Fully functionalized small-molecule probes for integrated phenotypic screening and target identification

Article abstract of DOI:10.1021/ja304213w

Phenotypic screening offers a powerful approach to identify small molecules that perturb complex biological processes in cells and organisms. The tendency of small molecules, however, to interact with multiple protein targets, often with moderate to weak affinities, along with the lack of straightforward technologies to characterize these interactions in living systems, has hindered efforts to understand the mechanistic basis for pharmacological activity. Here we address this challenge by creating a fully functionalized small-molecule library whose membership is endowed with: (1) one or more diversity elements to promote interactions with different protein targets in cells, (2) a photoreactive group for UV light-induced covalent cross-linking to interacting proteins, and (3) an alkyne handle for reporter tag conjugation to visualize and identify cross-linked proteins. A library member was found to inhibit cancer cell proliferation selectively under nutrient-limiting (low glucose) conditions. Quantitative chemoproteomics identified MT-ND1, an integral membrane subunit of the ～1 MDa NADH:ubiquinone oxidoreductase (complex 1) involved in oxidative phosphorylation, as a specific target of the active probe. We further demonstrated that the active probe inhibits complex 1 activity in vitro (IC ₅₀ = 720 nM), an effect that is known to induce cell death in low-glucose conditions. Based on this proof of principle study, we anticipate that the generation and integration of fully functionalized compound libraries into phenotypic screening programs should facilitate the discovery of bioactive probes that are amenable to accelerated target identification and mechanistic characterization using advanced chemoproteomic technologies.

Full text of DOI:10.1021/ja304213w

Communication
pubs.acs.org/JACS
Fully Functionalized Small-Molecule Probes for Integrated
Phenotypic Screening and Target Identification
Justin S. Cisar 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
First, hit compounds often bind to one or more protein
ABSTRACT: Phenotypic screening offers a powerful
approach to identify small molecules that perturb complex
biological processes in cells and organisms. The tendency
of small molecules, however, to interact with multiple
protein targets, often with moderate to weak affinities,
along with the lack of straightforward technologies to
characterize these interactions in living systems, has
hindered efforts to understand the mechanistic basis for
pharmacological activity. Here we address this challenge by
creating a fully functionalized small-molecule library whose
membership is endowed with: (1) one or more diversity
elements to promote interactions with different protein
targets in cells, (2) a photoreactive group for UV light-
induced covalent cross-linking to interacting proteins, and
(3) an alkyne handle for reporter tag conjugation to
visualize and identify cross-linked proteins. A library
member was found to inhibit cancer cell proliferation
selectively under nutrient-limiting (low glucose) condi-
tions. Quantitative chemoproteomics identified MT-ND1,
an integral membrane subunit of the ∼1 MDa
NADH:ubiquinone oxidoreductase (complex 1) involved
in oxidative phosphorylation, as a specific target of the
active probe. We further demonstrated that the active
probe inhibits complex 1 activity in vitro (IC50 = 720 nM),
an effect that is known to induce cell death in low-glucose
conditions. Based on this proof of principle study, we
anticipate that the generation and integration of fully
functionalized compound libraries into phenotypic screen-
ing programs should facilitate the discovery of bioactive
probes that are amenable to accelerated target identi-
fication and mechanistic characterization using advanced
chemoproteomic technologies.
targets with low to moderate affinity, and these relatively weak
interactions can be difficult to characterize using conventional
affinity chromatography methods, where the small molecule is
linked to a solid support to enrich binding proteins from cell
lysates.7,8 Recent advances in the synthesis of small-molecule
libraries with preinstalled linker elements9 and improvements in
quantitative proteomics10 have enhanced the scope, sensitivity,
and accuracy of such affinity enrichment experiments; but the
approaches still involve identifying protein targets from cell
lysates, and therefore, small molecule−protein interactions that
depend on the integrity of the native cellular environment11,12
remain difficult to characterize. Genetic approaches for target
identification can address this problem,13,14 but eliminating
protein expression may not replicate the action of small
molecules, which often produce effects through blockade (or
activation) of multiple protein targets and without the physical
removal of these proteins from cells. While small-molecule
libraries that possess electrophilic groups offer a way to trap
interacting proteins in situ through covalent reactivity,11 we felt
that this strategy, which depends on the fortuitous positioning
of complementary nucleophilic residues in the binding pockets
of target proteins, lacked generality for proteome-wide profiling
in phenotypic screens. With these considerations in mind, we
sought to develop a chemoproteomic strategy that could be
combined with cell-based screening to enable the global and
quantitative assessment of reversible small molecule−protein
interactions directly in living systems.
Our strategy involves the synthesis of a small-molecule
library that is “fully functionalized” to enable direct progression
from cell-based screening to target identification without
requiring synthetic modification of the biologically active hit
compounds. Each library member is equipped with a
photoreactive group that is embedded within its structure to
permit UV light-induced cross-linking to interacting proteins
directly in living cells. Each compound also contains an alkyne,
which serves as a click chemistry15,16 derivatizable handle for
visualization, enrichment, and identification of interacting
proteins.17−19 We synthesized an ∼30 member library based
on two scaffolds: the 5-benzoyl indole (BzIndole) and 7-
benzoyl-benzo-1,4-diazepin-2,5-dione (BzBD), where structural
diversity was introduced at multiple sites on the core backbone
of the molecules (Figure 1A and Figure S1, Supporting
Information (SI)). Before proceeding to a phenotypic screen,
primary goal of modern chemical biology is to create
Asmall-molecule probes that selectively perturb individual
biochemical pathways in cells and animals.1 Such probes can
help to elucidate the mechanistic basis for complex physiology
and disease as well as serve as starting points for drug
development to treat human disorders.2−5 Phenotypic screen-
ing with small-molecule libraries has played a prominent role in
the discovery of new chemical probes and drugs.6 However,
determining the biologically relevant targets of small-molecule
hits from phenotypic (cell or organismal) screens remains a
major technical challenge for multiple reasons.
Received: May 1, 2012
Published: June 5, 2012
© 2012 American Chemical Society
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we first evaluated the proteomic interactions of library
members in mammalian cells.
We next screened the probe library for antiproliferative
activity in MDA-MB-231 cells under both normal and low-
glucose conditions (8 and 1 mM glucose, respectively). The
former condition matches standard cell culture protocols, but
the latter may better mimic the nutrient-deprived state
encountered in solid tumors, which tend to be poorly
vascularized.20 While none of the compounds affected MDA-
MB-231 proliferation in normal glucose, one compound, 1,
profoundly inhibited proliferation in low glucose (Figure 2A
Figure 2. Antiproliferative activity of compound 1 in human cancer
cell lines. (A) Inhibition of cancer cell viability in low glucose (1 mM)
media after 1 treatment (24 h) as determined by the WST-1 assay. (B)
Comparison of cell viability in low (1 mM) and normal (8 mM)
glucose for cells treated with 10 μM active (1) or control (14) probes
or vehicle (DMSO). Each cell line’s viability was normalized to
DMSO-treated cells grown in 8 mM glucose. See Figure S3, SI, for
complete screening results.
and Figure S3, SI). The antiproliferative activity of 1 was also
observed in several other human cancer cell lines (Figure 2A)
with IC50 values ranging from 0.33 to 1.3 μM (Figure 2B and
Figure S3, SI).
Figure 1. A fully functionalized small-molecule library for integrated
cell-based screening and target identification. (A) BzIndole and BzBd
scaffolds and structures of representative library members. See Figure
S1, SI for structures of all synthesized members of the library. (B)
Proteomic profiles for library members (10 μM) following in situ
photo-cross-linking in MDA-MB-231 cells. Probe-labeled proteins
were visualized by click chemistry conjugation to an azide-rhodamine
tag, SDS-PAGE, and in-gel fluorescence scanning (fluorescent gels
shown in gray scale).
Compound 1 shares structural features with other inactive
library members, perhaps most notably compound 14, which
differs only in the position of the t-butoxycarbamoylpiperazine
carbonyl group (Figure 1A). We therefore surmised that a
comparison of the proteomic targets of 1 versus 14 could reveal
proteins relevant for the antiproliferative activity of the former
compound. We also synthesized a nonclickable analogue of 1
(compound 37, Figure 3A) for use in competitive profiling
experiments and confirmed that this agent retained anti-
proliferative activity (IC50 ∼ 5 μM, Figure S4, SI). Gel-based
profiling of proteomes from cells treated with 1, 14, or 1 + 5X
37 detected multiple proteins that were preferentially labeled
by 1 over 14 and competed by 37 (Figure 3B). To identify
these proteins, we performed quantitative proteomics experi-
ments using stable isotope labeling by amino acids in cell
culture (SILAC) methods10 as follows: MDA-MB-231 cells
were grown in media containing isotopically heavy- or light-
labeled amino acids and then treated with probes 1 and 14,
The human breast cancer cell line MDA-MB-231 was treated
with individual probes (10 μM, 40 min), cross-linked in situ
with 365 nm light (5 min) and then lysed, and their proteomes
were conjugated to rhodamine-azide16 under click-chemistry
conditions and visualized using SDS-PAGE and fluorescent
scanning. Individual probes showed markedly distinct protein
labeling events (Figure 1B), indicating that the diversity
elements introduced into the probe library were sufficient to
direct binding to different subsets of proteins in the proteome.
Interestingly, these in situ profiles also differed considerably
when compared to small molecule−proteome reactions
performed in vitro (Figure S2, SI), indicating that the probes
interact with different sets of proteins in living cells versus cell
lysates.
respectively, or probe 1
5X 37, respectively. We also
performed control experiments where both heavy- and light-
labeled cells were treated with probe 1 or with 1 and DMSO,
respectively. After in situ photo-cross-linking, cells were lysed
and their proteomes conjugated to a biotin-azide tag, and then
paired heavy and light proteomic samples were mixed in a 1:1
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Communication
a
Table 1. High-Confidence Protein Targets of Compound 1
1 vs 1 + 5X 37
experiment
1 vs 14 experiment
gene name
MW (kDa)
av ratio
SEM
av ratio
SEM
EPHX1
MT-ND1
APMAP
SCCPDH
PIGS
53
35.7
5.19
3.00
1.82
1.64
1.38
1.30
0.94
0.76
0.39
0.07
0.17
0.47
0.20
0.32
2.27
2.38
2.24
5.30
2.27
2.34
2.30
0.33
0.42
0.38
0.44
0.76
0.54
0.77
46.5/32.2
47.1
61.6
TRABD
HEBP1
42.3/37.1
21.1
a
Highlighted are two proteins that showed >2-fold higher SILAC
ratios in quantitative chemoproteomic studies of 1- versus 14-treated
and 1- versus 1 + 5X 37-treated MDA-MB-231 cells. Additional
protein targets that showed >2-fold higher SILAC ratios in versus 1 +
5X 37-treated MDA-MB-231 cells are also shown.
targets, MT-ND1 stood out, in our mind, as the most likely
candidate, considering that other complex 1 inhibitors are
known to impair cancer cell viability under low-glucose
conditions.29,30 We therefore assayed 1 and control compound
14 for their ability to inhibit complex 1 activity in purified
submitochondrial particles. Complex 1 activity was inhibited
more potently by 1 (IC50 = 720 nM) than 14 (IC50 = 6.1 μM)
(Figure 3C), consistent with the relative labeling of MT-ND1
by these compounds in cancer cells (Figure 3B,D). We also
confirmed that 1 selectively labels EPHX1 by recombinantly
expressing this protein in HEK293T cells (Figure S6, SI).
These data, taken together, demonstrate that the antiprolifer-
ative agent 1 selectively targets only a handful of proteins in
cancer cells, including MT-ND1, the inhibition of which would
be predicted to impair mitochondrial respiration and lead to
cancer cell death under nutrient-limiting conditions.
The importance of phenotypic screening as a source for new
chemical probe and drug discovery is well-recognized.6
However, determining the mechanism of action for hit
compounds from cell-based assays remains a major challenge
due, at least in part, to a lack of robust technologies for target
identification. Here, we have described a strategy for generating
small-molecule libraries that addresses this problem. Each
library member is “fully functionalized” to contain photo-
activatable and alkyne groups for in situ cross-linking to protein
targets and chemoproteomic characterization of these small
molecule−protein adducts, respectively. As a proof of principle,
we identified a small-molecule probe 1 that inhibits
hypoglycemic cancer cell proliferation and determined that
one of its major cellular targets is the complex 1 component
MT-ND1. We further validated that 1 inhibits the NADH
oxidase activity of complex 1, similar to other known inhibitors
of the complex that elicit glucose-dependent antiproliferative
effects in cancer cells. It is noteworthy that previous complex 1
inhibitors have been converted into radiolabeled, photoreactive
analogs to ascertain their subunit interactions and binding site
information;25−28 but these studies have often spanned many
years in duration, likely due to the technical challenges that
accompany incorporation of photo-cross-linkers and radio-
isotopes into bioactive compounds as well as the difficulties
inherent to the biochemical purification and analysis of
multisubunit integral membrane protein complexes. By using
a fully functionalized library of small molecules in which
photoreactive groups, alkyne affinity handles, and diversity
elements are embedded into the core structure of the library,
Figure 3. Chemoproteomic identification of proteins that interact with
probe 1 in cancer cells. (A) A nonclickable analog of 1 (compound
37) for competition profiling experiments (reagents: 1 atm H2, cat.
Pd−BaSO4, EtOH). (B) Gel profile comparing proteins labeled by 1
and 14 (10 μM) and 1 competed with 5X 37 (50 μM). Candidate
protein bands corresponding to selective targets of 1 identified by LC-
MS are denoted on right. (C) Extracted MS1 chromatographs for a
representative peptide (ILGYMQLR, 497.28114 m/z) from MT-ND1
shown for 1
5X 37 competition, 1 versus 1, 1 versus 14, and 1
versus DMSO (unlabeled) experiments. See Table 1 and Figure S5
and Tables S1 and S2, SI, for more complete presentations of the
quantitative proteomic data. (D) Assay for complex 1 NADH oxidase
showing selective inhibition by 1 over 14.
ratio, separated into soluble and membrane fractions, enriched
with avidin, digested on a bead with trypsin, and analyzed by
LC-MS/MS using an LTQ-Orbitrap instrument. Light and
heavy signals were quantified from the parent ion (MS1) peaks
using the CIMAGE software21 to generate SILAC ratios for
each detected peptide, and corresponding protein identities
were determined from product ion peptide profiles (MS2)
using the ProLuCID search algorithm.22
Two proteins fit the criteria of being labeled by 1
preferentially over 14 and competed at least 2-fold by 5X 37:
1) epoxide hydrolase-1 (EPHX1), a 50 kDa microsomal
enzyme that metabolizes exogenous and endogenous epoxide
metabolites23 (Table 1 and Figure S5 and Tables S1 and S2,
SI); 2) MT-ND1, an integral membrane subunit of the
ubiquinone oxidoreductase (complex 1) involved in mitochon-
drial respiration (Table 1, Figure 3C, and Table S1, SI).24 MT-
ND1 is predicted to be a 35 kDa protein based on amino acid
sequence but has been shown in several previous studies to
migrate as an ∼25 kDa protein by SDS-PAGE.25−28 Gel-based
profiling identified candidate 1-labeled bands that correspond
to both proteins (as deduced by their preferential labeling by 1
over 14 and competition by excess 37; Figure 3C). Gel-based
profiling also detected an additional ∼35 kDa target selectively
reactive with 1 over 14 (Figure 3C, asterisk), but a
corresponding protein target that matches this molecular
weight was not detected in our mass spectrometry analyses,
perhaps because the comparatively weak probe-labeling signals
for this protein precluded sufficient enrichment for its
identification. While it is possible that 1 produces its
antiproliferative effect through a combination of these protein
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Koehler, A. N.; Marcaurelle, L. A.; Golub, T. R.; Gould, R. J.;
Schreiber, S. L.; Carr, S. A. Proc. Natl. Acad. Sci. U.S.A. 2009, 106,
4617.
(11) Evans, M. J.; Saghatelian, A.; Sorensen, E.; Cravatt, B. F. Nat.
Biotechnol. 2005, 23, 1303.
we have provided a more streamlined platform for uniting the
steps of phenotypic screening and target identification.
Projecting forward, we should mention some challenges,
perhaps first and foremost the need to create fully function-
alized libraries of much greater size and complexity. Here, the
development of synthetic methods that allow efficient
incorporation of additional photo-cross-linking groups beyond
the benzophenone, such as diazirines or aryl azides, would be
helpful in expanding the structural diversity of libraries. Another
potentially confounding feature is photo-cross-linking yields,
which can vary considerably depending on the proximity of the
photoreactive group to interacting protein(s). We anticipate
that embedding the photoreactive group within the core
scaffold of libraries should help to maximize protein
interactions and cross-linking yields. Finally, we should
emphasize that hit compounds emerging from phenotypic
screens with fully functionalized libraries should be viewed as
tool compounds as opposed to drug candidates. But, by
defining the target profile required to produce a desired
biological effect as well as establishing a structure−activity
relationship for this activity, we anticipate that the described
strategy will set the stage for future medicinal chemistry efforts
to convert chemical probes into more drug-like small
molecules.
(12) Salisbury, C. M.; Cravatt, B. F. Proc. Natl. Acad. Sci. U.S.A. 2007,
104, 1171.
(13) Wacker, S. A.; Houghtaling, B. R.; Elemento, O.; Kapoor, T. M.
Nat. Chem. Biol. 2012, 8, 235.
(14) Ho, C. H.; Piotrowski, J.; Dixon, S. J.; Baryshnikova, A.;
Costanzo, M.; Boone, C. Curr. Opin. Chem. Biol. 2011, 15, 66.
(15) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B.
Angew. Chem., Int. Ed. Engl. 2002, 41, 2596.
(16) Tornoe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem. 2002,
67, 3057.
(17) Speers, A. E.; Cravatt, B. F. Chem. Biol. 2004, 11, 535.
(18) Speers, A. E.; Adam, G. C.; Cravatt, B. F. J. Amer. Chem. Soc.
2003, 125, 4686−4687.
(19) Wang, Q.; Chan, T. R.; Hilgraf, R.; Fokin, V. V.; Sharpless, K.
B.; Finn, M. G. J. Amer. Chem. Soc. 2003, 125, 3192−3193.
(20) Vaupel, P.; Kallinowski, F.; Okunieff, P. Cancer Res. 1989, 49,
6449.
(21) Weerapana, E.; Wang, C.; Simon, G. M.; Richter, F.; Khare, S.;
Dillon, M. B.; Bachovchin, D. A.; Mowen, K.; Baker, D.; Cravatt, B. F.
Nature 2010, 468, 790.
(22) Tayo, L. L.; Lu, B.; Cruz, L. J.; Yates, J. R., III J. Proteome Res.
2010, 9, 2292.
(23) Morisseau, C.; Hammock, B. D. Annu. Rev. Pharmacol. Toxicaol
2005, 45, 311.
(24) Brandt, U. Annu. Rev. Biochem. 2006, 75, 69.
(25) Murai, M.; Ishihara, A.; Nishioka, T.; Yagi, T.; Miyoshi, H.
Biochemistry 2007, 46, 6409.
ASSOCIATED CONTENT
* Supporting Information
Supplementary figures, experimental protocols, synthetic
schemes, and compound characterization. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
S
(26) Murai, M.; Sekiguchi, K.; Nishioka, T.; Miyoshi, H. Biochemistry
2009, 48, 688.
(27) Kakutani, N.; Murai, M.; Sakiyami, N.; Miyoshi, H. Biochemistry
2010, 49, 4794−4803.
(28) Murai, M.; Mashimo, Y.; Hirst, J.; Miyoshi, H. Biochemistry
2011, 50, 6901.
AUTHOR INFORMATION
Corresponding Author
cravatt@scripps.edu
■
(29) Ahmad, I. M.; Aykin-Burns, N.; Sim, J. E.; Walsh, S. A.;
Higashikubo, R.; Buettner, G. R.; Venkataraman, S.; Mackey, M. A.;
Flanagan, S. W.; Oberley, L. W.; Spitz, D. R. J. Biol. Chem. 2005, 280,
4254.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
(30) Han, M.; Im, D.-S. Arch. Pharm. Res. 2008, 31, 749.
We thank Takao and Akemi Yagi who generously provided
purified submitochondrial particles. This work was supported
by NIH grant GM090294, a Ray Kathren American Cancer
Society fellowship (J.S.C.), and a Ruth L. Kirschstein National
Research Service Award F32GM095245-01 (J.S.C.).
REFERENCES
■
(1) Paolini, G. V.; Shapland, R. H.; van Hoorn, W. P.; Mason, J. S.;
Hopkins, A. L. Nat. Biotechnol. 2006, 24, 805.
(2) Yagoda, N.; von Rechenberg, M.; Zaganjor, E.; Bauer, A. J.; Yang,
W. S.; Fridman, D. J.; Wolpaw, A. J.; Smukste, I.; Peltier, J. M.;
Boniface, J. J.; Smith, R.; Lessnick, S. L.; Sahasrabudhe, S.; Stockwell,
B. R. Nature 2007, 447, 864.
(3) Chen, S.; Do, J. T.; Zhang, Q.; Yao, S.; Yan, F.; Peters, E. C.;
Scholer, H. R.; Schultz, P. G.; Ding, S. Proc. Natl. Acad. Sci. U.S.A.
2006, 103, 17266.
(4) Petrascheck, M.; Ye, X.; Buck, L. Nature 2007, 450, 553.
(5) Guiguemde, W. A.; Shelat, A. A.; Garcia-Bustos, J. F.; Diagana, T.
T.; Gamo, F. J.; Guy, R. K. Chem. Biol. 2012, 19, 116.
(6) Swinney, D. C.; Anthony, J. Nat. Rev. Drug Discovery 2011, 10,
507.
(7) Leslie, B. J.; Hergenrother, P. J. Chem. Soc. Rev. 2008, 37, 1347.
(8) Burdine, L.; Kodadek, T. Chem. Biol. 2004, 11, 593.
(9) Ahn, Y. H.; Chang, Y. T. Acc. Chem. Res. 2007, 40, 1025.
(10) Ong, S. E.; Schenone, M.; Margolin, A. A.; Li, X.; Do, K.; Doud,
M. K.; Mani, D. R.; Kuai, L.; Wang, X.; Wood, J. L.; Tolliday, N. J.;
10388
dx.doi.org/10.1021/ja304213w | J. Am. Chem. Soc. 2012, 134, 10385−10388