experiments.We found that the most of the protein targets showed very low frequenciesof detection in the CRAPome repository (<5%) (Figure 4), suggesting that they are lower abundanceproteins. A handful of protein targets were much more commonly detectedin CRAPome searches (e.g., TUBB, >50% frequency of detection) (Figure 4), but, even for such abundant proteins, selectiveprobe labeling could be readily discerned over background signalsin our comparisons of test probes to probe 3 (Figure S4).


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)

Frequency of detection of probe targetsin the CRAPome database(http://www.crapome.org/).31 Targets show a broad distribution of detection frequencies, indicatingthat they span a wide range of abundances in human cells.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Frequency of detection of probe targetsin the CRAPome database(http://www.crapome.org/).31 Targets show a broad distribution of detection frequencies, indicatingthat they span a wide range of abundances in human cells.
Mentions: Protein targets of the probe library not only exhibiteddiversityin their structure and function, but also in their relative abundancein cells, as estimated by searches of the public CRAPome (ContaminantRepository for Affinity Purification) database,31 which inventories the detection frequency of proteins across∼400 negative-control affinity chromatography experiments.We found that the most of the protein targets showed very low frequenciesof detection in the CRAPome repository (<5%) (Figure 4), suggesting that they are lower abundanceproteins. A handful of protein targets were much more commonly detectedin CRAPome searches (e.g., TUBB, >50% frequency of detection) (Figure 4), but, even for such abundant proteins, selectiveprobe labeling could be readily discerned over background signalsin our comparisons of test probes to probe 3 (Figure S4).

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