Property-based design of a glucosylceramide synthase inhibitor that reduces glucosylceramide in the brain

Article abstract of DOI:10.1194/jlr.M021261

Synthesis inhibition is the basis for the treatment of type 1 Gaucher disease by the glucosylceramide synthase (GCS) inhibitor eliglustat tartrate. However, the extended use of eliglustat and related compounds for the treatment of glycosphingolipid storag

Full text of DOI:10.1194/jlr.M021261

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Property-based design of a glucosylceramide synthase
inhibitor that reduces glucosylceramide in the brain
1
,
†
†
Scott D. Larsen, * Michael W. Wilson,* Akira Abe, Liming Shu, Christopher H. George,*
§
§
Paul Kirchhoff,* H. D. Hollis Showalter,* Jianming Xiang, Richard F. Keep,
and James A. Shayman
1
,†
Vahlteich Medicinal Chemistry Core - Department of Medicinal Chemistry,* Department of Internal
†
§
Medicine, and Department of Neurosurgery, University of Michigan, Ann Arbor, MI 48109
Abstract Synthesis inhibition is the basis for the treatment
of type 1 Gaucher disease by the glucosylceramide synthase
GCS) inhibitor eliglustat tartrate. However, the extended
use of eliglustat and related compounds for the treatment of
glycosphingolipid storage diseases with CNS manifestations
is limited by the lack of brain penetration of this drug. Prop-
erty modeling around the D-threo-1-phenyl-2-decanoylamino-
chaperone therapy, bone marrow transplantation, or gene
therapy) (1). Enzyme replacement therapy is clinically ap-
proved for lysosomal storage diseases with peripheral man-
ifestations but is limited by the absence of distribution of
infused recombinant enzyme into the central nervous sys-
tem (CNS) and by the frequent development of auto-anti-
bodies to the protein in patients who carry null mutations.
The second strategy involves synthesis inhibition therapy
(2). Synthesis inhibition is a more recent therapeutic ap-
proach and has focused on identifying small molecule in-
hibitors of GCS. Two classes of these inhibitors have been
described, including imino sugars and analogs of D-threo-1-
phenyl-2-decanoylamino-3-morpholino-propanol (PDMP)
(
3
-morpholino-propanol (PDMP) pharmacophore was employed
in a search for compounds of comparable activity against
the GCS but lacking P-glycoprotein (MDR1) recognition.
Modifications of the carboxamide N-acyl group were
made to lower total polar surface area and rotatable bond
number. Compounds were screened for inhibition of GCS
in crude enzyme and whole cell assays and for MDR1 substrate
recognition. One analog, 2-(2,3-dihydro-1H-inden-2-yl)-N-
(
3). The imino sugar N-butyldeoxynojirimycin (NBDNJ) is
((1R,2R)-1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1-hydroxy-
3-(pyrrolidin-1-yl)propan-2-yl)acetamide (CCG-203586), was
limited by its micromolar level inhibitory activity and lim-
ited specificity against the synthase. The latter trait is as-
sociated with a high level of untoward effects resulting
from secondary sites of action unrelated to glycolipid syn-
thesis inhibition. These effects most notably include diar-
rhea, weight loss, and tremor, and limit its approved use in
the United States (4). However, one distinct advantage of
NBDNJ over the PDMP-based homologs reported to date
is its ability to distribute into the CNS.
identified that inhibited GCS at low nanomolar concentra-
tions with little to no apparent recognition by MDR1. Intrap-
eritoneal administration of this compound to mice for 3 days
resulted in a significant dose dependent decrease in brain glu-
cosylceramide content, an effect not seen in mice dosed in
parallel with eliglustat tartrate.—Larsen, S. D., M. W. Wilson,
A. Abe, L. Shu, C. H. George, P. Kirchhoff, H. D. H. Showalter,
J. Xiang, R. F. Keep, and J. A. Shayman. Property-based design
of a glucosylceramide synthase inhibitor that reduces glucosyl-
ceramide in the brain. J. Lipid Res. 2012. 53: 282–291.
The active lead compound that is currently in clinical
trials and is structurally related to PDMP is N-((1R,2R)-1-(2,
3-dihydrobenzo[b][1,4]dioxin-6-yl)-1-hydroxy-3-(pyrrolidin-
Supplementary key words Gaucher disease • blood-brain barrier •
glycosphingolipid • synthesis inhibition therapy • eliglustat tartrate
1-yl)propan-2-yl)octanamide (Genz-112638, eliglustat tar-
trate 3a) (5). Recent phase 2 clinical trials using this drug
for type 1 Gaucher disease demonstrate a level of effi-
cacy equal to or greater than that seen for recombinant
Two general strategies for the treatment of lysosomal
storage diseases exist. The first strategy includes the re-
placement or restoration of the defective or absent catabo-
lizing enzyme (e.g., the infusion of recombinant enzyme,
Abbreviations: BBB, blood-brain barrier; CNS, central nervous sys-
tem; GlcCer, glucosylceramide; GCS, glucosylceramide synthase; HBA,
number of hydrogen bond acceptors; HBD, number of hydrogen bond
donors; MDR1, P-glycoprotein; MDR/WT, the ratio of MDR-MDCKII
This work was supported by the National Institutes of Health grants
IC50 divided by WT-MDCKII IC ; MW, molecular weight; NBDNJ, N-
50
5
R21NS065492-02 and 5RO1DK055823-11, a pilot grant from the Michigan
butyldeoxynojirimycin; PDMP, D-threo-1-phenyl-2-decanoylamino-3-
morpholino-propanol; RotB, rotatable bonds; TPSA, topological polar
surface area; WT, wild-type.
Institute for Clinical and Health Research, and the National Tay-Sachs and
Allied Disease Foundation. Its contents are solely the responsibility of the authors
and do not necessarily represent the official views of the National Institutes of
Health or other granting agencies.
1
To whom correspondence should be addressed.
e-mail: sdlarsen@umich.edu (S.D.L.) or jshayman@umich.edu
J.A.S.)
Manuscript received 4 October 2011 and in revised form 3 November 2011.
(
Published, JLR Papers in Press, November 4, 2011
DOI 10.1194/jlr.M021261
The online version of this article (available at http://www.jlr.org)
contains supplementary data in the form of data, figures, and references.
Copyright © 2012 by the American Society for Biochemistry and Molecular Biology, Inc.
2
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ꢁ-glucocerebrosidase as evidenced by reversal of spleen
METHODS
and liver enlargement, correction of anemia, and improve-
ments in thrombocytopenia and bone density (6). Phase 3
trials with eliglustat tartrate are currently in progress. Ex-
perimental data also support a potential role for eliglustat
tartrate in the treatment of Fabry disease, another lyso-
somal storage disease with peripheral manifestations (7).
In theory, substrate inhibition might also work for six
other lysosomal storage diseases with CNS involvement
that include early and late onset Tay-Sachs disease, Sand-
hoff disease, GM1 gangliosidosis, and types 2 and 3 Gau-
cher disease. Indeed, an experimental model of genetic
epistasis demonstrates markedly improved survival in a
mouse model of Sandhoff disease that also lack GM2 syn-
thase (8). Unfortunately, drug distribution studies indicate
that eliglustat tartrate is not transported across the blood-
brain barrier (BBB) (5). A possible basis for the poor brain
distribution of eliglustat tartrate may be that the drug is a
substrate for the p-glycoprotein (MDR1) transporter. MDR1
is highly expressed at the BBB where it markedly limits the
brain penetration of many xenobiotics (9). We confirmed
this possibility and then embarked on the design of new
glycolipid synthesis inhibitors using the active pharma-
cophore of PDMP with a goal of retaining activity against
GCS but eliminating substrate specificity for the MDR1
protein. In this paper, we report on the design and synthe-
sis of a series of PDMP analogs that are active against GCS,
the evaluation of these compounds in in vitro models of
MDR1 transport, and the identification of a novel analog
that inhibits glucosylceramide (GlcCer) synthesis in both
brain and peripheral organs.
Materials
N-((1R,2R)-1-(2,3-dihydrobenzo-[b][1,4]dioxin-6-yl)-1-hydroxy-
3-(pyrrolidin-1-yl)propan-2-yl)octanamide (eliglustat tartrate, 3a)
3
14
was provided by Genzyme Corporation. [ H]Vinblastine and [ C]
mannitol were both purchased from American Radiolabeled Chem-
icals (St Louis, MO).
Inhibitor synthesis
The preparation of analogs of 3a is summarized in Fig. 1. The
3,4-ethylenedioxy (EDO) amino alcohol intermediate 1 was pre-
pared as previously described (10). Hydrogenolysis of 1 afforded
common intermediate amine 2. N-acylation of 2 to afford analogs
3b-i (Table 2) was effected with various acid chlorides or carboxy-
lic acids under standard EDC coupling conditions. Monomethyl
amide 3j was prepared in three steps from 1 by Eschweiler-Clarke
mono-N-methylation to afford 4, hydrogenolysis of the benzyl
group to afford N-methylamine 5, and N-acylation with hydroci-
nammic acid/EDC. The chemical structures, experimental pro-
cedures, and spectral characterization for all final analogs are
described fully in the supplementary data.
GCS activity
Enzyme activity was measured as described previously (11).
Madin-Darby canine kidney (MDCK) cell homogenates (120 ꢀg
of protein) were incubated with uridine diphosphate-[ H]glu-
cose (100,000 cpm) and liposomes consisting of 85 ꢀg of oc-
tanoylsphingosine, 570 ꢀg of dioleoylphosphatidylcholine, and
3
100 ꢀg of sodium sulfatide in a 200 ꢀl reaction mixture and kept
for 1 h at 37°C. PDMP derivatives dissolved in dimethyl sulfoxide
(final concentration <1%, which did not affect enzyme activity)
were dispersed into the reaction mixture after adding the
liposomes.
Fig. 1. General scheme for the synthesis of new inhibitors. The structure of compound 3h (CCG-203586),
2
1
-(2,3-dihydro-1H-inden-2-yl)-N-((1R,2R)-1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1-hydroxy-3-(pyrrolidin-
-yl)propan-2-yl)acetamide, is indicated in the rectangular box.
A CNS-active glycolipid synthesis inhibitor
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GCS inhibition in MDCKII cells
thelial flux, was calculated as described previously (13). To inves-
tigate the effect of experimental drugs on MDR-mediated
transport, those compounds were added to the apical chamber
Parental wild type-MDCKII (WT-MDCKII) cells and MDCKII
cells retrovirally transduced with human MDR-1 cDNA were ob-
tained from the Netherlands Cancer Institute. Both cell lines
were routinely maintained in medium consisting of Opti-MEM/
F12 (1:1), 5% FBS, 100U/ml of penicillin, 100 µg/ml streptomy-
cin, and 200 mM L-glutamine. MDCKII cells were newly thawed
from frozen ampules every 2 months. Protein levels of MDR1 in
MDR1-MDCKII cells were measured monthly, and MDR1-MD-
CKII cell passages were immediately terminated when a reduc-
tion of MDR1 levels was observed by Western blot using
anti-human MDR1 monoclonal antibody (Abcam C219).
(
1-100 µM) during vinblastine uptake and the uptake and
trasepithelial flux results expressed as a percentage of that
with vehicle alone.
In vivo studies
C57BL/6 mice were maintained on regular chow in specific-
pathogen-free facilities. All animal studies were performed under
the review of the University of Michigan Committee on the Use
and Care of Animals and conformed to the National Institutes of
Health Guide for the Care and Use of Laboratory Animals. Injec-
tion solutions were prepared from inhibitor-ethanol stock solu-
tion (100 mM). A portion of the stock solution was evaporated
Stock solutions of water-insoluble glycosphingolipid inhibitors
(100 mM) were prepared by dissolving each inhibitor into 100%
ethanol as previously described (3). The inhibitor-ethanol solu-
tions were then diluted 50× into 2 mM delipidated BSA-PBS solu-
tion to make water-soluble glycosphingolipid inhibitor-BSA
complexes. The inhibitor-BSA complexes were sterile-filtered
and stored at ꢂ20°C. Prior to use, portions of the inhibitor-BSA
complexes were further diluted with Opti-F12 to make treatment
solutions. Equal amounts of BSA and ethanol were added into
under a stream of N gas. Dried eliglustat tartrate was directly dis-
2
solved into 1X PBS. Compound 3h was dissolved with 250 µl of
water plus 13.7 µl of 0.5 N HCl by hand shaking and gentle vor-
texing resulting in 1 or 3 mg/ml of solution. The acidic solution
was neutralized by mixing 100 µl of 10× PBS with 636.3 µl of wa-
ter to bring the total volume to 1 ml. Inhibitor solutions were
sterilized by passage through a 0.2 µM filter. Inhibitor recovery
after filtration was confirmed by UV spectrometry and exceeded
99%. For control injections, PBS containing the same amount of
HCl was used. Inhibitors were given to 6- to 8-week-old female or
male C57BL/6 mice by intraperitoneal injection volume at 1% of
body weight.
5
the control cultures. WT and MDR1-MDCKII cells (5 × 10 ) were
seeded into 10 cm culture dishes containing 10 ml of Opti-F12
with 5% FBS. After 24 h, the medium was replaced with fresh se-
rum-free Opti-F12 medium, and cells were exposed to candidate
GCS inhibitors at concentrations of 0, 1, 3, 10, 30, 100, and 300
nM for 24 h.
Mouse tissue lipid analysis
Cell lipid analysis
Lipid extractions of liver, kidney, and brain were performed as
previously described (7). Briefly, frozen liver (ف0.5 g), two kid-
neys (ف0.3 g), and whole brain (ف0.4 g) were individually ho-
mogenized in sucrose buffer (250 mM sucrose, pH 7.4, 10 mM
Hepes and 1 mM EDTA), at 0.2 g tissue/1 ml of sucrose buffer,
with a Tri-R homogenizer. Each 0.8 ml of homogenate was
mixed with 2 ml of methanol and 1 ml of chloroform, bath soni-
cated for 1 min, and incubated at room temperature for 1 h.
Tissue debris were removed by centrifugation at 2,400 g for 30
min. The pellets were reextracted by mixing with 1 ml of metha-
nol, 0.5 ml of chloroform, and 0.4 ml of 0.9% NaCl (chloroform-
methanol-0.9% NaCl, 1:2:0.8), incubated at room temperature
for 1 h, and centrifuged at 2,400 g for another 30 min. Two
extracts were combined and mixed with 4.5 ml of chloroform
and 1.2 ml of 0.9% NaCl (chloroform-methanol-0.9% NaCl,
Following inhibitor treatment, whole cellular lipids of wild-
type and MDR1-MDCKII cells were extracted as previously de-
scribed in detail (12). Briefly, cells were washed with ice-cold
PBS, fixed by methanol and collected with rubber scraper. Chlo-
roform was then added to yield a theoretical ratio of chloroform-
methanol-water at 1:2:0.8 (v/v/v) to form a mono-phase. Cell
debris and proteins were removed by centrifugation at 2200 g
for 30 min. The supernatants were portioned by adding chloro-
form and 0.9% NaCl. The lower organic phases containing neu-
tral glycosphingolipids lipids were washed with methanol and
0
.9% NaCl and subjected to base- and acid-hydrolysis (12). A
portion of purified glycosphingolipids normalized to 100 nmol
of total phospholipids was analyzed by high-performance TLC.
The TLC separations were processed twice. The plate pretreated
with 1% sodium borate was first developed in a solvent system
consisting of chloroform-methanol (98:2, v/v). After air drying,
the plate was then developed in a solvent system containing
chloroform-methanol-water (70:30:4, v/v/v). The levels of glu-
cosylceramide were detected by charring with 8% cupric sulfate
in 8% phosphoric acid and quantified by densitometric scan-
ning using ImageJ, NIH Image. Image data was analyzed, and
the IC50 of each inhibitor was calculated using GraphPad Prism
2:1:0.8). After centrifugation at 800 g for 5 min, the lower layer
was washed with 3 ml of methanol and 2.4 ml of 0.9% NaCl. A
second washing was carried with 3 ml of methanol, 2 ml of water,
and 0.4 ml of 0.9% NaCl followed by a 5 min centrifugation at
800 g. The resultant lower phase was collected and dried under a
stream of N gas.
2
The analysis of neutral glycosphingolipids from mouse liver,
kidney, and brain was processed after alkaline methanolysis. Kid-
ney lipids were incubated with 2 ml of chloroform and 1 ml of
(
version 5.03).
0
.21N NaOH in methanol for 2 h (kidney) or 7.5 h (liver and
Vinblastine transport
MDR1- and WT-MDCKII cells were grown to confluence on
Transwell filters (12-well plates) in DMEM + 10% FBS. The media
brain) at room temperature. The lipid extract was normalized
to 0.5 µmol of total phospholipid phosphate (liver and kidney)
or 2 µmol of total phospholipid phosphate (brain) for high
performance TLC analysis. After alkaline methanolysis, the brain
lipids were passed through a silica gel column (7). Borate-im-
pregnated TLC plates were developed in a two solvent system.
Plates were first developed in chloroform-methanol (98:2, v/v).
The plates loaded with kidney and liver lipids were then devel-
oped in chloroform-methanol-water (64:24:4, v/v/v), and brain
lipids were further separated in chloroform-methanol-water
(60:30:6, v/v/v). GlcCer levels were quantified by comparison to
known standards.
3
was then replaced with fresh DMEM and [ H]vinblastine (0.5
µCi/ml; 10 µM final unlabeled vinblastine concentration) and
1
4
[
C]mannitol (0.25 µCi/ml; an extracellular space marker) was
added to the apical chamber. Uptake and, in a subset of experi-
ments, transepithelial flux, was measured over 2 h at 37°C. At
that time, the basal chamber was sampled and uptake was stopped
by washing each side of the membrane three times with ice-cold
PBS. Vinblastine uptake into the cells, after correction for any
remaining adherent extracellular contamination and transepi-
2
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into MDR1-MDCKII cells with eliglustat was also associated
with an increase in transepithelial flux (to 280 ± 23 and
558 ± 28% of vehicle control at 30 and 100 ꢀM, respec-
tively). In comparison, 30 ꢀM verapamil increased tran-
sepithelial flux of [ H]vinblastine to 490 ± 26% of that in
vehicle-treated cells.
The design of new PDMP analogs was based on attenu-
ating significant differences between key physical chemi-
cal properties of 3a (eliglustat) and successful CNS drugs.
Table 1 summarizes a comparison of selected computed
properties of 3a and known CNS and nonCNS drugs. Data
was derived from structures for 1,213 approved drugs as
reported in DrugBank (14). A number of physical chemi-
cal properties were calculated for the 198 CNS drugs and
the remaining 1,015 nonCNS drugs using the Molecular
Operating Environment software (MOE, version 2008.10,
Chemical Computing Group Inc., Montreal, Quebec, Can-
ada). Mean and SD of the computed properties are tabu-
lated in the table for the two groups of drugs.
RESULTS
The GlcCer content of WT- and MDR1-MDCKII cells
was measured as a function of inhibitor concentration to
confirm that eliglustat tartrate (3a) was a substrate for the
MDR1 transporter (Fig. 2A). A significant rightward shift
in the concentration response curve was observed for the
reduction in GlcCer levels in the MDR1 expressing cells.
The IC s for the WT and MDR1 expressing cells were 13.7
and 31.7 nM, respectively. The interaction of eliglustat
with MDR1 was further examined by measuring [ H]vin-
blastine (an MDR substrate) transport in MDR1- MDCKII
cells. Eliglustat produced a dose-dependent increase in
3
5
0
3
3
[
H]vinblastine uptake similar to that found for verapamil,
a known MDR1 inhibitor (Fig. 2B). This increase in uptake
A comparison of the computed property values for 3a
relative to the mean values for the approved CNS drugs
(
Table 1) highlights significant divergences (ꢃ1 SD) in
molecular weight (MW), topological polar surface area
TPSA), number of hydrogen bond acceptors (HBA),
(
number of hydrogen bond donors (HBD) and rotatable
bonds (RotB). Within the constraints of the established
pharmacophore for 3a (which requires maintenance of
the free OH, the tertiary amine, and the absolute relative
stereochemistry of the two chiral centers) we elected to
focus on reducing the number of rotatable bonds and
TPSA (Table 2). Rotatable bond number was most easily
reduced in the aliphatic amide side chain by incorpora-
tion of a phenyl group (3b–3i), whereas TPSA was reduced
by methylating the amide (3j).
The activity of the new analogs at inhibiting GlcCer pro-
duction in the disrupted cell assay is summarized in Table
2
. Benzyl carbamate analog 3b retained the enzyme inhibi-
tory activity of 3a, confirming a similar observation by
Jimbo et al. (15) in a related template. The corresponding
3
-propionamide 3c, lower in TPSA than 3b, was equally
potent. Adjusting the length of the tether to the phenyl
group revealed that a 3-carbon tether (3e) was optimal
(
IC = 80 nM). Conformational restriction of the tether to
50
further reduce the number of rotatable bonds was ex-
plored with analogs 3f – 3i. Among these, 2-indanylmethyl
TABLE 1. Calculated physical properties of eliglustat tartrate (3a)
versus CNS drugs
Fig. 2. A: Concentration dependent change in GlcCer content of
WT-MDCKII and MDR-MDCKII cells in response to eliglustat tar-
trate. MDCK cells were cultured in the presence of eliglustat tar-
trate as described in the Methods section. After exposure to the
inhibitor for 24 h, the cells were harvested and the lipids extracted
and analyzed also as described in the Methods section. The data
represent the mean ± SD. (n = 4). B. Effects of different concentra-
tions of Genz 112368 (3a; eliglustat) and verapamil (a known
a
b
b
Computed Property
Non-CNS Drugs
CNS Drugs
3a
MW
383 ± 210
2.1 ± 2.9
98 ± 84
4.8 ± 4.1
2.8 ± 3.3
6.2 ± 5.5
288 ± 88
2.8 ± 1.4
46 ± 26
2.6 ± 1.3
1.0 ± 0.9
4.0 ± 2.6
405
3.5
71
5
2
SLogP
TPSA (Å )
2
HBA
HBD
RotB
12
3
MDR1 inhibitor) on the uptake of [ H]vinblastine into MDR1-MD-
a
Values calculated using The Molecular Operating Environment
MOE), version 2008.10, Chemical Computing Group Inc., Montreal,
CKII cells. Values are expressed as a percent of vehicle-treated cells
(
3
and are given as means ± SE (n = 3). With vehicle treatment, [ H]
Quebec, Canada.
b
vinblastine uptake into MDR1-MDCKII cells was 13.6 ± 1.7% of that
in WT-MDCKII cells.
Mean values and standard deviations derived from 198 approved
CNS drugs and 1015 approved nonCNS drugs (DrugBank) (14).
A CNS-active glycolipid synthesis inhibitor
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analog 3h displayed a striking improvement in activity
IC = 27 nM) and, to our knowledge, represents the most
(
5
0
potent GCS inhibitor reported to date.
The activity of the compounds at inhibiting GlcCer pro-
duction in whole WT-MDCKII cells is also summarized in
Table 2. Eliglustat (3a) was almost 10-fold more potent in
intact cells than disrupted, possibly a result of intracellular
accumulation. This phenomenon, originally reported for
the D-threo-ethylenedioxyphenyl homologs of PDMP, was
also observed to varying degrees with most of the other
analogs (16). We found that the ratio of broken cell IC50
divided by whole cell IC is weakly correlated negatively
5
0
2
2
with MW (R = 0.58) and positively with SlogP (R = 0.50),
consistent with well established medicinal chemistry prin-
ciples that decreasing MW and increasing lipophilicity
each improve passive permeability into cells (17).
The MDR-MDCKII cell line was used to help predict
BBB permeability because it expresses an efflux trans-
porter that is physiologically relevant to the brain endothe-
lium (18, 19). We assumed that IC s for inhibition of
5
0
GlcCer production in these cells would correlate directly
with intracellular drug levels, thereby providing a conve-
nient and sensitive estimate of susceptibility to MDR-medi-
ated efflux when compared with IC s in WT-MDCKII
5
0
cells. Results are included in Table 2. Significantly, the ra-
tios of MDR-MDCKII IC50 divided by WT-MDCKII IC50
(
MDR/WT) varied widely among the group, presumably
reflecting a wide range of affinities for MDR1.
Although several replacements for the C8 acyl group
2
(
R ) of eliglustat were associated with the retention of
nanomolar range inhibition of GlcCer in the WT cell line,
only the –CH -indan-2-yl homolog 3h displayed a compa-
2
rable IC in the MDR1-MDCKII cells when compared with
5
0
WT (<2-fold increase in IC ). Remarkably, deletion of a
5
0
single endocyclic or exocyclic methylene group from the
indane (3e or 3i, respectively) was associated with signifi-
cant loss of activity in the MDR1-MDCKII line. A compari-
son of the dose-dependent changes in GlcCer levels for
these three homologs is shown in Fig. 3A. Representative
TLC plates of the extracted lipids from the WT-MDCKII
and MDR1-MDCKII cells are shown in Fig. 3B. Consistent
with many reports on the association of cellular GlcCer
content and MDR1 expression, the MDR1-MDCKII cells
had significantly higher concentrations of glucosylcer-
amide at baseline (20). However, the percent decrement
in GlcCer in the presence of increasing concentrations of
3
h was comparable for the two cell lines. The decrease in
GlcCer occurred in concert with a fall in lactosylceramide,
but in the absence of any apparent change in sphingomy-
elin and galactosylceramide, consistent with a specific ef-
fect of 3h on GlcCer synthase. This observation is consistent
with previous reports for eliglustat as well as the other
PDMP-based analogs.
The potential interactions of the novel compounds with
MDR1 was further examined by determining their effects
3
on [ H]vinblastine transport in MDR1-MDCKII cells (Fig.
3
C). Consistent with the marked rightward shifts in the
concentration response curves observed for analogs 3e
and 3i, a significant dose dependent increase in uptake in
2
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Fig. 3. Correlation between cellular GCS inhibition and inhibition
3
of [ H]vinblastine uptake in the presence of MDR1. A: A comparison
of the dose dependent changes in GlcCer for three homologs of
PDMP. B: Representative thin layer chromatography plates of WT-MD-
CKII and MDR1-MDCKII cells treated with 3h. SM, sphimgomyelin;
GalCer, galactosylceramide. C: Effects of different concentrations of
3
three novel GCS inhibitors on the uptake of [ H]vinblastine into
MDR1-MDCKII cells. Values are expressed as a percent of vehicle-
treated cells and are given as means ± SE (n = 3). 3h only differs from
3
i by a single methylene spacer and from 3e by a single bridging CH2,
but these changes result in marked changes in the interaction with
MDR. D: Effects of different GCS inhibitors on two parameters, [ H]
3
vinblastine uptake into MDR1-MDCKII cells expressed as a fold change
between 30 ꢀM of the inhibitor and vehicle (x axis) and the fold
change in GCS IC50 between MDR1- and WT-MDCKII cells (y axis).
For most compounds, there was a good correlation between the two
parameters (the line represents equal changes). There was a signifi-
cant correlation between both parameters although two compounds,
3
i and 3j, showed noticeably greater effects on the enzyme IC50 than 3f
3
and 3e despite similar magnitude effects on [ H]vinblastine uptake. As
assessed by both assays, 3h had the least interaction with MDR.
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3
[
H]vinblastine was observed. In contrast, the –CH -indan-
in the eliglustat or vehicle-treated mice. When a higher
dose of inhibitor was employed (30 mg/kg q 12 h for 3
days), a more significant fall in brain GlcCer levels was ob-
served (17%) in 3h inhibitor-treated mice without any
changes in the vehicle-treated or eliglustat-treated mice.
By contrast, eliglustat lowered liver and kidney GlcCer lev-
els, consistent with its previously reported effects. These
data confirmed the ability of 3h to cross the BBB and in-
hibit GCS. The mice tolerated treatment with both doses
of 3h well, displaying no weight loss or obvious gastrointes-
tinal, respiratory, or behavioral changes.
2
2
-yl homolog 3h resulted in no significant dose-depen-
dent change in vinblastine uptake. These results are
consistent with the absence of recognition of 3h as a sub-
strate for MDR1.
The full range of active carboxamide inhibitors was
compared using both the GCS inhibition and vinblastine
transport assays in WT- versus MDR1-MDCKII cells. There
was reasonable agreement between the two assays with re-
gard to changes in the presence of MDR1 (Fig. 3D; r =
0
.50). Based on these results and the IC50 against the cell
lysate synthase activity, 3h (CCG-203586) was chosen as a
lead compound for in vivo studies. Six-week-old wild-type
mice were initially treated with 10 mg/kg/day of 3h or ve-
hicle for 3 days and euthanized 12 h after the last injection
DISCUSSION
Property modeling of compounds retaining the well-
established PDMP pharmacophore and four screening as-
says were employed in an attempt to find GCS inhibitors
(
Fig. 4). A 10% decrease in the GlcCer levels in the brains
of 3h inhibitor-treated mice was observed without changes
Fig. 4. Effect of eliglustat and inhibitor 3h (CCG-203586) on organ GlcCer content after short term expo-
sure. Six- to eight-week-old C57BL/6 mice were treated with either vehicle or inhibitor for 3 days. Mice re-
ceived 10 or 60 mg/kg/day i.p. of the respective inhibitor and were then euthanized 12 h following the last
injection. The data are expressed as ꢀg of GlcCer normalized to 0.5 or 2 ꢀmoles of phospholipid (PL) and
represent the mean ± SD and * denotes P < 0.05 by student’s t-test versus vehicle treated control, n = 4 except
for 10 mg/kg/day treatment brain samples where n = 3.
2
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that would be active in the brain. Specific findings in-
cluded the following. First, a series of PDMP analogs were
designed to lower or eliminate their recognition as sub-
strates for the MDR1 transporter by comparison of selected
physical properties to drugs known to cross the BBB. Sev-
eral compounds retained nanomolar activity as GCS in-
hibitors in both crude cell enzyme and whole cell assays.
The activity of these compounds confirmed the utility of
the pharmacophore of PDMP as a scaffold for GCS inhibi-
tors. Second, one analog (3h), was demonstrated to be a
particularly poor substrate for the MDR1 protein relative
to the other analogs using two in vitro assays based on the
stable expression of MDR1. Third, the 3h analog demon-
strated significant in vivo activity in lowering brain GlcCer
levels after 3 days of intraperitoneal administration. The
decrease in brain GlcCer was in contrast to eliglustat tar-
trate, which had no discernable effect on brain GlcCer
when administered in parallel.
A wealth of scholarship exists about the differences in
the physical chemical properties of CNS versus nonCNS
drugs (21–23) and in fact, a number of computed proper-
ties vary significantly between the two groups. Compounds
possessing molecular parameters outside of the “ideal”
ranges for CNS drugs tend to either have poor passive dif-
fusion through cell membranes or to be substrates for MDR1
mediated efflux. The range of tolerated physical chemical
properties observed for CNS-active agents is significantly
narrower than the whole of oral therapeutics, a result of the
unique physical characteristics of the BBB (24, 25). Spe-
cifically, the endothelial cells of the cerebral capillaries have
extraordinarily tight junctions, requiring compounds to pass
through entirely via the transcellular route by passive dif-
fusion. Furthermore, xenobiotic efflux transporter MDR1
in the BBB efficiently expels a wide variety of substrates.
The retention of the pharmacophore derived from the
development of D-threo-PDMP led us to focus primarily on
modifications to the N-acyl group of the amide. Substitutions
were made to lower the number of rotatable bonds and total
polar surface area. Although a number of new PDMP ho-
mologs with nanomolar activity against GCS were identi-
fied (notably 3h), no overall correlations were apparent
between the ratio of inhibition of the synthase in MDR
versus WT MDCKII cells and any of the calculated parame-
ters in Table 2. However, some interesting pair-wise com-
parisons between closely related structures were observed.
MDR1 within this series is much more dependent on con-
formation than on small changes in lipophilicity or TPSA.
Indeed, when energy-minimized conformations of com-
pounds 3c, 3e, 3h, 3i, and 3j were overlaid, significant dif-
ferences were apparent (supplementary Fig. V) This is
consistent with previous reports that conformational restric-
tion can be successful at attenuating MDR1 efflux, presum-
ably due to locking compounds into conformations that are
no longer recognized by MDR1 (26).
The comparison in GCS inhibition between WT and
MDR-MDCKII cells also served as a facile and convenient
method for detecting recognition of GCS inhibitors by
MDR1. However, because of the importance of MDR1 in
determining brain penetration of many therapeutics (9), a
second assay was used to assess MDR1 interactions by de-
3
termining whether GCS inhibitors increased [ H]vinblas-
tine uptake into MDR-MDCKII cells. Vinblastine is an
MDR substrate and evidence indicates that MDR is the
sole/predominant efflux mechanism present for vinblas-
tine in MDR-MDCKII cells (27, 28). Under control condi-
3
tions, [ H]vinblastine levels were only 14 ± 2% of that
found in WT-MDCKII cells [which do express some canine
MDR (28)], and inhibiting MDR (e.g., with verapamil) re-
3
sults in an increase in [ H]vinblastine uptake. Such an in-
creaseinuptakewasfoundwithmanyoftheGCSinhibitors.
3
It should be noted, however, that an increase in [ H]vinblas-
tine uptake only indicates that the GCS inhibitor interacts
with MDR; it does not necessarily imply it is a substrate.
3
In general, there was good accord between the [ H]vin-
blastine and GCS assays. However, for two pairs of com-
pounds (3i/3j vs. 3e/3f) that showed similar effects on
3
[ H]vinblastine uptake, there were marked differences in
the MDR effects in the GCS assay (ف20-fold vs. ف5-fold).
3
This may reflect that the [ H]vinblastine assay measures
whole cell uptake, whereas the effective concentration for
inhibition of GCS is the drug level at the outer Golgi mem-
brane. However, these compounds are also cationic am-
phiphilic drugs that are protonated under the acidic
conditions and trapped in the lysosome where they may
exert other effects. For example, a secondary site of activ-
ity of PDMP and other cationic amphiphilic drugs is the
inhibition of group XV phospholipase A2 (29). This inhi-
bition may be the basis for some forms of drug-induced
phospholipidosis. Nevertheless, the use of an enzyme assay
in WT- and MDR-MDCKII cells to assess the MDR affinity
of enzyme inhibitors is novel and may potentially be ex-
tended to other types of enzyme inhibitors provided that
MDCKII cells (or other cells overexpressing MDR) express
the relevant target enzyme.
The final assay employed was measurement of GlcCer
levels in vivo. Although MDR is important for the efflux of
many therapeutics at the BBB, there are also other active
efflux mechanisms that impact brain penetration [e.g.,
breast cancer-related protein (9)]. The role of such trans-
porters can be examined using overexpressing cell lines
(as used here for MDR). However, because of the com-
plexity of the efflux systems present at the BBB, it was more
efficient to employ an in vivo assay to determine whether our
lead compound could penetrate the brain at sufficient
Replacing the carbamate oxygen of 3b with CH (3c) reduces
2
both TPSA and lipophilicity (SlogP), and appeared to re-
sult in less recognition by MDR1. N-methylating the amide
of 3c to afford 3j resulted in a dramatic increase in MDR/
WT, which is opposite to what one would expect from re-
moval of a hydrogen bond donor and reduced TPSA. Phenyl-
propionamides 3e and 3h differ only in a single bridging
CH that eliminates two rotatable bonds and conformation-
2
ally restricts the propyl chain. This resulted in both a signi-
ficant improvement in enzyme activity (27 nM vs. 80 nM) and
an apparent dramatic reduction in MDR efflux (MDR/WT
of 1.6 vs. 5.7). Finally, compound 3i, differing from 3h by
the absence of a single CH , was avidly recognized as a MDR1
2
substrate. Overall, these results suggest that recognition by
A CNS-active glycolipid synthesis inhibitor
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concentrations to impact GCS. Importantly, with short-
term dosing, inhibitor 3h was observed to significantly lower
brain GlcCer levels. This effect was specific for this analog
in that eliglustat failed to demonstrate any change in brain
cerebroside content under identical dosing conditions, even
though it significantly lowered liver and kidney glycolip-
ids. This finding confirmed that developing a high-affinity
GCS inhibitor (3h) with a lack of recognition by MDR1
is able to result in a pharmacological response in the brain.
In summary, the present study outlines a general strat-
egy for the design and testing of compounds that lack
MDR1 affinity and identifies a new PDMP analog that ap-
pears to satisfy the properties of high inhibitory activity
against GCS and limited MDR1 affinity. Because the con-
cept of synthesis inhibition for the treatment of glycosphin-
golipidoses by PDMP-based GCS inhibitors is now well
established on both experimental and clinical grounds,
the identification of a related compound that is active
within brain is encouraging. Specifically, the D-threo-
ethylendioxyphenyl analogs of PDMP are characterized by
inhibition of GCS at low nanomolar concentrations, high
specificity, and the absence of ꢁ-glucocerebrosidase bind-
ing. Recent phase 2 clinical data for eliglustat tartrate have
demonstrated a clinical response in type 1 Gaucher dis-
ease that is comparable to enzyme replacement therapy as
measured by reduction in spleen and liver volume, correc-
tion of anemia, and improvement in thrombocytopenia.
The untoward effects observed with NBDNJ, including
weight loss, diarrhea, and tremor, were not observed in
this clinical trial nor in an extension study. These observa-
tions are consistent with the high specificity of the drug
and its absence of CNS penetration.
3. Shayman, J. A., L. Lee, A. Abe, and L. Shu. 2000. Inhibitors of glu-
cosylceramide synthase. Methods Enzymol. 311: 373–387.
4
. Weinreb, N. J., J. A. Barranger, J. Charrow, G. A. Grabowski, H. J.
Mankin, and P. Mistry. 2005. Guidance on the use of miglustat for
treating patients with type 1 Gaucher disease. Am. J. Hematol. 80:
2
23–229.
5
. Shayman, J. A. 2010. Eliglustat tartrate. Drugs Future. 35: 613–621.
. Lukina, E., N. Watman, E. A. Arreguin, M. Banikazemi, M. Dragosky,
M. Iastrebner, H. Rosenbaum, M. Phillips, G. M. Pastores, D. I.
Rosenthal, et al. 2010. A phase 2 study of eliglustat tartrate (Genz-
6
1
12638), an oral substrate reduction therapy for Gaucher disease
type 1. Blood. 116: 893–899.
7. Abe, A., S. Gregory, L. Lee, P. D. Killen, R. O. Brady, A. Kulkarni,
and J. A. Shayman. 2000. Reduction of globotriaosylceramide in
Fabry disease mice by substrate deprivation. J. Clin. Invest. 105:
1
563–1571.
8
. Liu, Y., R. Wada, H. Kawai, K. Sango, C. Deng, T. Tai, M. P.
McDonald, K. Araujo, J. N. Crawley, U. Bierfreund, et al. 1999. A
genetic model of substrate deprivation therapy for a glycosphingo-
lipid storage disorder. J. Clin. Invest. 103: 497–505.
9
. Miller, D. S. 2010. Regulation of P-glycoprotein and other ABC
drug transporters at the blood-brain barrier. Trends Pharmacol. Sci.
3
1: 246–254.
1
0. Hirth, B. H., and C. Siegel. 2005. Synthesis of UDP-glucose:
N-acylsphingosine glucosyltransferase inhibitors. Patent no.
6,855,830. Genzyme Corporation, United States.
1
1. Shayman, J. A., and A. Abe. 2000. Glucosylceramide synthase: assay
and properties. Methods Enzymol. 311: 42–49.
1
2. Shu, L., and J. A. Shayman. 2003. Src kinase mediates the regula-
tion of phospholipase C-gamma activity by glycosphingolipids. J.
Biol. Chem. 278: 31419–31425.
1
3. Shu, C., H. Shen, N. S. Teuscher, P. J. Lorenzi, R. F. Keep, and D.
E. Smith. 2002. Role of PEPT2 in peptide/mimetic trafficking at
the blood-cerebrospinal fluid barrier: studies in rat choroid plexus
epithelial cells in primary culture. J. Pharmacol. Exp. Ther. 301:
8
20–829.
1
4. Wishart, D. S., C. Knox, A. C. Guo, D. Cheng, S. Shrivastava, D.
Tzur, B. Gautam, and M. Hassanali. 2008. DrugBank: a knowledge-
base for drugs, drug actions and drug targets. Nucleic Acids Res. 36:
D901–D906.
5. Jimbo, M., K. Yamagishi, T. Yamaki, K. Nunomura, K. Kabayama, Y.
Igarashi, and J. I. Inokuchi. 2000. Development of a new inhibitor
of glucosylceramide synthase. J. Biochem. 127: 485–491.
1
Although the absence of eliglustat tartrate distribution
into brain may be advantageous for glycosphingolipidoses
without CNS manifestations, including type 1 Gaucher
and Fabry diseases, our identification of a PDMP homolog
that crosses the BBB is of potential therapeutic benefit for
disorders such as GM2 gangliosidoses, Tay-Sachs, Sand-
hoff disease, and types 2 and 3 Gaucher disease. Excellent
murine models for these disorders exist and, following the
characterization of the pharmacokinetic profile of 3h, will
provide the basis for experimental proof of principle for
the use of synthesis inhibition in these disorders. Finally,
recent work has defined a mechanistic association between
loss of glucocerebrosidase activity, neuronal GlcCer accu-
mulation, and ꢄ-synuclein accumulation (30). These find-
ingsprovideapotentialexplanationforthehighassociation
of type 1 Gaucher disease with Parkinson’s disease. The
identification of a potent GCS inhibitor with CNS activity
provides a pharmacological tool for further assessing this
association and possible therapeutic intervention.
1
6. Lee, L., A. Abe, and J. A. Shayman. 1999. Improved inhibitors of
glucosylceramide synthase. J. Biol. Chem. 274: 14662–14669.
7. Lipinski, C. A., F. Lombardo, B. W. Dominy, and P. J. Feeney. 2001.
Experimental and computational approaches to estimate solubility
and permeability in drug discovery and development settings. Adv.
Drug Deliv. Rev. 46: 3–26.
1
1
1
2
8. Garberg, P., M. Ball, N. Borg, R. Cecchelli, L. Fenart, R. D. Hurst,
T. Lindmark, A. Mabondzo, J. E. Nilsson, T. J. Raub, et al. 2005.
In vitro models for the blood-brain barrier. Toxicol. In Vitro. 19:
2
99–334.
9. Wang, Q., J. D. Rager, K. Weinstein, P. S. Kardos, G. L. Dobson, J.
Li, and I. J. Hidalgo. 2005. Evaluation of the MDR-MDCK cell line
as a permeability screen for the blood-brain barrier. Int. J. Pharm.
2
88: 349–359.
0. Gouaze-Andersson, V., and M. C. Cabot. 2006. Glycosphingolipids
and drug resistance. Biochim. Biophys. Acta. 1758: 2096–2103.
21. Mahar Doan, K. M., J. E. Humphreys, L. O. Webster, S. A. Wring,
L. J. Shampine, C. J. Serabjit-Singh, K. K. Adkison, and J. W. Polli.
2
002. Passive permeability and P-glycoprotein-mediated efflux dif-
ferentiate central nervous system (CNS) and non-CNS marketed
drugs. J. Pharmacol. Exp. Ther. 303: 1029–1037.
22. Leeson, P. D., and A. M. Davis. 2004. Time-related differences
in the physical property profiles of oral drugs. J. Med. Chem. 47:
6
338–6348.
2
3. Pajouhesh, H., and G. R. Lenz. 2005. Medicinal chemical properties
of successful central nervous system drugs. NeuroRx. 2: 541–553.
4. Cecchelli, R., V. Berezowski, S. Lundquist, M. Culot, M. Renftel,
M. P. Dehouck, and L. Fenart. 2007. Modelling of the blood-brain
barrier in drug discovery and development. Nat. Rev. Drug Discov. 6:
650–661.
REFERENCES
2
1
2
. Barton, N. W., R. O. Brady, J. M. Dambrosia, A. M. Di Bisceglie, S.
H. Doppelt, S. C. Hill, H. J. Mankin, G. J. Murray, R. I. Parker, C.
E. Argoff, et al. 1991. Replacement therapy for inherited enzyme
deficiency–macrophage-targeted glucocerebrosidase for Gaucher’s
disease. N. Engl. J. Med. 324: 1464–1470.
. Radin, N. S. 1996. Treatment of Gaucher disease with an enzyme
inhibitor. Glycoconj. J. 13: 153–157.
25. Lundquist, S., M. Renftel, J. Brillault, L. Fenart, R. Cecchelli, and
M. P. Dehouck. 2002. Prediction of drug transport through the
blood-brain barrier in vivo: a comparison between two in vitro cell
models. Pharm. Res. 19: 976–981.
2
90
Journal of Lipid Research Volume 53, 2012

Supplemental Material can be found at:
http://www.jlr.org/content/suppl/2011/11/04/jlr.M021261.DC1
.
html
2
2
6. Raub, T. J. 2006. P-glycoprotein recognition of substrates and cir-
cumvention through rational drug design. Mol. Pharm. 3: 3–25.
7. Ito, S., C. Woodland, B. Sarkadi, G. Hockmann, S. E. Walker,
and G. Koren. 1999. Modeling of P-glycoprotein-involved
epithelial drug transport in MDCK cells. Am. J. Physiol. 277:
F84–F96.
affect P-glycoprotein mediated drug transport. Br. J. Pharmacol. 160:
1453–1463.
29. Abe, A., M. Hiraoka, and J. A. Shayman. 2007. A role for lysosomal
phospholipase A2 in drug induced phospholipidosis. Drug Metab
Lett. 1: 49–53.
30. Mazzulli, J. R., Y. H. Xu, Y. Sun, A. L. Knight, P. J. McLean, G. A.
Caldwell, E. Sidransky, G. A. Grabowski, and D. Krainc. 2011. Gaucher
disease glucocerebrosidase and alpha-synuclein form a bidirectional
pathogenic loop in synucleinopathies. Cell. 146: 37–52.
2
8. Kuteykin-Teplyakov, K., C. Luna-Tortos, K. Ambroziak, and W.
Loscher. 2010. Differences in the expression of endogenous ef-
flux transporters in MDR1-transfected versus wildtype cell lines
A CNS-active glycolipid synthesis inhibitor
291