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NaCl, 3 mM EDTA, and 0.005% Surfactant P20) and injected onto the sensor chip
for 150 s at 30 µl/min, and the response unit (RU) was recorded. After injecting the
analyte, HBS-EP buffer was poured over the chip for 420 s at 30 µl/min to allow the
bound analytes to dissociate from the immobilized X-peptide and dissociation
curves were obtained. The RU elicited by injecting 1% elution buffer (20 mM
HEPES, pH 7.0, 0.2 M NaCl, 10% glycerol, and 2 mM DTT) included HBS-EP
buffer and was used as the vehicle control. Biacore X-100 control software was used
to measure the changes in RU and to plot the binding curve. The curves obtained
from the surface plasmon resonance assay experiments were analyzed and the
acetate (50 mL). The organic solution was successively washed with saturated
aqueous Na CO solution and brine. The solvent was evaporated in vacuo to give
2 3
3,4-bis(benzyloxy)phenyl formate. NaOH (6 N) was added to a stirred solution of
crude 3,4-bis(benzyloxy)phenyl formate in MeOH (15 mL). After stirring at room
temperature for 10 min, 10% aq. HCl solution was added. The reaction mixture was
diluted with ethyl acetate (50 mL), washed with brine and dried over anhydrous
2 4
Na SO . The flash chromatography (7:3 hexane/ethyl acetate) furnished the
compound 3,4-bis(benzyloxy)phenol 3 as solid (0.77 g, 80 % yield). A solution of
3,4-bis(benzyloxy)phenol 3 (0.5 g, 1.63 mmol) in EtOH (6 mL) was treated with
potassium hydroxide (91 mg, 1.63 mmol) and epichlorohydrin (151 mg, 1.63
mmol) and continued stirring at ambient room temperature for 6 h and then
concentrated in vacuum. The residue was suspended in water, and the mixture
dissociation equilibrium constant (K
peptides was calculated and derived from the equation, K
are dissociation- and association-rate constants, respectively.
D
) of p62-D3-GST to immobilized biotinylated
D
= k /k , where k and k
d
a
d
a
2 4
extracted with ether and dried with anhydrous Na SO and evaporated in vacuum
to give 2-((3,4-bis(benzyloxy)phenoxy)methyl)oxirane 4 (414 mg, 70%). A solution
of 2-((3,4-bis(benzyloxy)phenoxy)methyl)oxirane 4 (15 mg, 0.004 mmol) in MeOH
Molecular modeling of p62. The 3D structure model of full-length human p62
4
7
protein was constructed using Modeller-9.12 with multiple sequence alignments
(
2 mL) with EZ-Link Pentylamine-Biotin (13 mg, 0.004 mmol) was refluxed for 6 h.
6
3, 64
and homology modeling protocol
. The models were further refined by clus-
After rotary evaporation of the mixture, to remove traces of EZ-Link Pentylamine-
Biotin azeotrope with toluene (2 × 5 mL) and the residue was dissolved in methanol
and added oxalic acid dehydrate. After evaporation, the residue was recrystallized
from methanol/ether to give XIE2008-Biotin (17 mg, 60%) as a white solid and the
final purity of the XIE2008-Biotin (>98%) was assessed by RP-HPLC on an ana-
lytical Vydac C18 column (4.6 mm × 250 mm, 300 Å, 5 μm particle size). The
molecular masses of purified XIE2008-Biotin confirmed by recording its mass
spectrum, by its molecular ion peak at m/z 691.74 [M + H]+ corresponds to
tering, docking and molecular dynamics (MD) simulation. For example, we first
performed 50 ns MD for conformational sampling. We also carried out a series of
p62 dockings with the reported interacting partners, including IDR-1, LC3, Keap1,
λPKC, and ubiquitin. As a result, the best 3D structure of p62 protein was then
used in virtual screening and molecular docking studies.
Virtual screening for chemical ligands to p62 ZZ domain. The putative binding
cavity of p62 ZZ-domain was predicted using Fast Connolly Type implemented in
the MOLCAD module in SYBYL-X 1.3 by a similar protocol as described pre-
50 4 6
molecular formula C38H N O S.
6
5
viously , before which the corresponding ZZ domain residues were first selected to
generate a protocol with the default threshold radius of 0.5. Several focal residues
involved in our predicted binding pocket included Asp129, Asn132, Val135,
Arg139, Cys142, Cys145, Asp147, Tyr148, Asp149, Cys151, Cys154, Lys157,
Leu159, His160, and His163. Docking virtual screening searches were carried out to
Soluble and insoluble fractionation. HeLa cells were transiently transfected with
plasmids encoding either wild-type (Q25) or mutant (Q103) huntingtin gene using
X-tremeGENE HP. Following transfection, cells were cultured for 8 h and treated
with DMSO, XIE62-1004, XIE2008 or rapamycin for 16 h. The cells were pelleted,
2
resuspended in a cell lysis buffer (20 mM HEPES pH 7.9, 0.2 M KCl, 1 mM MgCl ,
screen a pre-filtered in-house database of 540,000 compounds with our published
cell-based partition chemistry-space matrix calculation algorithm48, 66–68, in which
1 mM EGTA, 1% Triton X-100, 10% glycerol, protease inhibitor and phosphatase
inhibitor) and incubated on ice for 30 min. Soluble- and insoluble-fractions in 1%
Triton X-100 were obtained by centrifugation at 13,000 × g for 20 min at 4 °C. The
insoluble fraction was solubilized in a SDS-detergent lysis buffer (20 mM HEPES
d
the docking score was expressed in -log10(K ). The top-ranked compounds with the
docking scores greater than 7.0 were selected for further experimental validation
studies in vitro. Among all compounds tested, two small molecules (XIE62-1004
and XIE2008), which showed very high docking scores, were the most promising
compounds.
2
pH 7.9, 0.2 M KCl, 1 mM MgCl , 1 mM EGTA 1% Triton X-100, 1% SDS, 10%
glycerol, protease inhibitors, and phosphatase inhibitors). After sonication, the
samples were heated at 100 °C for 10 min in SDS sample buffer and subjected to
western blotting analysis.
Chemical synthesis and analytical data. Synthesis and analytical data of 3,4-Bis
(
(
benzyloxy)benzaldehyde 2. To the stirred solution of 3,4-dihydroxybenzaldehyde
3.00 g, 21.7 mmol) in dry DMF (50 mL) added anhydrous K CO (9.60 g, 69.4
Immunocytochemistry. HeLa cells were cultured on cover slips and fixed with 4%
paraformaldehyde in PBS (pH 7.4) for 10 min at room temperature. After washing
three times with PBS, the cells were permeabilized with ice-cold methanol for 2.5
min. After three washes with PBS, the cells were incubated with blocking solution
2
3
mmol) followed by benzyl bromide (7.65 g, 44.7 mmol). The resulted mixture was
stirred at room temperature for 2 h. Additional K CO (2.4 g, 17.3 mmol) was
added, and the mixture was heated to 70 °C for 30 min and then cooled to room
temperature. The mixture was partitioned between H O and ether (120 ml each).
The organic layer was separated, and the water layer was extracted with ether (3 ×
0 ml). The pooled organic layers were washed with H O (2 × 50 ml) and saturated
2
3
(
5% BSA in PBS) for 1 h and then with primary antibody overnight at 4 °C. The
2
next day, the cells were washed five times for 5 min each time with PBS and then
incubated with secondary antibody for 1–2 h. The cells were washed four times
with PBS for 5 min each time, and DAPI stained for 15 min. After three washes
with PBS, the coverslips were mounted on slides using Fluoro-GEL (Electron
Microscopy Sciences). Confocal images were taken by laser scanning confocal
microscope 510 Meta (Zeiss) and analyzed using Zeiss LSM Image Browser (ver.
5
2
aqueous NaCl (50 mL). The pale, straw-colored extracts were dried over anhydrous
sodium sulfate and concentrated to yield 3,4-Bis(benzyloxy)benzaldehyde 2 (6.57 g,
9
NMR (400 MHz, CDCl
1
5%) as a cream-colored solid after washing with hexanes (75 ml) and drying. 1
H
3
): δ 9.81 (s, 1H), 7.49–7.31 (m, 12H), 7.04 (d, J = 8.3 Hz,
4.2.0.121). To investigate the effect of XIE62-1004 and XIE2008 compounds on p62
H), 5.27 (s, 2H), 5.22 (s, 2H).
puncta formation, cells were incubated with different concentrations of XIE
compounds (0, 1, 2.5, 5, and 10 μM) for 12 h or incubated with 10 µM XIE com-
pound for varying time periods (0, 1, 3, 6, and 12 h). To analyze the physical
association of p62 puncta with autophagic vacuoles containing LC3, HeLa cells
were left untreated or treated with 10 µM XIE62-1004 or XIE2008 compound for
Synthesis and analytical data of 2-((3,4-bis(benzyloxy)benzyl)amino)ethan-1-ol
: 3,4-Bis(benzyloxy)benzaldehyde 2 (3.18 g, 10 mmol) was dissolved in dry
3
ethanol (20 ml), and ethanolamine (0.61 g, 10 mmol) was added. The reaction
mixture was stirred for 12 h at 60 oC. The reaction solution was cooled to room
4
temperature. NaBH (0.57 g, 15 mmol) was added slowly in portions, and the
resulting solution was stirred for another 12 h. The solvent was evaporated in
vacuum, and the residue was dissolved in water and extracted with ethyl acetate.
16 h followed by immunostaining with anti-p62 and LC3 antibodies.
The organic layers were combined and dried with anhydrous Na
2 4
SO , filtered, and
In vitro oligomerization assay. HEK293 cells were transiently transfected with a
plasmid DNA encoding p62-myc/his using Lipofectamine 2000. Approximately 24
h after transfection, cells were lysed with cell lysis buffer (50 mM HEPES, pH 7.4,
evaporated in vacuum. The residue was purified by flash column to generate the
desired product 2-((3,4-bis(benzyloxy)benzyl)amino)ethan-1-ol 3 (2.0 g, 56%). 1
H
NMR (400 MHz, CDCl
.17 (s, 2H), 3.71 (s, 2H), 3.64 (t, J = 4.8, 2H), 2.93 (s, 2H), 2.72 (t, J = 4.8, 2H).
Synthesis and analytical data of XIE62-1004: 2-((3,4-bis(benzyloxy)benzyl)
3
): δ 7.52–7.33 (m, 10H), 7.01–6.84 (m, 3H), 5.20 (s, 2H),
0.15 M KCl, 0.1% Nonidet P-40, 10% glycerol, protease inhibitors, and phosphatase
5
inhibitors). Following a cycle of freeze/thaw, the cell suspension was incubated on
ice for 1 h and centrifuged at 13,000 × g for 20 min at 4 °C. Protein concentration
was determined using the Bradford assay. For the p62 oligomerization experiments,
amino)ethan-1-ol 3 (1.0 g, 2.75 mmol) was dissolved in absolute methanol (25 mL),
and pumped HCl gas for 1 h. The mixture was stirred for another 2 h, evaporated
to about 1 mL and added hexane to get the solid, which was filtered and dried to
0.5 µg protein was diluted in oligomerization assay buffer (16.7 mM HEPES, pH
1
7.4, 0.05 M KCl, 0.033% Nonidet P-40, 3.33% glycerol, protease inhibitors, and
phosphatase inhibitors) and incubated with or without dipeptides (dissolved in
water at a final concentration of 0.5 or 1 M) for 2–4 h in the presence of 100 μM
bestatin at room temperature. Samples were mixed with a non-reducing loading
buffer containing 4% lithium dodecyl sulfate (LDS), heated at 95 °C for 10 min, and
resolved on 3% stacking and 12% separating SDS-PAGE. The monomer, oligomer,
and aggregate forms of p62 were detected with a mixture of p62 and myc anti-
bodies. To investigate the effect of XIE compounds on p62 aggregate formation,
HEK293 whole cell extracts containing ectopically expressed p62 were incubated
with XIE62-1004 at various concentrations (0, 10, 100, and 1000 μM) for 2 h at
room temperature. For LC–MS/MS analysis, myc/His tagged p62 was over-
expressed in HEK293 cells for 24 h. Two milligrams of total protein was subjected
give the final compound XIE62-1004 (720 mg, 65%). H NMR (400 MHz, DMSO-
d6): δ 8.83 (bs, 2H), 7.52–7.46 (m, 5H), 7.31–7.32 (m, 1H), 7.26–7.20 (m, 5H),
7
4
1
5
.12–7.10 (m, 1H), 7.05–7.03 (m, 1H), 5.24–5.22 (m, 1H), 5.14 (s, 2H), 5.11 (s, 2H),
.07 (s, 2H), 3.67–3.63 (m, 2H), 2.90 (s, 2H). 13C NMR (400 MHz, CDCl
3
): δ 149.0,
48.2, 137.4, 133.3, 128.5, 127.8, 127.5, 127.4, 121.3, 115.4, 115.2, 71.5, 71.3, 60.8,
+
3.2, 50.7. LC−MS (ESI): m/z 364.3 (M + H) .
Synthesis and analytical data of XIE2008-Biotin. m-Chloroperbenzoic acid
(
(
0.81 g, 4.71 mmol) was added to a solution of the above synthesized 3,4-bis
benzyloxy)benzaldehyde 2 (1.0 g, 3.14 mmol) in dichloromethane (15 mL). The
mixture was stirred at room temperature for 24 h and then diluted with ethyl
1
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