Cytosolic Delivery of Proteins by Bioreversible Esterification

Article abstract of DOI:10.1021/jacs.7b06597

Cloaking its carboxyl groups with a hydrophobic moiety is shown to enable a protein to enter the cytosol of a mammalian cell. Diazo compounds derived from (p-methylphenyl)glycine were screened for the ability to esterify the green fluorescent protein (GFP

Full text of DOI:10.1021/jacs.7b06597

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Cytosolic Delivery of Proteins by Bioreversible Esterification
Kalie A. Mix, Jo E. Lomax, and Ronald T Raines
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b06597 • Publication Date (Web): 04 Oct 2017
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†
,‡
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,†,‡,#
Kalie A. Mix, Jo E. Lomax, and Ronald T. Raines*
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†
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Department of Biochemistry, Program in Cellular and Molecular Biology, and Department of Chemistry, University of
‡
Wisconsin–Madison, Madison, Wisconsin 53706, United States; Department of Chemistry, Massachusetts Institute of
Technology, Cambridge, Massachusetts 02139, United States
Supporting Information
ABSTRACT: Cloaking its carboxyl groups with a hydro-
phobic moiety is shown to enable a protein to enter the
cytosol of a mammalian cell. Diazo compounds derived
from (p-methylphenyl)glycine were screened for the
ability to esterify the green fluorescent protein (GFP) in
an aqueous environment. Esterification of GFP with 2-
diazo-2-(p-methylphenyl)-N,N-dimethylacetamide was
efficient. The esterified protein entered the cytosol by
traversing the plasma membrane directly, like a small-
molecule prodrug. As with prodrugs, the nascent esters
are substrates for endogenous esterases, which regener-
ate native protein. Thus, esterification could provide a
general means to deliver native proteins to the cytosol.
the cytosolic delivery of a green fluorescent protein
1
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(GFP) conjugate to a cationic CPP. Additionally, sev-
eral natural and synthetic protein transduction domains
(e.g., penetratin, TP10, and pVEC) consist of cationic
and hydrophobic residues, which impart an amphipathic
7
e,7f,14
character.
Their hydrophobic residues are crucial
for mediating membrane translocation.
We envisioned a different strategy—one that invokes
a chemoselective reaction that remodels the protein sur-
face to become less anionic and more hydrophobic. The
surface of proteins displays cationic groups (i.e., guani-
dinium, ammonium, and imidazolium) and anionic
groups (carboxylates). We hypothesized that the
Approximately 20% of the drugs in today’s pharmaco-
1
peia are proteins. Essentially all of those proteins act on
extracellular targets. This limitation arises from an in-
2
trinsic inability of proteins to enter the cytosol. Alt-
hough viral vectors can be used to deliver DNA that en-
codes a protein of interest, this genetic approach lacks
regulation and can induce stress responses, carcinogene-
3
sis, or immunogenicity. In contrast, the direct delivery
of proteins into cells would enable temporal control over
cellular exposure and minimize deleterious off-target
4
effects.
Proteins can be delivered into cells by using site-
5
directed mutagenesis, irreversible chemical modifica-
6
tion, conjugation of transduction domains (such as cell-
7
8
penetrating peptides, CPPs), cationic lipid carriers, or
9
electroporation. Many of these strategies show promise
but also pose problems, such as inefficient escape
from endosomes or inapplicability in an animal.
2
,4
To cross the plasma membrane, proteins must over-
come two barriers: Coulombic repulsion from the anion-
ic glycocalyx and exclusion from the hydrophobic envi-
Figure 1. Bar graph showing the extent of esterification of
the superfolder variant of GFP with diazo compounds 1–6
(black) and the internalization of the ensuing esterified
GFPs into CHO-K1 cells (green). Values (± SD) were de-
termined by mass spectrometry and flow cytometry, re-
spectively. Parenthetical logP values were calculated with
software from Molinspiration (Slovensky Grab, Slovak
Republic).
1
0
ronment of the lipid bilayer. Natural and synthetic sys-
tems suggest means to overcome these barriers. For ex-
ample, mammalian ribonucleases are capable of cyto-
solic entry that is mediated by clusters of positively
1
1
charged residues. Cellular uptake can also be enhanced
1
2
by exogenous hydrophobic moieties. For example,
noncovalent complexation with pyrene butyrate enables
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esterification of its carboxyl groups could endow a pro- Then, we screened solution conditions for maximal
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tein with the ability to access the cytosol. In particular,
by cloaking negative charges with a hydrophobic moie-
ty, we might increase the nonpolar surface area while
enabling endogenous positive charges to manifest favor-
able Coulombic interactions with anionic cell-surface
components. The ensuing mode-of-action would resem-
ble that of small-molecule prodrugs, which have been in
protein esterification by our scaffold. We were aware
that the mechanism of esterification requires a protonat-
ed carboxyl group, which is encouraged by a low pH
and an organic cosolvent. Using GFP and diazo com-
pound 3, we found that an aqueous solution at pH 6.5
that contains 20% v/v acetonitrile gives a high yield of
esters (Figure S1). These conditions should be tolerable
by most proteins.
18
15
the pharmacopoeia for decades.
To effect our strategy, we employed diazo compounds
derived from (p-methylphenyl)glycine. We had shown
previously that the basicity of such diazo compounds
enables the efficient esterification of carboxylic acids in
Next, we evaluated diazo compounds 1–6 for their
ability to esterify a protein and facilitate its internaliza-
tion into a mammalian cell. We found that more polar
diazo compounds alkylated more carboxyl groups than
did less polar compounds (Figures 1 and S2). Then, we
treated live cells with esterified proteins and quantified
internalization with flow cytometry. We discovered that
the level of cellular internalization parallels the number
of labels per protein (Figure 1), which suggests that
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an aqueous environment. Now, we exploited the
modular nature of this scaffold. Specifically, we dei-
1
6,17
midogenated azide precursors
to access diazo com-
pounds 1–6, which span a range of hydrophobicity (Fig-
ure 1).
Figure 2. Images of the cellular internalization of GFP and its super-charged and esterified variants. CHO-K1 cells were
incubated with protein (15 µM) for 2 h at 37 or 4 °C. Cells were then washed, stained with Hoechst 33342 and wheat germ
agglutinin (WGA)–Alexa Fluor 647, and imaged by confocal microscopy (Hoechst 33342: ex. 405 nm, em. 450 nm; WGA–
Alexa Fluor 647: ex. 647 nm, em. 700 nm; GFP: ex. 488 nm, em. 525 nm). Scale bars: 25 µm.
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Figure 3. Images of the nuclear internalization of a protein that contains a nuclear localization signal and its esterified vari-
ant. CHO-K1 cells were incubated with nlsGFP or nlsGFP–1 (15 µM) for 2 h at 37 °C. Cells were then washed, stained with
Hoechst 33342 and WGA–Alexa Fluor 647, and imaged by confocal microscopy (Hoechst 33342: ex. 405 nm, em. 450 nm;
WGA–Alexa Fluor 647: ex. 647 nm, em. 700 nm; GFP: ex. 488 nm, em. 525 nm). Scale bars: 25 µm.
simply masking anionic groups is advantageous. Moreo-
ver, cellular fluorescence increases in a time-dependent
manner (Figure S3), as expected for a process based on
vectorial diffusion from the outside to the inside.
Of the six diazo compounds, compound 1 was the
most effective in engendering cellular uptake and was
selected for further study. On average, 11 of the 32 car-
boxyl groups in GFP were masked as neutral esters by
diazo compound 1 (Figure S2). Although the esterifica-
charged GFP. At 37 °C, treatment with GFP–1 elicited
diffuse fluorescence, suggestive of cytosolic localization
(Figure 2). Most remarkably, this pattern persisted at
4 °C, indicating that uptake does not rely on endocyto-
sis. In other words, GFP–1 appears to enter cells by
passing directly through the plasma membrane, like a
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small-molecule prodrug.
To enter the nucleus, a protein must pass through the
cytosol. To verify cytosolic entry, we reiterated a known
GFP variant bearing a nuclear localization signal
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tion of 11 carboxyl groups in GFP could produce 32C =
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1.2 × 10 different molecules, esters are most likely to
(nlsGFP; Figure S5) and esterified that variant with
form with solvent-accessible carboxyl groups that have a
high pK value (Table S1). That trend was apparent in
a
compound 1 (Figure S6). We then treated live cells with
either nlsGFP or esterified nlsGFP (nlsGFP–1) and visu-
alized the cells with confocal microscopy. In the ensuing
images (Figure 3), nlsGFP colocalizes with membrane
stain (Pearson’s r = 0.21) and is excluded from the nu-
cleus (r = –0.12). This result is expected, as GFP is im-
permeant but a nuclear localization signal is cationic and
can form salt bridges with the anionic glycocalyx. In
contrast, nlsGFP–1 not only exhibits diffuse staining like
GFP–1 (Figure 2), but also colocalizes with a nuclear
stain (r = 0.51) to an extent expected for this particular
1
8
tandem mass spectrometry data (Figure S4). This selec-
tivity has fortuitous consequences. An aspartate or glu-
tamate residue within a hydrophobic patch is a likely
target for esterification, which would extend the size of
the patch. Clustered anionic residues likewise have high
a
pK values, and their esterification would overcome a
strong deterrent to cellular uptake. In contrast, an aspar-
tate or glutamate residue within a salt bridge is unlikely
to be esterified, but a salt bridge manifests less Cou-
lombic repulsion with anionic cell-surface components
than do isolated or clustered anionic residues.
20
variant. These data indicate that nlsGFP–1 accesses the
nucleus and, thus, the cytosol.
We used confocal microscopy to visualize the uptake
of GFP by live mammalian cells. For calibration, we
compared the uptake of GFP with that of a “super-
charged” variant in which site-directed mutagenesis was
used to replace anionic residues with arginine (Figure
Finally, we investigated the bioreversibility of esterifi-
cation. Incubation of a model protein esterified with di-
azo compound 1 in a mammalian cell extract resulted in
the complete removal of labels (Figure S7). This finding
is consistent with an inability of de-esterified GFP–1
(i.e., GFP) to escape from the cytosol and thus accumu-
lating there (Figure S3). Thus, the esters formed upon
reaction with 1 are substrates for endogenous esterases,
like prodrugs. Moreover, the alcohol product of the
esterase-mediated hydrolysis is benign to mammalian
cells (Figure S8).
In summary, we have demonstrated that esterification
of protein carboxyl groups with a tuned diazo compound
can engender delivery of the protein across the plasma
5
a
S5). Unmodified GFP did not enter cells (Figure 2).
Super-charged GFP did enter cells, but produced a punc-
tate pattern of fluorescence that is suggestive of endo-
somal localization. At 4 °C, which is a temperature that
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precludes endocytosis, the fluorescence from super-
charged GFP was scant and localized to the plasma
membrane.
Images of cells treated with GFP–1 were in marked
contrast to those treated with unmodified GFP or super-
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cellular esterases. This delivery strategy provides an
unprecedented means to deliver native proteins into cells
for applications in the laboratory and, potentially, the
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ASSOCIATED CONTENT
Supporting Information
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Synthetic methods, cell biological methods, and additional
analytical data, including Table S1 and Figures S1–S8. This
material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*
rtraines@mit.edu
ORCID
Ronald T. Raines: 0000-0001-7164-1719
ACKNOWLEDGMENTS
We are grateful to Dr. K. A. Andersen for early observa-
tions, Dr. E. K. Grevstad for help with microscopy, L. B.
Hyman for technical advice, Dr. T. T. Hoang for supplying
FLAG–ANG, and Dr. C. L. Jenkins for contributive discus-
sions. K.A.M. was supported by Molecular Biosciences
Training Grant T32 GM007215 (NIH). J.E.L. was support-
ed by a National Science Foundation Graduate Research
Fellowship. This work was supported by grant R01
GM044783 (NIH) and made use of the National Magnetic
Resonance Facility at Madison, which is supported by grant
P41 GM103399 (NIH).
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Figure 1. Bar graph showing the extent of esterification of the superfolder variant of GFP with diazo
compounds 1–6 (black) and the internalization of the ensuing esterified GFPs into CHO-K1 cells (green).
Values (± SD) were determined by mass spectrometry and flow cytometry, respectively. Parenthetical logP
values were calculated with software from Molinspiration (Slovensky Grab, Slovak Republic).
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Figure 2. Images of the cellular internalization of GFP and its superꢀcharged and esterified variants. CHOꢀK1
cells were incubated with protein (15 µM) for 2 h at 37 or 4 °C. Cells were then washed, stained with
Hoechst 33342 and wheat germ agglutinin (WGA)–Alexa Fluor 647, and imaged by confocal microscopy
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Hoechst 33342: ex. 405 nm, em. 450 nm; WGA–Alexa Fluor 647: ex. 647 nm, em. 700 nm; GFP: ex.
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88 nm, em. 525 nm). Scale bars: 25 µm.
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Figure 3. Images of the nuclear internalization of a protein that contains a nuclear localization signal and its
esterified variant. CHOꢀK1 cells were incubated with nlsGFP or nlsGFP–1 (15 µM) for 2 h at 37 °C. Cells were
then washed, stained with Hoechst 33342 and WGA–Alexa Fluor 647, and imaged by confocal microscopy
(
Hoechst 33342: ex. 405 nm, em. 450 nm; WGA–Alexa Fluor 647: ex. 647 nm, em. 700 nm; GFP: ex. 488
nm, em. 525 nm). Scale bars: 25 µm.
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