The Effect of Small Molecules on Sterol Homeostasis: Measuring 7-Dehydrocholesterol in Dhcr7-Deficient Neuro2a Cells and Human Fibroblasts

Article abstract of DOI:10.1021/acs.jmedchem.5b01696

Well-established cell culture models were combined with new analytical methods to assess the effects of small molecules on the cholesterol biosynthesis pathway. The analytical protocol, which is based on sterol derivation with the dienolphile PTAD, was found to be reliable for the analysis of 7-DHC and desmosterol. The PTAD method was applied to the screening of a small library of pharmacologically active substances, and the effect of compounds on the cholesterol pathway was determined. Of some 727 compounds, over 30 compounds decreased 7-DHC in Dhcr7-deficient Neuro2a cells. The examination of chemical structures of active molecules in the screen grouped the compounds into distinct categories. In addition to statins, our screen found that SERMs, antifungals, and several antipsychotic medications reduced levels of 7-DHC. The activities of selected compounds were verified in human fibroblasts derived from Smith-Lemli-Opitz syndrome (SLOS) patients and linked to specific transformations in the cholesterol biosynthesis pathway.

Full text of DOI:10.1021/acs.jmedchem.5b01696

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Article
The Effect of Small Molecules on Sterol Homeostasis: Measuring 7-
Dehydrocholesterol in Dhcr7-deficient Neuro2a Cells and Human Fibroblasts
Zeljka Korade, Hye-Young H. Kim, Keri A. Tallman, Wei Liu, Katalin
Koczok, Istvan Balogh, Libin Xu, Karoly Mirnics, and Ned A. Porter
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01696 • Publication Date (Web): 20 Jan 2016
Downloaded from http://pubs.acs.org on January 25, 2016
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The Effect of Small Molecules on Sterol Homeostasis:
Measuring 7ꢀDehydrocholesterol in Dhcr7ꢀdeficient
Neuro2a Cells and Human Fibroblasts
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a,b‡Zeljka Korade, c‡Hye-Young H. Kim, cKeri A. Tallman, cWei Liu, eKatalin Koczok, eIstvan Balogh,
c,dLibin Xu, a,bKaroly Mirnics and a,cNed A. Porter*
aVanderbilt Kennedy Center for Research on Human Development, Vanderbilt University, Nashville,
b
Tennessee 37235. Department of Psychiatry. cDepartment of Chemistry and Vanderbilt Institute of
Chemical Biology, Vanderbilt University, Nashville, Tennessee 37235. dCurrent address: Department of
Medicinal Chemistry, University of Washington, Seattle, WA 98195. eDepartment of Laboratory Medicine,
Division of Clinical Genetics, University of Debrecen, 4032 Debrecen, Nagyerdei krt. 98, Hungary.
KEYWORDS: 7ꢀdehydrocholesterol, desmosterol, cholesterol, PTAD, UPLCꢀMS, GCꢀMS, DHCR7,
SmithꢀLemliꢀOpitz Syndrome, HTS.
‡These authors contributed equally to this publication
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ABSTRACT: Wellꢀestablished cell culture models were combined with new analytical methods to assess
the effects of small molecules on the cholesterol biosynthesis pathway. The analytical protocol, which is
based on sterol derivation with the dienolphile PTAD, was found to be reliable for the analysis of 7ꢀDHC
and desmosterol. The PTAD method was applied to the screening of a small library of pharmacologically
active substances and the effect of compounds on the cholesterol pathway was determined. Of some 727
compounds, over 30 compounds decreased 7ꢀDHC in Dhcr7ꢀdeficient Neuro2a cells. The examination of
chemical structures of active molecules in the screen grouped the compounds into distinct categories. In
addition to statins, our screen found that SERMs, antifungals, and several antiꢀpsychotic medications
reduced levels of 7ꢀDHC. The activities of selected compounds were verified in human fibroblasts derived
from SmithꢀLemliꢀOpitz syndrome (SLOS) patients and linked to specific transformations in the cholesterol
biosynthesis pathway.
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The biosynthesis of cholesterol is a complex process that involves multiple enzymes and intermediates.1
The pathway from lanosterol to cholesterol includes over a dozen metabolic intermediates, many of which
play important roles in cellular processes such as hedgehog signal transduction.2ꢀ4 Cholesterol is of critical
importance in myelination and normal brain cholesterogenesis is absolutely essential for normal embryonic
and perinatal development. Genetic errors affecting transformations that are early in the biosynthesis
pathway are generally lethal at preꢀimplantation or the very early embryonic stage while individuals with
inborn errors affecting the last steps of cholesterol biosynthesis can survive to birth, usually with severe
consequences. 5ꢀ16
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Figure 1 shows structures of three lateꢀstage sterols in
the postꢀlanosterol pathway. 7ꢀDehydrocholesterol (7ꢀ
DHC) and desmosterol are the immediate biosynthetic
precursors of cholesterol and there are known human
disorders associated with errors in the conversion of these
sterols to cholesterol.5,6 7ꢀDHC accumulates in patients
with SmithꢀLemliꢀOpitz syndrome (SLOS),7 a metabolic
disorder resulting from mutations in the gene encoding 7ꢀ
dehydrocholesterol reductase (DHCR7), the enzyme that
Figure 1. Lateꢀstage Sterols in Cholesterol
catalyzes the reduction of 7ꢀDHC to cholesterol.8,9
Biosynthesis
The pathophysiology of SLOS is the apparent result of changing the cholesterol/7ꢀDHC balance in these
individuals. The physiological concentration of 7ꢀDHC in healthy human plasma is very low (0.005 to 0.05
mg/dl) while in persons with SLOS it is greatly elevated (typically 10 mg/dl or greater) and cholesterol levels
are typically reduced substantially. Cholesterol supplementation is a standard clinical treatment for patients
with SLOS but in practice this treatment shows only limited efficacy in improving the behavioral phenotype:
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cholesterol from the systemic circulation does not penetrate the bloodꢀbrain barrier, thus limiting the
beneficial effects on the brain.
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Statins were also a logical, pathophysiologyꢀbased therapy for patients with SLOS – alone, or in
combination with cholesterol supplementation.17ꢀ19 Unfortunately, in clinical practice the benefit for the
patients is questionable, and several studies did not confirm the previously reported positive effect of
simvastatin treatment on anthropometric measures or behavior of patients. The biosynthetic pathway between
HMGꢀCoA reductase and DHCR7 is long and complex, with various intercalated metabolic pathways, and
inhibition of HMGꢀCoA reductase nonꢀselectively represses the whole cholesterol biosynthesis machinery,
potentially further reducing the already diminished cholesterol in SLOS patients.
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Recent evidence supports the notion that the lack of cholesterol is only partly responsible for the SLOS
pathophysiology and pathology. 7ꢀDHC is one of the most reactive lipids known toward free radical
peroxidation, its rate constant for propagation being 200 times that of cholesterol and 12 times greater than
arachidonic acid, a lipid generally thought to be highly reactive.20 The accumulating 7ꢀDHC is a highly
reactive lipid molecule, and it undergoes spontaneous free radical peroxidation, producing over a dozen
oxidation products (i.e., oxysterols) in vitro and in vivo. These 7ꢀDHCꢀderived oxysterols exert cytotoxicity,
reduce cell proliferation, and induce premature cell differentiation and lead to a host of gene expression
changes.21,22 At nanomolar concentrations in brain tissues of a Dhcr7-KO mouse, 7ꢀDHC oxysterols are
strongly neurotoxic. Indeed, oxysterols found in concentrations as high as 3 ꢀM in the KO mouse brain
dramatically accelerate differentiation and neurite outgrowth in neocortical neuronal cultures.22 The
accumulation of 7ꢀDHC leads to a brain regionꢀspecific formation of 7ꢀDHCꢀderived oxysterols that affect
expression of many transcripts,23,24 leading to premature neuronal differentiation, most likely due to oxysterol
formation in the brain tissue.
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In summary, recent studies reveal that the accumulation of 7ꢀDHC and its related oxysterols might
significantly contribute to the pathogenesis of SLOS, which points to a new direction of therapeutic approach
– inhibition of the formation and accumulation of 7ꢀDHC and restoring the 7ꢀDHC/cholesterol ratio.
Desmosterolosis, an autosomal recessive disorder, is even rarer than SLOS.11 The mutation in this
syndrome is in the gene encoding DHCR24, the enzyme that converts desmosterol to cholesterol.
Desmosterol accumulates in these patients and the diagnostic assessment of this syndrome and of SLOS is by
analysis of the blood sterol profile.12ꢀ16 Blood and skin cell sterol profile testing is also used for the diagnosis
of the AntleyꢀBixler, SC4MOL, CDPX2/Conradi Hunermann and lathosterolosis disorders, each of which
results from defective enzymes promoting a step in the biosynthetic pathway upstream from DHCR24 and
DHCR7.11
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Sterol Homeostasis and Small Molecules. Recent reports have noted that some prescribed pharmaceuticals
have a marked effect on sterol profiles, in some cases leading to plasma levels of 7ꢀDHC in individuals that
are as high as those found in some SLOS patients. One recent study examined the medical records of
patients with elevated levels of 7ꢀDHC in the absence of a DHCR7 mutation and found a number of
individuals who were SLOS false positives and who also had reported a history of treatments with
aripiprazole, an atypical antipsychotic, and trazodone, an antidepressant.25,26 Other small molecules have
been found to perturb sterol profiles in cell culture and some of these compounds are also prescribed
antipsychotic agents.3,27ꢀ30 AY9944, a small molecule synthesized as a potential cholesterolꢀlowering agent
was found to increase 7ꢀDHC and reduce cholesterol levels in rodents.31ꢀ40
What seems clear is that exposure to small molecules, some of which are a part of the US Pharmacopeia,
can have a profound effect on sterol profiles in vivo. Consideration of these previous studies also suggests
that a screening method to identify compounds that affect sterol homeostasis might find general use.41,42 We
report here the results of a preliminary screen of the compounds in the NIH Clinical Collection, a small
library of pharmacologically active molecules. The primary screening method relies on a liquid
chromatography mass spectrometry (LCꢀMS) analysis of lateꢀstage cholesterol biosynthetic intermediates
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including 7ꢀDHC, desmosterol, 7ꢀdehydrodesmosterol (7ꢀDHD) and lanosterol. Dhcr7ꢀdeficient Neuro2a
cells prove to be particularly convenient for the screen in a 384ꢀwell format. The LCꢀMS sterol analysis in
combination with these cells distinguishes not only small molecules that increase levels of 7ꢀDHC but also
those that lower levels of this potentially toxic cholesterol precursor. The activities of selected compounds
were tested in SLOS human fibroblasts to determine the utility of the approach in identifying compounds that
may prove beneficial and/or test the potential toxic sideꢀeffect of drugs that may be in therapeutic use in
SLOS patients.
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Results
We selected Neuro2a and Dhcr7ꢀdeficient Neuro2a for the highꢀthroughput screening. Neuro2a cells, a
mouse neuroblastoma cell line, are widely used in the neuroscience community as models of neuronal cell
culture.21,23,42ꢀ46 These cells express all of the
enzymes in the cholesterol biosynthesis
pathway and they survive in a lipid free
medium by endogenous biosynthesis. To
better
understand
the
molecular
consequences of DHCR7 deficiency in
neuronal cells we generated Dhcr7ꢀdeficient
Neuro2a cells using an shRNA approach.42
Dhcr7ꢀdeficient cells have downregulated
lipid and other transcripts (molecules that
play critical roles in proliferation and
Figure 2. PTAD, its 7-DHC Diels-Alder Adduct (7-DHC-
PTAD) and the Desmosterol Products of PTAD in
Methanol.
differentiation, intracellular signaling, vesicular transport, or are inserted into membrane rafts), and they have
greatly increased 7ꢀDHC and the 7ꢀDHC derived oxysterol DHCEO.23,41 Both control and Dhcr7ꢀdeficient
cells are sensitive to compounds that affect either Dhcr7 or Dhcr24 expression levels. These cells have
several benefits as the basis for a smallꢀmolecule screening program. The advantages also include fast
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proliferation as their doubling time is about 20 hrs. They grow well under a variety of cell culture conditions,
including with serumꢀdeficient and lipidꢀdeficient media. Although we used both cell types in the screening
procedure, Dhcr7ꢀdeficient cells were the better choice to monitor decreases of 7ꢀDHC levels since control
Neuro2a cells have very low levels of 7ꢀDHC compared to Dhcr7ꢀdeficient cells.47
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Sterol Assay. The LCꢀMS analysis relies on a derivatization strategy
that makes use of the reactive compound 4ꢀphenylꢀ1,2,4ꢀtriazolineꢀ
3,5ꢀdione (PTAD, see Figure 2) 47
. This dienophile reacts with 7ꢀDHC
in a DielsꢀAlder cycloaddition and in the process introduces five
heteroatoms into the adduct structure, 7ꢀDHCꢀPTAD,48ꢀ50 improving
the MS detection sensitivity for the sterol (Figure 2).51,52 The workup
for cultured cells involves lysis in methanol, agitation with a
PTAD/methanol solution for 20 min at RT followed by isocratic
UPLCꢀMS on C18 with methanol mobile phase. A typical UPLCꢀMS
run of Dhcr7-deficient Neuro2a cells in culture is shown in Figure 3.
Under our current conditions, 50ꢀ60 samples/hr can be processed from
a multiꢀwell culture plate. A deuterated 7ꢀDHC standard (d7-7ꢀDHC)
was synthesized permitting a quantitative assay with limits of
detection of 7ꢀDHCꢀPTAD less than 0.1 picograms, see peaks eluting
at 0.81 min in Figure 3.
Figure 3. UPLC-MS chromato-
gram of PTAD Assay for Dhcr7-
deficient Neuro2a cells. Shown are
selective reaction monitoring of 7ꢀ
DHC and its isotopic standard (d7-
7DHC), desmosterol (Des), lanosterol
(Lan) and their 13Cꢀisotopic standards
(313CꢀDes) and (313Cꢀ Lan).
Desmosterol, lanosterol and other sterols having a double bond at
C24ꢀ25 undergo reaction with PTAD by the “ene” reaction as shown in Figure 2 and these “ene” type
products are monitored in the UPLCꢀMS.53ꢀ57 Isotopicallyꢀlabeled standards for desmosterol (0.53 minutes)
and lanosterol (0.57 minutes) containing 13C carbons at C24, C25 and C26 were prepared and can be used for
analysis of these sterols, see Figure 3. We conclude further that UPLC separations and mass differences of
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the PTAD adducts permit quantitative assays of the sterols mentioned above. LanosterolꢀPTAD/MeOH, for
example, can be readily distinguished from other PTAD products by its mass.
Critical to the expansion of the assay to other sterols is the availability of isotopically labeled standards to
be added to cells, tissues or fluids before workup. The standard must be present in the sample before the
PTAD procedure is initiated so that standard and analyte undergo the same derivatizations.
Screens of Presumed “Active” Compounds.
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Our first studies were carried out in control
Neuro2a cells in 96ꢀwell plates. To determine
the utility of the PTAD assay in cultured cells
we measured 7ꢀDHC in a specified number
of both control and Dhcr7ꢀdeficient Neuro2a
cells in 96ꢀwell plates. We found that 7ꢀDHC
could be reliably measured in about 100
Dhcr7ꢀdeficient cells while ~2,000 control
Neuro2a cells were required to reach
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Figure 4. PTAD screening of Neuro2a Cells (control cells)
in 96-well plates. Examples of the effect on 7ꢀDHC of some
common pharmaceuticals at 1, 10 and 100 nM in cultured
control Neuro2a cells. AY9944 is used in a rat SLOS
pharmacological model, aripiprazole, haloperidol and
trazodone are widely used atypical antipsychotics and antiꢀ
depressants. n>3; **p<0.001, ***p<0.0001.
acceptable signal to noise levels.
To
establish the screening method, we used
control Neuro2a and a set of compounds that
included antipyschotics and antidepressants
that are suspected of increasing levels of 7ꢀ
DHC in humans.25,26,58,59 We also included in
these studies AY9944, the small molecule that is the basis of the SLOS pharmacological rodent model.31ꢀ40
AY9944, aripiprazole, trazodone and haloperidol caused elevated levels of 7ꢀDHC in control Neuro2a,
results that are consistent with clinical findings. At 100 nM, aripiprazole, the top selling drug in 2013,60
caused an increase in 7ꢀDHC levels in control Neuro2a by over 40ꢀfold, from 0.08 to 3.2 ng/
ꢀ
g protein,
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Figure 4. An effect was also observed of these common pharmaceuticals on levels of desmosterol and
lanosterol in these experiments. Compounds that increase 7DHC tended to lower levels of desmosterol and
lanosterol (data not shown). The 7ꢀDHC z’value61 determined for 100 nM aripiprazole vs DMSO treatment
in control Neuro2a cells ranged from 0.2 to 0.7, an acceptable level for a screen, but one suggesting that
followꢀup validation of “hits” should be carried out.
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Drugs that lower 7ꢀDHC levels were also detected in this assay but screening libraries of small molecules
Figure 5. HTS screening. 7ꢀDHC (pg/cell) in a 384ꢀwell plate of Dhcr7ꢀdeficient Neuro2a cells plated
(10,000 cells/well) on 281 of the 727 compounds in the NIH Clinical Collection. 1 ꢀM of a compound
was preꢀdeposited in each well and cells were incubated for 24 h at 37oC. 7ꢀDHC levels expressed as
pg/number of cells. The left four entries are for DMSO controls (n=19) aripiprazole, bazedoxifene, and
clomiphene at 200 nM, also shown colorꢀcoded with n>3; ***p<0.0001.
with Dhcr7ꢀdeficient Neuro2a cells proved more useful for finding compounds that reduce levels of the 7ꢀ
DHC and as a consequence, its toxic oxysterol derivatives. These experiments have the advantage that 7ꢀ
DHC levels in the deficient cells (33 ng/ꢀ
g protein)47 are about 150 times higher than in control Neuro2a
cells and compounds that produce lower levels of 7ꢀDHC are readily identified in incubations of the deficient
cells.
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Screening the NIH Clinical Collection and Dhcr7-Deficient Cells. About 13,000 Dhcr7ꢀdeficient Neuro2a
cells were added to each well of a 384 plate with the “test compound” concentration in the well set at 1 ꢀM.
In addition to test compounds, each plate had 30 control wells (DMSO only) and additional wells with three
drugs (aripiprazole, bazedoxifene and clomiphene) that were shown to have an effect on 7ꢀDHC in
preliminary incubations. These compounds were included in the 384ꢀwell screen to test the reproducibility of
the assay (5 wells each at 20 and 200 nM). The plates were then incubated for 24 hr (37°C) at which time a
nuclear stain (Hoechst) was added and the cells were imaged by automated microscope imager.
Subsequent to the removal of media, cells were lysed in methanol that contained isotopically labeled
standards and the cell lysate was transferred robotically to another plate that had the PTAD reagent preꢀ
deposited. The plate was immediately sealed, then agitated for 20 min at room temperature, at which time it
was ready for MS analysis. The MS UPLC injector acquired samples automatically from the 384ꢀwell plate
and the chromatography solvent was straight methanol (with no solvent programming) that allowed analysis
of 50ꢀ60 samples/hr. We report here only data for 7ꢀDHC. Followꢀup screens carried out in a 96ꢀwell format
included acquisition of data for 7ꢀDHC, lanosterol and desmosterol as a function of small molecule
treatment.
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The output of the screen for a given test compound was the PTAD value in picogram measured in a well
divided by cells counted per well. Figure 5 shows output for 7ꢀDHC in a screening of some 40% of the
compounds in the NIH Clinical Collection on one 384ꢀwell plate. Note that of the 727 compounds tested,
most of them have no significant effect on 7ꢀDHC levels in Dhcr7ꢀdef cells (Figure 5), but 33 compounds on
the plate gave increases in 7ꢀDHC outside of 3
σ
of the DMSO controls. Another 40 compounds assayed on
criterion.
the plate decreased 7ꢀDHC levels in the cells by the same 3
σ
The effect of the test compound aripiprazole on 7ꢀDHC levels in Dhcr7ꢀdef Neuro2a was readily detected,
200 nM of the compound doubled the level of 7ꢀDHC in the cells. As shown in Figure 5, bazedoxifene and
clomiphene reduced 7ꢀDHC with statistical significance at 200 nM and clomiphene’s effect was observed at
20 nM (data not shown).
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Validation of HTS hit compounds. We carried out followꢀup assays on the compounds from the NIH
Collection that decreased 7ꢀDHC levels in Dhcr7ꢀdeficient cells in these cells and in human skin fibroblasts
from SLOS affected individuals. We also examined compounds that are structurally or therapeutically
related to the “actives” in the NIH Collection. These followꢀup experiments were carried out in triplicate and
included quantitative analysis of 7ꢀDHC, desmosterol, lanosterol and cholesterol, which was included in the
assay described in Figure 3 by extending the UPLC run to 1.2 minutes with a flow rate of 550 ꢀL/ min and
including d7-cholesterol in the workup. Under the conditions of this assay, cholesterol elutes at 0.93 minutes.
GCꢀMS was also used in these followꢀup experiments to include zymosterol (Zym), zymostenol (Zymꢀe),
lathosterol (Lath), 7ꢀdehydrodesmosterol (DHD), 4,4ꢀdimethylzymostenol, 24ꢀdihydrolanosterol (diHLan),
7ꢀDHC, desmosterol (Des) and cholesterol (Chol) in the analysis.
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Figure 6 shows the postꢀlanosterol sterol profiles determined by a combination of LCꢀMS and GCꢀMS
methods for three compounds that reduce levels of 7ꢀDHC in Dhcr7ꢀdeficient Neuro2a cells.
The
compounds shown are representative of subꢀsets of compounds, see below, that lead to similar sterol profiles
based upon their effect on specific steps in the cholesterol biosynthesis pathway. Structures and names of
compounds that showed a reproducible effect on the level of one of the cholesterol precursor sterols in
Dhcr7ꢀdeficient Neuro2a cells or in human fibroblasts are presented in Table 1. Of the compounds in Table
1 that decrease levels of 7ꢀDHC, there are subꢀsets of compounds that have similar structures or are
prescribed for similar purposes (Table 2). Compounds affecting 7ꢀDHC are statins (Row 1, AꢀE), selective
estrogen receptor modulators (Row 1E and 2AꢀF), antipyschotics (Row 3 AꢀF), antimycotic/ꢀ
antibacterial/antimalarial drugs (Row 4AꢀF) and steroids (Row 6CꢀF). Thus, the statins, estrogen receptor
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B. Progesterone
A. Control
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D. Econazole
C. Fluphenazine
Figure 6. Sterol profiles for Dhcr7-deficient Neuro2a cells treated with 1 ꢀM representative
compounds. Mole fraction of the major postꢀlanosterol sterols detected by LCꢀMS and GCꢀMS. A:
DMSO control, B: progesterone inhibits DHCR24 and leads to increases in desmosterol (Des) and
dehydrodesmosterol (DHD), C: fluphenazine inhibits EBP (ꢁ8ꢀ7 isomerase), and leads to increases in
zymosterol (Zym) and zymostenol (Zymꢀe), D: econazole inhibits CYP51A1 demethylase and leads to
increases in lanosterol (Lan) and dihydrolanosterol (diHLan). Statins (data not shown) inhibit HMGCoA
reductase, resulting in a decrease of all sterol intermediates.
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modulators (SERMs), and antifungals were found to decrease levels of 7ꢀDHC, providing a structureꢀactivity
validation of the general screening approach. Statins affect cholesterol biosynthesis at HMG CoAꢀreductase,
a stage in biosynthesis before isoprenoid formation occurs, with the consequence that all sterol levels are
reduced, from lanosterol to cholesterol. Because of their well characterized function we independently
verified in our system only two compounds from this group: lovastatin and pitavastatin. There have been
reports in the literature that artemisin derivatives like those shown in Row 6, A and B, also affect cholesterol
biosynthesis at the HMG CoAꢀreductase step, in this case by downꢀregulation of the corresponding
gene.62SERMs decrease 7ꢀDHC and alter levels of other cholesterol intermediates. The initial HTS screen
identified four SERMs that decrease 7ꢀDHC in Dhcr7ꢀdeficient cells, consistent with literature reports.29,30,63ꢀ
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65
In addition to the four (clomiphene, toremifene, tamoxifen, raloxifene), we obtained several other
commercially available SERMs (bazedoxifene, levormeloxifene, and lasofoxifene) along with the selective
estrogen receptor downregulator fulvestrant and compared their effects on cholesterol biosynthesis in control
and Dhcr7ꢀdeficient Neuro2a cells in an independent set of experiments that utilized both the PTAD method
and GCꢀMS analysis. All of the SERMs studied were effective at decreasing 7ꢀDHC in both control and
Dhcr7ꢀdeficient Neuro2a cells. Assays for other postꢀlanosterol sterols in these experiments showed that
biosynthetic steps other than Dhcr7 were affected by the compounds, consequently reducing 7ꢀDHC levels in
the cells. Tamoxifen, clomiphene and toremifene appear to have their major effect on the
with increased levels of zymostenol and zymosterol being observed while 7ꢀDHC and cholesterol levels are
reduced. Raloxifene and lasofoxifene effect both the 7 isomerase and the Cꢀ24 reductase with increased
levels of zymosterol and desmosterol found in the 1 M treatment. Levormeloxifene appears to be one of the
ꢁ8−7 isomerase
ꢁ8−
ꢀ
more potent compounds, exerting its affect solely on Dhcr24 with the consequent increase of desmosterol
and 7ꢀdehydrodesmosterol in the cells.
Clomiphene is marketed as a mixture of geometric stereoisomers and we observed that both isomers were
active, the separated Z and E compounds showing somewhat greater efficacy than the mixture of the two.
We note that toremifene and tamoxifen are also obtained as stereoisomeric mixtures and our studies were
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carried out on the isomeric mixtures. It seems likely that the effect of concentration on various steps on
cholesterol biosynthesis will be variable for the different compounds studied, including steroisomeric
mixtures and, as a result, the distribution of sterols will depend both on the particular SERM studied and its
concentration.
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Psychiatric medications alter cholesterol biosynthesis.25 Several compounds found to significantly
decrease 7ꢀDHC in the screen (3AꢀF) are also prescribed as antipsychotics and antidepressants. Thus, 3AꢀD
in Table 1 reduce 7ꢀDHC levels and all are typical antidepressants having common structural features.
Complete sterol analysis of these compounds found them to act in a way that parallels the action of the
SERMs,63 increasing levels of zymosterol and zymostenol. Selected sterol analysis data is presented for these
compounds in Supporting Information. Another set of antipsychotics/antidepressants including aripiprazole,
trazodone and haloperidol were among the compounds that increase 7ꢀDHC levels in the 384ꢀwell assay
shown in Figure 5. It is noteworthy that all of these compounds are used in the treatment of depression,
bipolar disorder, and schizophrenia. Indeed, of the compounds in our primary screen of the NIH Clinical
Collection in Dhcr7ꢀdeficient Neuro2a cells that were shown to affect cholesterol biosynthesis in one way or
another, over 20% are medications prescribed for bipolar disorder, depression and the like.
Compounds with antimycotic/antibacterial/antimalarial properties decrease 7-DHC. Compounds in this
category have wellꢀdescribed pharmacological properties. Several of them act as 14ꢀα demethylase inhibitor
(Figure 9), a step in postꢀsqualene cholesterol biosynthesis. Chloroxine and hexachlorophene decrease 7DHC
and desmosterol, and increase lanosterol and cholesterol. Chloroxine has bacteriostatic, fungistatic and
antiprotozoan properties. Hexachlorophene has been used in soaps and toothpaste and also as a fungicide,
plant bactericide and acaricide. Both compounds at 500 nM greatly decrease cell proliferation of Dhcr7ꢀ
deficient Neuro2a cells (Supporting Information).
Steroids and other active compounds. While progesterone’s action on DHCR24 is well documented,66
several compounds identified in this screen have, to our knowledge, not been previously associated with an
effect on cholesterol biosynthesis. These include trimebutine, homoharringtonine, and imatinib. Trimebutine,
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an antimuscarinic and mu opioid agonist with spasmolytic effects, decreased 7ꢀDHC and increased
desmosterol and lanosterol with no change in cholesterol in our cell culture at 100 nM. Imatinib, and
homoharringtonine are protein tyrosine kinase inhibitors used for the treatment of chronic myeloid leukemia.
Homoharringtonine is relatively toxic in our cultures, stopping proliferation of Dhcr7ꢀdeficient cells at 10
nM and at higher concentration leading to cell death (Supporting Information).
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Validation of HTS hits in SLOS human fibroblasts. While Dhcr7ꢀdeficient Neuro2a are convenient for use
Figure 7. SERMs alter cholesterol metabolism in SLOS human fibroblasts. SLOS HF were grown in
96ꢀwell plates in delipidated serum in presence of different SERMs for five days. The cell viability was
assessed by Cell Titer and the sterol measurements were normalized to the absorbance readings. All
SERMs (100 nM) decrease 7DHC (A) with variable effects on desmosterol (B), and cholesterol (C).
Different SERMs have variable effects on fibroblasts viability with Fulvestrant being the most toxic (D).
At 5 µM, Clomiphene is the only SERM without an effect on viability. TAM=tamoxifen,
CLO=clomiphene, TOR=toremifene, RAL=raloxifene, BAS=basedoxifene, LEV=levormeloxifene,
FUL=fulvestrant.
in the HTS screening process, we examined the “hit” compounds in human SLOS fibroblasts to validate the
biological response in a different and more relevant cell type. Considering the biological diversity and
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complexities associated with the use of human derived cells we examined the effect of “active” small
molecules in several different SLOS fibroblasts.
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In fibroblasts from SLOS patients we found that 10 nM of clomiphene, tamoxifen, raloxifene, fulvestrant,
levormeloxifene and bazedoxifene resulted in a statistically significant reduction of 7ꢀDHC without a
measurable decrease in cholesterol levels in the cells. Toremifene was somewhat less effective, with 100 nM
of this compound required to cause a significant reduction of 7ꢀDHC in SLOS HF. The results from this
study are presented in Figure 7. Complete data sets for the analysis of SLOS HF and control and Dhcr7-
deficient cells treated with SERMs is also presented in Supporting Information. Figure 8 shows compounds
that consistently decreased 7ꢀDHC in all SLOS fibroblasts. Since different SLOS HF have different 7ꢀDHC
levels the numbers shown are expressed as a percentage of 7ꢀDHC present in the cells grown in the absence
of compounds. The verified hits in SLOS HF suggest that exploring the utility of these compounds may be
worthwhile in efforts to normalize lipid metabolism in these cells.
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B.
A.
Figure 8. Selection of small molecules that decrease 7-DHC in SLOS human fibroblasts. SLOS
HF were grown in 96ꢀwell plates in delipidated serum in presence of different compounds for five
days in three different SLOS fibroblasts. All of these compounds (100 nM) decrease 7DHC (A) with
only small effects on cholesterol (B). Pitavast=pitavastatin, Perphen=perphazine,
Trifluop=Trifluoperphanazine, Hydrox=hydroxyzine, Trimeb=trimebutine, Budes=budesonide.
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Screening Control Neuro2a Cells. As noted, one limitation of the use of Dhcr7ꢀdeficient Neuro2a cells is
that they are less sensitive to small molecules that increase 7ꢀDHC than are control Neuro2a cells (since they
already have 7ꢀDHC levels 330 times more than control cells). Thus, while 200 nM aripiprazole results in
doubling 7ꢀDHC levels in Dhcr7ꢀdeficient Neuro2a, at the same concentration the compound increases 7ꢀ
DHC in control Neuro2a by a factor of over 40. Control Neuro2a were also found to be more sensitive than
the Dhcr7ꢀdeficient cells to trazadone, haloperidol and AY9944 although these compounds were identified as
“actives” in the deficient cell screen. A screen of the entire NIH Collection in control Neuro2a cells is
ongoing and the results of that effort will be reported in due course.
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Discussion
Our screens of Dhcr7ꢀdeficient Neuro2a cells show that dozens of compounds of clinical importance can
affect one of the steps in postꢀlanosterol cholesterol biosynthesis (Figure 9) and calls attention to the fact that
pharmacological intervention can be used to reproduce, or in theory to counteract disorders involving
disruption of sterol biosynthesis. These disorders are rare but the consequences of mutations in the enzymes
in the cholesterol biosynthesis pathway can be profound.11,67
Our assay of control Neuro2a cells with several compounds known to affect the conversion of 7ꢀDHC to
Figure 9. Selected Transformations of Post-lanosterol Cholesterol Biosynthesis
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cholesterol (DHCR7 in Figure 1) proved to be exquisitely sensitive and readily detects an increase in levels
of 7ꢀDHC in the cells at concentrations as low as 10 nM for aripiprazole, trazodone, haloperidol and
AY9944. For reference, patient plasma concentrations of aripiprazole, trazodone and haloperidol can be well
above these levels.68ꢀ70 The effect of AY9944 on the cells was observed even at 1 nM, see Figure 4.
Aripiprazole, trazodone, haloperidol and AY9944 were all presumed “active” small molecules that affect
sterol homeostasis at the DHCR7 step25,26 and indeed, the assay identifies these compounds in the screen.37,71
AY9944 has been used as the basis for one SLOS rodent model.38 In this protocol, pregnant rats were
exposed to carefully programmed levels of AY9944 during gestation and the exposure was continued in the
pups for a period of up to 3 months. While the administration of high levels of AY9944 during pregnancy
resulted in severe fetal malformation and miscarriage, carefully controlled maternal exposure of rats gave
offspring that displayed many of the biochemical hallmarks and some of the phenotypic features of
SLOS.32,34,38 Ratios of 7ꢀDHC to cholesterol as high as 2:1 were found in the plasma and tissues of these
SLOS pharmacological rat models. AY9944 failed as a cholesterolꢀlowering drug because it was found to be
a teratogen, presumably due to its effect on cholesterol biosynthesis at the 7ꢀDHC to cholesterol
transformation. We note that other small molecules act in a way parallel to AY9944 and these compounds
should be considered suspect teratogens based upon common metabolism.72
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The Dhcr7ꢀdeficient Neuro2a cells demonstrate their primary utility as a first screen to identify compounds
that decrease the formation of cellular 7ꢀDHC. Table 2 shows the pharmaceutical action and prescription
indication for those compounds identified in the NIH Clinical Collection that are approved for human use
and that cause a decrease in 7ꢀDHC in our screen of Dhcr7ꢀdeficient cells. We also included a few additional
compounds in the study that are not in the NIH collection if there were reports of known activity or if they
were structurally related to active compounds. Our screen confirms the notion that SERMs affect cholesterol
biosynthesis by inhibiting the
ꢁ8ꢀ7 isomerase and/or the DHCR24 since the sterol intermediates that
accumulate upon treatment are those in the biosynthesis that are formed before the
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Table 2. Action, Indication and FDA pregnancy designation for drugs that decrease 7ꢀDHC in Dhcr7ꢀ
deficient Neuro2a.
Name
Action
Indication
FDA
Category
Simvastatin
Lovastatin
Statin, HMGCR inhibitor
Statin, HMGCR inhibitor
Hyperlipidemia
Hyperlipidemia
X
X
X
X
X
D
X
D
X
X
N
N
N
N
N
Mevastatin
Pravastatin
Pitavastatin
Tamoxifen
Clomiphene
Toremifene
Statin, HMGCR inhibitor
Statin, HMGCR inhibitor
Statin, HMGCR inhibitor
Hyperlipidemia
Hyperlipidemia
Hyperlipidemia
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Selective estrogen receptor modulator
Selective estrogen receptor modulator
Selective estrogen receptor modulator
Selective estrogen receptor modulator
Selective estrogen receptor modulator
Selective estrogen receptor modulator
Typical antipsychotic, D1/D2 antagonist
Typical antipsychotic, D1/D2 antagonist
Typical antipsychotic, D1/D2 antagonist
Typical antipsychotic, D2 antagonist
Hormoneꢀreceptorꢀcancer treatment
Ovulation induction
Hormoneꢀreceptorꢀcancer treatment
Breast cancer and osteoporosis
Postmenopausal osteoporosis
Breast cancer and osteoporosis
Positive symptoms of schizophrenia
Positive symptoms of schizophrenia
Positive symptoms of schizophrenia
Raloxifene
Bazedoxifene
Lasofoxifene
Perphenazine
Fluphenazine
Trifluoperazine
Prochlorperazine
Antiemetic, antipsychotic,
antivertiginoic
Doxepin
Tricyclic antidepressant, SNRI class
Depression, anxiety disorders
N
N
Molindone
Atypical antipsychotic, D2, 5HT1Aꢀ2A,
Psychoses and conduct disorder
M1
Hydroxyzine
Antihistaminic, anxiolytic
Sedative, allergy, treat hives, runny
nose
C
Econazole
Isoconazole
Antimycotic, 14ꢀα demethylase inhibitor
Antimycotic, 14ꢀα demethylase inhibitor
Dermatomycoses
Treatment of foot and vaginal
infections
C
N
75
Bifonazole
Clotrimazole
Chloroxine
Antimycotic, 14ꢀα demethylase inhibitor
Antimycotic, 14ꢀα demethylase inhibitor
Antibacterial, not well understood
Organochlorine
Antimalarial, ferriprotoporphyrin IX
Antimalarial, ferriprotoporphyrin IX
Tetracycline
Topical fungal infections
Topical fungal infections
C
C
C
Dandruff and seborrheic dermatitis
Disinfectant, topical antibacterial
Malaria treatment
Hexachlorophene
Artemether
C
Artemisinin
Doxycycline
Trimebutine
Malaria treatment
Broadꢀspectrum antibiotic
Irritable bowel syndrome
C
D
C76
Spasmolytic Antimuscarinic and muꢀ
opioid agonist
Homoharringtonine
Imatinib
Cephalotaxine, protein synthesis
inhibitor tyrosine kinase inhibitor
Tyrosineꢀkinase inhibitor
Hematological malignancies
D
D
Hematological malignancies
Treatment of multiple cancers
Budesonide
Magestin
Progesterone
Acitretin
Glucocorticoid
Asthma, skin disorders, rhinitis, colitis
C
X
B
X
N
Progestational hormone
Progestational steroid
Retinoid, RXR and RAR
Reproductive cancer, anorexia
Hormone replacement
Treatment of psoriasis
Experimental
U18666A
Categories: A, No risk in controlled human studies: Adequate and wellꢀcontrolled human studies have failed to demonstrate a risk to the fetus in the
first trimester of pregnancy (and there is no evidence of risk in later trimesters). B, No risk in other studies: Animal reproduction studies have failed to
demonstrate a risk to the fetus and there are no adequate and wellꢀcontrolled studies in pregnant women OR Animal studies have shown an adverse
effect, but adequate and wellꢀcontrolled studies in pregnant women have failed to demonstrate a risk to the fetus in any trimester. C, Risk not ruled
out: Animal reproduction studies have shown an adverse effect on the fetus and there are no adequate and wellꢀcontrolled studies in humans, but
potential benefits may warrant use of the drug in pregnant women despite potential risks. D, Positive evidence of risk: There is positive evidence of
human fetal risk based on adverse reaction data from investigational or marketing experience or studies in humans, but potential benefits may
warrant use of the drug in pregnant women despite potential risks. X, Contraindicated in Pregnancy: Studies in animals or humans have
demonstrated fetal abnormalities and/or there is positive evidence of human fetal risk based on adverse reaction data from investigational or
marketing experience, and the risks involved in use of the drug in pregnant women clearly outweigh potential benefits. N, FDA has not yet classified
the drug into a specified pregnancy category.
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affected enzyme or enzymes (see Figure 9).63,64,73ꢀ77 The selectivity and specificity of action is dependent on
the SERM structure and the cell type treated. Thus, as illustrated in Figure 7, treatment of SLOS skin
fibroblasts with each of the SERMs resulted in a decrease of 7ꢀDHC, but the levels of sterols such as
desmosterol, 7ꢀdehydrodesmosterol, zymosterol and zymostenol are affected to different extents by the
different compounds. This can be understood by a more detailed consideration of the postꢀlanosterol
cholesterol biosynthesis scheme, see Figure 9. Levormefoxifene, bazedoxifene and raloxifene increase levels
of desmosterol and/or 7ꢀdehydrodesmosterol in SLOS fibroblasts, indicating that DHCR24 is inhibited by
these compounds. Clomiphene, tamoxifen and toremifene increase levels of zymostenol and/or zymosterol
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confirming that the ꢁ8ꢀ7 isomerase is inhibited. The main mechanism of action of SERMs is as estrogen
receptor agonists in some tissues (bone, liver, and cardiovascular system), antagonists in other tissues (breast
and brain), and mixed agonists/antagonists in the uterus. SERMs are primarily used for the prevention and
treatment of breast cancer, osteoporosis and menopausal symptoms. In addition to their endocrine actions on
peripheral tissues and estrogenꢀdependent tumors, SERMs also affect the nervous system. They have been
used in animal studies of different experimental models of neuronal dysfunction including traumatic CNS
and PNS injury, multiple sclerosis, and stroke. Based on the limited information available from human
studies some of SERMs may have a positive effect on mood and cognition.65 It has also been shown that
SERMs affect several signaling proteins including MAPK, PI3K, Akt, CREB, NFkB and protein kinase C
with the positive net effect in the animal studies of neuronal dysfunction. Through their effects on signaling
proteins, SERMs have been shown to have beneficial effects on synaptic transmission, oxidative stress,
apoptosis and inflammation in the nervous system. They also bind to the heteroꢀoligomer of DHCR7 and
DHCR24 (identified as the microsomal antiꢀestrogen binding site).63 It is of some interest that side effects
associated with some SERM therapies have resulted in withdrawal of a drug from the clinic.
Levormeloxifene, for example, was discontinued during a phase III trial due to a significant incidence of
gynecologic adverse events, although the cause of those events was not ascertained.78
The set of phenothiazines shown in Table 1 in Row 3(AꢀD) and hydroxyzine (3E) (antipsychotics) have
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activities on sterol biosynthesis that parallel those of the SERMs. At subꢀmicroM levels, each of these
4
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compounds reduce levels of 7ꢀDHC in Dhcr7ꢀdeficient Neuro2a cells as well as in fibroblasts from SLOS
6
7
patients. The apparent pharmacological activity of these closely related compounds lies primarily at the ꢁ8ꢀ7
8
9
isomerase, and to a lesser extent the 24 reductase (Figure 9) since elevated levels of zymostenol and
zymosterol are detected in incubations that include these compounds. The effect of antipsychotics on
cholesterol and fatty acid metabolism is well documented.25 It has been suggested that some of antipsychotics
have class II cationic amphiphilic properties similar to amphiphile U18666A and their cytotoxic effects are
mediated by acting on cholesterol homeostasis.79 It is also of interest that prochloperazine, fluphenazine and
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trifluoperazine, all of which affect the ꢁ8ꢀ7 isomerase, have been shown to be inhibitors of hepatitis C viral
(HCV) entry by increasing target cell membrane fluidity.80,81 In a similar way, the SERMs toremifene and
clomiphene inhibit ebola viral cell entry by a mechanism that affects the triggering of virion membrane
fusion.82 It seems reasonable to suggest that the perturbation of sterol homeostasis and the consequential effect
on membrane properties may underlie the mechanism of action for the phenothiazines with HCV and the
SERMs with ebola.
Summary: We conclude that an HTS screen of small molecules that affect sterol biosynthesis in control
and Dhcr7ꢀdeficient Neuro2a cell cultures is feasible and our method provides a basis for the further
exploration of compounds that alter sterol homeostasis. While the PTADꢀLCꢀMS approach is selective and
sensitive, the method does have limitations. Screens of large libraries at a range of concentrations is not
possible with the current protocol since each assay requires about 1.5 minutes of instrument time. On the
other hand, standard HTS instruments can be used in the assay, the workup of samples is straightforward, and
multiple important sterol intermediates are determined in a single run. Because of these advantages, use of
the PTADꢀLCꢀMS approach proves useful when coupled with Dhcr7ꢀdeficient cells to identify compounds in
small libraries that affect sterol biosynthesis at steps other than Dhcr7 while screens of control Neuro2a
reveal compounds that affect the conversion of 7ꢀDHC to cholesterol. The approach may link small
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molecules that exert cellular toxicities with a specific disruption of cholesterol biosynthesis and it may lead
to the identification of molecules that normalize cholesterol biosynthesis in disorders that disrupt normal
cholesterol homeostasis.
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Figure 10 provides a list of compounds that decrease 7ꢀDHC at 1 ꢀM in Dhcr7ꢀdeficient Neuro2a cells as
well as the sterol transformation affected by the compound. A screen of the NIH Collection at higher
compound concentrations as well as a screen of larger libraries of pharmaceuticals and environmental agents
that pose a threat to exposed populations seems likely to find other small molecules that affect sterol
homeostasis.
Figure 10. Summary of Compounds and Sterol Transformations Affected
Experimental
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Materials. Unless otherwise noted, all chemicals were purchased from SigmaꢀAldrich Co (St. Louis, MO).
HPLC grade solvents were purchased from Thermo Fisher Scientific Inc. (Waltham, MA). All cell culture
reagents were from Mediatech (Manassas, VA), Life Technologies (Grand Island, NY), and Greiner BioꢀOne
GmBH (Monroe, NC). Standard d7ꢀChol was purchased from Medical Isotopes, Inc. and d7ꢀ7ꢀDHC was
synthesized as previously described.41 13C3ꢀLan and 13C3ꢀDes were synthesized as described in the
Supporting Information.
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Compound Purity. New compounds are characterized by HPLCꢀMS, 13C and 1H NMR spectroscopy and are
>98% purity by these methods of analysis. The NIH Clinical Collection of biologically active compounds is
supplied by the NIH through the Molecular Libraries Roadmap Initiative, it is curated by the Vanderbilt
High Throughput Screening Facility and distributed in DMSO with >95% purity. The compound molecular
ion is identified by MS and the samples are analyzed by HPLC with evaporative light scattering or UV
detection. This collection is used widely in the screening community as a standard library.
Cell Cultures: Neuro2a and human fibroblasts. The neuroblastoma cell line Neuro2a was purchased from
American Type Culture Collection (Rockville, MD). Dhcr7ꢀdeficient Neuro2a cells were generated as
previously described.42 All cells were subcultured once a week, and the culture medium was changed every
two days. Control (GM05399, GM05565, GM05758) and SLOS (GM05788, GM03044) human fibroblasts
were purchased from the Coriell Institute, UMB#727 was obtained from NICHD Brain and Tissue Bank and
8019 and 35878 originated from SLOS patients. All cell lines were maintained in DMEM supplemented
with Lꢀglutamine, 10% fetal bovine serum (FBS; Thermo Scientific HyClone, Logan, UT), and
penicillin/streptomycin at 37ºC and 5% CO2. All cultured SLOS and control human fibroblasts used were
passage 8ꢀ20. Delipidated FBS medium did not have detectable cholesterol level. At the end of experiment,
cells were washed with iceꢀcold PBS two times, scraped into 5 mL of iceꢀcold PBS, centrifuged at 200X g
for 10 min at +4°C, PBS was removed and the cell pellets frozen at ꢀ80°C until analysis.
The screening method. Chemicals were deposited to 384 cell culture plate (Greiner BioꢀOne) using Labcyte
Echo 550/555 (Sunnyvale, CA) liquid handler equipped in the Vanderbilt University High Throughput
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Screening Facility. 25 ꢀL of control and Dhcr7ꢀdeficient Neuro2a cells in reduced FBS media (0.5%) with a
density of 10,000 and 13,000 cells/well respectively, were dispensed to the wells using a Multidrop Combi
(ThermoScientific) instrument. The cells were placed in an incubator for 24 h at which time 10 ꢀL Hoechst
dye (40 ng/ꢀL) (Molecular Probes) was added using the Multidrop Combi. The cells were incubated at room
temperature for 30 min in the dark and imaged using an ImageXpress Micro XL (Molecular Devices,
Sunnyvale, CA) with a 10X objective. After removing the media, 80 ꢀL MeOH containing internal standards
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(6.5 ng for d7ꢀ7ꢀDHC, 100 ng for C3ꢀDes and 75 ng for C3ꢀLano/well) was added using the Multidrop
Combi. The plate was placed on an Orbital shaker for 20 min at room temperature and centrifuged for 10 min
using a Sorvall swing rotor. The supernatant was transferred by automated liquid handler Agilent Velocity 11
Bravo to a PTADꢀpredeposited plate (Waters #186002631, 100 ꢀL flat bottom). The plates were immediately
sealed with Easy Pierce Heat Sealing Foil (ThermoScientific ABꢀ1720) followed by 20 min agitation on an
Orbital shaker at room temperature. The sealed plates were kept in ꢀ80°C until LCꢀMS analysis.
LC-MS (SRM) analysis. The sealed plates were placed in an Acquity UPLC system equipped with ANSIꢀ
compliant well plate holder. 10 ꢀL was injected on to the column (Acquity UPLC BEH C18, 1.7 ꢀm, 2.1 x
50 mm) with 100% MeOH (0.1% acetic acid) mobile phase for 1 min runtime at a flow rate of 300 µL/min.
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The monitored transitions included: 7ꢀDHC 560ꢀ365, d7ꢀ7ꢀDHC 567ꢀ372, desmosterol 592ꢀ365, C3ꢀ
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desmosterol 595ꢀ368, lanosterol 634ꢀ602, C3ꢀlanosterol 637ꢀ605 with retention times of 0.8, 0.5, and
0.6 min, respectively.
Lipid extraction, separation, and GC-MS analyses of sterols in cells. Verification of the hits obtained from
the initial screening was carried out with varying concentrations of the compounds of interest. In addition, a
more complete sterol profile was determined. The experiment was carried out in 96 well plates as described
above for the 384 well plates. After cells were counted using the ImageXpress Micro XL with 10X
objective, the medium was removed. The cells were washed with PBS, the buffer removed and the plates
were stored at ꢀ80°C until taken for sterol analysis. The protocol for analysis was as follows: to each well
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was added 10 µL BHT/TPP solution (2.5 mg TPP and 1 mg BHT in 1 mL EtOH), 10 µL internal standard
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solution (0.87 nmol for d7ꢀChol, 0.033 nmol for d7ꢀ7ꢀDHC, 0.25 nmol for C3ꢀDes and 0.23 nmol for C3ꢀ
Lan/well), and 200 µL MeOH. The plate was agitated on an Orbital shaker for 20 min at room temperature.
An aliquot (100 µL) of the supernatant was transferred to a PTADꢀpreꢀdeposited plate, sealed with Easy
Pierce Heat Sealing Foil followed by 20 min agitation on an Orbital shaker at room temperature, and
analyzed by LCꢀMS as described above. If the LC flow rate was increased to 500 µL/min with a run time of
1.5 min, cholesterol could be included in the analysis (Chol 369, d7ꢀChol 376).
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For GCꢀMS analysis, the remaining sample in each well was transferred to a vial and concentrated on a
SpeedVac concentrator. To each vial was added N,Oꢀbis(trimethylsilyl)ꢀtrifluoroacetamide (BSTFA, 50 µL),
the sample vortexed well, and allowed to react for 30 min. The sample (5 µL) was injected onto the column
(SPBꢀ5, 0.25 µm, 0.32 mm x 30 m) with a temperature program of 220ꢀ300° (5 min) at 20°/min and helium
flow rate of 2.0 mL/min. The data was collected in full scan mode and the following ions extracted for
quantitation relative to d7ꢀChol: 458 for cholesterol, zymostenol, lathosterol; 456 for zymosterol,
dehydrolathosterol; 349 (Mꢀ105) for dehydrodesmosterol; 393 (Mꢀ105) for lanosterol; 395 (Mꢀ105) for
dihydrolanosterol; 486 for dimethylzymostenol; and 465 for d7ꢀcholesterol. Desmosterol and 7ꢀDHC were
not analyzed by GCꢀMS because they coꢀelute. Any sterol not listed above was not detected by GCꢀMS. All
data was normalized to cell count. All calculations of variability, standard deviation (SD) and normalized
SD were performed in Microsoft Excel or GraphPad Prism.
Acknowledgment. The authors thank Dr. H. Ronald Zielke and UMB Brain and Tissue Bank/NIH
NeuroBioBank for donation of UMB727 (http://medschool.umaryland.edu/btbank/). The National Institutes
of Health (NICHD R01 HD064727 to NAP, NIEHS R01 ES024133 to NAP and ZK, R21 ES024666 to
NAP) and the Hungarian Research Fund OTKA K109076 to IB supported this work. Assay development and
preparation was carried out with assistance from Paige Vinson and Joshua Bauer in the Vanderbilt Highꢀ
throughput Screening Core Facility, which receives institutional support through the Vanderbilt Institute of
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Chemical Biology. The NIH Clinical Collection is provided through the National Institutes of Health
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Molecular Libraries Roadmap Initiative.
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Conflict of interest. The authors declare no competing financial interests.
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ABBREVIATIONS: 7ꢀDHC, 7ꢀdehydrocholesterol; Des, desmosterol; Lan, lanosterol; Chol, cholesterol;
dHLan, 24ꢀdihydrolanosterol; Zym, zymosterol; Zyme, zymostenol; dMZyme, 4,4ꢀdimethylzymostenol;
dHLath, 24ꢀdehydrolathosterol; Lath, lathosterol; DHD, 7ꢀdehydrodesmosterol; PTAD, 4ꢀphenylꢀ1,2,4ꢀ
triazolineꢀ3,5ꢀdione; SLOS, SmithꢀLemli Opitz syndrome; DHCR7, 7ꢀdehydrocholesterol reductase;
DHCR24, 24ꢀdehydrocholesterol reductase; EBP, emopamil binding protein; HMGꢀCoA, hydroxymethyl
glutarylꢀcoenzyme A; MS, mass spectrometry; APCI, atmospheric pressure chemical ionization; SRM,
selected reaction monitoring; UPLCꢀMS, ultraꢀhigh pressure liquid chromatographyꢀmass spectrometry;
HPLCꢀUV, high pressure liquid chromatographyꢀultraviolet spectroscopy; MeOH, methanol; NMR, nuclear
magnetic resonance; TIC, total ion current; FBS, fetal bovine serum; DMEM, Dulbecco’s Modified Eagle
Medium; PBS, phosphate buffered saline; DMSO, dimethylsulfoxide; HTS, high throughput screening;
SERM, selective estrogen receptor modulator; HF, human fibroblasts; CNS, central nervous system; PNS,
peripheral nervous system; MAPK, mitogenꢀactivated protein kinase; PI3K, phosphoinositide 3ꢀkinase;
CREB, cAMP response elementꢀbinding protein, NFkB, nuclear factor kappaꢀlightꢀchainꢀenhancer of
activated B cells.
Corresponding Author Information: Ned A. Porter email: n.porter@vanderbilt.edu. Telephone: 615ꢀ343ꢀ
2693
Supporting Information. Synthesis of standards, sterol profiles and cell viability for selected compounds
in Dhcr7ꢀdeficient Neuro2a cells, verification of screening hits in SLOS human fibroblasts.
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(20) Xu, L.; Davis, T. A.; Porter, N. A. Rate constants for peroxidation of polyunsaturated fatty
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