REVIEWS

Visualizing biologically active small

molecules in cells using click chemistry
Tatiana Cañeque1,2, Sebastian Müller1,2 and Raphaël Rodriguez   1*
Abstract | Natural products and synthetic small molecules can be used to perturb, dissect and
manipulate biological processes, thereby providing the basis for drug development. Over the
past decades, the evolution of molecular biology protocols and microscopy techniques has made
it possible to visually detect proteins in living systems with valuable spatiotemporal resolution, in
which dynamic topological information has proved to be insightful. By contrast, although small
molecules have become essential for biological studies, general methods to track them in cells
remain underexplored. In this Review, we discuss how bioorthogonal chemistry , and click
chemistry in particular, can be exploited to label and visualize almost any biologically active small
molecule in cells and tissues. We review recent developments, highlighting cases in which
visualizing small molecules has provided crucial mechanistic insights. This methodology is facile
to implement, is versatile and is illuminating.

Natural products are essential to the central dogma of molecular biology (reviewed in depth elsewhere25,26).
molecular biology1. In contrast to large biopolymers, In particular, we focus on the chemical labelling of small
such as DNA, RNA and proteins, these small molecules molecules in cells to identify their subcellular localiza-
can interfere with biological processes as diverse as tion (by imaging) and to understand their mechanism of
membrane trafficking2,3, cell signalling4, regulation of action. Finally, we discuss the scope and limitations
the cell cycle5, regulation of cell division6, translation7, of bioorthogonal chemistry, including experimental
transcription8, DNA replication9, chromatin remodel- strategies that can be adapted to different scenarios, and
ling10 and DNA repair11. Inspired by nature, chemists we document other emerging approaches for visualizing
and biologists have used natural products and synthetic small molecules in cells.
small molecules to dissect and characterize cellular
processes in great detail12,13. Small molecules exhibit Development of bioorthogonal chemistry
unique properties that make them valuable tools to Technologies designed to fluorescently label endog-
study cell biology. For example, small molecules can enous proteins have had a transformative effect on
provide a high level of spatiotemporal control over research in the life sciences by enabling the direct
cellular processes14, inhibit specific functions of mul- visual detection of proteins in cells and organisms27–30
tifunctional proteins15, inhibit enzymatic activity10,16 or (Fig. 1). Structurally distinct molecules such as nucleic
the binding of proteins to various substrates17,18 (such as acids, oligosaccharides and lipids are not suitable sub-
post-translationally modified residues, proteins, DNA strates for these methodologies, which has prompted
and lipid membranes), activate functions19,20 and target the rapid development of bioorthogonal chemistry. As
proteins for degradation21–23. Furthermore, biologically bioorthogonal chemistry enables the functionalization
active small molecules can be used to identify drugga- of almost any class of molecules with high yield, mild
ble targets and thus provide the basis for drug develop- reaction conditions and little or no by-products, its
1
Institut Curie, PSL Research ment24. Despite the ever-growing use of small molecules use in chemistry and biology has grown exponentially
University, CNRS UMR3666– in biological studies and preclinical investigations, the since its description two decades ago. For example, click
INSERM U1143, Paris, inability to visually detect cellular sites of action of small reactions have been used in materials science and drug
France.
molecules is a substantial impediment to fully delin- discovery programmes (comprehensively reviewed else-
2
These authors contributed eating the phenotypes they induce and to effectively where25,26). Bioorthogonal chemistry refers to chemical
equally: Tatiana Cañeque,
Sebastian Müller.
exploiting these drugs. In this Review, we provide a his- reactions that involve complementary functional groups
torical perspective of the development and evolution of that are not present in living systems, that are inert
*e-mail: raphael.rodriguez@
curie.fr bioorthogonal reactions, including click chemistry, and towards biological components and that are prone to
https://doi.org/10.1038/ provide a concise overview of their use in numerous react with one another (for example, by in situ ligation)
s41570-018-0030-x applications spanning materials science to advanced in biocompatible conditions (for example, aqueous

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 Reviews

Original report on azide–alkyne reactions (Michael)129 1893 Chemistry Biology

Reaction of azide with phosphine (Staudinger)130 1919

Original report on azide-strained alkyne reaction (Wittig–Krebs)131 1961

Pioneering work on azide–alkyne cycloadditions (Huisgen)132 1963

1972
Insulin-derived affinity chromatography to isolate
putative insulin receptors133. Small-molecule-derived
affinity reagents to identify mTOR12 and HDACs10
1996
O O

O R2 N3 NHR2
O
1998
R1 P Staudinger R1 P Introduction of bioorthogonal chemistry. Labelling of proteins
Ph ligation Ph
Ph Ph in living cells27. Cell-surface chemical engineering34
Modified Staudinger reaction for bioorthogonal ligation (Bertozzi)34 2000

Introduction of click chemistry (Kolb–Finn–Sharpless)31 2001

Incorporation of azides into recombinant proteins for

Copper(I)-catalysed azide–alkyne cycloaddition (Meldal–Sharpless)35,36 2002 chemoselective modifications64. Activity-based protein
profiling in vivo using click chemistry67. Chemical
N R2 remodelling of cell surfaces in vivo63. Click chemistry
R2 N3 N N
in situ to generate selective protein inhibitors69.
1,3-dipolar azide– 2004
R1 Original report on strained alkynes for copper-free
alkyne cycloaddition R1
azide–alkyne ligation in vivo56

Mechanistic investigation of the Staudinger ligation (Bertozzi)134 2005

Development of DIFO for copper-free click chemistry (Bertozzi)57 2007 Original report on copper-free click chemistry for dynamic
in vivo imaging57. Development of NGS135–137. Original report
O describing the use of reversible terminator chemistry based
on a water-compatible Staudinger reaction underlying
CO2H 2008 Solexa/Illumina NGS technology (patented in 1998)65,66
F
F HO
DIFO DIBO Unbiased methodology combining quantitative proteomics
2009 (SILAC) with affinity enrichment to identify putative targets
Introduction of tetrazine–trans-cyclooctene as a fast copper-free of small molecules76
bioorthogonal reaction (Fox)37
Original report on small-molecule-mediated affinity isolation
2010 of genomic DNA targets107. Original reports of labelling small
R1
Inverse electron demand R1 molecules that target proteins and nucleic acids in cells for
N
Diels–Alder cycloaddition imaging93–95,108. Original example combining click
N
N 2012 chemistry-affinity enrichment with quantitative proteomics
N N
N to identify proteins that bind to epigenetic marks68. Click
R2
R2 chemistry in situ to generate selective RNA
N N
or DNA G-quadruplex-binding small molecules71
H H N2 2013
R1 R1
N N Original report on small-molecule-mediated affinity isolation
NH N of chromatin targets followed by NGS for genome-wide
H localization of small molecules (chem–seq)77,78. Report on a
R2 R2 2014 photocrosslinking-free strategy coupled to click
chemistry-affinity enrichment and mass spectrometry to
Proposed mechanism involving a dinuclear copper intermediate in identify protein targets of a small molecule99
copper(I)-catalysed azide–alkyne cycloadditions (Fokin)138 2017
Integrated approach combining click chemistry, flow
Introduction of sulfur(vI) fluoride exchange as a promising new class of cytometry, mass spectrometry and NGS (click–seq) to
click chemistry reaction (Sharpless)55 characterize drug-induced phenotypes in cells and in vivo102.
2018
Report of in cellulo labelling of a small molecule targeting a
metal ion for cell imaging119

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 Reviews

◀ Fig. 1 | Timeline of major innovations in bioorthogonal chemistry and associated endogenously produced proteins48, DNA60, RNA61 and
biological discoveries. The innovations and discoveries are discussed in detail in the oligosaccharides62, which has provided unprecedented
text. Note that this is not an exhaustive list. In the left column, the observations, biological insights.
discoveries and development of chemical reactions that are suitable for bioorthogonal The development of a modified Staudinger reac-
chemistry , including the Staudinger reaction, copper-catalysed azide–alkyne tion to covalently functionalize glycoconjugates at the
cycloaddition and strain-promoted cycloaddition, are indicated. These reactions, plasma membrane of mammalian cells revolutionized
the invention of new reagents that are suitable for strain-promoted azide–alkyne
chemical biology. In this study, an azide-containing
cycloaddition and proposed reaction mechanisms are listed in chronological order.
In the right column, some of the most important biological applications that relied on saccharide metabolically incorporated into larger oli-
biorthogonal chemistry , including imaging of biomolecules, protein profiling, affinity gosaccharides (that were predicted to be displayed at
isolation, next-generation sequencing (NGS) and imaging of small molecules, are the cell surface) was ligated with a specifically designed
described in chronological order. For clarity , single-isomer products are shown for triarylphosphine containing an ester at the ortho posi-
applicable chemical reactions. DIBO, dibenzocyclooctyne; DIFO, difluorinated tion, which trapped the intermediate aza-ylide to form
cyclooctyne; HDACs, histone deacetylases; mTOR , mechanistic target of rapamycin; a stable amide bond between the two substrates 34.
SIL AC, stable isotope labelling by amino acids in cell culture. Remarkably, this bioorthogonal ligation strategy
operated both in cell culture and living animals63. In
addition, the incorporation of an azido-containing
media, pH (4.5–7.2) and biologically relevant temper- unnatural amino acid in recombinant proteins enabled
atures) with favourable kinetics. Bioorthogonal reac- chemical modifications of proteins using the Staudinger
tions have been developed to produce stable adducts ligation64. The technology underlying next-generation
quantitatively and with high selectivity to reduce back- sequencing (NGS) initially developed by Solexa/
ground signals that are linked to cross reactivity with Illumina was also based on bioorthogonal chemistry
biological entities31–33. Highly useful chemical reac- involving a Staudinger reaction65,66. This technology
tions include the Staudinger ligation34 and click chem- relies on base-by-base nucleotide incorporation in a
istry reactions such as azide–alkyne cycloadditions35,36 stepwise manner using 3ʹ-O-azidomethyl-2ʹ-deoxynu-
and trans-cyclooctene (TCO)–tetrazine inverse elec- cleoside trisphosphates, each labelled with a distinct
tron demand Diels–Alder (IEDDA) cycloadditions37 removable fluorophore. This so-called terminator
(Fig. 1). Other reactions that were developed to fulfil the chemistry enables the addition of all four modified
stringent requirements of biological systems include nucleotides (A, T, G and C) simultaneously during
nitrone–cyclooctyne 38, nitrile oxide–norbornene 39, strand elongation on a solid support while avoiding the
azide–oxanorbornadiene40,41, isonitrile–tetrazine42, nor- risk of misincorporation. After each cycle of incorpora-
bornene–tetrazine43,44, cyclopropene–tetrazine45, bicyc- tion using engineered polymerases that can accommo-
lononyne–tetrazine46–48, tetrazole–alkene photoclick49, date the modified nucleotides, the chemical identity of
fluorosydnone–alkyne50 and quadricyclane cycloaddi- each base is determined from the emission wavelength
tions51, as well as aminothiol–cyanobenzothiazole con- following laser-induced excitation of the incorporated
densations52, sulfo-click involving the condensation of fluorophore. Then, fluorophores loaded on the elongat-
thioacids generated in situ with sulfonyl azides53,54 and ing strand are cleaved using a water-soluble phosphine
sulfur fluoride exchange (SuFEx)55, which can involve through a Staudinger reaction, which simultaneously
a variety of nucleophiles. Given that these reactions releases the free hydroxyl group at the 3ʹ position for
have distinct experimental advantages and limitations, incorporation of the next base. Thus, this ‘sequenc-
they are inherently complementary. In particular, the ing by synthesis’ technology enables ultrafast genome
Staudinger ligation uses phosphine-derived reagents decoding in microfluidic systems, which represents an
that, although not toxic to living organisms, rapidly important milestone towards personalized medicine
oxidize in biological systems, thereby becoming inert based on complete genome sequences.
and refractory to ligation. By contrast, copper-catalysed An original activity-based protein profiling strategy
azide–alkyne cycloaddition (CuAAC) uses more reli- that is based on CuAAC, involving the reaction of an
able functional groups. However, the requirement for alkyne-containing rhodamine with an azide-derivatized
copper at concentrations that are toxic to mammalian phenyl sulfonate ester group, has been developed and was
cells makes this reaction unsuitable for chemical label- used to target specific proteins in living cells and mice.
ling in living cells. To address this problem, copper-free Discrete azide-bearing probes yield a less biased probe
variants of azide–alkyne cycloaddition using structur- distribution in living cells than sterically bulkier tags
ally strained alkynes instead of terminal alkynes have such as fluorophores, which can adversely affect the
been developed56–58 (Fig.  1), such as TCO–tetrazine binding of the probe to its targets67. Consistent with
IEDDA cycloaddition59. Whereas these two methods this idea, a similar approach to profile proteins that
do not rely on a metal catalyst, the use of bulkier rea- interact with post-translational modifications was
gents can more drastically alter biological structures developed68. In this approach, alkyne-derived peptides
and their associated functions. Furthermore, the use of containing specific sequences with selected modifica-
intrinsically more-reactive strained reagents can result tions, such as methylated lysine residues as well as a
in increased nonspecific labelling. Nonetheless, these photoreactive benzophenone, were used to isolate pro-
reactions, together with the metabolic incorporation teins that bind to these modifications from cell lysates.
of unnatural reactive building blocks (for example, A biotin-containing azide was reacted by means of click
genetically encoded unnatural amino acids, nucleotides chemistry, which, together with affinity isolation and
and saccharides), now enable the labelling of quantitative proteomics using stable isotope labelling

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 Reviews

by amino acids in cell culture (SILAC), helped iden- sensors of large biomolecules and electrolytes. For
tify proteins that selectively bind to methylated lysines, example, the DNA-minor-groove-binding chemical
demonstrating the power of this approach. A distinct 4ʹ,6-diamidino-2-phenylindole (DAPI) is routinely
strategy termed in situ click chemistry69 was designed used in cell biology to visualize genomic DNA. Binding
to selectively synthesize potent inhibitors using the of DAPI to AT-rich regions in the minor groove of
target protein (for example, acetylcholinesterase) to DNA gives rise to a light-switch effect that increases
assemble the inhibitors in situ via irreversible cova- its fluorescence81, making it a useful turn-on DNA
lent bond formation between azide-containing and probe (Fig. 2b,c). By contrast, the inherent fluorescence
alkyne-containing building blocks — an approach of the cyanine moiety of a merocyanine-containing
that is conceptually similar to dynamic combinatorial picolylamine is substantially reduced by its chelation
chemistry70. In situ click chemistry was later exploited to copper, making it an excellent turn-off probe for
to produce a small molecule that selectively targets RNA copper(ii) in live-cell imaging experiments82 (Fig. 2d).
G-quadruplexes over those in DNA, a small molecule Furthermore, sodium-binding benzofuran isophtha-
that likely could not have been rationally designed by late (SBFI), a hybrid crown-ether–cryptand embedded
other means71. within two aromatic dyes, was developed to selec-
Although numerous studies have demonstrated its tively interact with sodium rather than potassium
utility, CuAAC is not suitable for labelling in live cells, in cells. Remarkably, the fluorescence properties of
except perhaps to label molecules that target organelles SBFI are drastically altered by chelation of sodium,
in which there is sufficient reactive copper(i) to catalyse which increases the ratio of excitation efficiency at
the desired reaction. Consequently, a variant of CuAAC 330–345 nm to that at 370–390 nm with emission
termed copper-free click chemistry was developed, detected at 450–550 nm, thus allowing for ratiometric
which uses a geometrically strained trans-cyclooctyne, ­fluorometry83 (Fig. 2e).
in which destabilization of the ground state of the alkyne As for biopolymers, most biologically active small
triple C–C bond accelerates the rate of the reaction molecules are not fluorescent, and thus alternative strat-
with azides56. In addition to many other applications, egies, including chemical labelling in cells, are required
copper-free click chemistry has been used extensively for their detection. Differences between endogenously
for in vivo imaging of glycan trafficking57 and for vis- produced biopolymers and biologically active small
ualizing glycans and the sialome during zebrafish molecules that can be artificially introduced into bio-
embryogenesis72. logical systems pose distinct challenges for labelling.
Unlike biomolecules, small molecules can be chemically
Visualizing small molecules in cells engineered in the flask and labelled before treatment of
Delineating the mechanisms of action of small mol- cells. However, the presence of bulky functional groups
ecules can be achieved using affinity reagents to iso- (for example, fluorophores) on small molecules can
late putative targets from complex cell lysates73–75. drastically alter pharmacological profiles, metabolism,
Bioorthogonal chemistry enables the attachment of affin­ target engagement and chemotypes. Bioorthogonal
ity reagents to alkyne-containing or azide-containing chemistry offers an interesting alternative that ena-
biologically active small molecules in treated cells or bles the chemical labelling of small molecules in cells
cell lysates. Small molecules can be chemically edited following treatment and phenotypic induction. This
at strategic positions to implement a functional hook strategy can reveal the subcellular localization and sites
suitable for isolating targets, which can then be char- of action of a small molecule that remains in proxim-
acterized by proteomic analysis for proteins68,76 or NGS ity to its mechanistic target after labelling. The free
for nucleic acids77,78. Cellular detection of biologically diffusion of small molecules in solution at rates that
active small molecules and probes (either intrinsically far exceed those of larger biomolecules is a potential
fluorescent or chemically labelled in cells) can provide limitation. Furthermore, the preparation of such small
information about both their mechanism of action molecules that are prone to bioorthogonal labelling
and the physiological state of cells. For example, the may be hampered by the lack of a suitable anchor point
natural angucycline marmycin A was proposed to to which a reactive functional group (for example,
induce cancer cell death by targeting genomic DNA79; alkyne, alkene, azide and tetrazine) can be introduced
structural similarities with other angucyclines that without altering biological properties. In addition,
were known to interact with DNA in vitro and with these functional groups must remain accessible to
the anthraquinone-containing DNA-intercalating chemical reagents once the molecule is bound to its
drug doxorubicin supported this hypothesis (Fig. 2a). targets to enable efficient labelling. Successful exam-
However, imaging studies exploiting the intrinsic flu- ples include the labelled small molecules that target
orescence of marmycin A revealed that it accumulates the DNA repair enzyme poly(ADP-ribose) polymer-
predominantly in lysosomes80 (Fig. 2b), highlighting ase (PARP)84, diverse protein kinases85, the Argonaute
the fact that mechanisms of action cannot be directly protein Piwi-like protein 3 (PIWIL3)86, the epigenetic
inferred from molecular structures and emphasizing the reader bromodomain-containing protein 4 (BRD4)87,
value of visualizing small molecules in cells. Topological genomic DNA88, RNA89 and components of mitochon-
information indicated that although marmycin A may dria90 and lysosomes91. Similarly, chemically modified
be able to interact with nucleic acids in vitro, DNA was lipids that can be artificially introduced into living cells
not a mechanistic substrate in cells. Photon microscopy and are suitable for bioorthogonal labelling have been
has also been used extensively to detect fluorescent reported92. Thus, bioorthogonal labelling of biologically

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active small molecules can be exploited in various set- structural variant of BMS-790052 containing a biotin
tings. The experimental versatility of protocols, general tag was successfully used to isolate NS5A from a rele-
applicability and substantial potential of bioorthogonal vant cellular model, validating this scaffold as a func-
labelling are discussed further below. tional surrogate of BMS-790052 (ref.94). Introducing a
boron dipyrromethene (BODIPY) fluorophore instead
A small molecule that targets a viral protein. of the biotin tag to this surrogate (Fig. 3a) interfered
Chemical proteomics identified NS5A, a hepatitis C with targeting, as defined by the absence of colocaliza-
virus (HCV)-encoded zinc-binding phosphoprotein, tion with NS5A. By contrast, an azide-functionalized
as the target of the viral replication inhibitor BMS- analogue, which was more potent at inhibiting viral
790052, which is a candidate for the treatment of replication in cells than the BODIPY-labelled probe,
HCV 93. To determine the subcellular localization colocalized with NS5A after chemical labelling in situ
of NS5A, a fluorescent derivative was designed. First, a with a propargyl amine-functionalized Alexa Fluor
488 (Fig.  3b) . This early study demonstrated that
pre-labelling a small molecule can substantially alter
a b its subcellular localization, whereas in situ labelling
O OH O OH
can overcome the various physicochemical barriers
OH that might prevent the drug from interacting with
its target.
O O OH O
10 μm Small molecules that target a protein network.
O Similarly, a TCO derivative of the anticancer drug and
H2N Doxorubicin microtubule-stabilizing agent paclitaxel (Taxol) was
OH
developed to enable its visual detection in living cells95
Doxorubicin
(Fig. 4a). In this study, coupling tetrazine to a series of
O structurally distinct fluorophores was found to quench
intrinsic fluorescence. Interestingly, IEDDA cycload-
H dition of tetrazine-conjugated dyes with TCO restored
O the fluorescence of the dyes, making it a powerful
NH O turn-on labelling strategy with reduced background
HO from excess unreacted dye. Thus, copper-free labelling
Marmycin A DAPI of Taxol–TCO revealed its localization to microtubule
Marmycin A networks, as defined by immunostaining of α-tubulin
in fixed cells (Fig. 4b). By contrast, a pre-labelled Taxol
–
c NH e O2C derivative did not exhibit a microtubule staining pat-
H2N – tern, demonstrating the value of in situ labelling versus
CO2
pre-labelling. Later, a similar strategy was used to label
a Taxol derivative using copper-mediated click chem-
O istry96. In this study, a cell-permeable hybrid reagent
HN
based on a copper(i)-stabilizing ligand embedded within
O an azide-containing fluorophore was developed and
N successfully used to label an alkyne-containing Taxol
O O
derivative in living cells.
H2N NH
O N O Small molecules that target nuclear proteins. The
DAPI
premature ageing syndrome Hutchinson–Gilford
Progeria Syndrome (HGPS) is caused by mutations
d in LMNA, which encodes the lamin A and lamin C
O
components of the nuclear lamina, leading to nuclear
N N –
CO2 envelope defects, misshapen nuclei and disrupted chro-
matin organization. The aberrant nuclear shapes were
N+
I– proposed to directly correlate with the ageing pheno-
N –
CO2 type97,98. Owing to mutations in LMNA, developing
Merocyanine dye SBFI drugs that are capable of correcting nuclear lamina
defects in HGPS is challenging. Nuclear shape defects
Fig. 2 | Visualizing fluorescent natural products and probes in cells. a | Molecular and chromatin compaction were used as readouts to
structure of doxorubicin (top) and marmycin A (bottom) containing a common screen for small molecules that are capable of amelio-
anthraquinone core (red). b | Fluorescence microscopy images of doxorubicin (red) and
rating ageing phenotypes. This discovery-based strat-
marmycin A (red) in U2OS human osteosarcoma cells. 4´,6-Diamidino-2-phenylindole
(DAPI; blue) stains nuclear DNA. Magnified images are 5×. c | Molecular structure of the egy led to the development of remodelin, a synthetic
fluorescent DNA turn-on probe DAPI. d | Molecular structure of a merocyanine-derived small molecule that rescues nuclear shape and chroma-
fluorescent copper(ii)-specific turn-off probe. e | Molecular structure of a ratiometric tin compaction, thus improving the cellular fitness of
sodium-binding benzofuran isophthalate (SBFI) fluorescent probe. Part b adapted from cells derived from patients with HGPS99. In this study,
ref.80, Springer Nature Limited. a biotinylated derivative of remodelin was produced

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 Reviews

a
N
O
B F
N
F HN

S
O

O H
Labelled probe derived from BMS-790052 N
N
O
O O
O
HN
N
O

b N3

O
– –
SO3 SO3
+
H2N O NH2 O

O H
N
– N
CO2
O
O O
O
HN
N O N
H O

Alexa Fluor 488-PA Azide probe derived from BMS-790052

488 N N
N
In situ labelling of BMS-790052-derived
azide-containing probe using click chemistry
O

O H
N
N
O
O O
O
HN
N
O

Fig. 3 | Visualizing a clickable small molecule targeting the viral protein NS5A. a | Molecular structure of a
fluorescently labelled drug surrogate derived from BMS-790052. b | Chemical labelling of a BMS-790052-derived
azide-containing drug surrogate with a propargyl amine-functionalized Alexa Fluor 488 (Alexa 488-PA) using click
chemistry.

in situ using CuAAC and was used to successfully iso- the authors proposed that destabilizing the microtu-
late N-acetyltransferase 10 (NAT10). Chemical label- bule network could release mechanical forces, decrease
ling of an alkyne-containing remodelin analogue in nuclear shape defects, reduce DNA damage and rescue
cells revealed a nuclear distribution that was altered by cellular fitness99.
small interfering RNA-mediated knockdown of NAT10 Bromodomain and extraterminal (BET) family pro-
(Fig. 5a,b). Together, these experiments validated NAT10 teins have a role in many cellular processes and are poten-
as a mechanistic target of remodelin. Consistent with tially druggable targets for the clinical management
NAT10 shuttling outside of the nucleus, further investi- of cancer. In particular, BRD4 is an epigenetic reader
gation indicated that NAT10 modulates the microtubule that recognizes acetylated lysines on chromatin and has
network, suggesting a pathophysiological mechanism been linked to cancer100,101. The synthetic small mole-
whereby physical forces applied to a weakened nuclear cules I-BET and (+)-JQ1 are protein–protein interac-
envelope contribute to chromatin disorganization and tion (PPI) inhibitors that prevent the binding of BRD4
DNA lesions, which have implications in ageing. Thus, to chromatin, which promotes the differentiation of

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Reviews

a click chemistry, protein target pull-down and quan-

O
O
titative proteomics, termed click proteomics, showed
N
O that structurally distinct inhibitors targeted identical
AcO O O H
N N
protein complexes, which is consistent with the similar
B biological effects of the inhibitors. Remarkably, flow
O NH O
N
F F
cytometry analysis of cells isolated from mice treated
N
O O
N
H
in vivo and high-resolution microscopy of tissues were
H O
OH OH OBz OAc N N used to assess the heterogeneity of drug activity within
Taxol–TCO Tetrazine–BODIPY fluorescent label
tumour cells located in different tissue compartments
and to evaluate differences in the intracellular level of
drugs in haematopoietic cells from healthy versus AML
In situ labelling mice. Thus, click chemistry enabled a thorough preclin-
N2
O
of Taxol–TCO ical evaluation of BET inhibitors, providing a general
NH framework for the study of other molecules that target
O OAc
O epigenetic modifications.
O
O O These two examples demonstrate the general appli-
HO O
N
cability of labelling small molecules in cells regard-
H O less of protein function, such as enzymatic activity or
HO NH
BzO H
chromatin-docking properties.
N
AcO O

Small molecules that target genomic DNA. Although

double-stranded DNA or replicating DNA can readily
O be detected using dyes such as DAPI or the metabolic
F
HN F incorporation of 5-ethynyl-2ʹ-deoxyuridine (EdU),
B
N
N respectively, methods to detect other DNA structures
are rare. G-quadruplexes are alternative, non-Watson–
b Crick base-paired nucleic acid structures arising
from specific G-rich sequences103. Guanine residues
interact with one another within the same strand by
means of hydrogen-bonding, electrostatic and stack-
ing interactions to produce higher-order architectures
that are virtually four-stranded. A broader definition
of G-quadruplexes also encompasses intermolecular
structures that are formally composed of more than
one strand. The prevalence of G-rich sequences that
are predicted by bioinformatics and biophysical analy-
ses to fold into G-quadruplexes in genomic DNA104,105
30 μm has prompted the search for putative biological func-
tions of G-quadruplexes and the physiological conse-
Fig. 4 | Visualizing a clickable small molecule that stabilizes microtubules. quences of their occurrence. To this end, the synthetic
a | Chemical labelling of a paclitaxel (Taxol)–trans-cyclooctene (TCO) derivative small molecule pyridostatin was designed to selec-
with a non-fluorescent tetrazine–boron dipyrromethene (BODIPY) fluorescent label tively interact with G-quadruplexes by supramolecular
by copper-free strain-promoted cycloaddition to yield fluorescently labelled Taxol interactions106,107 (Fig. 6a). Chromatin immunoprecip-
in cells. b | Fluorescence microscopy images of the labelled Taxol surrogate (white) in itation of the DNA damage marker γH2AX, which
PtK2 kangaroo kidney epithelial cells. Part b adapted with permission from ref.95, is induced by treatment with pyridostatin, followed
Wiley-VCH. by NGS (ChIP–seq) indicated that pyridostatin pro-
moted the formation of DNA breaks at specific regions
of the genome, including in gene bodies that contain
cancer cells and thereby limits cancer progression17,18. clusters of DNA sequences that are prone to fold into
In a comprehensive study, functionally conserved G-quadruplexes in vitro108. Chemical labelling of a ter-
alkyne and TCO-containing analogues of BRD4 minal alkyne-containing pyridostatin surrogate, termed
inhibitors were used in various settings to delineate pyridostatin-α, in cells using click chemistry revealed a
in depth the mechanisms of action of these drugs in striking colocalization of labelled pyridostatin-α with
a mouse model of acute myeloid leukaemia (AML)102 the G-quadruplex ATP-dependent DNA helicase PIF1
(Fig.  5c) . For example, the genome-wide occupancy in cancer cell nuclei (Fig. 6a,b). This study provided
of BET inhibitors was established using a combina- direct evidence of the existence of G-quadruplex struc-
tion of click chemistry, chromatin fragment isolation tures in human genes. From a technical standpoint, the
and NGS, termed click-seq, which enabled the direct clickable pyridostatin analogue was used in fixed cells
identification of genes that are affected by these inhib- before labelling and co-staining of PIF1, indicating
itors. Furthermore, clickable derivatives could be vis- that pyridostatin did not induce G-quadruplex fold-
ually detected in cell nuclei and colocalized with their ing in cells but instead targeted pre-folded structures.
mechanistic target BRD4 (Fig. 5d). A combination of This finding was later corroborated using specific

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 Reviews

a S
and DNA replication by forming intrastrand and inter-
S
CN Cl strand DNA crosslinks (DNA–Pt), leading to DNA
N N N
N
N N damage and cell death. However, some cancer cells
H
are refractory or develop resistance to these drugs.
An azide-containing cisplatin surrogate, 2(aminome-
Remodelin Remodelin analogue
thyl)pyridinedichloroplatinum(ii) azide (APPA), was
b NAT10 Click Merge engineered to detect DNA–cisplatin crosslinks in vitro
siCT by using a strained dibenzocyclooctyne (DIBO)-
functionalized fluorophore in a copper-free reaction
or a terminal alkyne-containing fluorophore (Alexa
Fluor 488) for visual detection of DNA–Pt in cells113
(Fig. 6c,d). APPA was used in living cells, whereas the
fluorophore was reacted with APPA by click chemistry
in fixed cells for evaluation of the phenotypes induced
10 μm by APPA treatment. Interestingly, labelling DNA–Pt in
APPA-treated cells revealed a pan-nuclear staining pat-
siNAT10 tern. The inclusion of excess cisplatin with APPA treat-
ment substantially reduced the fluorescence intensity
of labelled DNA–Pt, indicating that cisplatin and APPA
target similar genomic loci. This system was exploited
to screen for small molecules that alter the pattern of
DNA–Pt produced by APPA, as these small molecules
could potentially synergize with cisplatin and its deriv-
atives. For example, the histone deacetylase inhibitor
and anticancer drug vorinostat caused a redistribution
c N N
N N of DNA–Pt, which formed defined foci that are pre-
N O N
H
N
H
N O sumably clusters of platinated lesions. Furthermore,
S S
DNA–Pt foci colocalized with proliferating cell nuclear
N O N O O
antigen (PCNA) and E3 ubiquitin-protein ligase
RAD18, suggesting that translesion synthesis (TLS)
was activated at these sites (Fig. 6e). Although the TLS
machinery enables lesion bypass and resistance in nor-
Cl Cl mal conditions114, co-treatment of cells with APPA or
(+)-JQ1 JQ1–TCO
cisplatin and vorinostat induced a RAD18-dependent
apoptotic response, indicating that these drugs act
d DAPI Click BRD4 Merge via a mechanistic synergy. These findings revealed a
role for chromatin in genome targeting with cispla-
tin drugs, providing an explanation for the effects of
these drugs.

Small molecules that target metal ions. During cancer

10 μm progression, a small population of cancer cells, known
as cancer stem cells, can shift between distinct phe-
Fig. 5 | Visualizing clickable small molecules that target proteins with enzymatic notypic states that are characterized by epithelial and
activity or chromatin-docking properties. a | Molecular structure of remodelin and a mesenchymal traits. This plasticity enables tumour
clickable remodelin surrogate. b | Fluorescence microscopy image of the labelled cells to disseminate to distant locations and form
remodelin surrogate (green) in U2OS cells. Colocalization with N-acetyltransferase 10 metastases. Cancer stem cells exhibit self-renewal and
(NAT10; red) is shown. Control small interfering RNA (siCT) and that against NAT10 tumour seeding properties115–117. These cells are also
(siNAT10) were used as indicated. The peripheries of the nuclei are highlighted by
refractory to conventional treatments and are associ-
dashed lines. c | Molecular structure of (+)-JQ1 and the clickable surrogate JQ1–trans-
cyclooctene (TCO). d | Fluorescence microscopy image of the colocalization between ated with cancer relapse. The natural product salin-
labelled JQ1–TCO (green) and bromodomain-containing protein 4 (BRD4; red) in HeLa omycin (Fig. 7a) was identified in a high-throughput
human cervical cancer cells. 4´,6-Diamidino-2-phenylindole (DAPI; blue) stains nuclear screen as an inhibitor of breast cancer stem cell pro-
DNA. Part b adapted with permission from ref.99, AAAS. Part d adapted with permission liferation118. The mechanism of action of salinomycin
from ref.102, AAAS. underlying its selectivity against cancer stem cells was
initially thought to involve the transport of alkali met-
als (such as sodium and potassium) in cells and to alter
antibodies raised against G-quadruplexes and using the ionic balance, leading to cell death. The inability to
NGS data109–111. visually detect salinomycin in cells precluded a robust
Cisplatin and its derivatives are commonly used characterization of a mechanism of action underlying
Pt-based chemotherapeutic agents for the treatment of its selective killing of cancer stem cells. Using a series
cancer112. Pt-based drugs form covalent bonds with gua- of clickable alkyne-containing derivatives, it was shown
nine residues in DNA and interfere with transcription that molecules derived from salinomycin, including a

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Reviews

more potent synthetic compound named ironomycin, two approaches can provide further topological and
predominantly accumulate in lysosomes119 (Fig. 7a,b). mechanistic insights.
In particular, salinomycin and ironomycin interacted Importantly, detection of labelled surrogates at sites
with and sequestered iron in this organelle, leading of action can be prevented by the presence of more
to the production of reactive oxygen species (ROS) abundant and functionally less relevant cellular com-
via Fenton chemistry and to ferroptotic cell death120. ponents that interact with the clickable surrogate. In
Importantly, this study revealed that cancer stem cells this case, a pre-extraction step with detergent before
have greater iron requirements than normal cells, fixation will effectively remove some of these com-
suggesting a role for this metal in the maintenance of ponents108,113. In addition, proteinase K or RNase A
these cells and opening up unprecedented therapeutic treatment can be combined with pre-extraction to
opportunities. release clickable surrogates that are bound to soluble
biomolecules and improve visualization of function-
Scope and experimental considerations ally relevant sites of action. Click labelling of untreated
Insights into the mechanism of action of a small mole- fixed cells enables the quantification of the extent of
cule can be inferred from topological information. To nonspecific background fluorescence linked to a given
this end, biologically active surrogates that are suitable protocol and fluorophore. Interestingly, the residual
for bioorthogonal chemistry can be used to determine background is usually lower for copper-mediated click
the subcellular localization of drugs. Most biologically chemistry than for copper-free reactions. Further­
active small molecules are amenable to chemical func- more, copper-mediated click chemistry is sensitive
tionalization without noticeable alterations in target to oxygen and thus the quality of reagents and the
engagement or biological activity in cells. The chemical labelling efficiency should be evaluated using meta-
synthesis and design of these functionalized surrogates bolic incorporation of EdU in live cells followed by
may be influenced by existing structural information click staining, which can be used as a positive con-
(for example, X-ray or NMR structures of the drug trol108. In line with this, a biologically inactive click-
bound to its target) or by structure–activity relation- able surrogate can be used to assess the functional
ship data17,102. If these data are not available, a series relevance of the fluorescence readout obtained for its
of analogues can be obtained by molecular editing of biologically active counterpart. Similarly, competition
the drug or through total synthesis of the analogue, experiments using a small excess of a drug together
depending on synthetic tractability119. Nevertheless, the with its clickable surrogate may induce changes in the
biological activity of clickable surrogates must be thor- fluorescence that are characteristic of a given mecha-
oughly investigated to ensure that it fully phenocopies nism113. Click labelling of small molecules can also be
the activity of the parent small molecule. Although combined with standard immunostaining to identify
cell viability assays provide rudimentary biological or rule out putative protein targets. In this case, the
information for cytotoxic compounds, they must be click labelling can be carried out before or after immu-
complemented by more sophisticated phenotypic nostaining. Validation of potential protein targets can
readouts102. also be achieved using target-specific RNA interfer-
Various chemistries are amenable to either live-cell ence. For example, the fluorescence pattern of labelled
imaging or fixed-cell fluorescence microscopy, as small molecules can be altered following depletion of
shown for the above-described examples. Live-cell their protein targets99.
imaging does not necessarily provide more insights In target identification studies, click labelling of
than studies on fixed cells95,96, except perhaps for drugs small molecules together with cell imaging may be
involving mechanisms of action that directly rely on particularly informative, as it can provide information
trafficking. In the majority of cases, the mechanism on a suitable position to functionalize the bait with an
of action of small molecules can be studied using affinity reagent (for example, biotin) without altering
fixed-cell imaging coupled to time course analyses of target engagement99. Chemical labelling has proved to
the distribution of labelled small molecules in cells by be applicable to a diverse range of cell lines and to struc-
fluorescence microscopy119. Various fixatives, such as turally diverse small molecules with distinct mecha-
paraformaldehyde, glutaraldehyde or methanol, can nisms of action. Except in a few cases, copper-mediated
be used in parallel to evaluate their influence on the click chemistry is unsuitable for live-cell fluorescence
observed subcellular distribution of the labelled drug microscopy but provides an almost quantitative read-
or induced morphological alterations. Furthermore, out owing to the high specificity of the reaction and
clickable surrogates can be used on fixed cells and yields that are linked to the use of reagents in excess.
live cells before chemical labelling and cell imaging to Importantly, copper can alter the fluorescence of the
provide different types of information108. Treatment of green fluorescent protein (GFP) tag in GFP fusion
fixed cells with clickable surrogates provides a readout proteins, an issue that can be circumvented using
that can be compared with that obtained using primary anti-GFP antibodies108. By comparison, methods based
antibodies against a protein of interest, specifically pro- on strained alkynes or alkenes are suitable for live-cell
viding information about the site of action of the drug imaging but are less specific and less quantitative.
and its target. By contrast, live-cell treatment before In particular, a drawback of strained alkynes is their
fixation, click labelling and imaging provides infor- potential cross reactivity, whereas strained alkenes,
mation on dynamic events that are associated with such as TCO, can isomerize and become inert for
the phenotype of interest113. Comparing the data from the ­chemical labelling.

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 Reviews

H2N HN

O O

O O O O
N N
O NH HN O O NH HN O
H2N NH2 H2N NH2
N N N N

Pyridostatin Pyridostatin-α

b
GFP–PIF1 Click Merge c
Cl Cl
10 μm Pt
H3N NH3

Cisplatin

Cl
Cl Pt NH2
N

N3
APPA

d A T A T A T
A T A T A T 488

A T A T A T N
N
N3
A T A T N3 A T N

A T A T A T
Cl N
N N
C G Pt C G C G
Pt Pt
C G Cl NH2 C G C G
NH2 NH2

A T APPA A T A T
A T Treatment of A T In situ A T
A T live cells A T click labelling A T
A T A T A T

e Click RAD18 PCNA Merge

20 μm

NaTuRE REviEwS | ChemiSTry volume 2 | SEPTEMBER 2018 | 211

 Reviews

◀ Fig. 6 | Visualizing clickable small molecules that target genomic DNA. Spontaneous Raman microscopy and stimulated
a | Molecular structure of pyridostatin and the clickable surrogate pyridostatin-α. Raman scattering microscopy are emerging techniques
b | Fluorescence microscopy image of the colocalization of a green fluorescent to track small molecules in cells. However, these meth-
protein (GFP)-tagged nuclear version of the G-quadruplex ATP-dependent DNA ods are limited by a lower resolution and sensitivity than
helicase PIF1 (green) with labelled pyridostatin-α (red) in U2OS cells. GFP-tagged
other technologies125. Interestingly, alkynes and azides
nuclear PIF1 was detected using an anti-GFP antibody. Magnified images are 5×.
The peripheries of the nuclei are highlighted by dashed lines. c | Molecular structures
are suitable functional groups to enhance the sensitivity
of cisplatin and the clickable surrogate 2-(aminomethyl)pyridine dichloroplatinum(ii) of Raman spectroscopy, which enables visual detection of
azide (APPA). d | Schematic representation of the formation of a platinated DNA biologically active small molecules without the need
lesion and chemical labelling of the lesion using copper-catalysed azide–alkyne for further cell fixation, permeabilization and labelling
cycloaddition. e | Fluorescence microscopy image of labelled APPA (green) in U2OS by click chemistry and thus can be carried out in liv-
cells pre-treated with vorinostat. White arrows indicate foci of labelled APPA that ing cells126. However, the resolution is inferior to that of
colocalize with E3 ubiquitin-protein ligase RAD18 (red) and the replication protein click chemistry coupled to fluorescence microscopy. In
proliferating cell nuclear antigen (PCNA ; blue). Magnified image is 2×. Part b adapted addition, the concentrations required for detection are
from ref.108, Springer Nature Limited. Part e adapted with permission from ref.113, typically higher than those used in other techniques and
Wiley-VCH.
are physiologically less relevant.

Conclusions
Alternative detection strategies The studies discussed above emphasize the value of
Methodologies that are conceptually distinct from visualizing small molecules in cells and highlight how
click chemistry for visualizing biologically active small click chemistry is useful in this context. Clickable small
molecules in cells have been developed with the aim to molecules are amenable to diverse techniques, includ-
provide mechanistic insights. ing proteomics, NGS, flow cytometry and fluorescence
A tritiated G-quadruplex-binding small-molecule microscopy, which can be applied independently or in
analogue of 360A was used to identify the sites of action combination to delineate mechanisms of action in rele­
of 360A in genomic DNA121. In this study, metaphase vant disease models. The versatility of experimental
chromosome spreads of cultured cells were incubated protocols is such that the methodology can be adapted
with 3H-360A and visualized by autoradiography; to address specific questions, including the functional
3
H-360A preferentially accumulated at telomeres, the evaluation of a clickable surrogate, the validation of
ends of chromosomes that contain DNA sequences that putative mechanistic protein targets and the unbiased
are prone to fold into G-quadruplexes. This study deliv- identification of synergistic treatments. The studies dis-
ered some of the earliest visual evidence of G-quadruplex cussed here also demonstrate that labelling small mole­
targeting with small molecules using structured genomic cules using click chemistry together with cell imaging
DNA. However, as labelling was carried out in the is broadly applicable to structurally diverse and com-
absence of most other cellular components, this may plex small molecules that interact with a wide range of
not reflect G-quadruplex folding and drug binding cellular targets, involving diverse biological processes
in physiologically relevant conditions. Although this and various subcellular compartments. For example,
method can be useful given that it relies on a structur- click chemistry has been used for in cellulo detection of
ally closely related analogue of the parent small mole- small molecules that interact with nuclear DNA or pro-
cule, the resolution typically achieved using fluorescence teins with diverse functions as well as small molecules
microscopy has not been obtained, and it involves the that target metal ions in lysosomes. Although imaging
synthesis of radiolabelled small molecules, a non-trivial data obtained from fixed cells were used in most of these
step of the process. Similarly, isotopically labelled drugs studies, live-cell imaging can provide additional insights
can be detected with isotope-selective nanoscale second- about the cellular uptake mechanisms, subcellular distri-
ary ion mass spectrometry (nanoSIMS). For example, bution, dynamics of target engagement and interactions
multi-elemental analysis revealed that cisplatin deriv- with other molecules that mediate a phenotype of inter-
atives preferentially colocalized with sulfur clusters in est127. Importantly, small molecules can be refractory
the cytosol and phosphorus-rich chromatin regions to in situ labelling approaches owing to poor reactivity
in the nucleus, which is consistent with off-target effects of bioorthogonal functionalities embedded within a
of Pt drugs that cross-react with and are inhibited by drug derivative, increased steric hindrance imposed by
cysteine residues as well as genomic DNA targeting, the environment surrounding the reactive group once the
respectively122. Furthermore, using 15N-labelled cis- surrogate drug binds to its target or weak drug–target
platin, displacement of the nitrogen ligands of Pt was binding that is susceptible to signal loss during washing
observed in nucleoli. By contrast, a ruthenium-based steps. Therefore, additional efforts to identify alternative
drug accumulated at the plasma membrane of cancer chemical reactions will be pivotal for establishing new
cells and selectively lost a carbon-based Pt ligand but protocols for labelling small molecules in living cells
not its phosphine ligand, providing further insights into that use mild conditions, that preserve the integrity of
how this class of drug interacts with putative targets123. cells and that use reagents that are cell-permeable and
Thus, this technology, usually used in combination with biocompatible. Furthermore, electron microscopy128
other techniques such as electron microscopy or fluores- and super-resolution microscopy coupled with chem-
cence microscopy124, can provide otherwise unobtaina- ical labelling of small molecules in cells should pro-
ble insights but requires a demanding infrastructure that vide unprecedented structural detail about the effect of
is not always easily accessible. drugs in cells and organisms. We anticipate that these

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 Reviews

a
OH O OH O
O O
O O O O
H H O H H O
OH OH
OH NH
O O O O
H H
OH OH

Salinomycin Ironomycin

b Click Lysotracker DAPI Merge

10 μm

Fig. 7 | Visualizing a clickable small molecule that targets lysosomal iron. a | Molecular structure of salinomycin and
the clickable surrogate ironomycin. b | Fluorescence microscopy image of the colocalization of labelled ironomycin
(green) with the lysosomal marker Lysotracker (red) in HMLER human mammary cells. 4´,6-Diamidino-2-phenylindole
(DAPI; blue) stains nuclear DNA in the merged image. Magnified image is 6×. Part b adapted from ref.119, Springer
Nature Limited.

technologies will expand considerably to encompass clinically approved drug derivatives in patient samples
the study of a greater diversity of molecular structures (for example, patient-derived xenografts) could be used
and mechanisms of action and will be used beyond as a predictor of drug response.
standard cell culture to include patient-derived tissue
samples and live animals. It is conceivable that labelling Published online 16 August 2018

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