Spotlight

pubs.acs.org/acschemicalbiology

READING RNAS IN THE CELL

the in situ reads mapped to mRNAs. As expected, many of these

were highly abundant genes and were markers for broblasts.
In this proof-of-principal study, the authors showed that
their sequencing preparation method could work in tissues
such as brain slices or mouse embryos. This opens the door
for the FISSEQ technique to take on challenging problems in
complex organisms like cell-type specic transcription or RNA
processing.
Jason G. Underwood,, Ph.D.

A POWERFUL AND SELECTIVE TYPE I TYROSINE

KINASE INHIBITOR

From Lee, J. H., et al., Science, 2014, 343, 1360. Reprinted with
permission from AAAS.

In the past decade, high-throughput sequencing technologies

have inspired a wide range of novel techniques to interrogate
the genome and transcriptome. By altering the biochemical
steps of sample preparation, sequencing data is now used to
determine the structure of genomes or RNAs, observe ribosome
positioning on the mRNA, or determine the methylation status
of the DNA. These and many other seq methods have
emerged during the recent technology boom, but they all share
the common thread of observing sequences as they are copied
in the instrument. Now, a group has taken that observation back
to the cell by performing uorescent in situ RNA sequencing, or
FISSEQ.
Instead of the usual avor of RNA-seq where cDNA is read
while immobilized on a solid support in the sequencer, Lee
et al. (Science, 2014, 343, 13601363) generated the cDNA
segments inside of cells by cross-linking, circularization, and
rolling circle amplication. The resulting cells were subjected to
the same ligation-mediated sequencing chemistry used by the
Applied Biosystems SOLiD sequencer. Instead of imaging on
the usual instrument, however, confocal uorescence microscopy was used to see 2730 base pair reads growing into small
foci approximately 600 nm in diameter. Analysis of the data
conrmed the known subcellular locations for both cytoplasmic
and nuclear RNAs while proling expression of thousands of
genes. Using human primary broblasts, an impressive 43% of
2014 American Chemical Society

Smith, C. C., et al., Proc. Natl. Acad. Sci. U.S.A. DOI: 10.1073/
pnas.1320661111. Copyright 2014 National Academy of
Sciences, U.S.A.

Targeted cancer therapies are critical tools for the treatment of

specic cancers. Tyrosine kinase inhibitors (TKIs) make up an
important class of these treatments because they antagonize
enzymes that promote cellular growth. But developing these
drugs is dicult, and TKIs typically show at least one of two
common aws: they may interact with enzymes other than their
intended targets and thereby cause toxicity, or cancer cells may
easily acquire resistance to them. Now Smith et al. have shown
that a new TKI for acute myeloid leukemia (AML), crenolanib,
is both selective for its target, FLT3, and is invulnerable to the
most common mechanism of resistance (Proc. Natl. Acad. Sci.
U.S.A., 2014, DOI: 10.1073/pnas.1320661111).
Researchers have typically found that TKIs that bind to
inactive forms of their tyrosine kinase target (called type II
inhibitors) are the most selective, and most of the most
successful TKIs used in cancer therapy fall into this category,
with imatinib representing the prototypic example. Although
type II inhibitors can have very selective binding proles, cancer
Published: April 18, 2014
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Spotlight

Key to the success of their approach was the presence of

sucient cellular holdase activity, created by ATP depletion of
the lysed cell, which converts chaperones to holdases that bind
avidly to protein folding intermediates. Cellular holdase activity
prevents folding probe-associated folding equilibrium shifts.
This study oers insight into the intricacies that govern cellular
proteostasis and provides a blueprint for creating uorogenic
folding probes, probes that can be used in a plate reader to
quantify the folded fraction of a protein-of-interest to explore
how protein folding dynamics inuence cellular function.

cells can often develop resistance to these drugs over time by

acquiring mutations that favor the active kinase conrmation.
Therefore, small molecules that target the active conformation
of the kinase (Type I inhibitors) yet retain a high degree
of selectivity are particularly appealing. Crenolanib is a type I
inhibitor, that binds to and inhibits the active conformation
of select class III receptor tyrosine kinases of which FLT3,
the most commonly mutated gene in AML, is a member. Smith
and Lasater et al. investigated the specicity and activity of
crenolanib toward FLT3.
Initial studies demonstrated that crenolanib selectively
inhibited FLT3 over KIT, a closely structurally related class
III RTK. Given its specicity for FLT3 inhibition, crenolanib
was screened against a panel of FLT3 mutants known to confer
resistance to other FLT3 TKIs and was found to maintain
activity against all of these mutants. Crenolanibs lack of
vulnerability to known TKI resistant mutations prompted the
authors to search broadly for any possible drug-resistant
mutations. Crenolanib showed remarkably limited vulnerability
to mutations in FLT3 that could lead to resistance, thereby
warranting its designation as a pan-FLT3 inhibitor. Therefore,
crenolanib may represent an important step forward for
achieving deep and durable remissions in AML patients who
have activating FLT3 mutations.

Eva J. Gordon,, Ph.D.

HARNESSING THE HEXOSAMINE PATHWAY

Sarah A. Webb,, Ph.D.

PROBING PROTEIN FOLDING IN CELLS

Reprinted from Cell, 156, Denzel, M. S., et al., Hexosamine

Pathway Metabolites Enhance Protein Quality Control and
Prolong Life, 11671178. Copyright 2014, with permission
from Elsevier.

Liu, Y., et al., Proc. Natl. Acad. Sci. U.S.A., 111, 44494454.
Copyright 2014 National Academy of Sciences, U.S.A.

Though strung together as a linear collection of amino acids,

proteins must fold into intricate three-dimensional structures in
order to function properly. Through this process, various states
of properly folded, misfolded, and aggregated proteins can
coexist. It is relatively simple to distinguish aggregated, insoluble
protein from soluble protein; it is less straightforward to discern
between soluble folded and soluble misfolded protein species in
complex cellular environments. To gain insight into this tangled
dynamic, Liu et al. (Proc. Natl. Acad. Sci. U.S.A., 2014, 111,
44494454) present the design, synthesis, and utility of
uorescent small molecule probes that are highly selective for
a folded and functional protein-of-interest over their soluble, but
misfolded, counterparts.
The authors test their approach with two proteins, a de novo
designed retroaldolase and the thyroid hormone binding
protein transthyretin. They use mutated, destabilized versions
of these proteins, because an increased proportion of the proteinof-interest is present in a soluble misfolded statefacilitating an
investigation of the distribution of folded and soluble misfolded
proteins in cells. Using small molecule uorescent folding probes,
they were able to quantitate the fraction of folded and functional
protein in cell lysates at a given point in time and discover the
dependence of this fraction on the cellular folding environment.

Aging, the extraordinarily complex but inevitable process that

so many seek to elude, devolves through an accumulated
decline in function at the cellular, organ, and whole organism
level. Protein quality control systems, which regulate protein
synthesis, folding, maturation, and removal in the cell, play
a key role in longevity, and misregulation in these systems is
associated with a variety of age-related diseases. The nematode
Caenorhabditis elegans has been a pioneering model system to
study the inuence of protein quality control on longevity,
as exemplied by numerous long-lived mutant strains that are
able to sustain protein homeostasis to older ages. Exploring
this remarkable phenomenon further, Denzel et al. (Cell, 2014,
156, 11671178) discover that certain mutations in an enzyme
called GFAT-1 lead to diminished susceptibility to aging
disorders and increased lifespan.
The authors developed a screen to identify C. elegans mutants
resistant to treatment with tunicamycin, an inhibitor of the
synthesis of N-glycans, which are essential for the proper maturation and folding of many secreted proteins. Certain mutations
that activate GFAT-1, a key enzyme in the hexosamine pathway
responsible for synthesizing precursors for N-linked and O-linked
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glycans, conferred resistance to tunicamycin. Activation of the

hexosamine pathway, as well as supplementation with the
N-glycan precursor N-acetylglucsoamine, led to lifespan extension
and protection from certain age-related neurotoxicities. Though
an association between protein quality control and longevity
is well established, this study uncovers an unexpected link
between hexosamine metabolites and the protein quality control
system that was previously unappreciated. These ndings suggest
that enhancing the capacity of the cells protein quality control
mechanisms through hexosamine pathway activation or supplementation with N-acetylglucosamine may be a therapeutic
strategy for various diseases related to aging or protein misfolding.
Eva J. Gordon,, Ph.D.

A PHOTO-OP FOR GPCRS

Approximately one-quarter of marketed drugs target G-protein
coupled receptors (GPCRs), underscoring their value (both
therapeutic and nancial) in medicine. New molecular tools for
investigating GPCR biology will deepen our understanding of
how these proteins function and could lead to the development
of new and improved therapeutics for a variety of conditions.
Opioid receptors, which control the perception of various
sensations including pain and euphoria, belong to Family A of
the GPCRs. Also in this family are the opsin photoreceptors,
such as rhodopsin, which control vision. Rhodopsin is activated
upon a light-induced cistrans isomerization of a covalently
bound ligand, retinal. Schonberger et al. (Angew. Chem., Int. Ed.,
2014, 53, 32643267) cleverly impart this light-sensitive property
onto the u-opioid receptor (MOR) by designing and synthesizing
a photochromic ligand derived from the MOR agonist fentanyl.
The photochromic ligand, referred to as PF2, undergoes a
double-bond isomerization upon exposure to light. Specically,
in the dark or exposure to 420480 nm light, the transconguration predominates, but irradiation with 360 nm light
induces conversion to the cis-isomer. trans-PF2 is an eective
opioid receptor agonist, but cis-PF2 is much less active. Thus,
receptor activity can be manipulated by switching between 360
and 480 nm light. Indeed, using electrophysiology to measure
receptor activity, the authors show that under 480 nm light,
a potassium inux is triggered, but the inux can be stopped
by switching to 360 nm light. This photoactivation was stable
and repeatable. This method for optically controlling MOR
activity oers a new approach for exploring Family A GPCRs.
Moreover, it provides a blueprint for pharmacological development of light-activated drugs.
Eva J. Gordon,, Ph.D.

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