S. Lee et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4858–4864
4863
Table 2
HPLC characterization of dynasore derivatives for purity determination
References and notes
1. Abazeed, M. E.; Blanchette, J. M.; Fuller, R. S. J. Biol. Chem. 2005, 280, 4442.
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McNiven, M. A. Nat. Cell Biol. 2005, 7, 483.
3. Damke, H.; Baba, T.; Warnock, D. E.; Schmid, S. L. J. Cell Biol. 1994, 127, 915.
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5. Nabi, I. R.; Le, P. U. J. Cell Biol. 2003, 161, 673.
Compounds
Retention time (min)
Purity (%)
Dynasore
DD-6
DD-11
9.65
8.97
14.32
95.4
93.6
96.3
6. Nichols, B. J. Cell Sci. 2003, 116, 4707.
7. Roux, A.; Uyhazi, K.; Frost, A.; De Camilli, P. Nature 2006, 441, 528.
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McCluskey, A. Bioorg. Med. Chem. Lett. 2004, 14, 3275.
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ChemMedChem 2009, 4, 1182.
12. Quan, A.; McGeachie, A. B.; Keating, D. J.; van Dam, E. M.; Rusak, J.; Chau, N.;
Malladi, C. S.; Chen, C.; McCluskey, A.; Cousin, M. A.; Robinson, P. J. Mol.
Pharmacol. 2007, 72, 1425.
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for DD-6, 60.0607 5.7320 for DD-11, 40.5516 1.4943 for dyna-
sore). Interestingly, DD-20 which didn’t exhibit inhibitory activity
without preincubation, showed a comparable and reversible inhi-
bition on transferrin uptake after 30 min preincubation (Fig. 4).
To study in more detail the effects of DD-6 and DD-11 compared
to dynasore on the formation of clathrin-coated pits, total internal
reflection microscopy (TIRFM) was performed in COS-7 cells trans-
fected with clathrin (mRFP-LCa).21 TIRFM showed that mRFP-LCa is
recruited to the plasma membrane in a punctate pattern which ap-
pears and disappears and have life spans ranging between 20 and
60 s (Fig. 5a). Upon incubation for 30 min with 10 lM dynasore
(which is suboptimal concentration for dynamin inhibition), some
of mRFP-LCa spots become stable for long periods but many of
them are still able to bud off from the membrane (Fig. 5b). In con-
14. Kirchhausen, T.; Macia, E.; Pelish, H. E. Methods Enzymol. 2008, 438, 77.
15.
A
mixture of 2-naphthohydrazide or 3-hydroxy-2-naphthohydrazide
(0.5 mmol), appropriate benzaldehyde (0.5 mmol) and ceric ammonium
nitrate (0.1 mmol) in 50 mL of anhydrous ethanol was refluxed at 65 °C for
3 h. The solvent was removed under reduced pressure, and the residue was
partitioned between saturated sodium bicarbonate (100 mL) and CHCl3
(3 ꢁ 30 mL). The organic layer was dried on Na2SO4 and evaporated under
vacuum, and the residue was purified by silica gel column chromatography
(CHCl3/MeOH, 20:1) to afford the product as solid (DD-4–11).
trast, upon incubation for 30 min with 10 lM DD-6 or DD-11, most
of mRFP-LCa coats remain locked at their starting positions on the
membrane, suggesting DD-6 and DD-11 block dynamin-mediated
membrane fission more potently than dynasore (Fig. 5c and d).
Compared to conventional genetic or immunological tools,
small chemical molecule dynamin inhibitors have the potential
to study the dynamic nature of endocytic events in cells. Dynasore
was discovered by chemical genetics discovery approach and has
proven to be a useful tool for studying membrane traffic.22–26 We
have designed a series of dynasore derivatives, and found that
introduction of chlorine atoms or dimethyl substitution at 40 and
50 position on phenyl ring completely lost the dynasore’s inhibitory
effect on dynamin GTPase. In contrast, the substitution of hydroxyl
group of 3-position on the naphthyl ring in dynasore to hydrogen
(DD-6) and introduction of hydroxyl group at 30 and methoxy
group at 40 on phenyl ring (DD-11) significantly increase inhibitory
potency on dynamin GTPase (Table 2). Using TIRF microscopy, we
further showed that DD-6 and DD-11 stabilize clathrin-coated
16. To the 2-naphthoic acid or 3-hydroxy-2-naphthoic acid (0.5 mmol) in 50 mL of
absolute dichloromethane was added 4-dimethylaminopyridine (0.05 mmol),
EDCI (0.6 mmol) and corresponding 2-(substituted phenyl) ethyl amine
(0.5 mmol). The mixture was stirred at room temperature for 1 h and the
solvent was removed under reduced pressure. The residue was partitioned
between saturated sodium bicarbonate (100 mL) and CHCl3 (3 ꢁ 30 mL). The
organic layer was dried on Na2SO4 and evaporated under vacuum, and the
residue was purified by silica gel column chromatography (CHCl3/MeOH, 20:1)
to afford the product as solid (DD-12–19).
17. All starting materials were purchased from Aldrich (St. Louis, MO, USA) and TCI
(Tokyo, Japan). Proton nuclear magnetic resonance spectroscopy was
performed on a JEOL JNM-LA 300WB spectrometer, and spectra were taken
in CDCl3 or DMSO-d6. Unless otherwise noted, chemical shifts are expressed as
ppm downfield from internal tetramethylsilane, or relative ppm from NMR
solvent. Data are reported as follows: chemical shift, multiplicity (s, singlet; d,
doublet; t, triplet; m, multiplet; b, broad; app., apparent), coupling constants,
and integration. Mass spectroscopy was carried out on MALDI-TOF and LC–MS
instruments.
spots on the plasma membrane at the concentration of 10 lM,
18. For dynamin inhibition efficiency test of dynasore and derivatives, starved
which is suboptimal concentration for dynasore to inhibit dynam-
in. They did not induce any noticeable toxicity to the cells even
with 12 h incubation (Supplementary Fig. 2). Besides, DD-6 has
comparable solubility in aqueous solution to that of dynasore (Sup-
plementary Fig. 3). Although DD-11 is less soluble (Supplementary
Fig. 3) but its IC50 for transferrin uptake is 3 times less than that of
dynasore. All in all, our results display considerable promise as
leads for the development of more potent analogues for dynamin
inhibition.
cells were incubated in Dulbecco’s PBS (DPBS) containing 20
transferrin (Invitrogen, Eugene, OR) and various concentration (10–80
l
g/ml Texas red
l
M)
dynasore and derivatives for 10 min at 37 °C with or without preincubation.
Cells were washed with cold DPBS three times and removed surface binding
Texas red transferrin in acid stripping solution (150 mM NaCl, 2 mM CaCl2
and 25 mM CH3COONa, pH 4.5) and fixed in 4% paraformaldehyde. To study
the reversible effect of these molecules in transferrin uptake, cells were
preincubated with dynasore and derivatives (80
DD-13; 40 M for DD-6, 20 M for DD-11) for 30 min at 37 °C and washed
with DPBS for various 0 min, 10 min, 30 min. After washing the cells, 20 g/ml
lM for dynasore, DD-20 and
l
l
l
Texas red transferrin was treated to these cells and incubated for 10 min at
37 °C.
19. Images were obtained with an Olympus IX-71 inverted microscope (Olympus
Optical, Tokyo, Japan) with a 40ꢁ, 1.0 N.A. oil lens using a CoolSNAP-Hq CCD
camera (Roper Scientific, Tucson, AZ) driven by MetaMorph imaging software
(Universal Imaging Corporation, West Chester, PA) with a Texas red optimized
filter set (Omega Optical, Brattleboro, VT). Light from a mercury lamp was
shuttered using a VMM1 Uniblitz shutter (Vincent Associates, Rochester, NY).
Analysis and quantification of data were performed using MetaMorph software
and SigmaPlot 8.0 (Systat Software, Point Richmond, CA), and data were
presented as mean SE.
20. Native dynamin I purified from mouse brain. Briefly, Amphiphysin I-SH3-GST
immobilized agarose bead incubated with brain lysate for 2 h and eluted only
dynamin I from Amphiphysin I-SH3-GST using high salt elution buffer (1.2 M
NaCl, 20 mM PIPES, 1 mM DTT, pH 6.5). The eluted dynamin I protein was
Acknowledgments
This work was supported by Grants from the Korea Science and
Engineering Foundation (R01-2006-000-10818-0) and the Brain
Research Center of the 21st Century Frontier Research Program
(M103KV010009-06K2201-00910) to S.C. funded by the Ministry
of Education, Science and Technology, Republic of Korea. TIRF
microscopy data for this study were acquired and analyzed in the
Biomedical Imaging Center at Seoul National University College
of Medicine.
placed in
a diluting buffer (30 mM Tris/HCl, 100 mM NaCl pH 7.4) and
quantified by SDS–PAGE. Human Drp1 and SNX26 RhoGAP domain were
subcloned in-frame into pGEX-4T-1 vector (GE Biosciences, Piscataway, NJ)
and transformed into Escherichia coli BL-21 and the cells were cultured in 2X-
YT medium supplemented with ampicillin. After overnight induction with
0.5 mM IPTG at 25 °C, the cells were sonicated in lysis buffer (1% Triton X-100,
0.5% sodium deoxy-cholate, 20 mM Tris, pH 8.0, 150 mM NaCl, 1 mM MgCl2,
Supplementary data
Supplementary data associated with this article can be found, in
Lee, Suho
