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ChemComm
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DOI: 10.1039/C5CC06325F
COMMUNICATION
Journal Name
S. W. Altmann, K. T. Chapman and N. A. Thornberry, Proc.
Natl. Acad. Sci. U.S.A., 2005, 102, 8132-8137.
E. D. Labonte, P. N. Howles, N. A. Granholm, J. C. Rojas, J.
P. Davies, Y. A. Ioannou and D. Y. Hui, Biochim. Biophys.
Acta, 2007, 1771, 1132-1139.
J. J. Hulce, A. B. Cognetta, M. J. Niphakis, S. E. Tully and B.
F. Cravatt, Nat. Methods, 2013, 10, 259-264.
C. Wolfrum, S. Shi, K. N. Jayaprakash, M. Jayaraman, G.
Wang, R. K. Pandey, K. G. Rajeev, T. Nakayama, K.
Charrise, E. M. Ndungo, T. Zimmermann, V. Koteliansky,
M. Manoharan and M. Stoffel, Nat. Biotechnol., 2007, 25,
1149-1157.
B. Pitard, N. Oudrhiri, J. P. Vigneron, M. Hauchecorne, O.
Aguerre, R. Toury, M. Airiau, R. Ramasawmy, D.
Scherman, J. Crouzet, J. M. Lehn and P. Lehn, Proc. Natl.
Acad. Sci. U.S.A., 1999, 96, 2621-2626.
S. B. Sato, K. Ishii, A. Makino, K. Iwabuchi, A. Yamaji-
Hasegawa, Y. Senoh, I. Nagaoka, H. Sakuraba and T.
Kobayashi, J. Biol. Chem., 2004, 279, 23790-23796.
J. Zhang, S. Cai, B. R. Peterson, P. M. Kris-Etherton and J.
P. Heuvel, Assay Drug Dev. Tech., 2011, 9, 136-146.
D. Wustner, Chem. Phys. Lipids, 2007, 146, 1-25.
D. Wustner, L. Solanko, E. Sokol, O. Garvik, Z. G. Li, R.
Bittman, T. Korte and A. Herrmann, Chem. Phys. Lipids,
2011, 164, 221-235.
F. R. Maxfield and D. Wustner, Methods Cell Biol., 2012,
108, 367-393.
M. Holtta-Vuori, R. L. Uronen, J. Repakova, E. Salonen, I.
Vattulainen, P. Panula, Z. G. Li, R. Bittman and E. Ikonen,
Traffic, 2008, 9, 1839-1849.
S. Sankaranarayanan, G. Kellner-Weibel, M. de la Llera-
Moya, M. C. Phillips, B. F. Asztalos, R. Bittman and G. H.
Rothblat, J. Lipid Res., 2011, 52, 2332-2340.
B. R. Peterson, Org. Biomol. Chem., 2005, 3, 3607-3612.
D. Hymel and B. R. Peterson, Adv. Drug Deliv. Rev., 2012,
64, 797-810.
For these assays, cellular fluorescence was converted to
molecular equivalents of fluorescein (MEFL) using fluorescent
bead standards. For the Jurkat cell line, this analysis indicated
7.
that treatment with
4 or 5 at a concentration of 2 µM for 5
minutes loads 0.5–1.5 × 106 molecules into the plasma
membrane of individual cells. This rapid and massive increase
in cellular fluorescence suggested that an enzyme may be
actively inserting these compounds into the cellular plasma
membrane. Further analysis of time-dependent fluorescence
8.
9.
resulting from treatment with
Mechaelis-Menten model of enzyme kinetics is shown in
Figure 4. These studies revealed that the cellular uptake of
, and to a lesser extent 13, is highly efficient, with 1.8–5.0 ×
4, 5, 8, and 13 using the
10.
4,
5
105 molecules incorporated per minute per cell, consistent
with a catalytic process. Kinetic values of KM and Vmax from this
analysis are shown in Table 1.
11.
12.
In conclusion, we identified novel structure-activity
relationships that govern binding of fluorescent cholesterol
mimics to the surface of living mammalian cells. New
cholesterol-mimetic membrane anchor motifs of 4, 5, and 13
13.
14.
were identified that engage a rapid cellular uptake pathway,
consistent with a receptor-mediated process, that catalytically
inserts these compounds into the plasma membrane. Although
the receptor or enzyme targeted by these compounds has not
yet been identified, it is unlikely to be NPC1L1, the classical
pharmacological target of ezetimibe, because this protein is
not highly expressed outside of the liver and intestine,29 and
the active metabolite ezetimibe-glucuronide, prepared as
15.
16.
17.
previously reported,30 does not inhibit binding of
4 to Jurkat
cell surfaces (data shown in Figure S2 of the supporting
information). Given that cholesterol trafficking and distribution
involves dynamic receptor-mediated and vesicular processes
that are not completely understood,31 these compounds have
potential as novel probes and tools for the delivery of
impermeable molecules into mammalian cells.
18.
19.
20.
21.
22.
23.
24.
25.
Q. Sun, S. Cai and B. R. Peterson, Org. Lett., 2009, 11, 567-
570.
Q. Sun, S. Cai and B. R. Peterson, J. Am. Chem. Soc., 2008,
130, 10064-10065.
Z. R. Woydziak, L. Fu and B. R. Peterson, Synthesis-
Stuttgart, 2014, 46, 158-164.
L. F. Mottram, E. Maddox, M. Schwab, F. Beaufils and B. R.
Peterson, Org. Lett., 2007, 9, 3741-3744.
L. F. Mottram, S. Boonyarattanakalin, R. E. Kovel and B. R.
Peterson, Org. Lett., 2006, 8, 581-584.
This work was supported by the National Institutes of
Health (R01-CA83831 and P20-GM103638) and the University
of Kansas Cancer Center.
1.
2.
3.
J. L. Goldstein and M. S. Brown, Arterioscler. Thromb.
Vasc. Biol., 2009, 29, 431-438.
L. J. Wang and B. L. Song, Biochim. Biophys. Acta, 2012,
1821, 964-972.
S. W. Altmann, H. R. Davis, Jr., L. J. Zhu, X. Yao, L. M. Hoos,
G. Tetzloff, S. P. Iyer, M. Maguire, A. Golovko, M. Zeng, L.
Wang, N. Murgolo and M. P. Graziano, Science, 2004, 303,
1201-1204.
C. A. Strott and Y. Higashi, J. Lipid. Res., 2003, 44, 1268-
1278.
26.
27.
A. P. Wade, Clin. Chim. Acta, 1970, 27, 109-116.
R. G. Gould, R. J. Jones, G. V. LeRoy, R. W. Wissler and C.
B. Taylor, Metabolism, 1969, 18, 652-662.
L. J. Wang and B. L. Song, Biochim. Biophys. Acta., 2012,
1821, 964-972.
H. R. Davis, Jr. and S. W. Altmann, Biochim. Biophys. Acta,
2009, 1791, 679-683.
L. Kvaerno, T. Ritter, M. Werder, H. Hauser and E. M.
Carreira, Angew. Chem. Int. Ed. Engl., 2004, 43, 4653-
4656.
4.
A. B. Weinglass, M. Kohler, U. Schulte, J. Liu, E. O. Nketiah,
A. Thomas, W. Schmalhofer, B. Williams, W. Bildl, D. R.
McMasters, K. Dai, L. Beers, M. E. McCann, G. J.
Kaczorowski and M. L. Garcia, Proc. Natl. Acad. Sci. U.S.A.,
2008, 105, 11140-11145.
28.
29.
30.
5.
6.
L. Ge, J. Wang, W. Qi, H. H. Miao, J. Cao, Y. X. Qu, B. L. Li
and B. L. Song, Cell. Metab., 2008, 7, 508-519.
M. Garcia-Calvo, J. Lisnock, H. G. Bull, B. E. Hawes, D. A.
Burnett, M. P. Braun, J. H. Crona, H. R. Davis, Jr., D. C.
Dean, P. A. Detmers, M. P. Graziano, M. Hughes, D. E.
Macintyre, A. Ogawa, A. O'Neill K, S. P. Iyer, D. E. Shevell,
M. M. Smith, Y. S. Tang, A. M. Makarewicz, F. Ujjainwalla,
31.
D. B. Iaea and F. R. Maxfield, Essays Biochem., 2015, 57,
43-55.
4 | J. Name., 2012, 00, 1-3
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