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

Article abstract of DOI:10.1021/ja505517t

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.

Full text of DOI:10.1021/ja505517t

Article
pubs.acs.org/JACS
Open Access on 07/21/2015
Mapping the Protein Interaction Landscape for Fully Functionalized
Small-Molecule Probes in Human Cells
Tohru Kambe, Bruno E. Correia, Micah J. Niphakis, and Benjamin F. Cravatt*
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
S
* Supporting Information
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 spectrom-
etry-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.
interactions with different proteins in cells and “constant”
photoreactive and alkyne groups for covalent modification and
detection/identification of protein targets, respectively.7 These
probes have the advantage of forging covalent bonds with protein
targets directly in living cells under the same assay conditions
used for phenotypic screening. Subsequent target enrichment
and identification are then achieved by copper-catalyzed azide−
alkyne cycloaddition (CuAAC or “click” 9,10) chemistry-enabled
biotin−(strept)avidin chromatography and mass spectrometry
(MS) analysis. Nonetheless, the incorporation of photoreactive
and alkyne groups presents synthetic challenges that may limit
the structural diversity of fully functionalized probes and impinge
on the scope of their protein interactions in cells. Here, we set out
to address these challenges by first devising an efficient synthetic
route to create a structurally diverse library of fully functionalized
probes and then applying these probes in combination with
quantitative MS-based proteomic methods to globally assess
probe-target interactions in human cancer cells. These studies
uncovered a broad protein interaction landscape for fully
functionalized probes, including enzymes, channels, adaptor
and scaffolding proteins, and proteins of uncharacterized
function. Many of these proteins lack known ligands, indicating
INTRODUCTION
■
The phenotypic screening of small-molecule libraries is a central
component of academic and industrial programs aimed at
pharmacologically perturbing and understanding biochemical
pathways in living systems.1,2 The output of phenotypic
screening can be new chemical probes for basic research and
drugs for treating human disorders. Identifying the targets of
phenotypic screening hits, however, remains a major technical
hurdle and a critical goal for elucidating the mechanism(s) of
action of bioactive compounds.3,4 Several strategies have been
introduced to facilitate target identification,4−6 and each
approach has a distinct set of advantages and limitations: some
identify targets through direct binding to small molecules, but at
the cost of requiring the prior lysis of cells and attachment of
compounds to a solid support; others facilitate target
identification in situ but rely on indirect (e.g., genetic or
metabolic) evidence to forge functional connections between
compounds and proteins. We,7 and others,8 have introduced
complementary approaches where the small-molecule library
itself possesses design features intended to facilitate target
identification. These features can include small-molecule libraries
with embedded linker elements so that the point of contact to
solid support for biochemical target enrichment is already
defined prior to phenotypic assay.8 We have alternatively
described small-molecule libraries that are fully functionalized,
in that they contain “variable” binding elements to promote
Received: June 2, 2014
Published: July 21, 2014
© 2014 American Chemical Society
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Figure 1. Synthesis and representative members of a fully functionalized small-molecule probe library. (A) Ugi-azide multicomponent condensation
reaction used to synthesize a library of 1,5-disubstituted tetrazoles. (B) Components used in Ugi-azide reaction for library synthesis. (C) Structures of
representative library members highlighting diazirine and alkyne groups for protein photocrosslinking and ligation of reporter tags to probe-bound
targets, respectively. Also see Figure S1 for a complete list of probe structures.
that fully functionalized probes can provide pharmacological
access to underexplored portions of the human proteome.
alized” probe library (Figure 1C and Figure S1). We then set out
to globally map the proteomic interactions of this probe library in
human cells.
RESULTS
Target Profiling in Cells. We initially evaluated protein
targets of the probe library by treating the human PC-3 prostate
cancer cell line with each library member (10 μM, 30 min, 37
°C), followed by exposure to UV light for 10 min at 4 °C, cell
lysis, and separation of soluble and membrane proteomes by
centrifugation. Probe-labeled proteins in these proteomic
fractions were coupled to a rhodamine−azide (Rh−N3) reporter
tag14 using CuAAC chemistry,9,10 and separated and detected by
SDS-PAGE and in-gel fluorescence scanning, respectively. The
resulting profiles revealed a striking diversity of in situ protein
labeling events, with most probes showing distinct labeling
patterns across the cancer cell proteome, and, conversely, the
protein targets exhibited different preferences for individual
probes (Figure 2 and Figure S2). Virtually all protein labeling was
UV-light-dependent, with the exception of a 55 and 60 kDa
proteins that were labeled by a subset of probes in no-UV-light
control experiments (e.g., probes 10 and 20, Figure 2A). The
aggregate structure−activity relationship (SAR) for in situ
probe−protein interactions enabled selection of a subset of
probes3, 8, 22, 24, 26, 31, and 55 (Figure 2B)for in-depth
MS studies based on their complementary profiles.
In our initial MS studies, we measured the relative protein
enrichments for each test probe versus a common comparison
probe 3, which showed a protein-interaction profile that included
most of the proteins that interacted broadly with the probe
library, but limited evidence of unique protein-labeling events.
Quantification of relative protein enrichments was achieved by
SILAC (Stable isotope labeling by amino acids in cell culture15)
methods, following previously described protocols.7,16 In brief,
we treated heavy-isotope-labeled (“heavy”) and light-isotope-
labeled (“light”) PC3 cells with the test probe (8, 22, 24, 26, 31,
or 55) and probe 3 (10 μM of each probe), respectively,
following the general protocol described for gel-based profiling
■
Library Design and Synthesis. We previously reported a
first-generation fully functionalized library constructed using a
benzophenone photocrosslinking element.7 While this library
proved effective for coupled phenotypic screening and target
identification, its structural diversity and, by extension, protein-
interaction potential were limited by the size of the
benzophenone group and synthetic chemistry challenges facing
its derivatization. Toward the goal of developing an efficient
synthetic strategy to produce a more structurally varied library,
we applied the Ugi-azide multicomponent condensation
reaction11 to afford rapid assembly of 1,5-disubstituted tetrazoles
from amine, isocyanide, and aldehyde/ketone building blocks
(Figure 1A,B). With the goal of minimizing the steric footprint of
the photocrosslinking element, we chose the diazirine group to
forge the covalent bond between probes and protein targets.
Upon UV irradiation, diazirines extrude nitrogen to form a highly
reactive carbene species that is capable of inserting into C−H,
N−H, and O−H bonds on a protein, forming a covalent
adduct.12 This mechanism differs from that of benzophenones,
which react via a triplet diradical.13 Diazirine and benzophenone
probes may therefore exhibit distinct crosslinking efficiencies for
individual amino acids, although how such differences ultimately
affect labeling at the protein level is unclear. We also
incorporated a terminal alkyne into each library member to
allow visualization and enrichment of probe-labeled proteins
following CuAAC with azide-bearing reporter groups.14 We first
prepared a collection of diazirines that possess either an amine or
a carbonyl, either to serve as one component of the Ugi-azide
condensation reaction or to allow immediate functionalization of
the corresponding Ugi-azide product. Using these, along with a
larger collection of commercially available reactants (Figure 1B),
we prepared a structurally diverse, ∼60-member “fully function-
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with some probes showing a restricted interaction profile (e.g.,
24, which selectively enriched a single protein PEBP1 relative to
probe 3) and others exhibiting a larger number of targets (e.g.,
31, which enriched nine protein targets relative to probe 3)
(Figure 3A). Interestingly, many of the protein targets could be
matched with confidence to the gel-based profiles on the basis of
their predicted molecular weights and selective interactions
across the test probes (Figure S3). Control experiments with
representative probes confirmed that targets showed approx-
imately 1:1 ratios in comparisons of heavy and light cells treated
with the same probe and that target enrichment was, in general,
dependent on UV-light exposure (Figure 3B and Table S1).
Analysis of Protein Targets. In total, 24 preferred protein
targets were identified for the six test probes and included diverse
classes of enzymes (kinases, peptidases, metabolic enzymes),
adaptor proteins, scaffolding proteins, and proteins of unchar-
acterized function (Table 1). A search of the literature revealed
that the targets near-equally distributed into proteins with and
without known ligands. Among the proteins with known ligands
were NUDT1, a metabolic enzyme that has recently been shown
to repair damaged bases in cancer cells17,18 and found here to
interact preferentially with 31 relative to other probes; PEBP1, a
protein binding partner and negative regulator of Raf kinase19
and found here to interact preferentially with 24; and tubulin, a
classical target of many anti-tumorigenic drugs20 and found here
to interact preferentially with 55. Among the proteins that lack
known ligands, it is interesting to note that several could be
viewed as traditionally difficult to “drug”, including adaptor and
scaffolding proteins (e.g., BCAR3,21 CUTA22). We also
performed a more limited analysis of membrane proteomes
from probe-treated cells and identified a distinct set of proteins
that showed differential probe labeling in comparisons of 3 and
Figure 2. Gel-based profiles of in situ protein labeling events of PC3 cells
treated with fully functionalized probes. (A) Gel profiles for
representative probes (10 μM) incubated with PC3 cells for 30 min
prior to UV light exposure (10 min), lysis, conjugation to an Rh−N3
reporter tag by CuAAC, and analysis by SDS-PAGE and fluorescence
scanning. Shown are soluble PC3 proteomes; see Figure S2 for gel
profiles of additional probes and membrane proteomes of PC3 cells. (B)
PC3 cell gel-based profiles of probes selected for in-depth MS studies.
except that the heavy and light proteomic samples were mixed
prior to CuAAC conjugation to an azide-biotin tag. Biotinylated
proteins were enriched using streptavidin chromatography,
digested on-bead with trypsin, and the resulting tryptic peptide
mixture analyzed by liquid chromatography−mass spectrometry
(LC-MS) using an LTQ-Orbitrap mass spectrometer. Proteins
that exhibited heavy:light SILAC ratios ≥3 were designated as
preferred targets of the test probe. These SILAC experiments
identified a distinct set of preferred targets for each test probe,
Figure 3. Quantitative proteomic data for representative targets of the probe library. (A) SILAC plots for total proteins identified in experiments
comparing cells treated with test probe (8, 22, 24, 26, 31, and 55) versus 3 (10 μM probes, 30 min). Proteins with median SILAC ratios ≥3 (test probe/
3) are designated as preferred targets of the test probe (red dashed line marks 3-fold enrichment). Results are a combination of duplicate proteomic
experiments performed in PC3 cells. See Table S1 for full proteomic data sets. (B) Representative MS1 peptide traces for protein 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 shown as heavy:light.
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Table 1. Probe Targets, Defined as Proteins That Showed 3-
fold or Greater Enrichment in Quantitative Proteomic
a
Comparisons of Test Probes versus Probe 3
protein
name
preferred
known
ligands
probe(s) competition
protein type
enzyme
(dehydrogenase)
ALDH1A3
31
partial
yes
BCAR3
26
8
yes
yes
adaptor protein
no
no
C21orf33
putative enzyme
(amidotransferase)
Figure 4. Frequency of detection of probe targets in the CRAPome
database (http://www.crapome.org/).31 Targets show a broad
distribution of detection frequencies, indicating that they span a wide
range of abundances in human cells.
CTSD
31
yes
enzyme (peptidase)
scaffolding protein
enzyme
yes
no
yes
no
no
no
yes
CUTA
DCTPP1
EPDR1
HADH
KPNA2
LDHA
8, 31
22, 55
31
yes
yes
partial
ND
yes
uncharacterized
enzyme (metabolic)
importin
55
31
26
no
enzyme
(dehydrogenase)
LTA4H
NENF
26
55
31
22
24
partial
ND
yes
enzyme (hydrolase)
neurotrophic factor
enzyme (phosphatase)
enzyme (peptidase)
yes
no
NUDT1
OTUB1
PEBP1
yes
no
yes
yes
kinase/lipid-binding
protein
yes
PLD3
31
26
31
26
26
26
31
55
8
partial
no
enzyme (lipase)
enzyme (peroxidase)
proteasome activator
ribosomal protein
enzyme (kinase)
enzyme (kinase)
enzyme (peptidase)
tubulin
no
no
no
no
yes
yes
no
yes
yes
PRDX3
PSME1
RPS3
no
yes
RPS6KA1
RPS6KA3
SCPEP1
partial
yes
yes
b
Figure 5. Probe−protein interaction profiles. (A) Heat map showing
enrichment ratios for various probe−protein interactions as determined
by quantitative proteomic experiments in PC3 cells comparing test
probes to probe 3. (B) Recombinantly expressed (recom) and
endogenous (endog) forms of CUTA and PEBP1 show similar probe-
interaction profiles in cells. Recombinant, Myc-tagged proteins were
assayed for probe labeling in transiently transfected HEK293T cells and
the profiles compared to those of mock-transfected cells.
TUBB
ND
partial
TXNRD1
enzyme (reductase)
a
For competition data, designations of “yes” and “partial” correspond
to competition ratios of less than or equal to 0.5 and 0.5−0.67,
respectively; ND, not determined. References for known ligands
(defined as compounds that have been shown to affect the biochemical
or cellular activity of the target): ALDH1A3,23 CTSD,24 DCTPP1,25
LDHA,26 LTA4H,27 NUDT1,17,18 PEBP1,28 RPS6KA1/3,29 TUBB,20
and TXNRD1.30 Multiple tubulin variants were enriched by 55 and
b
these proteins shared overlapping and distinct peptides. For the sake of
clarity, we have listed these variants as a single entry, but the data for
all variants can be found in Table S1.
protein could be assigned a preferred probe (Table 1). We
confirmed these preferential probe interactions for representa-
tive protein targets by recombinantly expressing these proteins in
HEK293T cells by transient transfection and testing the
transfected cells against the test probe set (Figure 5B). We
then generated “non-clickable” alkane (or alkene) versions of
each probe for use in competitive profiling experiments to
estimate target engagement in cells (Figure 6A and Figure S5).
Initial competition experiments were performed by treating PC3
cells with probes (10 μM) and 2× competitors (20 μM) or
DMSO for 30 min, followed by UV-light exposure (10 min at 4
°C), cell lysis, coupling of probe-labeled proteins to Rh−N3 by
CuAAC, and analysis by gel-based profiling. Several probe-
labeled proteins showed reduced signals in competitor-treated
versus DMSO-treated cell preparations (Figure S5).
We next treated isotopically heavy and light cells with probe
(10 μM) and either 2× competitor (20 μM; heavy cells) or
DMSO (light cells) as described above, coupled probe-labeled
proteins to biotin-N3 by CuAAC, and analyzed the samples by
streptavidin chromatography combined with quantitative LC-
MS-based proteomics. Proteins that showed heavy:light ratios of
0.5 or less were considered substantially engaged by the
competitor, while those showing light:heavy ratios between 0.5
and 0.67 were considered partially engaged by the competitor.
26 (Table S1). These proteins included transmembrane
channels, enzymes, and proteins of uncharacterized function.
Protein targets of the probe library not only exhibited diversity
in their structure and function, but also in their relative
abundance in cells, as estimated by searches of the public
CRAPome (Contaminant Repository for Affinity Purification)
database,31 which inventories the detection frequency of proteins
across ∼400 negative-control affinity chromatography experi-
ments. We found that the most of the protein targets showed
very low frequencies of detection in the CRAPome repository
(<5%) (Figure 4), suggesting that they are lower abundance
proteins. A handful of protein targets were much more
commonly detected in CRAPome searches (e.g., TUBB, >50%
frequency of detection) (Figure 4), but, even for such abundant
proteins, selective probe labeling could be readily discerned over
background signals in our comparisons of test probes to probe 3
(Figure S4).
Evidence That Protein Targets Are Substantially
Engaged by Probes in Cells. Based on their different
interaction profiles with the test probes (Figure 5A), each
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Figure 6. Blockade of probe−protein interactions in cells by nonclickable competitor analogues. (A) Structure of probes 24 and 31 and their
nonclickable competitor agents 60 and 64, respectively. (B) SILAC plots for total proteins identified 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 marks a light:heavy ratio of 0.5; protein
ratios at or below this line indicate substantial competition. See Figure S5 and Table S2 for competition data for additional probes. (C) Representative
MS1 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 test probe-versus-3 and test probe-versus-competitor experiments. A good correlation
is observed between the test probe showing the highest target enrichment and the corresponding nonclickable analogue showing the highest
competition (depicted using the inverse of the competition ratio shown in panel B and Figure S5) of target−probe interactions.
Most of the 24 protein targets exhibited clear evidence of partial
or substantial engagement by the competitor analogue of its
preferred interacting probe in cells (Figure 6B,C and Table 1;
also see Figure S5 and Table S2). In some cases, the competitor
showed greater than the anticipated 2-fold competition, which
could reflect superior binding of the target to the non-clickable
analogue compared to the parent alkyne probe. Importantly, for
protein targets that were detected across the majority of probe-
versus-3 data sets, we confirmed that the extent of probe
enrichment correlated with the extent of competition by the
corresponding competitor agent (Figure 6D). This finding
indicates that the distinct probe−protein interaction profiles
reflect authentic differences in binding as opposed to variations
in photocrosslinking efficiency among the probes.
A second set of proteins emerged from our quantitative
proteomic experiments that exhibited an initially curious profile
of competition without preferential probe interactions (Table
S3). These proteins also showed strong enrichment in probe-
versus-no UV light control experiments (Tables S1 and S3),
confirming that they are direct targets of the probe library. We
believe that these proteins represent avid and promiscuous
ligand-binders, which would explain their lack of preferential
interactions in probe-versus-3 experiments and pervasive
competition in probe-versus-competitor experiments.
alized libraries provide a technically straightforward means to
achieve this objective, since probe−protein interactions can be
trapped in situ by exposing probe-treated cells to UV light. With
these considerations in mind, we set forth two primary goals for
this study: (1) to synthesize a library of fully functionalized
probes that displays substantial structural diversity, while also
preserving features for protein capture and enrichment, and (2)
to map the protein interactions for this library in human cells.
The first objective was accomplished using Ugi-azide
condensation chemistry to install variable recognition elements
into the probe library in conjunction with sterically minimized
photoreactive (diazirine) and affinity (alkyne) groups. Combin-
ing these probes with quantitative proteomics uncovered a
striking diversity of protein targets in human cancer cells. Most of
these proteins showed unique SARs across the probe library, and
their interactions were, in general, blocked by treatment with
excess non-clickable analogues of the probes. We are encouraged
by these features of specific binding events, inasmuch as they
forecast the potential for the ligands to affect protein activity in
cells. However, it is important to qualify that we do not yet know
the functional consequences of the numerous specific probe−
protein interactions characterized in this study and whether they
reflect binding to active, allosteric, or ineffectual sites on proteins.
This question should be addressable in future studies where the
binding sites and biochemical effects of ligands are characterized
for individual proteins.
CONCLUSIONS
■
We believe that our results support the future implementation
of fully functionalized libraries in phenotypic screens, where the
respective pharmacological effects and protein interaction
profiles of library members can be integrated to facilitate the
target identification process. Such efforts often benefit from the
discovery of structurally related active and inactive control
probes, the selection of which can be guided by chemical
proteomic methods.7,35 A preliminary survey of the probe−
protein interactions mapped so far also indicates that probes with
It seems logical to surmise that the general utility of a compound
library for phenotypic screening would be coupled to the scope of
its target interactions in cells. Despite this expectation, few if any
compound libraries, to our knowledge, have been systemically
examined for protein interactions in cells. We, and others, have
assessed the targets of small-molecule libraries bearing electro-
philic functionalities that impart covalent reactivity with proteins
in cells,32−34 but the target landscape of reversibly acting small-
molecule probes remains poorly understood. Fully function-
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AUTHOR INFORMATION
Corresponding Author
cravatt@scripps.edu
■
Notes
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The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
This work was supported by the NIH (CA132630, DA032541)
and Ono Pharmaceuticals.
■
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