Parallel Chemistry Approach to Identify Novel Nuclear Receptor Ligands Based on the GW0742 Scaffold

Article abstract of DOI:10.1021/acscombsci.7b00066

We describe the parallel synthesis of novel analogs of GW0742, a peroxisome proliferator-activated receptor δ (PPARδ) agonist. For that purpose, modified reaction conditions were applied, such as a solid-phase palladium-catalyzed Suzuki coupling. In addition, tetrazole-based compounds were generated as a bioisostere for carboxylic acid-containing ligand GW0742. The new compounds were investigated for their ability to activate PPARδ mediated transcription and their cross-reactivity with the vitamin D receptor (VDR), another member of the nuclear receptor superfamily. We identified many potent PPARδ agonists that were less toxic than GW0742, where ～65 of the compounds synthesized exhibited partial PPARδ activity (23-98%) with EC₅₀ values ranging from 0.007-18.2 μM. Some ligands, such as compound 32, were more potent inhibitors of VDR-mediated transcription with significantly reduced PPARδ activity than GW0742, however, none of the ligands were completely selective for VDR inhibition over PPARδ activation of transcription.

Full text of DOI:10.1021/acscombsci.7b00066

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Article
A parallel chemistry approach to identify novel nuclear
receptor ligands based on the GW0742 scaffold
Kelly A. Teske, Ganesha Rai, Premchendar Nandhikonda, Preetpal Sidhu, Belaynesh D.
Feleke, Anton Simeonov, Adam Yasgar, Ajit Jadhav, David J. Maloney, and Leggy A. Arnold
ACS Comb. Sci., Just Accepted Manuscript • DOI: 10.1021/acscombsci.7b00066 • Publication Date (Web): 21 Aug 2017
Downloaded from http://pubs.acs.org on August 23, 2017
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A parallel chemistry approach to identify novel
nuclear receptor ligands based on the GW0742
scaffold
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Kelly A. Teske,1,# Ganesha Rai,2 Premchendar Nandhikonda,1 Preetpal S. Sidhu,1 Belaynesh
Feleke,1 Anton Simeonov,2 Adam Yasgar,2 Ajit Jadhav,2 David J. Maloney,2,ⱡ and Leggy A.
Arnold 1,*
1Department of Chemistry and Biochemistry, Milwaukee Institute for Drug Discovery,
University of Wisconsin, Milwaukee, Milwaukee, Wisconsin 53211, United States
2NIH Chemical Genomics Center, National Center for Advancing Translational Sciences
(NCATS), National Institutes of Health, Bethesda, Maryland 20892ꢀ3370, United States
KEYWORDS
Nuclear Receptor (NR), Vitamin D Receptor (VDR), Peroxisome ProliferationꢀActivated
Receptors α, δ, γ (PPAR α, δ, γ), GW0742, steroid receptor coactivator 2
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ABSTRACT
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We describe the parallel synthesis of novel analogs of GW0742, a peroxisome proliferatorꢀ
activated receptor δ (PPARδ) agonist. For that purpose, modified reaction conditions were
applied, such as a solidꢀphase palladiumꢀcatalyzed Suzuki coupling. In addition, tetrazoleꢀbased
compounds were generated as a bioisostere for carboxylic acidꢀcontaining ligand GW0742. The
new compounds were investigated for their ability to activate PPARδ mediated transcription and
their crossꢀreactivity with the vitamin D receptor (VDR), another member of the nuclear receptor
superfamily. We identified many potent PPARδ agonists that were less toxic than GW0742,
where ~65 of the compounds synthesized exhibited partial PPARδ activity (23ꢀ98%) with EC50
values ranging from 0.007 – 18.2 ꢁM. Some ligands, such as compound 32, were more potent
inhibitors of VDRꢀmediated transcription with significantly reduced PPARδ activity than
GW0742, however, none of the ligands were completely selective for VDR inhibition over
PPARδ activation of transcription.
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INTRODUCTION
Nuclear receptors (NRs) are ligandꢀactivated transcription factors representing one of the
largest classes of proteins targeted for drug intervention.1 Thus far, 48 identified proteins belong
to this superfamily, which is typically subdivided into three classes that share similar protein
structure and ligand binding. These classes are steroid receptors, retinoid X receptor (RXR)
heterodimers, and orphan receptors.2 Regardless of their class, NRs are modular proteins that
contain a DNAꢀbinding domain (DBD) and a ligandꢀbinding domain (LBD). The LBD is a
highly structured region that contains the ligandꢀdependent activation function or AFꢀ2, which
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acts as the major interface for the recruitment of coactivators or corepressors. In general, the NRꢀ
LBD consists of a threeꢀlayered αꢀhelical sandwich containing up to 12 helices designated as
H1–H12. In the absence of endogenous ligand, corepressor proteins of the SMRT/NCoR family
might associate with NRꢀLBD and block transcription.3 In the presence of ligand, NRs undergo a
conformational change that both stabilizes the binding of the ligand and accommodates
coactivator proteins such as SRCꢀ1 that bind the hydrophobic cleft formed by rearranged H12.4
The vitamin D receptor (VDR) and peroxisome proliferatorꢀactivated receptor δ (PPARδ)
form RXR heterodimers. The biological role of PPARδ has only recently been characterized, in
part, due to its broad tissue expression and the lack of selective chemical probes to distinguish
the pharmacology between the PPAR isoforms (δ, α, γ). To help reveal the biological function
of PPARδ, GlaxoSmithKline (GSK) used combinatorial chemistry and structureꢀbased drug
design to develop GW0742 and GW501516, both highly selective and potent PPARδ agonists
(EC50 = ~0.001 ꢁM).5 Since their discovery, it has been revealed that PPARδ plays a role in lipid,
lipoprotein, and glucose homeostasis thus making it a suitable target for hypertension,6 diabetes7,
obesity, inflammation8 and cancer9 therapies. Although GW501516 has shown beneficial effects
in primate models of metabolic disorders,5b, 10 only PPARα and PPARγ ligands have been
approved for human use. Fibrates (PPAR α ligands) and thiazolidinethiones (PPARγ ligands) are
prescribed for the treatment of hypercholesterolemia and diabetes mellitus type 2, respectively,
but have shown adverse side effects after longꢀterm use possibly due to poor isoform selectivity
and offꢀtarget effects.11 Due to its complementary role in lipid homeostasis and energy
metabolism, a strong interest in developing selective PPARδ ligands without adverse side effects
has emerged. VDR, on the other hand, mediates the biological action of the active form of
vitamin D, 1α,25ꢀdihydroxyvitamin D3 (1,25(OH)2D3) and regulates calcium homeostasis,
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immunity, and cellular growth and differentiation.12 Although a lot of research has been directed
towards developing new VDR modulators, there still remains a need for potent and VDR
selective drugs to treat diseases like osteoporosis, cancer, inflammation and cardiovascular
disease that do not cause detrimental side effects such as hypercalcemia.13
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High throughput screening (HTS) has quickly evolved over the last decades to become
one of the main methods for the identification of lead compounds in drug discovery.14 The
advantages of using HTS to identify hit compounds includes the quick retrieval of quality data
from large libraries of compounds in a costꢀeffective manner.15 Previously we reported our
collaborative work conducted with the NIH Chemical and Genomics Center (NCGC) to identify
VDR ligands that did not possess the secosteroid scaffold of 1,25(OH)2D3 in order to overcome
hypercalcemia induced by these endogenous ligands.16 By using a fluorescence polarization (FP)
assay employing VDRꢀLBD and a fluorescently labeled peptide, SRC2ꢀ3, we derived GW0742
as a nonꢀsecosteroid inhibitor of VDR (IC50 = 27 ± 2.7 ꢁM) out of a library of ~390,000
compounds (PubChem Assay Identifier (AID): 504847). We found GW0742 possessed moderate
inhibitory effects for VDR and the androgen receptor (AR) in addition to greatly activating
PPARδ. Evaluation of VDRꢀtarget gene regulation in LNCaP cells in the presence of GW0742
resulted in negative regulation of CYP24A1, IGFBPꢀ3 and TRPV6 when stimulated with
1,25(OH)2D3. Additionally, GW0742 was capable of inhibiting (IC50 = 37.6 ꢁM) cell
differentiation induced by 1,25(OH)2D3 in HLꢀ60 cells, a process governed by VDR gene
expression.
Herein, we discuss the medicinal chemistry approach used to optimize GW0742 as a
potent VDR antagonist with decreased PPARδ activity. Four major regions of GW0742 were
modified as described in Figure 1. The SAR included the replacement of the phenyl ring (pink
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region) with substitutedꢀaryl or heteroaryl groups, exchanging of the methyl (cyan region) with a
hydrogen, substitution of the linker atoms (green region) with oxygen, nitrogen or sulfur, and
bioisosteric substitution of the carboxylic acid (blue region).
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Figure 1. Design of GW0742 derivatives.
RESULTS AND DISCUSSION
Synthesis. Over 100 compounds were synthesized based on the GW0742 core scaffold using a
parallel chemistry approach that efficiently produced the desired compounds at sufficient yields.
Monoꢀ, polyꢀ, and aromaticꢀsubstituted GW0742 analogues were synthesized according to
reaction Scheme 1. Sodium borohydride was used to reduce ethyl 2ꢀbromoꢀ4ꢀmethylthiazoleꢀ5ꢀ
carboxylate to the corresponding primary alcohol 1a. Subsequent reaction with thionyl chloride
afforded 1b, which was coupled with 4ꢀhydroxyꢀ3ꢀmethylthiophenol in the presence of cesium
carbonate to give 1c. Suzuki coupling methodology was applied to enable diversity in this
position via different boronic acids and a unique solid supported diphenylphosphine palladium
(II) heterogeneous catalyst that could be recovered and used again. The resulting esters were
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then cleaved with trifluoroacetic acid in CH2Cl2 to afford the final carboxylic acid products (1ꢀ
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78).
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Scheme 1. General synthetic route for mono, poly, and aromaticꢀsubstituted ligands.
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i) NaBH4, EtOH, R.T., 66%, ii) SOCl2, CH2Cl2, R.T., 86%; iii) a) 4ꢀhydroxyꢀ3ꢀ
methylthiophenol, Cs2CO3, MeCN, R.T.; b) tert. butyl bromoacetate, Cs2CO3, MeCN, R.T.,
overall yield = 89%; iv) Boronic acid Na2CO3, SiliaCat® DPPꢀPd, DME, 150 °C, MW, 0.5 h,
33ꢀ60%; v) TFA, CH2Cl2, R.T., 1 h, ~100%.
Bioisosteric substitution is a common approach in medicinal chemistry to improve
potency, selectivity and/or metabolic stability while maintaining similar physical (e.g. size,
shape, polarity) and chemical properties (e.g. pKa). In our case, a tetrazole was introduced in
place of the carboxylate as depicted in Scheme 2. 2ꢀmethylꢀ4ꢀthiocyanatophenol and 2ꢀ
bromoacetonitrile were coupled under basic conditions to produce 2a. Reaction with Na2S
yielded the free thiol that was sequentially coupled to 1b to yield 2c. The cyanoꢀsubstituted
analogues 2dꢀ2g were formed by Suzuki coupling and subsequently treated with sodium azide to
form tetrazoles analogues (79ꢀ82) by [2+3] cycloaddition.
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Scheme 2. General synthetic route for bioisostereꢀsubstituted analogues.
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i) Cs2CO3, MeCN, R.T., 12 h, 42%; ii) Na2S, EtOH•H2O, R.T., 3 h, 91%; iii) 1b, Cs2CO3,
MeCN, R.T., 1 h, 75%; iv) Boronic acid, Na2CO3, SiliaCat® DPPꢀPd, DME, 150 °C, MW, 0.5 h;
36%ꢀ45%; v) NaN3, NH4Cl, DMF, 100 ⁰C, 12 h, 35ꢀ69%.
The synthesis of linkerꢀmodified GW0742 analogues (87, 92 and 93) started with
reduction of ethyl 2ꢀbromoꢀ4ꢀmethylthiazoleꢀ5ꢀcarboxylate to obtain 1a.17 This compound was
coupled with methyl 3ꢀ(4ꢀhydroxyphenyl)propionate under Mitsunobu reaction conditions
yielding thiazole ester 3a.18 A microwaveꢀassisted Suzuki reaction allowed for the introduction
of the third aromatic ring. Conveniently, the carbonꢀcarbon bond formation and hydrolysis
occurred under microwave conditions to form the final carboxylic acids 87, 92 and 93.
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Scheme 3. General synthetic route for linkerꢀsubstituted GW0742 analogues.
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i) PPh3, DIAD, CH2Cl2, methyl 3ꢀ(4ꢀhydroxyphenyl) propionate, R.T., 2 h, 30%; ii) PdCl2ꢀ
(PPh3)2, Na2CO3ꢂH2O, DME/H2O/EtOH, 160 oC, 10 minutes, MW, 36%ꢀ45%.
Biological Evaluation of GW0742 Analogues. GW0742 analogues were characterized using
several biochemical and cellꢀbased assays to determine their activity towards VDR and PPARδ.
Cytotoxicity of each compounds was also investigated in HEK293T cells. This included a highꢀ
throughput FP assay investigating the interaction between VDRꢀLBD and coactivator SRC2ꢀ3,
VDRꢀ and PPARδꢀmediated transcription assays and a cytotoxicity assay to differentiate
between inhibition of transcription and cytotoxicity.16, 19 We report the results in the following
tables: monoꢀsubstituted analogues (Table 1), polyꢀsubstituted analogues (Table 2), aromaticꢀ
substituted analogues (Table 3), tetrazoleꢀsubstituted analogues (Table 4) and linkerꢀsubstituted
analogues (Table 5).
The monoꢀsubstituted GW0742 analogues exhibited a wide range of potencies (EC50
0.009 – >100 ꢁM) with respect to PPARδ activation in cells (Table 1).
=
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Table 1. Evaluation of monoꢀsubstituted GW0742 analogues
Compound
R
PPARδ EC50 (ꢁM) a,b
Toxicity CC50 (µM)c
H
oꢀCH3
mꢀCH3
pꢀCH3
oꢀCl
mꢀCl
pꢀCl
oꢀF
mꢀF
pꢀ F
oꢀCF3
mꢀCF3
0.26 ± 0.35 (90 ± 7.5)
0.60 ± 0.17 (66 ± 5.2)
1.62 ± 0.41 (74 ± 4.3)
0.05 ± 0.02 (104 ± 12)
0.58 ± 0.20 (71 ± 4.8)
0.50 ± 0.16 (74 ± 4.5)
0.13 ± 0.04 (66 ± 5.8)
0.26 ± 0.08 (94 ± 5.9)
0.43 ± 0.23 (96 ± 12)
0.16 ± 0.04 (93 ± 3.3)
2.4 ± 0.95 (39 ± 2.8)
1.1 ± 0.6 (79 ± 14)
>100
>100
>80
>80
>80
28.8 ± 5.0
>100
>80
>100
Nonꢀtoxic
>100
>50
>33
>100
>80
1
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7
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10
11
12
pꢀCF3
oꢀOCH3
mꢀOCH3
mꢀOCF3
pꢀOCF3
mꢀCN
pꢀCN
pꢀN(CH3)2
pꢀNHCH3
pꢀCO2Et
mꢀmethylsulfinyl
pꢀmethylsulfonamido
pꢀNHCOCH3
mꢀCONH(CH2)3N(CH3)2
pꢀCONH(CH2)2N(CH3)2
pꢀ(4ꢀmethylpiperazinyl)
methanone
pꢀpiperazinyl
Morpholinoꢀmethanone
0.013 ± 0.004 (95 ± 3.3)
2.3± 0.70 (27 ± 4.2)
2.36 ± 0.67 (96 ± 7.2)
0.45 ± 0.15 (40 ± 2.4)
0.014 ± 0.007 (49 ± 2.2)
3.2 ± 1.0 (74 ± 5.4)
0.12 ± 0.42 (126 ± 11)
0.16 ± 0.02 (140 ± 14)
0.57 ± 0.24 (83 ± 7.4)
0.009 ± 0.002 (123 ± 5.8) >80
6.7 ± 3.2 (59 ± 4.9)
>100
3.9 ± 2.3 (97 ± 4.7)
2.4 ± 1.3 (70 ± 7.5)
2.1 ± 1.4 (65 ± 11)
13 (GW501516)
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17
18
19
20
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22
23
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25
26
27
>50
>50
>80
>80
>50
Nonꢀtoxic
Nonꢀtoxic
Nonꢀtoxic
>100
Nonꢀtoxic
Nonꢀtoxic
6.9 ± 5.53 (70 ± 13)
Nonꢀtoxic
28
16.8 ± 5.7 (37 ± 3.4)
3.8 ± 1.5 (37 ± 2.6)
>80
Nonꢀtoxic
29
30
aTwoꢀhybrid assay using a CMVꢀPPARδꢀLBDꢀGAL4ꢀDBD plasmid and a 6xGal4ꢀluc reporter
vector. The maximum concentration used for this assay was 100 ꢁM of each compound;
bEfficacy in PPARδ assay in respect to full activation with GW0742 are in parentheses; Cellꢀ
c
TiterGlo (Promega). The maximum concentration used for transcription and toxicity assay was
100 ꢁM of each compound. Data were analyzed using a nonlinear regression with a variable
slope (GraphPad Prism).
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Three monoꢀsubstituted compounds modulated transcription with high potency. They were 13
(pꢀCF3), 17 (pꢀOCF3), and 22 (pꢀCO2Et) with EC50 values lower than 15 nM. Compound 13, also
known as GW501516, was confirmed as a potent PPARδ agonist.5a The other two compounds
have not been reported but possess similar electron donating properties and significant
hydrophobicity. Notably, the positioning of substituents around the phenyl ring affected the
ligand’s ability to activate PPARδ mediated transcription. A para positioned substituent, in most
cases, resulted in a more potent PPARδ agonist than compounds that bear the same group in the
ortho or meta position. This relationship was observed with methyl (2ꢀ4), trifluoromethyl (11ꢀ
13), trifluoromethoxy (16ꢀ17) and cyano (18ꢀ19) substituents. However, ligands with halide
substituents, like Cl (5ꢀ7) and F (8ꢀ10), showed no significant activity difference between ortho,
meta, or para positioning possibly due to their atomic size or change in orientation. Compounds
with paraꢀpositioned fluorine and chlorine substituents had similar but relatively high EC50
values of ~0.14 µM (compounds 10 and 7). The least active PPARδ agonist was polar
sulfonamide 24. As evidenced by the crystal structure of PPARδ complexed with GW0742
(PDB: 3TKM), the binding pocket accommodates the pink region of Figure 1 of the ligand which
is relatively large and hydrophobic due to residues V245, V305, V312, L317, and I328 (Figure
2).5a, 20 Therefore, depending on its position within the LBP, it is expected that extra hydrogen
bonding capability is not favorable in this region of the ligand. Compound 29 bearing a
piperazine was another example where polarity reduced activity towards PPARδ with an EC50 =
16.8 ± 5.7 ꢁM.
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Interestingly, while many ligands exhibited partial agonistic activity towards PPARδ, we
did not observe a trend between structure and efficacy. This is significant because some nuclear
receptor partial agonists possess superior pharmacodynamics due to reduced side effects and
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moderate activation.21 Finally, most compounds exhibited CC50 values, determined in the
presence of HEK293 kidney cells, of 80 ꢁM and higher. An exception was compound 6 (mꢀCl)
with an CC50 of 28.8 µM.
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Polyꢀsubstituted ligands found in Table 2 were also capable of activating PPARδ
mediated transcription (EC50 = 0.0035 – 6.6 ꢁM).
Table 2. Evaluation of polyꢀsubstituted GW0742 analogues.
PPARδ
Compound
R
R1
R2
R3
R4
Toxicity CC50 (µ
EC50 (ꢁM)a, b
31
H
H
CF3
F
H
0.0035 ± 0.0003 (97 ± 4.4) >50
(GW0742)
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CF3
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H
H
H
H
H
H
H
H
H
H
Cl
H
H
CF3
H
H
H
H
H
CF3
H
H
Cl
Cl
F
H
H
F
H
F
F
CN
H
Cl
H
Cl
CF3
Cl
H
Cl
H
H
H
Cl
H
H
H
H
H
H
H
F
0.99 ± 0.42 (46 ± 5.4)
0.20 ± 0.09 (50 ± 2.8)
0.028 ± 0.013 (88 ± 2.8)
0.007 ± 0.003 (75 ± 5.4)
0.009 ± 0.004 (69 ± 3.1)
0.020 ± 0.010 (100 ± 6.3)
1.06 ± 0.47 (44 ± 4.6)
0.074 ± 0.031 (112 ± 5.2)
0.050 ± 0.028 (91 ± 7.9)
0.49 ± 0.31 (116 ± 14)
0.042 ± 0.009 (122 ± 7.3)
0.046 ± 0.019 (106 ± 19)
1.07 ± 0.59 (150 ± 24)
>50
>50
>50
>33
>50
CF3
CF3
CF3
OCF3
OCF3
H
>50
OCH3 OCH3
F
>100
>100
>50
>50
>80
>100
>100
>50
OCH3
OCH2CF3
F
F
F
F
Cl
Cl
CN
H
H
H
H
H
H
H
CF3
H
F
H
H
H
H
F
Cl
Cl
H
H
Cl
H
H
CF3 0.18 ± 0.12 (63 ± 5.3)
H
Cl
H
H
0.692 ± 0.137 (79 ± 4.5)
0.066 ± 0.04 (108 ± 11)
0.17 ± 0.05 (62 ± 3.4)
0.60 ± 0.28 (23 ± 2.3)
0.71 ± 0.27 (78 ± 6.0)
6.6 ± 4.8 (84 ± 11)
>50
>100
>50
>33
>50
>100
>100
>80
48
49
50
51
52
53
H
Cl
Cl
Cl
0.028 ± 0.017 (78 ± 7.5)
1.40 ± 0.74 (75 ± 2.1)
Benzylꢀ
morpholine
54
H
F
H
H
4.3 ± 1.9 (50 ± 4.4)
>100
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aTwoꢀhybrid assay using a CMVꢀPPARδꢀLBDꢀGAL4ꢀDBD plasmid and a 6xGal4ꢀluc reporter
vector. The maximum concentration used for this assay was 100 ꢁM of each compound;
3
4
5
6
c
bEfficacy in PPARδ assay in respect to full activation with GW0742 are in parentheses; Cellꢀ
7
8
9
TiterGlo (Promega). The maximum concentration used for transcription and toxicity assay was
100 ꢁM of each compound. Data were analyzed using a nonlinear regression with a variable
slope (GraphPad Prism).
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35
36
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41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The least active compound in our series was compound 51 (o,o’ꢀCl) with an EC50 value of
greater than 5 µM, indicating that two substituents occupying both ortho positions of the phenyl
ring is not favorable for PPARδ activation. Interestingly, by moving just a chloride to the R3
position (compound 52) activity of this ligand is greatly increased by 230ꢀfold when compared to
51. The positioning of groups like CF3, Cl, F and OCF3 on phenyl ring positions gave some
insight about the PPARδ ligand binding pocket. For example, by switching the pꢀCF3 and mꢀF
substituents on GW0742 to make compound 41 (mꢀCF3, pꢀF) a 140ꢀfold decrease in ligand
potency was observed. When mꢀF of GW0742 is exchanged for a mꢀCl (compound 35), no
significant change in ligand potency was observed. When comparing compound 35 (pꢀCF3, mꢀ
Cl) to compound 34 (pꢀCF3, oꢀCl), only a small decrease in PPARδ activation was observed (4ꢀ
fold change). When pꢀCF3 of GW0742 was replaced with pꢀOCF3 regardless of a Cl or F in the
meta position (compounds 36 and 37, respectively), PPARδ activation was observed at low
nanomolar concentrations. With respect to all fluorine substituents, it appeared that two fluorine
substituents were better than one regardless of their positioning. The same trend was observed
for chlorine substituents. The toxicity of polyꢀsubstituted GW0742 analogues was, in general,
more pronounced than their monoꢀsubstituted counterparts, however none of them exhibited
toxicity below 50 ꢁM.
Aromatic substituents were also coupled to the Cꢀ2 position of the thiazole ring and their
biological activity is summarized in Table 3.
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35
36
37
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 3. Evaluation of substituted GW0742 analogues
Compo
und
55
56
57
Toxicity CC50
(µM) c
>100
>100
>80
>80
>80
>50
>50
R
PPARδ EC50 (ꢁM) a,b
3,5ꢀdimethylisoxazolꢀ4ꢀyl
1Hꢀindazolꢀ4ꢀyl
4ꢀpyridine
0.29 ± 0.12 (103 ± 17)
0.052 ± 0.035 (101 ± 14)
9.1 ± 3.6 (81 ± 6.4)
2ꢀ(benzofuranꢀ2ꢀyl)
2,3ꢀdihydrobenz[1,4]dioxinꢀ6ꢀyl
Naphthaleneꢀ1ꢀyl
Benzo[1,3]dioxolꢀ5ꢀyl
Benzo[1,2,5]oxadiazolꢀ5ꢀyl
Furanꢀ2ꢀyl
0.061 ± 0.028 (97 ± 9.1)
0.72 ± 0.23 (127 ± 6.2)
1.69 ± 0.34 (36 ± 1.7)
0.26 ± 0.12 (52 ± 3.8)
0.16 ± 0.04 (35 ± 2.2)
6.0 ± 2.7 (96 ± 11)
58
59
60
61
62
63
>100
>100
>100
Pyridinꢀ3ꢀyl
8.1 ± 5.6 (87 ± 12)
64
65
Piperazinꢀ1ꢀyl
0.57 ± 0.39 (46 ± 9.6)
>100
Pyrimidinꢀyl
1Hꢀindazolꢀ6ꢀyl
Benzothiazolꢀ6ꢀyl
Isoquinolinꢀ4ꢀyl
6ꢀfluoropyridinꢀ3ꢀyl
1ꢀmethylꢀ1Hꢀindazolꢀ6ꢀyl
6ꢀ(trifluoromethyl)pyridinꢀ3ꢀyl
1ꢀmethylꢀ1Hꢀindolꢀ6ꢀyl
1Hꢀpyrazolꢀ4ꢀyl
1Hꢀindazolꢀ5ꢀyl
Benzothiophenꢀ5ꢀyl
1Hꢀindolꢀ4ꢀyl
1.57 ± 0.62 (51 ± 3.8)
3.18 ± 1.80 (122 ± 36)
0.073 ± 0.053 (99 ± 23)
0.35 ± 0.18 (51 ± 6.7)
Nonꢀtoxic
>100
100
66
67
68
69
70
71
72
73
74
75
76
77
78
Nonꢀtoxic
0.192 ± 0.082 (110 ± 9.5) Nonꢀtoxic
0.58 ± 0.34 (99 ± 6.7)
0.013 ± 0.006 (91 ± 4.9)
0.52 ± 0.29 (103 ± 5.0)
3.9 ± 1.7 (74 ± 5.7)
1.44 ± 0.66 (64 ± 5.5)
0.14 ± 0.08 (95 ± 12)
>100
>100
>100
>100
Nonꢀtoxic
Nonꢀtoxic
>50
Nonꢀtoxic
1Hꢀindolꢀ2ꢀyl
0.035 ± 0.015 (108 ± 5.5) >50
aTwoꢀhybrid assay using a CMVꢀPPARδꢀLBDꢀGAL4ꢀDBD plasmid and a 6xGal4ꢀluc reporter
vector. The maximum concentration used for this assay was 100 ꢁM of each compound;
bEfficacy in PPARδ assay in respect to full activation with GW0742 are in parentheses; Cellꢀ
c
TiterGlo (Promega). The maximum concentration used for transcription and toxicity assay was
100 ꢁM of each compound. Data were analyzed using a nonlinear regression with a variable
slope (GraphPad Prism).
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Five compounds activated PPARδ with EC50 values less than 75 nM (56, 58, 68, 72 and 78). Of
these, all but one had a bicyclic aromatic ring structure. This result confirmed earlier
observations that the LBD of PPARδ is spacious enough to accommodate such ligands, possibly
through a unique orientation unlike GW0742. It is worth noting that when compared to VDR,
PPARδ has a larger Yꢀshaped ligand binding pocket which can make contact with the ligand in
three different regions, thus possibly explaining the accommodation for large ring systems.20
From Table 1 we concluded that nonꢀsubstituted phenyl ring structures such as compound 1
made poor PPARδ agonists. Similarly, substitution of the phenyl ring with a pyridine, as in
compound 64, resulted in a poor binding. However, when the pyridine ring contained a
trifluoromethyl group like compound 72, a 620ꢀfold increase in transcriptional activation was
observed when compared to 64. The positioning of heterocyclic rings may be important
considering the effect compounds 77 and 78 have on PPARδ activation. Both have an indole
ring attached to Cꢀ2 of the thiazole but the 1Hꢀindolꢀ4ꢀyl group (77) was nearly 3,000 times less
potent than the 1Hꢀindolꢀ2ꢀyl substituent (78).
10
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19
20
21
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23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Bioisoteric substitution of the carboxylate group with a tetrazole ring was used for
several analogues to determine its effect on PPARδꢀmediated transcription (Table 4).
Table 4. Evaluation of tetrazoleꢀsubstituted GW0742 analogues
Com
Entry for
Corresponding
Carboxylic Acid
Toxicity
poun R1 R2 R3 R4 PPARδ EC50 (ꢁM) a, b
d
CC50 (µM)c
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CF
3
31 (GW0742)
79
H
Cl
H
H
F
H
0.04 ± 0.018 (29 ± 1.5)
>33
H
Cl
H
H
CF
0.594 ± 0.435 (29 ± 5.2) >50
80
81
53
46
Cl
0.51 ± 0.18 (51 ± 4.8)
>33
3
Cl Cl
H
0.24 ± 0.11 (26 ± 1.9)
>33
82
49
aTwoꢀhybrid assay using a CMVꢀPPARδꢀLBDꢀGAL4ꢀDBD plasmid and a 6xGal4ꢀluc reporter
vector. The maximum concentration used for this assay was 100 ꢁM of each compound;
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47
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50
51
52
53
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55
56
57
58
59
60
bEfficacy in PPARδ assay in respect to full activation with GW0742 are in parentheses; Cellꢀ
c
TiterGlo (Promega). The maximum concentration used for transcription and toxicity assay was
100 ꢁM of each compound. Data were analyzed using a nonlinear regression with a variable
slope (GraphPad Prism).
In general, increased toxicity and decreased potency was observed for tetrazole analogues when
compared to their carboxylate counterparts. The tetrazole substitution of GW0742, resulting in
compound 79, had an 11ꢀfold decrease in potency and a twoꢀfold increase of toxicity. Although
similar in acidity, the spacial ability of this particular tetrazole to form hydrogen bond
interactions is different from a carboxylic acid. It is known that both oxygens on the carboxylate
group make hydrogen bonds with key residues like His413 (helix 10/11), Try437 (helix 12),
His287 (helix 7) of PPARδ LBP and are implicated with maintaining the locked conformation of
helix 12 (Figure 2).20 Thus, the inability of the tetrazole to form all necessary hydrogen bonds
might be responsible for low efficacy of these compounds ranging between 26ꢀ51%.
The replacement of the sulfur and oxygen linkers of GW0742 allowed structureꢀactivity
relationship investigations into the central part of PPARδ agonists (Table 5).
Table 5. Evaluation of linkerꢀsubstituted GW0742 analogues.
Comp R1
R2
Y
R
X
PPARδ EC50 (ꢁM) a,b
Toxicity CC50
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ound
83
84
85
86
87
88
89
90
91
92
93
(µM)c
>100
>100
>100
>100
>100
>100
>100
>80
H
F
F
F
F
F
F
F
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
H
S
S
S
H
H
H
CH3
H
H
CH3
H
H
H
CH2
ꢀꢀ
O
O
CH2
S
O
O
CH2
CH2
CH2
0.039 ± 0.022 (87 ± 4.3)
0.018 ± 0.010 (70 ± 3.7)
0.026 ± 0.008 (84 ± 5.1)
0.02 ± 0.02 (73 ± 11)
0.032 ± 0.021 (77 ± 5.0)
0.27 ± 0.023 (75 ± 5.0 )
0.18 ± 0.12 (61 ± 5.0)
0.16 ± 0.045 (78 ± 6.0)
0.058 ± 0.021 (95 ± 7.0)
18.2 ± 2.8 (47 ± 1.8)
O
O
O
N
N
N
O
O
10
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23
24
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35
36
37
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
F
>80
Nonꢀtoxic
>100
CH2OH
H
CF3
H
0.70 ± 0.11 (111 ± 6.5)
aTwoꢀhybrid assay using a CMVꢀPPARδꢀLBDꢀGAL4ꢀDBD plasmid and a 6xGal4ꢀluc reporter
vector. The maximum concentration used for this assay was 100 ꢁM of each compound;
bEfficacy in PPARδ assay in respect to full activation with GW0742 are in parentheses; Cellꢀ
TiterGlo (Promega). The maximum concentration used for transcription and toxicity assay was
100 ꢁM of each compound. Data were analyzed using a nonlinear regression with a variable
slope (GraphPad Prism).
c
In addition, the role of the ortho methyl group (R) was investigated in terms of favorable PPARδ
binding. Compound 85 is structurally similar to GW0742 except for the lack of methyl group in
the R position: it was 7 times less potent than GW0742 and surprisingly, behaved as a partial
agonist (84% in comparison with GW0742). A large decrease in potency (~200ꢀfold) was
observed for compound 93 when Y was replaced with a smaller oxygen atom and X with a CH2
in the absence of a fluoride and methyl group in positions R1 and R, respectively. When
compared to compound 87, bearing a fluorine in the R1 position, the activity difference was less,
suggesting an additional hydrophobic interactions between 87 and PPARδ LBP. This behavior
was not observed for Y = nitrogen when comparing 89 and 90. The presence or absence of an R
positioned methyl had little effect on activation of transcription thus producing agonists that
were ~50 times less potent than GW0742. However, CH2 in place of oxygen (91) improved
PPARδ binding when compared to 89 and 90. The least potent compound was 92, bearing a mꢀ
CH2OH substituent on the phenyl ring with linkers similar to that of 93. This compound was
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approximately 5,000 times less potent than GW0742 confirming that hydrogen bond donor and
acceptor moieties in the pink region of Figure 1 of ligands are not favorable for PPARδ binding.
In addition to GW0742 and GW501516, compounds 7, 10, 32, 46, and 83 were patented
by GSK.22 I was reported that most of the GW0742ꢀlike compounds showed ~50% activation of
hPPARδ at concentrations of 10 nM or less in a βꢀgalactosidase transactivation assay using CVꢀ1
cells. Other than compounds 32 and 46, this correlates well with what we have observed in our
PPARδꢀmediated transcription assay. Also, these compounds were reported being at least 10ꢀfold
selective for hPPARδ over hPPARα and PPARγ.
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52
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58
59
60
The SAR study of GW0742 analogues has given us some insight into the structural
requirements for these highly potent PPARδ ligands. In addition, structural elements have been
identified that significantly reduce the ability of ligands to activate PPARδꢀmediated
transcription. The in vitro data was further supported by molecular modeling using a crystal
structure of GW0742 bound to PPARδ LBD (PDB: 3TKM). Using the Schrödinger software to
perform standard precision gridꢀbased ligand docking with energetics (Glide) studies, we were able
to show that 32 aligns favorably with GW0742. However, occupation of the highly hydrophobic
region on the left side of the pocket (Figure 2) differs significantly. It is evident that the addition
of two bulky trifluoromethyl groups in both meta positions orients the pink region of Figure 1 of
the molecule downwards therefore causing a 283ꢀfold decrease in PPARδ activity (GW0742,
EC50 = 0.0034 μM and 32, EC50 = 0.99 μM).
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Figure 2. Computer docking of 32 (blue) in the ligand binding pocket of PPARδ with GW0742
(green) (PDB: 3TKM). Major nonpolar and hydrogen bonding interactions have been labeled.
Having identified several PPARδ ligands with EC50 values greater than 1 µM, we next set
out to test their ability to bind to VDR. All “poor” PPARδ ligands were investigated with respect
to their ability to inhibit the interaction between VDR and coactivator peptide SRC2ꢀ3 and their
ability to inhibit VDRꢀmediated transcription in cells. The results are summarized in Table 6.
Table 6. Evaluation of “poor” PPARδ ligands (EC50 > 1ꢁM) in respect to VDR binding.
PPARδ EC50 (ꢁM) a,b
VDRꢀSRC2ꢀ3 interaction IC50 VDR transcription IC50
Entry
(ꢁM)a
(ꢁM)b
40.11 ± 9.26
32.27 ± 5.28
45.18 ± 5.65
>100
inactive
25
11
14
18
3.9 ± 2.3 (97 ± 4.7)
2.4 ± 0.95 (39 ± 2.8)
2.3± 0.70 (27 ± 4.2)
3.2 ± 1.0 (74 ± 5.4)
31.5 ± 9.1
31.4 ± 8.1
25.1 ± 10.0
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>100
>100
inactive
>100
15.0 ± 4.7
>50
23.7 ± 4.7
33.0 ± 7.2
>50
inactive
26.3 ± 0 8.3
>100
>50
>50
23
26
27
32
38
44
51
53
57
63
30
54
63
64
66
67
74
75
77
3
6.7 ± 3.2 (59 ± 4.9)
2.4 ± 1.3 (70 ± 7.5)
2.1 ± 1.4 (65 ± 11)
0.99 ± 0.42 (46 ± 5.4)
1.06 ± 0.47 (44 ± 4.6)
1.07 ± 0.59 (150 ± 24)
6.6 ± 4.8 (84 ± 11)
1.40 ± 0.74 (75 ± 2.1)
9.1 ± 3.6 (81 ± 6.4)
1.69 ± 0.34 (36 ± 1.7)
3.8 ± 1.5 (37 ± 2.6)
4.3 ± 1.9 (50 ± 4.4)
6.0 ± 2.7 (96 ± 11)
8.1 ± 5.6 (87 ± 12)
1.57 ± 0.62 (51 ± 3.8)
3.18 ± 1.80 (122 ± 36)
3.9 ± 1.7 (74 ± 5.7)
1.44 ± 0.66 (64 ± 5.5)
>100
30.28 ± 4.5
52.03 ± 12.79
9.088 ± 0.982
>100
53.53 ± 15.95
40.28 ± 8.07
14.47 ±2.21
43.88 ± 8.54
20.19 ± 1.89
>100
68.79 ± 13.04
>100
>100
>100
57.78 ± 8.69
>100
10
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22
23
24
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26
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32
33
34
35
36
37
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
>100
inactive
inactive
inactive
inactive
inactive
38.2 ± 8.6
19.0 ± 6.01
26.3 ± 6.9
24.5 ± 6.1
inactive
inactive
51.14 ± 5.55
28.93 ± 4.69
47.45 ± 6.61
24.5 ± 2.27
48.82 ± 6.56
>100
1.62 ± 0.41 (74 ± 4.3)
1.1 ± 0.6 (79 ± 14)
2.36 ± 0.67 (96 ± 7.2)
6.9 ± 5.53 (70 ± 13)
16.8 ± 5.7 (37 ± 3.4)
18.2 ± 2.8 (47 ± 1.8)
12
15
28
29
92
>100
inactive
aVDRꢀLBD concentration used was 0.1 µM. Inhibition of VDRꢀSRC2ꢀ3 interaction in the
presence of LG190178 (0.75 µM). The maximum concentration used for this assay was 300 ꢁM
b
of each compound; Transcription assay using a CMVꢀVDR plasmid and a luciferase reporter
plasmid under control of a 24ꢀhydroxylase promoter with GW0742 analogues. The maximum
concentration used for this assay was 100 ꢁM of each compound. Data were analyzed using a
nonlinear regression with a variable slope (GraphPad Prism).
Among the ligands investigated only 32, bearing a CF3 group in the meta position instead of the
para position like GW0742, exhibited an IC50 value below 10 µM in the VDR FPꢀbased assay.
Many of the other ligands were not active. The activity of 32 was confirmed in cells with an IC50
of 15 µM for VDRꢀmediated transcription. Notably, most ligands including GW0742 in light of
the FPꢀbased assay and the VDRꢀmediated transcription assay, showed full antagonistic activity
when compared to the DMSO control (Figures S2 and S3). Compounds whose IC50 values were
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greater than 100 μM appeared to be trending towards full antagonistic activity although higher
concentrations would need to be tested to confirm this. Unfortunately, none of the synthesized
ligands exhibited high affinity for VDR. Interestingly, other identified VDR ligands that bear a
carboxylic acid function such as lithocholic acid23 or calcitroic acid24 exhibited binding constants
toward VDR in the same range (1ꢀ10 µM); thus the carboxylic acid function of these molecules
might be inferior to the two hydroxyl groups of 1,25(OH)2D3 to the bind VDR.25 On the other
hand, 1,25(OH)2D3 interacts with histidine residues via it’s tertiary alcohol. The only ligand
bearing a hydroxyl function among the ligands generated is compound 92 that interacted with
neither PPARδ nor VDR. Thus, the current investigation illustrates that the change of
substituents introduced here to design new VDR ligands based on the GW0742 scaffold is not
sufficient. In the future, we will change the length of this scaffold by introducing smaller ring
structures and linkers in addition to other carboxylic acid bioisosteres. However, it is
encouraging that the simple removal of a para substituent and the addition of meta substituents
on the phenyl ring (pink region, Figure 1), as in 32, does greatly reduce PPARδ activity. Thus, an
introduction of a 3,4ꢀbis(hydroxymethyl)phenyl group as shown for VDR ligands CD442026
might be advantageous to improve VDR binding and reduce PPARδ activity. On the other hand,
we did identify many new potent PPARδ agonists and partial agonists during this study,
however, further studies are needed to determine the PPAR subtype selectivity of these ligands.
This will be essential to develop new drug candidates for lowering density lipoprotein
therapeutics based on PPARδ binding.
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EXPERIMENTAL PROCEDURES
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Chemistry. General Information for Compounds 1-86 and 88-91: All reagents and solvents used
in this study were obtained from commercial suppliers and used without further purification.
Preparative purification was performed on a Waters semiꢀpreparative HPLC system using a
Phenomenex Luna C18 (5 micron, 30 x 75 mm) at a flow rate of 45 mL/min. A gradient of 10%
to 50% acetonitrile in water over 8 minutes (each containing 0.1% trifluoroacetic acid) was used
as a mobile phase during the purification. Fraction collection was triggered by UV detection
(220 nm). Analytical analysis was performed on an Agilent LC/MS (Agilent Technologies,
Santa Clara, CA). Method t1: A 7 minute gradient of 4% to 100% Acetonitrile (containing
0.025% trifluoroacetic acid) in water (containing 0.05% trifluoroacetic acid) was used with an 8
minute run time at a flow rate of 1 mL/min. A Phenomenex Luna C18 column (3 micron, 3 x 75
mm) was used at a temperature of 50 °C. Method t2: A 3 minute gradient of 4% to 100%
Acetonitrile (containing 0.025% trifluoroacetic acid) in water (containing 0.05% trifluoroacetic
acid) was used with a 4.5 minute run time at a flow rate of 1 mL/min. A Phenomenex Gemini
Phenyl column (3 micron, 3 x 100 mm) was used at a temperature of 50° C. Method t3: Analysis
was performed on an Agilent 1290 Infinity Series HPLC. UHPLC Long Gradient Equivalent 4%
to 100% acetonitrile (0.05% trifluoroacetic acid) in water over 3.5 minutes run time of 4 minutes
with a flow rate of 0.8 mL/min. > 95 % Purity was determined using an Agilent Diode Array
Detector for both Method t1, Method t2 and Method t3. Mass determination was performed
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using an Agilent 6130 mass spectrometer with electrospray ionization in the positive mode. H
NMR spectra were recorded on Varian 400 MHz spectrometer. Chemical shifts are reported in
ppm with DMSO at 2.49 ppm as internal standard. High resolution mass spectrometry was
recorded on Agilent 6210 TimeꢀofꢀFlight LC/MS system. Confirmation of molecular formula
was accomplished using electrospray ionization in the positive mode with the Agilent
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Masshunter software (version B.02). The synthesis of these compounds is described in more
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detail in the Supporting Information.
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General Information for Compounds 87, 92-93: All starting reagents were purchased from
SigmaꢀAldrich. Anhydrous solvents were purchased in sureꢀseal bottles and handled under dry
o
conditions using syringe technique. All glassware was dried overnight at 100 C before use.
Thin layer chromatography was performed on preꢀcoated silica gel 60 F254 plates (Fisher
Scientific). Microwave reactions were performed using a CEM Discover SP instrument.
Synthesized compounds were purified by normal phase flash chromatography (SPI Biotage,
silica gel 230ꢀ400 mesh) and concentrated under vacuum. All pure compounds were stored as
solids at ꢀ20 °C. Molecular masses and >95 % purities were determined with a Shimadzu 2020
LCꢀMS (single quadrupole) instrument. NMR spectra were recorded on a Bruker 500 MHz or
300 MHz instrument with samples diluted in either CDCl3 or DMSOꢀD6. Chemical shifts are
reported in δ (parts per million: ppm) by reference to the hydrogenated residues of the deuterated
solvent as an internal standard (CDCl3: δ= 7.24 ppm (1HNMR) and δ= 77 ppm (13CNMR) and
DMSOꢀD6: δ= 2.50 ppm (1HNMR) and δ= 39.5 ppm (13CNMR). The synthesis of these
compounds is described in more detail in the Supporting Information.
Biology. Reagents and Instrumentation. LG190178 (a VDR agonist) was synthesized following
a published procedure27 and used as a positive control. 3ꢀdibutylaminoꢀ1ꢀ(4ꢀhexylꢀphenyl)ꢀ
propanꢀ1ꢀone (a VDRꢀcoactivator inhibitor) was used as a second positive control and was
synthesized using a previously published method.28 1,25(OH)2D3 was purchased from
Endotherm. The VDRꢀLBDmt DNA was provided by D. Moras29 and cloned into pMALꢀz2X
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vector (New England Biolabs). For a details on the expression and purification of VDRꢀLBD,
see ref30. AlexaFluor 647 ꢀLabeled SRC2ꢀ3 was synthesized by coupling SRC2ꢀ3
(CKKKENALLRYLLDKDDTKD) with cysteineꢀreactive AlexaFluor 647 probe followed by
reverse phase HPLC purification using a C18 column. VDRꢀCMV and CYP24A1ꢀluciferase
reporter plasmids were provide by D.D. Bikle.
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VDR Binding Determined by a Fluorescence Polarization (FP) Assay. Was conducted in 384ꢀ
well black polystyrene microplates (Corning, #3573). The assay solution contained buffer
(25mM PIPES, 50 mM NaCl (Fisher), and 0.01% NPꢀ40, at pH 6.75), 0.1 ꢁM VDRꢀLBD and
7.5 nM Alexa Fluor 647ꢀlabeled SRC2ꢀ3. For competitive inhibition, 0.75 ꢁM LG190178 was
also added to the solution. 10 mM stock solutions of synthesized compounds made in DMSO
were serially diluted (1:3), and four 14 ꢁL aliquots of each compound concentration was
transferred to opaque 384ꢀwell polypropylene plates for storage. Small volume transfers were
performed by a Tecan Freedom EVO liquid handling system using a 50H hydrophobic coated
pin tool that carried 100 nL (V&P Scientific). To obtain a maximum concentration of 300 ꢁM,
600 nl of each compound concentration was transferred into the 20 µL assay solution. Agonistic
binding was determined in the absence of LG190178 while competitive inhibition binding was
determine in the presence of LG190187. Fluorescence polarization was detected after 30
minutes at an emission/excitation wavelength of 635/685 nm (Alexa Fluor 647). LG190178 (Ki
= 150 nM) and DMSO were used as positive and negative controls in the agonistic binding
assay, respectively. In the competitive inhibition binding assay 3ꢀdibutylaminoꢀ1ꢀ(4ꢀhexylꢀ
phenyl)ꢀpropanꢀ1ꢀone was the positive control while DMSO was used as a negative control.
Controls were measured within each plate to determine the z’ factor, which assed the quality of
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the assay, and enable data normalization. Three independent experiments were carried out in
quadruplicate, and data was analyzed using nonlinear regression with a variable slope
(GraphPrism). Data reported in the Supporting Information.
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VDRꢀ and PPARδꢀ Mediated Transcription Assays. Human embryonic kidney (HEK) 293T cells
were purchased (ATCC) and cultured in 75 cm2 flasks (CellStar) coated in matrigel (BD
Bioscience, #354234). Cells were grown in DMEM/High Glucose (Hyclone, #SH3024301)
media to which nonꢀessential amino acids (Hyclone, #SH30238.01), 10 mM HEPES (Hyclone,
#SH302237.01), 5 x 106 units of penicillin and streptomycin (Hyclone, #SV30010), and 10% of
heat inactivated fetal bovine serum (Gibco, #10082147) were added. For the assay, cells at 70ꢀ
80% confluency were transfected by lipidꢀbased methods where 2 mL of untreated DMEM/High
Glucose media (without additives) containing: 1) transcription assay: 0.7 ꢁg of VDRꢀCMV
plasmid, 16 ꢁg of a CYP24A1ꢀluciferase reporter gene, LipofectamineTM LTX (75 ꢁL, Life
Technologies, #15338020), and PLUSTM reagent (25 ꢁL) or 2) 2ꢀ Hybrid Assay: 1.5 ꢁg of CMVꢀ
PPARδꢀLBDꢀGAL4ꢀDBD plasmid, 16 ꢁg of a 6xGal4ꢀ luciferase reporter gene,
LipofectamineTM LTX (75 ꢁL) and PLUSTM reagent (25 ꢁL) was added to the flask. After 16
hours of incubation at 37 ⁰C with 5% CO2, the cells were harvested with 3 mL of 0.05% Trypsin
(Hyclone, #SH3023601), added to 10mL of the assay buffer, DMEM/High Modified buffer
without phenol red (Hyclone, #SH30284.01) containing all the above mentioned additives plus
10 mM sodium pyruvate and 2% percent charcoal treated FBS (Invitrogen, #12676ꢀ011) instead
of HI FBS, and spun down for 2 minutes at 1000 rpm. The media was removed and cells were
reꢀsuspended in the assay buffer, DMEM/High Modified buffer without phenol red. Prior to
adding cells to sterile white, optical bottom 384ꢀwell plates (NUNC, #142762), plates were
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treated with 20 ꢁL per well of a 0.25% Matrigel solution. To each well, 20 ꢁL of cells were
added to yield a final concentration of 15,000 cells per well. After 4 hours, plated cells were
treated with 200 nL of small molecules (1:3 serial dilution, with maximum final concentration at
100 µM) and controls (VDR: DMSO and 10 nM 1,25(OH)2D3 [EC50 = 1 nM] and PPARδ:
DMSO and 30 nM GW0742 [PPARδ EC50 = 3.5 nM]) which were added using a Tecan
Freedom EVO liquid handling system with a 50H hydrophobic coated pin tool. In the
competitive inhibition assay, 1,25(OH)2D3 (10 nM) was also added to the assay wells containing
small molecule. After 16 hours of incubation at 37 ⁰C with 5% CO2, 20 ꢁL of BrightꢀGlo™
Luciferase Assay Kit (Promega, Madison, WI) was added to each well and luminescence was
read. Controls were measured within each plate to determine the z’ factor and to enable data
normalization. At least two independent experiments were performed in quadruplicate and data
was analyzed using nonlinear regression with variable slope (GraphPrism).
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Cytotoxicity Assay. Hek293T cells were plated according to the transcription assay protocol and
200 nL of serially diluted (1:3 in DMSO) small molecules (final maximum concentration at 100
µM) were transferred using a Tecan Freedom EVO liquid handling system with a 50H
hydrophobic coated pin tool. The controls for the cytotoxicity assay used were 3ꢀdibutylaminoꢀ
1ꢀ(4ꢀhexylꢀphenyl)ꢀpropanꢀ1ꢀone (100 ꢁM in DMSO, positive) and DMSO (negative). After 18
hours, cell viability was evaluated by adding 20 µL of Cell TiterꢀGlo™ Luminescence Assay Kit
(Promega, Madison, WI) to each well and reading luminescence on a Tecan Infinite M1000 plate
reader. Controls were measured within each plate to determine the z’ factor and to enable data
normalization. At least two independent experiments were performed in quadruplicate and data
was analyzed using nonlinear regression with variable slope (GraphPrism).
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ASSOCIATED CONTENT
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Supporting Information.
The supporting information is available free of charge on the ACS publication website at DOI:
It includes detailed descriptions of the chemical synthesis and compound characterization as well
as VDR activities for all compounds.
AUTHOR INFORMATION
Corresponding Author
*Email: arnold2@uwm.edu
Present Addresses
#Kelly A. Teskeꢀ Department of Pharmaceutical Sciences, University of Connecticut, 69 North
Eagleville Road, Unit 3092, Storrs, Connecticut 06269ꢀ3092, United States
ⱡDavid J. MaloneyꢀInspyr Therapeutics, 31200 Via Colinas, Suite 200, Westlaske Village, CA,
91362
Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval
to the final version of the manuscript.
Funding Sources
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This work was supported by the University of WisconsinꢀMilwaukee (UWM) (L.A.A.), the
UWM Research Growth Initiative (RGI Grant) (L.A.A.), National Institutes of Health Grant
R03DA031090 (L.A.A.), the UWM Research Foundation (Catalyst grant), the Lynde and Harry
Bradley Foundation (L.A.A.), the Richard and Ethel Herzfeld Foundation (L.A.A.), and the
Intramural Research Program of the National Center for Advancing Translational Sciences
(NCATS), National Institutes of Health (NIH) (G.R., A.S., A.J., A.Y., and D.M.)
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ABBREVIATIONS
NR, Nuclear Receptor; VDR, Vitamin D Receptor; PPARδ, γ, α, Peroxisome Proliferatorꢀ
Activated Receptor δ, γ, α;; SAR, structureꢀactivity relationship, DBD, DNAꢀBinding Domain;
LBD, LigandꢀBinding Domain; 1,25(OH)2D3, 1α, 25ꢀdihydroxyvitamind D3, HTS, High
Throughput Screening; FP, Fluorescence Polarization; AR, Androgen Receptor.
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