Fluorescent mimics of cholesterol that rapidly bind surfaces of living mammalian cells

Article abstract of DOI:10.1039/c5cc06325f

Mammalian cells acquire cholesterol, a critical membrane constituent, through multiple mechanisms. We synthesized mimics of cholesterol, fluorescent N-alkyl-3β-cholesterylamine-glutamic acids, that are rapidly incorporated into cellular plasma membranes compared with analogous cholesteryl amides, ethers, esters, carbamates, and a sitosterol analogue. This process was inhibited by ezetimibe, indicating a receptor-mediated uptake pathway.

Full text of DOI:10.1039/c5cc06325f

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Fluorescent Mimics of Cholesterol that Rapidly Bind Surfaces of
Living Mammalian Cells
Received 00th January 20xx,
Accepted 00th January 20xx
David Hymel, Sutang Cai, Qi Sun, Rebecca S. Henkhaus, Chamani Perera, and Blake R. Peterson*
DOI: 10.1039/x0xx00000x
www.rsc.org/
Mammalian cells acquire cholesterol, a critical membrane constituent,
through multiple mechanisms. We synthesized mimics of cholesterol,
fluorescent N-alkyl-3β-cholesterylamine-glutamic acids, that are rapidly
incorporated into cellular plasma membranes compared with analogous
cholesteryl amides, ethers, esters, carbamates, and a sitosterol analogue.
This process was inhibited by ezetimibe, indicating a receptor-mediated
uptake pathway.
Cholesterol (1) is a critical constituent of membranes of
mammalian cells. Cells acquire exogenous forms of this sterol
through multiple mechanisms involving structurally distinct
cell surface receptors. Lipoprotein particles such as low-
density lipoprotein (LDL) and high-density lipoprotein (HDL)
carry cholesteryl esters (2) and associated protein and lipid
components throughout the bloodstream.1 Cells expressing
LDL and HDL receptors actively internalize these natural
nanoparticles via receptor-mediated endocytosis. In contrast,
Niemann-Pick C1 Like 1 protein (NPC1L1) plays key roles in the
cellular uptake of dietary (unesterified) cholesterol (
found in mixed micelles.2 This receptor was identified in 20043
as target of ezetimibe ), drug used to treat
1), as
a
(
3
a
hypercholesterolemia. More recent studies suggest that
although NPC1L1 is a primary target of ezetimibe and its active
glucuronide metabolite,4-6 other proteins, such as the HDL
receptor SR-BI, can also be inhibited by this drug.7 Recent
proteomics experiments have identified over 250 cholesterol-
binding proteins, including receptors, channels, and enzymes.8
Derivatives of cholesterol have numerous biological
applications. These compounds have been used to facilitate
the delivery of small inhibitory RNA (siRNA),9 enhance DNA
transfection,10 probe cellular membrane subdomains,11 and
assay cholesterol transport processes.12-17 Cholesteryl
carbamates have been extensively investigated, and cellular
uptake of cholesteryl carbamate-conjugated siRNA has been
reported to be similar to uptake of cholesteryl esters, requiring
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Fig. 2. Specific binding of compounds 4–13 to plasma membranes of living Jurkat
lymphocytes in media containing 10% serum. Cells were treated with 4–13 for 5
min at 37 °C and analyzed by flow cytometry with and without excess ezetimibe (3,
200 µM) in saturation binding experiments. The linear non-specific binding
component was subtracted from the total binding data followed by curve fitting
with a one-site binding model (GraphPad Prism 6).
derived intermediates,21 were
reported. The novel building block 3
prepared from sitosterol using methodology described for the
synthesis of The 4-carboxy
-cholesterylamine.20
prepared as previously
-sitosterylamine was
β
3β
Pennsylvania Green fluorophore was prepared as previously
described.22 Full synthetic details are provided in Scheme S1
and Scheme S2 of the supporting information. These probes
were designed to systematically compare membrane anchors
derived from N-alkyl-3
analogue ( ), a N-acyl-3
11
13) linked to the hydrophobic Pennsylvania Green23, 24
β
-cholesterylamines (
4-7), a sitosteryl
8
β-cholesterylamine (10), or cholesterol
(
9,
–
Fig. 1. Differential interference contrast (DIC) and confocal laser scanning
microscopy of living Jurkat lymphocytes in media containing 10% serum. Cells were
treated with fluorescent compounds 4–13 (2 µM) at 37 °C for 5 min or 1 h. In panel
C, ezetimibe (3, 200 µM in 0.2% DMSO) was included to illustrate competitive
inhibition of uptake of 4. Scale bar = 7.5 microns.
(PG) fluorophore through amino acid subunits. We
hypothesized that the carbonyl linked to the steroid in amide
10, ester 11 or carbamates 12 and 13 would be similar to
natural cholesteryl esters, and this structural modification
might correspondingly affect their ability to bind cell surfaces.
As shown in Figure 1, confocal laser scanning microscopy
binding to HDL or LDL,9 followed by internalization via HDL or
LDL receptors. This initial lipoprotein-binding step can slow was employed to compare living human Jurkat lymphocyte
cellular uptake, and the presence of high concentrations of cells after treatment with 13 These experiments
serum (e.g. 10%) in media typically reduces the activity of demonstrated that a brief (5 minute) treatment of cells with
4–
.
4
these compounds, likely because of competition between or
5
(2 µM) at 37 °C results in robust fluorescent staining of
serum lipoproteins and cognate cell surface receptors.
cellular plasma membranes. Examination of these cells after 1
In an effort to mimic the molecular recognition properties hour showed enhanced cellular binding, uptake of the probe,
of free cholesterol ( ), we hypothesized that the protonated and localization in transferrin-positive early/recycling
secondary amino group of N-alkyl-3
as found in compounds , might function as a bioisostere for information). Binding of
the 3 -hydroxyl group of cholesterol. Thus, in contrast to predominantly receptor-mediated as evidenced by up to 80%
cholesteryl esters, cholesteryl carbamates, and structurally inhibition upon coaddition with excess (200 µM) ezetimibe (
1
19
β
-cholesterylamines,18,
endosomes (Figure
1
and Figure S1 of the supporting
(and ) to cell surfaces was
4-7
4
5
β
3,
related compounds, which may require lipoprotein-mediated Compare Figure 1A and 1C, and see Figure S3 of the supporting
cellular uptake, these compounds might bind to receptors on information). This inhibition indicates that proteins on cell
cell surfaces that recognize free cholesterol or structurally surfaces may recognize these compounds as mimics of free
similar metabolites through alternative mechanisms. We cholesterol or related cholesterol metabolites. These
further hypothesized that the addition of anionic amino acids metabolites might include structurally related cholesterol
might affect binding to serum proteins and increase the sulfate25 and cholesterol glucuronide,26 which are present in
affinity of these compounds for cells.
To investigate how structural features affect recognition of Comparison of
cholesterol derivatives and related compounds by proteins on linker region revealed that the anionic moiety of
the surface of cells, we synthesized the fluorescent molecular rapid high affinity/efficacy binding to cells (Figure 1, compare
micromolar concentrations in the bloodstream of animals.
with lacking the glutamic acid residue in the
is critical for
4
6
4
probes 4-13. For probes derived from 3β-cholesterylamine, panels B and G). Moreover, despite the presence of a
this steroid building block,20 and some cholesterylamine-
2 | J. Name., 2012, 00, 1-3
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Fig. 3. Total binding (A) and specific binding (B) of compounds 4, 5, 8, and 13 to
three mammalian cell lines. Cells in media containing 10% serum were treated with
compounds, without (A) and with (B) excess ezetimibe (3, 200 µM), in triplicate at
22 °C for 5 min. Jurkat, HeLa, and HEK-293 cells were suspended in media prior to
treatment and analysis of fluorescence by flow cytometry.
Fig. 4. Analysis of the kinetics of cellular uptake of compounds 4, 5, 8, and 13.
Panels A-D: Living Jurkat lymphocytes in media containing 10% serum were treated
with compounds in triplicate at 22 °C, aliquots were sampled at the times shown
and fluorescence analyzed by flow cytometry. Panel E: Values of MEFL/min,
obtained by linear regression of the data shown in A-D, was analyzed with a
Michaelis-Menten model (GraphPad Prism 6).
structurally analogous glutamic acid, the cholesteryl
carbamate 12 showed low cellular binding and cellular uptake
in media containing 10% serum. Data from saturation binding
compared with 4, supporting the hypothesis that carbamates experiments after treatment for five minutes, designed to limit
engage a mechanistically distinct cellular uptake pathway. cellular uptake by endocytosis, are shown in Figure 2, Table 1,
However, this loss of activity of 12 could be at least partially and the supporting information. The high affinity and efficacy
rescued by addition of a second glutamic acid, as found in 13
Using ezetimibe ( , 200 µM, 0.2% DMSO) as a specific revealed that N-alkyl cholesterylamines bearing a anionic
competitor, we quantified the relative affinities (Kd, app) and functional group and a spacer residue such as -alanine most
efficacies (Bmax) of rapid binding of 13 to Jurkat lymphocytes efficiently bind cell surfaces, a critical initial step for initiation
of endocytosis and delivery of linked agents. The absence of
substantial cellular binding of the sitosterolamine analogue ( ),
differing from by the presence of an additional ethyl group in
.
of binding of 4 and 5 to cell surfaces compared to 6-12
3
β
4
-
Compd.
Kd, app
(µM)
Bmax (MEFL KM (µM) Vmax
106)
(MEFL /
min
105)
8
×
4
×
the tail of the sterol, further supports a specific receptor-
mediated uptake mechanism. This result is consistent with
limited receptor-mediated27, 28 uptake of phytosterols such as
sitosterol by mammalian cells.
4
1.4 ± 0.5 0.9 ± 0.1
1.2 ± 0.2 2.1 ± 0.2
1.5 ± 0.3
2.6 ± 0.7
ND
1.8 ± 0.2
5.0 ± 0.8
ND
5
6
NC
NC
7
7.7 ± 4.1 3.1 ± 1.3
ND
ND
8
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
1.8 ± 0.5
ND
0.2 ± 0.1
ND
To investigate whether other cell types show differential
9
effects, we examined rapid (5 min) binding of 4, 5, 8, and 13 to
10
11
12
13
ND
ND
three different human cell lines. Human Jurkat cells, grown in
suspension were compared with the human cell lines HeLa and
HEK-293, which were suspended in media prior to treatment.
As shown in Figure 3, HeLa cells bound the fluorescent
cholesterol mimics to the greatest extent. Comparison of HeLa
with Jurkat and HEK-293 cells revealed 3.3-fold to 8.1-fold
more specific binding to HeLa cell surfaces compared to the
other cell lines. These results might be explained by higher
expression by HeLa cells of a specific receptor that interacts
with these compounds.
ND
ND
ND
ND
3.2 ± 0.6 2.34 ± 0.28
5.8 ± 2.2
1.9 ± 0.5
Table 1. Left columns: Apparent affinity (Kd, app) and efficacy (Bmax) of
binding of 4–13 to plasma membranes of living Jurkat cells. Cells were treated
with compounds at 37 °C for 5 min in media containing 10% FBS (± SEM).
Non-specific binding was quantified with ezetimibe (200 µM) as a competitor;
vehicle = 0.2% DMSO; NC: Not calculated due to low efficacy. Right columns:
Values of KM and Vmax calculated from Michaelis-Menten analysis of rapid
time-dependent cellular uptake at 22 °C (Figure 3). ND: Not determined.
MEFL: molecules of equivalent fluorescein. Data, based on measurements in
triplicate, is reported as mean ± SEM.
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the active metabolite ezetimibe-glucuronide, prepared as
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4 to Jurkat
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