From the Division of Nephrology, Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor, Michigan 48109
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Previous work has led to the identification of
inhibitors of glucosylceramide synthase, the enzyme catalyzing the
first glycosylation step in the synthesis of glucosylceramide-based
glycosphingolipids. These inhibitors have two identified sites of
action: the inhibition of glucosylceramide synthase, resulting in the
depletion of cellular glycosphingolipids, and the inhibition of
1-O-acylceramide synthase, resulting in the elevation of
cell ceramide levels. A new series of glucosylceramide synthase
inhibitors based on substitutions in the phenyl ring of a parent
compound, 1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (P4), was
made. For substitutions of single functional groups, the potency of
these inhibitors in blocking glucosylceramide synthase was primarily
dependent upon the hydrophobic and electronic properties of the
substituents. An exponential relationship was found between the
IC50 of each inhibitor and the sum of derived hydrophobic ( GlcCer1 is the precursor
of hundreds of different glycosphingolipids. This cerebroside is
synthesized from uridine diphosphate-glucose and ceramide by a
glucosyltransferase, GlcCer synthase. GlcCer-based sphingolipids have
been identified as important mediators of a variety of cellular
functions, including proliferation, differentiation, development, and
cell-cell recognition (1). The
(R,R)-(D-threo)-isomer of
1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) and its
homologues are potent inhibitors of GlcCer synthase. These compounds
have been used extensively to study the metabolism and function of
glycosphingolipids in living cells (2-6).
In previously reported work, a series of PDMP homologues and analogues
was synthesized (4). Replacing the decanoyl moiety with a palmitoyl
moiety enhanced the effectiveness of PDMP. In addition, replacing the
morpholino ring with a pyrrolidino ring, forming
DL-threo-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (DL-threo-P4), also enhanced the inhibitory
activity. It was also noted that the DL-threo-P4
derivative possessing a p-methoxy substituent on the phenyl
group increased the inhibitory activity further. This latter
observation led to the present study, an evaluation of other phenyl
group substitutions in which the phenyl group of the P4 compound was
modified by various electron-donating or -withdrawing groups. As
expected, only the D-threo-enantiomers among P4
or P4 derivatives specifically inhibited the enzyme activity. The
potency of these compounds in inhibiting GlcCer synthase was quantitatively related to the hydrophobic and electronic properties of
the phenyl group substitutions of single substituents. This association
resulted in the design of a new PDMP homologue (4'-hydoxy-P4) that was
significantly more potent than those studied to date.
Materials
The acetophenones and amines were from Aldrich, Lancaster
Synthesis Inc., and Maybridge Chemical Co. Silica gel for column chromatography (70-230 mesh ASTM) and silica gel thin-layer
chromatography plates were purchased from Merck. The reagents and their
sources included non-hydroxy fatty acid ceramide from bovine brain and delipidated bovine serum albumin from Sigma,
dioleoylphosphatidylcholine from Avanti, DL-dithiothreitol
from Calbiochem, and uridine diphosphate-[1-3H]glucose
from NEN Life Science Products. Octanoylsphingosine was prepared as
described previously (7).
General Synthesis of Inhibitors
The aromatic inhibitors were synthesized by the Mannich reaction
from 2-N-acylaminoacetophenone, paraformaldehyde, and
pyrrolidine, followed by reduction with sodium borohydride as described
previously (2, 4). The reaction produced a mixture of four isomers, due
to the presence of two asymmetric carbons. For syntheses in which
phenyl-substituted starting materials were used, the
chloroacetophenone, methoxyacetophenone, methylenedioxyacetophenone,
and methylacetophenone were brominated and converted to the primary
amine. Brominations of the methoxyacetophenone, dimethoxyacetophenone,
and 3',4'-(methylenedioxy)acetophenone were performed in chloroform at
room temperature, and the products were recrystallized from ethyl
acetate and hexane.
The synthesis of
1-(4'-hydroxy)phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol is
described in detail in Fig. 1. This
synthesis differed from those of the other compounds because of the
need for the placement of a protecting group on the free hydroxyl
(product 2) and its subsequent removal (product 8). All other syntheses employed a similar synthetic scheme.
) and electronic (
) parameters. This relationship demonstrated that substitutions that increased the electron-donating characteristics and decreased the lipophilic characteristics of the homologues enhanced
the potency of these compounds in blocking glucosylceramide formation.
A novel compound was subsequently designed and observed to be even more
active in blocking glucosylceramide formation. This compound,
D-threo-4'-hydroxy-P4, inhibited
glucosylceramide synthase at an IC50 of 90 nM.
In addition, a series of dioxane substitutions was designed and tested.
These included 3',4'-methylenedioxyphenyl-, 3',4'-ethylenedioxyphenyl-,
and 3'4'-trimethylenedioxyphenyl-substituted homologues.
D-threo-3',4'-Ethylenedioxy-P4-inhibited
glucosylceramide synthase was comparably active to the
p-hydroxy homologue. 4'-Hydroxy-P4 and ethylenedioxy-P4
blocked glucosylceramide synthase activity at concentrations that had
little effect on 1-O-acylceramide synthase activity. These
novel inhibitors resulted in the inhibition of glycosphingolipid
synthesis in cultured cells at concentrations that did not
significantly raise intracellular ceramide levels or inhibit cell growth.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (16K):
[in a new window]
Fig. 1.
Synthetic pathway for
1-(4'-hydroxy)phenyl-2-
palmitoylamino-3-pyrrolidino-1-propanol.
Synthesis of 1-(4'-Hydroxy)phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol
4'-Benzyloxyacetophenone Formation (Product
2)--
4'-Hydroxyacetophenone (compound 1; 13.62 g, 100 mmol), benzyl
bromide (17.1 g, 100 mmol), and cesium carbonate (35.83 g, 100 mmol)
were added to tetrahydrofuran at room temperature and stirred
overnight. The product was concentrated to dryness and recrystallized
from ether and hexane to yield 15 g of 4'-benzyloxyacetophenone, which appeared as a white powder. An RF of 0.42 was
observed when resolved by thin-layer chromatography using methylene
chloride. 1H NMR (ppm, CDCl3) 7.94 (2H, d,
8.8 Hz, O-Ar-C(O)), 7.42 (5H, m,
Ar'CH2O-), 7.01 (2H, d, 8.8 Hz,
O-Ar-C(O)), 5.14 (2H, s,
Ar'CH2O-), and 2.56 (3H, s,
CH3).
Bromination of 4'-Benzyloxyacetophenone (Product 3)--
Bromine
(80 mmol) was added dropwise over 5 min to a stirred solution of
4'-benzyloxyacetophenone (70 mmol) in 40 ml of chloroform. This mixture
was stirred for an additional 5 min and quenched with saturated sodium
bicarbonate in water until the pH reached 7. The organic layers were
combined, dried over MgSO4, and concentrated to dryness.
The crude mixture was purified over a silica gel column and eluted with
methylene chloride to yield 2-bromo-4'-benyloxyacetophenone. An
RF of 0.62 was observed when resolved by thin-layer chromatography using methylene chloride. 1H NMR (ppm,
CDCl3) 7.97 (2H, d, 9.2 Hz, O-Ar-C(O)),
7.43 (5H, m, Ar'CH2O-), 7.04 (2H, d, 9.0 Hz,
O-Ar-C(O)), 5.15 (2H, s,
Ar'CH2O-), and 4.40 (2H, s,
CH2Br).
2-Amino-4'-benzyloxyacetophenone HCl Formation (Product
4)--
Hexamethylenetetramine (methenamine; 3.8 g, 23 mmol) was
added to a stirred solution of 2-bromo-4'-benyloxyacetophenone (6.8 g,
23 mmol) in 100 ml of chloroform. After 4 h, the crystalline adduct was filtered and washed with chloroform. The product was dried
and heated with 150 ml of methanol and 8 ml of concentrated HCl in an
oil bath at 85 °C for 3 h. Upon cooling, the precipitated hydrochloride salt (2.5 g) was removed by filtration. The filtrate was
left at 20 °C overnight, and additional product (2.1 g) was isolated. The yield was 4.6 g (82.6%). [M + H]+:
242 for C15H16NO2. 1H
NMR (ppm, CDCl3)
8.38 (2H, bs, NH2), 7.97 (2H, d, 8.8 Hz, O-Ar-C(O)), 7.41 (5H, m,
Ar'CH2O-), 7.15 (2H, d, 8.6 Hz,
O-Ar-C(O)), 5.23 (2H, s,
Ar'CH2O-), and 4.49 (2H, s,
CH2NH2).
2-Palmitoylamino-4'-benyloxyacetophenone Formation (Product
5)--
Sodium acetate (50% in water, 29 ml) was added in three
portions to a stirred solution of 2-amino-4'-benzyloxyacetophenone HCl
(4.6 g, 17 mmol) and tetrahydrofuran (200 ml). Palmitoyl chloride (19 mmol) in tetrahydrofuran (25 ml) was added dropwise over 20 min,
yielding a dark brown solution. The mixture was stirred overnight at
room temperature. The aqueous fraction was removed by use of a
separatory funnel, and chloroform/methanol (2:1, 150 ml) was added to
the organic layer, which was then washed with water (50 ml). The yellow
aqueous layer was extracted once with chloroform (50 ml). The organic
solutions were then pooled and rotoevaporated until near dryness. The
residue was redissolved in chloroform (100 ml) and crystallized by the
addition of hexane (400 ml). The flask was cooled to 4 °C for 2 h. The crystals were filtered, washed with cold hexane, and dried in a
fume hood overnight. The product yield was 3.79 g (8 mmol). An
RF of 0.21 was observed when resolved by thin-layer
chromatography using methylene chloride. [M + H]+: 479 for C31H45NO3. 1H NMR
(ppm, CDCl3) 7.96 (2H, d, 8.8 Hz,
O-Ar-C(O)), 7.40 (5H, m, Ar'CH2O-),
7.03 (2H, d, 8.8 Hz, O-Ar-C(O)), 6.57 (1H, bs,
NH2), 5.14 (2H, s, Ar'CH2O-), 4.71 (2H, s, C(O)CH2NHC(O)), 2.29 (2H, t, 7.4 Hz,
C(O)CH2(CH2)13CH3),
1.67 (2H, m,
C(O)CH2(CH2)13CH3), 1.26 (24H, m, C(O)CH2(CH2)13CH3), and
0.87 (3H, t, 6.7 Hz,
C(O)CH2(CH2)13CH3).
1-(4'-Benzyloxy)phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol Formation (Products 6 and 7)-- 2-Palmitoylamino-4'-benyloxyacetophenone (3.79 g, 8.0 mmol), paraformaldehyde (0.25 g, 2.7 mmol, equivalent to 8.1 mmol of formaldehyde), pyrrolidine (0.96 ml, 11.4 mmol), and ethanol (70 ml) were stirred under nitrogen. Concentrated HCl (0.26 ml) was added through the condenser, and the mixture was heated to reflux for 16 h. The resultant brown solution was cooled on ice, and then sodium borohydride (1.3 g, 34 mmol) was added in three portions. The mixture was stirred at room temperature overnight, and the product was dried in a solvent evaporator. The residue was redissolved in dichloromethane (130 ml) and hydrolyzed with 3 N HCl (pH ~4). The aqueous layer was twice extracted with dichloromethane (50 ml). The organic layers were pooled, washed twice with water (30 ml) and twice with saturated sodium chloride (30 ml), and dried over anhydrous magnesium sulfate. The dichloromethane solution was rotoevaporated to a semisolid and purified by use of a silica rotor using a solvent consisting of 10% methanol in dichloromethane. This yielded a mixture of DL-threo- and DL-erythro-enantiomers (2.53 g, 4.2 mmol). RF values of 0.43 for the erythro-diastereomers and 0.36 for the threo-diastereomers were observed when resolved by thin-layer chromatography using methanol/methylene chloride (1:9). [M + H]+: 565 for C36H56N2O3.
1-(4'-Hydroxy)phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol
Formation (Product 8)--
A suspension of 20% palladium/carbon (40 mg) in acetic acid (15 ml) was stirred at room temperature under a
hydrogen balloon for 15 min.
1-(4'-Benzyloxy)phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol (420 mg, 0.74 mmol) was added, and the solution was stirred overnight. The suspension was filtered through a glass frit, and the filter was
rinsed with acetic acid/methylene chloride (1:1, 5 ml). The filtrate
was concentrated in vacuo and crystallized to yield a pale
yellow semisolid (190 mg, 0.4 mmol). An RF of 0.21 was observed when resolved by thin-layer chromatography using methanol/methylene chloride (1:9). [M + H]+: 475 for
C29H50N2O3.
1H NMR (ppm, CDCl3) 7.13 (4H, m,
ArCHOH-), 7.14 (1H, d, 6.9 Hz, -NH-), 5.03 (1H, d, 3.3 Hz,
CHOH-), 4.43 (1H, m,
c-(CH2CH2)2NCH2CH), 3.76 (2H, m,
c-(CH2CH2)2N-), 3.51 (1H, m,
c-(CH2CH2)2NCH2-),
3.29 (1H, m,
c-(CH2CH2)2NCH2-),
2.97 (3H, m,
c-(CH2CH2)2N- and
ArC(OH)H-), 2.08 (6H, m,
-C(O)CH2(CH2)13CH3
and c-(CH2CH2)2N-),
1.40 (2H, m,
C(O)CH2CH2(CH2)12CH3),
1.25 (24H, m,
-C(O)CH2CH2(CH2)12CH3), and 0.87 (3H, t, 6.7 Hz,
C(O)CH2(CH2)13CH3).
Synthesis of D-threo-1-(3',4'-Ethylenedioxy) phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol
2-Amino-3',4'-(ethylenedioxy)acetophenone HCl-- Hexamethylenetetramine (methenamine, 5.4 g, 39 mmol) was added to a stirred solution of ethylenedioxyphenacyl bromide (10.0 g, 39 mmol) in 200 ml of chloroform. After 2 h, the crystalline adduct was filtered and washed with chloroform. The product was then dried and heated with methanol (200 ml) and concentrated HCl (14 ml) in an oil bath at 85 °C for 2 h. On cooling, the precipitated ammonium chloride was removed by filtration, and the filtrate was left in a freezer overnight. After filtration, the crystallized aminoacetophenone HCl was washed with cold isopropyl alcohol and then with ether. The yield of this product was ~7.1 g (81%).
2-Palmitoylamino-3',4'-(ethylenedioxy)acetophenone-- Aminoacetophenone HCl (7.1 g, 31 mmol) and tetrahydrofuran (300 ml) were placed in a 1-liter three-neck round-bottom flask with a large stir bar. Sodium acetate (50% in water, 31 ml) was added in three portions to this suspension. Palmitoyl chloride (31 ml, 10% excess, 36 mmol) in tetrahydrofuran (25 ml) was then added dropwise over 20 min to yield a dark brown solution. This mixture was stirred for an additional 2 h at room temperature. The resultant mixture was poured into a separatory funnel to remove the aqueous solution. Chloroform/methanol (2:1, 150 ml) was then added to the organic layer and washed with water (50 ml). The yellow aqueous layer was extracted once with chloroform (50 ml). The organic solutions were pooled and rotoevaporated until almost dry. The residue was redissolved in chloroform (100 ml) and crystallized by the addition of hexane (400 ml). The flask was then cooled to 4 °C for 2 h. The crystals were filtered and washed with cold hexane until they were almost white and then dried in a fume hood overnight. The yield of the product was 27 mmol (11.6 g).
D-threo-1-(3',4'-Ethylenedioxy)phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol-- Palmitoylaminoacetophenone (11.6 g, 27 mmol), paraformaldehyde (0.81 g, 9 mmol), pyrrolidine (3.6 ml, 42 mmol), and ethanol (250 ml) were added to a 500-ml round-flask under nitrogen flow. Concentrated HCl (0.8 ml) was added to this mixture through the reflux condenser, and the mixture was refluxed for 16 h. The brown solution was cooled in an ice bath. Sodium borohydride (2.28 g, 60 mmol) was added in three portions. This mixture was stirred at room temperature for 3 h and then rotoevaporated. The residue was dissolved in 130 ml of dichloromethane, and the borate complex was hydrolyzed with HCl (3 N) until the pH was ~4. The aqueous layer was extracted twice with 50 ml of dichloromethane. The organic layers were pooled, washed twice with H2O (30 ml) and saturated NaCl (30 ml), and dried over anhydrous MgSO4. The dichloromethane solution was rotoevaporated to a viscous oil, which was purified by use of a Chromatotron with a solvent consisting of 10% methanol in dichloromethane to obtain a mixture of DL-threo- and erythro-enantiomers (2.24 g, 4 mmol).
Resolution of Inhibitor Enantiomers
High performance liquid chromatography (HPLC) resolution of the
DL-threo- and
DL-erythro-enantiomers was performed using a preparative HPLC column (Chirex 3014, (S)-Val-(R)-1-(-naphthyl)ethylamine, 20 × 250 mm; Phenomenex Inc.) eluted with
hexane/1,2-dichloroethane/ethanol/trifluoroacetic acid
(64:30:5.74:0.26) at a flow rate of 8 ml/min. The column eluant was
monitored at 254 nm in both the preparative and analytical modes. The
isolated products were reinjected until pure by analytical HPLC
analysis using an analytical Chirex 3014 column (4.6 × 250 mm)
and the same solvent mixture at a flow rate of 1 ml/min.
GlcCer Synthase Activity
The enzyme activity was measured as described previously (8). Madin-Darby canine kidney (MDCK) cell homogenates (120 µg of protein) were incubated with uridine diphosphate-[3H]glucose (100,000 cpm) and liposomes consisting of 85 µg of octanoylsphingosine, 570 µg of dioleoylphosphatidylcholine, and 100 µg of sodium sulfatide in a 200-µl reaction mixture and kept for 1 h at 37 °C. P4 and P4 derivatives dissolved in dimethyl sulfoxide was dispersed into the reaction mixture after adding the liposomes. The final concentration of dimethyl sulfoxide was <1%. At this concentration, there was no inhibition of the enzyme activity.
Cell Culture and Lipid Extraction
MDCK cells (5 × 105) were seeded into 10-cm dishes containing 8 ml of serum-free supplemented Dulbecco's modified Eagle's medium (9). After 24 h, the medium was replaced with 8 ml of the medium containing 0, 11.3, 113, or 1130 nM D-threo-P4, D-threo-3',4'-ethylenedioxy-P4, or D-threo-4'-hydroxy-P4. The GlcCer synthase inhibitors were added to the medium as a 1:1 molecular complex with delipidated bovine serum albumin (4, 6). The cells were incubated for 24 or 48 h with the inhibitors. After the incubation, the cells were washed twice with 8 ml of cold phosphate-buffered saline and fixed with 2 ml of cold methanol. The fixed cells were scraped and transferred to a glass tube. An additional 1 ml of methanol was used to recover the remaining cells in the dish.
Three ml of chloroform were added to the tube and briefly sonicated
using a water bath sonicator. After centrifugation at 800 × g for 5 min, the supernatant was transferred into another glass tube. The residues were reextracted with chloroform/methanol (1:1). After the centrifugation, the resultant supernatant was combined
with the first one. The residues were air-evaporated and kept for
protein analysis by the bicinchoninic acid method. NaCl (0.9%) was
added to the supernatant, and the ratio of chloroform/methanol/water was adjusted to 1:1:1. After centrifugation at 2000 × g for 5 min, the upper layer was discarded. The lower layer
was washed with methanol/water (1:1) in an volume equal to that of the
lower layer. The resultant lower layer was transferred into a glass tube and dried under a stream of nitrogen gas. A portion of the lipid was used for lipid phosphate determination (10). The remainder was analyzed using high performance thin-layer chromatography.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Resolution of PDMP Homologues by Chiral Chromatography--
The
structures of the parent compound, D-threo-P4,
and the phenyl-substituted homologues including the new
dioxy-substituted and 4'-hydroxy-P4 homologues are shown in Fig.
2. Initially, the effect of each P4
isomer separated by chiral chromatography on GlcCer synthase activity
was determined (Fig. 3A). Four
peaks were observed for the chiral separation of P4. Peaks 1 and 2 represent the erythro-diastereomers, and peaks 3 and 4 represent the threo-diastereomers as determined by a
sequential separation of the P4 mixture by reverse-phase chromatography
followed by the chiral separation. The enzyme activity was specifically
inhibited by the fourth peak, the D-threo-isomer
(Fig. 3B). This specificity for the
D-threo-enantiomer was consistent with the
previous results observed in PDMP and PDMP homologues (2-4). The
IC50 of D-threo-P4 was 0.5 µM for GlcCer synthase activity measured in the MDCK cell
homogenates.
|
|
Effects of P4 and P4 Derivatives with a Single Phenyl Group Substitution on GlcCer Synthase Activity-- In previous work, the addition of a p-methoxy group to DL-threo-P4 was found to enhance the effect of the inhibitor on the enzyme activity (4). This improved efficacy was confirmed for the D-threo-enantiomer. The enzyme activity was potently inhibited by D-threo-p-methoxy-P4 (IC50 = 0.2 µM) (Fig. 3C). Chiral chromatography of the four p-methoxy-P4 enantiomers failed to resolve completely to base line each enantiomer (Fig. 3A). A slight inhibition of the enzyme activity by p-methoxy-P4 in a combined mixture of D-erythro- and L-threo-enantiomers (peaks 2 and 3) was observed; this might have been due to contamination of the D-threo-isomer (peak 4) into these fractions, although this is not apparent on the separation shown.
Next, a series of D-threo-P4 derivatives
containing other single substituents on the phenyl group was
investigated. The potency of these phenyl-substituted compounds as
GlcCer synthase inhibitors was inferior to that of
D-threo-P4 or
p-methoxy-D-threo-P4 (Table I). For many drugs, the effect of
aromatic substitutions on the biological activity has been
characterized and is often predictive. Generally, the IC50
can be described by the following equation (11):
log(1/IC50) = a (hydrophobic parameter ()) + b (electronic parameter (
)) + c (steric
parameter) + d (another descriptor) + e, where
a-e are the regression coefficients.
|
The hydrophobic effect () is described by the equation
= log
PX
log PH, where
PX is the partition coefficient of the substituted
derivative and PH is that of the parent compound,
measured as the distribution between octanol and water. The electronic
substituent parameter (
) was originally developed by Hammett (12)
and is expressed as
= log KX
log
KH, where KX and
KH are the ionization constants for a
para- or meta-substituted derivative and benzoic acid, respectively. Positive
values represent electron-withdrawing properties, and negative
values represent electron-donating properties.
The potency of D-threo-P4 and P4 derivatives as
inhibitors is mainly dependent upon two factors (hydrophobic and
electronic properties) of a substituent of the phenyl group (Table I).
Surprisingly, a linear relationship was observed between
log(IC50) and +
(Fig.
4). These findings suggest that the more
negative the value of
+
, the more potent are
D-threo-P4 derivatives made as GlcCer synthase
inhibitors.
|
The p-Hydroxy-substituted Homologue Is a Significantly Better
GlcCer Synthase Inhibitor--
The strong association between +
and GlcCer synthase inhibition suggested that a still more potent
inhibitor could be produced by increasing the electron-donating
properties and decreasing the lipophilic properties of the phenyl group
substituent. A predictably negative
+
value would be observed
for the p-hydroxy homologue. This compound was synthesized
(see "Experimental Procedures"), and the
D-threo-enantiomer was isolated by chiral
chromatography. An IC50 of 90 nM for GlcCer
synthase inhibition was observed (Fig. 5), suggesting that the
p-hydroxy homologue was twice as active as the
p-methoxy compound. Moreover, the linear relationship
between log(IC50) and
+
was preserved (Fig. 4).
|
Effects of D-threo-3',4'-Dioxy-P4 Derivatives on GlcCer
Synthase Activity--
To further evaluate the potential effects of
structurally similar but more complex substitutions, new
D-threo-P4 derivatives with methylenedioxy,
ethylenedioxy, and trimethylenedioxy substitutions on the phenyl group
were designed (Fig. 2). The enzyme activity was most strongly inhibited
by D-threo-3',4'-ethylenedioxy-P4, with an
IC50 of 100 nM (Fig.
6). On the other hand, the
IC50 values for
D-threo-3',4'-methylenedioxy-P4 and
D-threo-3',4'-trimethylenedioxy-P4 were ~500
and 600 nM, respectively.
|
Interestingly, D-threo-3',4'-dimethoxy-P4 was
inferior to these dioxy derivatives, even to
D-threo-P4 or m- or
D-threo-p-methoxy-P4, as an inhibitor
(Fig. 6). As the parameters m and
p (the Hammett
constants for the meta- and para-substitutions,
respectively) and
for a single methoxy substituent are 0.12,
0.27, and
0.02, respectively (11), the value of
+
for
D-threo-dimethoxy-P4 is presumed to be negative.
Therefore, based only on the electronic substituent parameters,
D-threo-dimethoxy-P4 deviates quite far from the
correlation observed in Fig. 4. This may be due to a repulsion between
two methoxy groups in the dimethoxy-P4 molecule that induces a steric
effect that was negligible in mono-substituted D-threo-P4 derivatives studied in Fig. 4. GlcCer
synthase is thought to possess a domain that interacts with
D-threo-PDMP and PDMP homologues and that
modulates the enzyme activity (2, 6). The steric effect generated by an
additional methoxy group may affect one or more of these interaction
domains. As a result, the potency of the dimethoxy homologue as a
GlcCer synthase inhibitor was markedly decreased.
Distinguishing between Inhibition of GlcCer Synthase and
1-O-Acylceramide Synthase--
Prior studies on PDMP and related
homologues revealed that both the threo- and
erythro-diastereomers were capable of increasing cell
ceramide and inhibiting cell growth despite the observation that only
the D-threo-enantiomers blocked GlcCer synthase
(4). An alternative pathway for ceramide metabolism was subsequently identified (the acylation of ceramide at the 1-hydroxyl position) that
was blocked by both threo- and
erythro-diastereomers of PDMP. The specificities of
D-threo-P4,
D-threo-3',4'-ethylenedioxy-P4, and
D-threo-4'-hydroxy-P4 for GlcCer synthase were
studied by assaying the transacylase. Although there was a significant
difference in activity among
D-threo-3',4'-ethylenedioxy-P4,
D-threo-4'-hydroxy-P4, and
D-threo-P4 in inhibiting GlcCer synthase, the
D-threo-enantiomers of all three compounds
demonstrated comparable activity in blocking 1-O-acylceramide synthase (Fig.
7).
|
To determine whether inhibition of 1-O-acylceramide synthase was the basis for inhibitor-mediated ceramide accumulation, the ceramide and diradylglycerol levels of MDCK cells treated with D-threo-P4, D-threo-3',4'-ethylenedioxy-P4, and D-threo-4'-hydroxy-P4 were measured (Table II). Significant increases in both ceramide and diradylglycerol occurred only in cells treated with inhibitor concentrations in excess of 1 µM. This was ~30-fold lower than the concentration required for inhibition of the 1-O-acylceramide synthase assayed in the cellular homogenates. This disparity in concentration effects most likely reflects the ability of the more potent homologues to accumulate within intact cells (6).
|
Effects of D-threo-P4,
D-threo-4'-Hydroxy-P4, and
D-threo-Ethylenedioxy-P4 on GlcCer Synthesis and Cell
Growth--
To confirm the cellular specificity of
D-threo-3',4'-ethylenedioxy-P4 and
D-threo-4'-hydroxy-P4 as compared with
D-threo-P4, MDCK cells were treated with different
concentrations of the inhibitors. Approximately 66 and 78% of the
GlcCer was lost from the cells treated with 11.3 nM
D-threo-4'-hydroxy-P4 and
D-threo-ethylenedioxy-P4, respectively (Fig.
8, B and C). By
contrast, only 27% depletion of GlcCer occurred in cells exposed to
D-threo-P4 (Fig. 8A). A low level of
GlcCer persisted in the cells treated with a 113 or 1130 nM
concentration of either compound. This may be due to the contribution,
by degradation, of more highly glycosylated sphingolipids or the
existence of another GlcCer synthase that is insensitive to the
inhibitor.
|
On the other hand, there was little difference in the total protein content between untreated cells and those treated with 11.3 or 113 nM D-threo-4'-hydroxy-P4 and D-threo-ethylenedioxy-P4 (Fig. 8, B and C). A significant decrease in total protein was observed in the cells treated with a 1130 nM concentration of either P4 homologue. In addition, the level of ceramide in the cells treated with 1130 nM D-threo-ethylenedioxy-P4 and D-threo-4'-hydroxy-P4 was two times higher than that measured in the untreated cells (Table II). There was no change in ceramide or diradylglycerol levels in cells treated with 11.3 and 113 nM concentrations of either compound. Similar patterns for GlcCer levels and protein content were observed after 48-h incubations (data not shown).
The phospholipid content was unaffected at the lower concentrations of
either D-threo-ethylenedioxy-P4 or
D-threo-4'-hydroxy-P4. The ratios of cell
protein to cellular phospholipid phosphate (µg of protein/nmol of
phosphate) were 4.94 ± 0.30, 5.05 ± 0.21, 4.84 ± 0.90, and 3.97 ± 0.29 for 0, 11.3, 113, and 1130 nM
D-threo-ethylenedioxy-P4, respectively, and
4.52 ± 0.39, 4.35 ± 0.10, and 3.68 ± 0.99 for 11.3, 113, and 1130 nM
D-threo-4'-hydroxy-P4, respectively, suggesting that the changes in GlcCer content were truly related to inhibition of
GlcCer synthase activity. These results strongly indicate that two new
inhibitors, D-threo-4'-hydroxy-P4 and
D-threo-3,4-ethylenedioxy-P4, are able to
potently and specifically inhibit GlcCer synthesis in intact
cells at low nM concentrations without any inhibition of
cell growth.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Since the original description of an inhibitor of GlcCer synthesis by Vunnam and Radin (13), the pharmacological blockade of glycosphingolipid synthesis has proven to be a valuable approach to understanding the metabolism and function of glycosphingolipids. Previous work with the parent GlcCer synthase inhibitor (PDMP) identified two concurrent effects in cells. These included the time-dependent depletion of all GlcCer-based glycosphingolipids and the accumulation of ceramide. Originally, the ceramide accumulation was believed to be the result of substrate accumulation. However, it was discovered that the erythro-enantiomers of pyrrolidino-substituted compounds raised cell ceramide independent of GlcCer depletion (4). In addition, homologues with aliphatic substitutions depleted GlcCer with minimal effects on ceramide levels. The current data with the new phenyl-substituted inhibitors are consistent with this observation. No growth inhibition was observed at low nM concentrations of either D-threo-ethylenedioxy-P4 or D-threo-4'-hydroxy-P4, but was observed at higher concentrations at which ceramide levels increase. These findings suggested a second site of action for these inhibitors that is independent of the inhibition of GlcCer synthase.
The search for another site of inhibition of ceramide metabolism in the presence of either threo- or erythro-diastereomers of P4 led to the identification of a novel pathway for ceramide metabolism, the acylation of ceramide at the 1-hydroxyl position. The formation of this lipid is catalyzed by a novel phospholipase A2. In the presence of ceramide as an acceptor, 1-O-acylceramide synthase can transacylate ceramide utilizing the sn-2-fatty acid of phosphatidylethanolamine or phosphatidylcholine (14). This transacylase has recently been purified (15). By using the erythro-diastereomers of inhibitors that increase ceramide to the exclusion of blocking GlcCer formation, it has been determined that the growth inhibitory effects of these homologues are mediated by ceramide accumulation and not GlcCer depletion.
The dissociation of GlcCer depletion from ceramide accumulation is an important and necessary finding if one is to consider the development of GlcCer synthase inhibitors as potential drugs for inherited glycosphingolipid storage diseases. Ideally, such a drug should exhibit little or no cellular toxicity. The growth inhibitory and proapoptotic effects of ceramide should ideally be eliminated. Recently, support for the concept of treating sphingolipid storage disorders by inhibition of GlcCer synthase was reported by the reversal of the Tay-Sachs phenotype in knockout mice treated with a structurally unrelated inhibitor of GlcCer synthesis, N-butyldeoxynojirimycin (16). This inhibitor is significantly less potent and less specific than the compounds characterized in the present report.
Previous refinements of the parent structure of PDMP have resulted in
compounds with greater activity against GlcCer synthase. However, these
substitutions of the fatty acyl chain and cyclic amine moieties were
designed empirically. In the present study, single phenyl substitutions
permitted the analysis of inhibitor activity based on constants derived
by Hansch and others many years ago (18). Parameters of both
lipophilicity and electronegativity were observed to be predictive of
inhibitory activity. Specifically, the design and synthesis of
D-threo-4'-hydroxy-P4 yielded the most potent
GlcCer synthase inhibitor to date. These parameters, however, could be
applied only to simple substitutions and were inadequate to explain the
structure activity profile of more complex ring structures.
Nevertheless, the further application of these principles may result in
newer glycolipid synthase inhibitors with even greater activity and specificity.
![]() |
ACKNOWLEDGEMENT |
---|
We gratefully acknowledge the role of Norman Radin in reviewing this manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants RO1 DK41487 and RO139255 and by a merit review research award from the Veterans Affairs Medical Center (to J. A. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Established Investigator of the American Heart Association. To
whom correspondence should be addressed: Nephrology Div., Dept. of
Internal Medicine, University of Michigan, P. O. Box 0676, Rm. 1560 MSRBII, 1150 West Medical Center Dr., Ann Arbor, MI 48109-0676. Tel.:
734-763-0992; Fax: 734-763-0982; E-mail: jshayman{at}umich.edu.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: GlcCer, glucosylceramide; PDMP, 1-phenyl-2-decanoylamino-3-morpholino-1-propanol; P4, 1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol; HPLC, high performance liquid chromatography; MDCK, Madin-Darby canine kidney.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|