Structure-Activity Relationship of 2,4-Dichloro-N-(3,5-dichloro-4-(quinolin-3-yloxy)phenyl)benzenesulfonamide (INT131) Analogs for PPARγ-Targeted Antidiabetics

Article abstract of DOI:10.1021/acs.jmedchem.6b01727

Peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear receptor central to fatty acid and glucose homeostasis. PPARγ is the molecular target for type 2 diabetes mellitus (T2DM) therapeutics TZDs (thiazolidinediones), full agonists of PPARγ with

Full text of DOI:10.1021/acs.jmedchem.6b01727

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Article
Structure-Activity Relationship of 2,4-dichloro-N-(3,5-
dichloro-4-(quinolin-3-yloxy)phenyl)benzenesulfonamide
(INT131) Analogs for PPAR#-Targeted Antidiabetics
Rebecca L Frkic, Beatriz B. Rodriguez, Mi Ra Chang, Dana Kuruvilla, Anthony Ciesla,
Andrew D. Abell, Theodore M. Kamenecka, Patrick R. Griffin, and John B Bruning
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 09 May 2017
Downloaded from http://pubs.acs.org on May 10, 2017
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StructureꢀActivity Relationship of 2,4ꢀdichloroꢀ
Nꢀ(3,5ꢀdichloroꢀ4ꢀ(quinolinꢀ3ꢀ
yloxy)phenyl)benzenesulfonamide (INT131)
Analogs for PPARγꢀTargeted Antidiabetics
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Rebecca L. Frkica, Beatriz B. Rodriguezc, Mi Ra Changb, Dana Kuruvillab Anthony
Cieslab, Andrew D. Abellc, Theodore Kameneckab, Patrick R. Griffinb, and John B.
Bruninga,*
aSchool of Biological Sciences
cDepartment of Chemistry
The University of Adelaide
Adelaide, South Australia, 5005, Australia
b Department of Molecular Therapeutics, The Scripps Research Institute, Scripps Florida , Jupiter,
Florida 33458, United States.
*To whom correspondence should be addressed. John B. Bruning, School of Biological Sciences,
Adelaide, South Australia, Australia, Tel.: +61 (08) 8313ꢀ5218; Fax: +61 (08) 8313ꢀ4362; Eꢀmail:
john.bruning@adelaide.edu.au
ABSTRACT
Peroxisome ProliferatorꢀActivated Receptor γ (PPARγ) is a nuclear receptor central to fatty
acid and glucose homeostasis. PPARγ is the molecular target for type 2 diabetes mellitus
(T2DM) therapeutics TZDs (thiazolidinediones), full agonists of PPARγ with robust antidiabetic
properties, which are confounded with significant side effects. Partial agonists of PPARγ, such
as INT131 (1), have displayed similar insulinꢀsensitizing efficacy as TZDs, but lack many sideꢀ
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effects. To probe the structureꢀactivity relationship (SAR) of the scaffold 1, we synthesized 14
analogs of compound 1 which revealed compounds with higher transcriptional potency for
PPARγ and identification of moieties of the scaffold 1 key to high transcriptional potency. The
sulfonamide linker is critical to activity, substitutions at position 4 of the benzene ring A were
associated with higher transcriptional activity, substitutions at position 2 aided in tighter
packing and activity, and the ring type and size of ring A affected the degree of activity.
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INTRODUCTION
The Peroxisome ProliferatorꢀActivated Receptor γ (PPARγ) is a transcription factor and member of
the multiꢀdomain ligandꢀmodulated nuclear receptor superfamily. PPARγ performs its function in part
through heterodimerization with the nuclear receptor Retiniod X Receptor α (RXRα) and binding to
DNA response elements in the proximal promoter region of target genes to regulate their expression.
PPARγ target genes include proteins involved in peripheral insulin sensitivity, adipogenesis, fatty acid
uptake and storage, glucose homeostasis, and metabolism of lipids and carbohydrates 1ꢀ4. The array of
PPARγ target genes makes this receptor essential for normal insulin sensitivity and proper regulation
of blood glucose. For instance, dominant negative partial loss of function mutations in PPARγ cause
severe insulin resistance and are often accompanied by the onset of type 2 diabetes 5. Adipose depots
secrete various cytokines and fat cell specific hormones called adipokines including adiponectin,
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adipsin, and resistin, many of which are under direct or indirect transcriptional control of PPARγ .
These proteins modulate insulin sensitivity of muscle, liver and adipose depots. An imbalance in this
process leads to the development of insulin resistance and eventually type 2 diabetes.
The key role of PPARγ in metabolism has made it an appealing target for therapeutics of type
2 diabetes. The thiazolidinedione (TZD) class of PPARγ modulators, rosiglitazone (Avandia,
GlaxoSmithKline) and Pioglitazone (Actos, Takeda), bind tightly within the ligand binding pocket of
PPARγ and fully agonize the receptor by driving the interaction of the receptor with transcriptional
coꢀactivator proteins. Although TZDs afford robust insulin sensitization and normalization of blood
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glucose in T2DM patients, treatment with TZDs has been linked to an array of adverse side effects
which has significantly reduced their utility. TZD side effects include weight gain, increased
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adipogenesis, renal fluid retention, loss of bone density, congestive heart failure, and plasma volume
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expansion leading to hemodilution
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exhibited by some patients using TZD antidiabetics have not yet been elucidated to date. Given that
PPARγ is a promiscuous binder of low affinity fatty acids and other metabolic signaling molecules,
one possibility is that side effects arise from disruption of the natural signaling processes by a very
high affinity agonist such as rosiglitazone, hyperactivation of target genes yet unidentified, or offꢀ
target interactions. However, ligands of PPARγ that only partially agonize the receptor have been
shown to have reduced sideꢀeffect profiles in preclinical species and in some cases in clinical trials,
yet they maintain robust insulinꢀsensitizing properties 9, 10. These observations suggest that insulin
sensitization can be separated from some if not all of the adverse effects associated with TZDs.
Previous structural analysis of partial agonists of PPARγ demonstrated that their unique
1,
transcriptional output is attributed to distinct binding mechanisms 11, suggesting that structural
properties of PPARγ ligands can govern their therapeutic index as T2DM therapeutics.
The
PPARγ
compound
2,4ꢀdichloroꢀNꢀ(3,5ꢀdichloroꢀ4ꢀ(quinolinꢀ3ꢀ
yloxy)phenyl)benzenesulfonamide 1 (INT131) is a highly potent ligand of PPARγ, with a Ki of 10nM
in ligandꢀdisplacement direct binding assays, sufficient to displace rosiglitazone from the ligand
binding pocket 11. 1 is highly potent in cell based transcriptional activation assays, with an EC50 value
of 4nM and a maximal transcriptional activation of reporter genes of approximately 30% as compared
to rosiglitazone. Distinct coregulatory recruitment profiles of 1, as compared to full agonists such as
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rosiglitazone, have been shown through cell based functional assays
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In vivo studies in rodents
have shown that 1 lowers blood glucose levels by over 30% with only a 0.3mg/kg dose compared to
the 3mg/kg dose required for a similar effect using rosiglitazone. Significantly, 1 showed less total
weight gain, heart weight gain, and lung weight gain than that observed with rosiglitazone
administration, supporting the idea that 1 leads to less adipogenesis and fluid retention/edema.
Adipocyte differentiation was not enhanced in cultured cells treated with 1 in contrast to significant
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induction with rosiglitazone treatment. Phase 1 studies with 1 (4 studies) showed favorable
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pharmacokinetic and pharmacodynamic properties . Phase 2a studies have demonstrated through a
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multicenter, double blind, placebo controlled study with T2DM patients that 1 is generally well
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tolerated while displaying antidiabetic effects
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related to phase 2b or phase 3 studies of this compound.
While partial agonists such as 1 have shown great promise as insulin sensitizers, their
mechanism of action has been less forthꢀcoming. Antidiabetic effects have been shown to be
correlated to blocking of phosphorylation of the receptor at position Ser273 of the ligand binding
domain (LBD) by CDK5ꢀactivated ERK. Blockage of pS273 normalizes PPARγ target genes that are
repressed in the diabetic and obese state 7, 14. However, the structural mechanism of this phenomenon
is not well understood.
PPARγ is a multiꢀdomain protein containing a highly conserved DNA binding domain and a
structurally conserved LBD. The LBD of PPARγ is comprised of 13 αꢀhelices (H1ꢀH12 and H2’) and
a small βꢀsheet. The binding pocket within the LBD is large enough (approximately 1200 Å3) to
accommodate binding of a wide range of structurally distinct ligands, and the exact nature of the
endogenous ligand remains controversial 15. Within the LBD is the AF2 surface which is formed by
helices 3ꢀ5 and helix 12, and is important for ligandꢀdependent cofactor binding 1, 2, 16. The TZD head
group of full agonists such as rosiglitazone have been shown to stabilize helix 12 and the AF2 by
means of a tight hydrogen bond network allowing for coꢀactivator binding, as well as making
hydrophobic contacts with H3 and stabilizing hydrogen bonding with the betaꢀsheet. Interestingly,
partial agonists of the receptor have been shown to require no stabilization of helix 12 for
transcriptional activation 1.
In order to better define the specific chemical epitopes of 1 responsible for high
transcriptional activity, we developed a SAR platform correlating chemistry, potency based on
activity, and protein structure. Here we present fourteen analogs of 1. These analogs were tested for
their ability to transcribe target genes (potency) in a cellular reporter assay allowing us to compare
activity to structure by means of EC50 values. An Xꢀray crystal structure was obtained for one of the
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analogs, 4ꢀbromoꢀNꢀ(3,5ꢀdichloroꢀ4ꢀ(quinolinꢀ3ꢀyloxy)phenyl)ꢀ2,5ꢀdifluorobenzenesulfonamide (10),
bound to the PPARγ ligand binding domain to a resolution of 2.2Å. The binding mode of the other 13
analogs was deciphered by means of in silico docking methods. The analogs displayed a wide range
of activity from an EC50 of 2nM to no binding at all, allowing us to define several important moieties
in the structureꢀactivity of the compound 1 scaffold. These analogs allowed us to define the
sulphonamide linker, positions 2 and 4 of benzene ring A, as well as the nature and position of the
substituents in ring A as being most important for defining high potency.
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RESULTS & DISUSSION
Design and activity of compound 1 analogs. Compound 1 is a 514 Da sulfonamide
compound comprised of three major aromatic moieties, denoted A, B, and C (Figure 1). A is a 2,4 diꢀ
Cl benzene linked by a sulphonamide to a 3, 5 diꢀCl aniline moiety (with aniline nitrogen as atom 1)
denoted B. Ring system C is a quinoline moiety joined to B by an ether linker. The coꢀcrystal
structure of 1 bound to PPARγ has been solved 11, which shows that, unlike TZDs, 1 does not contact
and stabilize helix 12 but instead wraps around helix 3 to stabilize helix 3 and the βꢀsheet region 11
(Figure 2). The ligand forms two hydrogen bonds with Tyr327 of helix 5, which donates a hydrogen
to the S=O group of 1 and accepts a hydrogen from the suflonamide NꢀH in the center of the
compound. A displaced parallel pi-pi interaction between the head group ring (ring A) of 1 and
Phe363 of helix 7 situated 3.7Å away further promotes ligand binding. The apolar region generated by
Ile341, Cys285, Gly284, and Phe363 is also important for its interaction with the aromatic rings of 1.
Previous structureꢀactivity data has indicated that the composition of the C ring system of 1
had profound effects on CYP450 inhibition (CYP3A4). Specifically, the presence of a pyridine ring
in the C position of 1 led to potent CYP3A4 inhibition (IC50 1nMꢀ1ꢀM) 4. However, changing the C
ring from pyridine to quinoline showed the highest affinity PPARγ direct binding with significantly
reduced CYP3A4 inhibition. Additionally, Cl substitution of positions R5 and R6 of the B ring aniline
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demonstrated the highest affinity PPARγ direct binding and transactivation . In order to define the
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structure activity relationship of compound 1 analogs, we focused on altering the substitution of the A
ring given the need for a quinoline group in the C ring for drug metabolism reasons and the need for a
dichloroꢀaniline ring at the B position for affinity reasons. The purpose of our SAR is to precisely
define what chemical epitopes of 1 and, conversely, what regions of the PPARγ LBD need to be
stabilized for optimum affinity as well as transactivation. Additionally, given the appeal of
antagonists of PPARγ as therapeutic agents, compound 1 analogs with reduced transactivation
potential are likely to be required for an attractive therapeutic profile with a very low or no incidence
of side effects.
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14 analogs of 1 (as well as 1) were synthesized with various alterations of the A ring (Table
1) including one previously reported compound, (4ꢀbromoꢀNꢀ(3,5ꢀdichloroꢀ4ꢀ(quinolinꢀ3ꢀ
yloxy)phenyl)ꢀ2ꢀ(trifluoromethoxy)benzenesulfonamide, described in our assays as compound 34.
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Synthesis was performed following reported protocols as in the materials and methods
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Substitutions of the benzene ring included Br, F, CF3, OꢀCH3, as well as CH3 at varying positions.
The benzene ring was substituted for rings of a different nature in two of the compounds:
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naphthalene ring system (Nꢀ(3,5ꢀdichloroꢀ4ꢀ(quinolinꢀ3ꢀyloxy)phenyl)naphthaleneꢀ2ꢀsulfonamide
(12) and a thiophene ring Nꢀ(3,5ꢀdichloroꢀ4ꢀ(quinolinꢀ3ꢀyloxy)phenyl)thiopheneꢀ2ꢀsulfonamide (15).
These substituents were chosen to probe the effect of having a ring other than benzene in the A ring
position of 1. All compounds contained a sulfonamide linker to the A ring with the exception of Nꢀ
(3,5ꢀdichloroꢀ4ꢀ(quinolinꢀ3ꢀyloxy)phenyl)thiopheneꢀ2ꢀcarboxamide (7) containing an amide linker to
the A ring in order to probe the effect on binding and activity of substitution at this position.
All compounds were tested for activity against PPARγ in a cell based transcriptional reporter
assay to measure both potency (EC50) as well as the maximal transcriptional output as normalized to
the model full agonist rosiglitazone (transactivation = 100%). Potencies of the compounds ranged
from 2nM (3) to no activity (7). All EC50 and maximal transactivation values can be found in Table 1
for all compounds. The maximum transactivation level for 1 was 24% and our analogs displayed
maximum transactivation levels in the range of 2ꢀ34%, indicating that all compounds were partial
agonists.
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Structural analysis of compound 1 analogs in complex with PPARγ. In order to probe the
structural basis of 1 analog potency, we performed coꢀcrystallization experiments of compound 1
analogs with PPARγ. Coꢀcrystals were only obtained for 4ꢀbromoꢀNꢀ(3,5ꢀdichloroꢀ4ꢀ(quinolinꢀ3ꢀ
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yloxy)phenyl)ꢀ2,5ꢀdifluorobenzenesulfonamide (10) in complex with the PPARγ LBD. The PPARγ
LBD bound to 10 was solved to a resolution of 2.2Å and the phase problem was overcome by
molecular replacement. Data processing and refinement statistics can be found in Table 2. The
asymmetric unit contained two subunits of PPARγ (homodimer), conforming to the canonical PPARγ
LBD fold. The 10 bound LBD structure revealed high global similarity to previously solved
structures with a 0.84Å RMSD with the LBD from the full agonist rosiglitazone bound structure (over
256 Cα atoms, PDB:2PRG), a 0.69Å RMSD with the LBD from the apo structure (over 258 Cα
atoms, PDB:1PRG), and a 0.88Å RMSD with the partial agonist 1 bound LBD (over 254 Cα atoms,
PDB:3FUR) 11, 17. A ribbons diagram of the 10 bound LBD can be seen in Figure 2A. The 10 ligand
was easily visible in the electron density and modelled into the difference Fourier electron density. A
reduced model bias electron density map of 10 can be viewed in Figure S1.
Compound 10 occupies the ligand binding pocket of PPARγ and is centered at and bends
around helix 3; the scaffold location of 10 is similar to 1 (Figure 2B and 2C). Several weak
electrostatic interactions are formed between the sulfonamide linker of 10 and residues of or near the
AF2. One oxygen atom of the sulfonamide moiety of 10 is in weak hydrogen bonding distance to
Tyr327 side chain oxygen atom (3.5Å), Lys367 side chain nitrogen atom (2.9Å), and His449 side
chain nitrogen atom (3.5Å). This leaves Tyr473 of the AF2 helix 12 unstabilized and not within
hydrogen bonding distance to His449 (4.6Å) as seen in full agonist bound structures. Tyr327 accepts a
hydrogen atom from the sulfonamide NꢀH in the center of the 10 compound to form a 3.2Å hydrogen
bond analogous to 1 (2.8Å). A comparable set of pi-pi interactions between the A ring of 10 and
Phe363 exists and likely contributes to ligand binding affinity. 10 engages in hydrophobic contacts
with Ile341 (beta sheet), Cys285 (H3), and Gly284 (H3), similarly to 1 (Figure 2C). 10 contains a Br
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atom at position 4 of the A ring (in contrast to a Cl in 1) which is in weak halogen bonding distance to
the main chain nitrogen atom of Phe282 (3.7Å).
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In silico docking of all other ligands (those other than 10) shown to bind to the PPARγ LBD
was carried out using a Monte Carlo method in the Molsoft ICM Software suite to identify the binding
mechanisms of these compounds. As a positive control, 1 was docked into the PPARγ LBD and
compared to the experimentally derived Xꢀray crystal structure. The binding mode of the two
structures was nearly identical and a superimposition of the two molecules is located in Figure S2.
Docking of the 1 analogs revealed a similar overall binding mode for them all, with the C and B rings
exhibiting very high positional similarity with only minor differences in the A rings due to
substitution. A superimposition of the ligands docked to the receptor can be found in Figure 3.
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The sulfonamide moiety is essential for binding to PPARγ. The A ring of 1 is located in the
hydrophobic pocket between H3 and H7, making not only hydrophobic interactions with Cys285 of
H3 but more importantly several critical pi-pi interactions with Phe363. The A ring of 1 is connected
to the 1 scaffold via the S atom of the sulfonamide linker. The sulfur atom displays tetrahedral
geometry which places the connected A ring in ideal position to interact with Phe363 in a stacking
manner (Figure 2B).
To probe if the sulfonamide linker of 1 is necessary for activity we synthesized 7. Table 1
shows that 7 shows no significant transactivation activity and contains an amide linker instead of the
sulfonamide of the other ligands. This substitution makes the atom which connects the A ring to the
compound 7 scaffold, C of the C=O, in a planar Sp2 hybridized state. As shown in Figure 4, this
places the A ring in a location closer to the AF2 surface and incapable of making the favorable pi-pi
interactions with Phe363 as well as the favorable hydrophobic interactions in the surrounding pocket.
The sulfonamide moiety absent in 7 also means that there is no atom in appropriate distance to act as a
hydrogen bond donor or acceptor with the side chain of Tyr327. Taken together, our data thus
suggests that the A ring linker geometry, and to a lesser degree interaction with Tyr327, is essential
for significant receptor binding and activity. This is critical for potential drug design, as the inclusion
of a sulfonamide moiety will substantially promote compound binding to PPARγ.
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Higher affinity is achieved with Br at position 4 of benzene ring A. Ligands with the nine highest
potencies (2ꢀ957nM) all have a Br atom at position 4 of the A ring (4ꢀbromoꢀNꢀ(3,5ꢀdichloroꢀ4ꢀ
(quinolinꢀ3ꢀyloxy)phenyl)ꢀ3ꢀmethylbenzenesulfonamide (2), 3, 4ꢀbromoꢀ2ꢀchloroꢀNꢀ(3,5ꢀdichloroꢀ4ꢀ
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(quinolinꢀ3ꢀyloxy)phenyl)benzenesulfonamide
(4),
4ꢀbromoꢀNꢀ(3,5ꢀdichloroꢀ4ꢀ(quinolinꢀ3ꢀ
yloxy)phenyl)ꢀ3ꢀ(trifluoromethyl)benzenesulfonamide (5), 4ꢀbromoꢀNꢀ(3,5ꢀdichloroꢀ4ꢀ(quinolinꢀ3ꢀ
yloxy)phenyl)ꢀ2ꢀ(trifluoromethyl)benzenesulfonamide (6), 4ꢀbromoꢀNꢀ(3,5ꢀdichloroꢀ4ꢀ(quinolinꢀ3ꢀ
yloxy)phenyl)ꢀ2ꢀfluorobenzenesulfonamide
(8),
4ꢀbromoꢀNꢀ(3,5ꢀdichloroꢀ4ꢀ(quinolinꢀ3ꢀ
yloxy)phenyl)ꢀ3ꢀfluorobenzenesulfonamide (9), 10, and 4ꢀbromoꢀNꢀ(3,5ꢀdichloroꢀ4ꢀ(quinolinꢀ3ꢀ
yloxy)phenyl)benzenesulfonamide (11). In addition, the five lowest affinity ligands (3314nMꢀno
significant activity) all lack Br at his position. Superimposition of Brꢀcontaining compound 1 analogs
at different positions in the A ring can be seen in Figure 5. Structural analysis reveals that the Br
atom is located in nearly identical positions in all of these structures. This places the Br atom within
distance to form a halogen bond with the back bone nitrogen atom of Phe282 (Figure 5) and allows
for better packing within the ligand binding pocket through Van der Waals interactions. The halogen
bond is indeed weak (4Å) and improved activity from substitution at this site with Br is perhaps
associated with tighter packing of the pocket in this region. This is clearly an important observation
for ongoing drug design efforts with 1 which would therefore ideally incorporate Br at this position or
place a hydrogen bonding partner extending from position 4 of the A ring in proximity to the back
bone nitrogen atom of Phe282.
Tight packing within the ligand binding pocket is associated with higher activity. Examination of
space filling representations of the compound 1 analogs within the ligand binding pocket of PPARγ
shows that the highest affinity ligands are distinguished from lower affinity ligands by means of
tighter molecular packing. The five ligands (3, 4, 6, 8 and 10) with the highest affinities (2ꢀ957nM)
present a substituent at position 2 on benzene ring A. These substituents contribute steric bulk to the
ligand and more tightly occupies the space of the binding pocket between helices 3 and 7, increasing
the strength and number of van der Waals interactions. Substitutions at position 2 of the benzene ring
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A of these compounds include –F, ꢀCF3, ꢀCl, and –OCF3. Van der Waals volumes for these groups
range from 13Å3 to 100Å3, with a general trend of the smaller van der Waals volume substitutions
correlating with the lower activity. As shown in Figure 6A, position 2 of the benzene ring A is most
amenable to substitution for improved packing such that compounds with substitutions on other
positions do not pack as favorably in the binding pocket (Figure 6B) and induce lower activation rates
of the receptor. Despite 3 and 6 having very similar structures (– OCF3 versus – CF3), 3 is
significantly more potent than 6 because the addition of the oxygen enables the triflouro group to pack
deeper in the binding pocket between helices 3, 7 and 11 (Figure 6C). Furthermore, this extension also
enables two of the fluorine atoms to be within halide bond distance (both approximately 3Å) of the
side chain nitrogen atom of Gln286, which further increases the compound’s affinity for PPARγ.
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Divergence from aromatic benzene ring A does not favor high activity. Two of the compound 1
analogs (12 and 15) differ from the otherwise conserved Aꢀring containing a single aromatic benzene.
These two ligands have very low potencies (4289nM and ND respectively). This suggests that the sixꢀ
membered aromatic ring is optimal for PPARγ activity. 12 has a naphthalene moiety, sizably
enhancing its hydrophobicity at this position as compared to the other analogs (Figure 7). Despite
having many more interactions with hydrophobic residues of the binding pocket, its potency is poor,
with an EC50 of 4289nM. This may be due to the nature of the aromatic system. Rings B and C of 12
are in nearly identical positions to the B and C rings of 1 and other analogs (Figure 3), suggesting that
differences in activity lie solely in the A ring. Additionally, the naphthalene moiety of 12 is in a
parallel displaced stacking interaction with Phe363 positioned similarly to ring A of the other analogs.
While the naphthalene group is larger than the benzene ring A of the other analogs, it packs well
without clashing into the hydrophobic pocket of the LBD between H3 and H7 (Figure 6D). Although
12 can form stacking interactions with Phe363, the activity is much lower than the other compound 1
analogs implying that differences in affinity may lie in the absence of substitutions on the naphthalene
group. 12 is the only compound that lacks a substituent on the A ring. The Hunter/Sanders model has
demonstrated that electron withdrawing substituents, such as the halides, diminish electron density in
the pi cloud of the ring leading to enhanced piꢀstacking 18. This is consistent with our data in which
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the electron withdrawing substitutions of the A ring such as F, Br, and Cl show stronger activity. In
agreement with this hypothesis, is compound Nꢀ(3,5ꢀdichloroꢀ4ꢀ(quinolinꢀ3ꢀyloxy)phenyl)ꢀ2,4,6ꢀ
trimethylbenzenesulfonamide (14) which is substituted with no electron withdrawing groups, only
methyl groups (electron donating), which also shows diminished activity as compared to the analogs
with electron withdrawing substitutions.
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Compound 15, where the benzene ring was replaced by a thiophene moiety, showed no data
in terms of potency for the receptor. Importantly, 15 does not make pi-pi interactions with Phe363,
with the component thiophene known to be less aromatic than benzene, which provides less
availability for pi-pi interaction, likely decreasing binding affinity. It could be postulated that the
interaction with Phe363 favors higher affinity ligands. Critically, structural analysis also demonstrated
15 does not pack as well in the PPARγ LBD due to the lack of substitution patterns characteristic of
the higherꢀaffinity compounds. This includes a halide at position 4, and steric bulk in positions 2 or 3.
The fiveꢀmembered thiophene ring does not pack as tightly in the hydrophobic pocket as the other
analogs due to lack of substitution as well as the smaller, fiveꢀmembered ring size. This, taken
together, suggests that a single aromatic benzene forms the best scaffold for ring A.
Substitutions at benzene ring A of compound 1 have modest effects on the degree of agonism.
Given the promise of PPAR antagonists as a novel class of antidiabetics with little or no side effects,
decreasing the degree of agonism of 1 would be ideal. There is some variation in the level of
transactivation of the receptor among all of the compound 1 analogs we surveyed as maximum
transactivation rates were in the range of 2ꢀ34%. While this is a similar transactivation rate from the
compound 1 parent compound (24% maximal transactivation) further decreases in transactivation will
require more SAR efforts. Our data suggests that modifying the ligands at the A ring moiety affects
only their affinities for the receptor, and not their level of agonism. Comparing the binding
mechanisms of the current compounds with full agonists demonstrates that they bind in different
positions and this governs their level of transcriptional output. The consistent binding mechanisms of
the partial agonists can be attributed to the presence of the conserved regions of rings B and C. It is
important for design of T2DM therapeutics using the compound 1 scaffold to include these moieties,
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so that the compound will have high affinity binding while not fully agonizing the receptor. Lessons
from other scaffolds have revealed that reducing agonism can be achieved through destabilization of
the AF2 surface and H12. Hence, future compound 1 based therapeutics with lessoned transcriptional
activation may be achieved through substitution of the quinolone ring extending to H12 to form
destabilizing contacts.
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CONCLUSION
We have described here the synthesis of 14 analogs of the antidiabetic compound 1 by means of
chemical alteration of the A ring. This study includes protein structural studies which when combined
with transcriptional activation assays (which measure potency of the compound 1 analogs) allow the
derivation of precise structureꢀactivity relationships of these compounds. This SAR study defined the
effects that substituents of the A ring, position of substituents on the A ring, and the type of ring at
position A have on transcriptional activity and, more importantly, how these substituents affect
interaction of the compound 1 scaffold with the PPARγ receptor. SAR of compound 1 analogs
revealed seven ligands with increased potency for PPARγ. These maintained the sulfonamide moiety
and a bromine atom at position 4 on the aromatic benzene ring A. They differ from 1 in their
substitutions which enable better lockꢀandꢀkey fitting in the binding pocket of PPARγ, mediated by
the presence of a bulky substitution at position 2 of benzene A. Additionally, the SAR data
demonstrates the importance of a sulfonamide linker as well as a substituted, 6ꢀmembered benzyl ring
in position A. Taken together, these results present a clearer picture of the molecular mechanism of 1
and how future drug discovery efforts for new therapeutics targeting PPARγ can be tailored for higher
potency.
EXPERIMENTAL SECTION
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Transactivation assay. 4.5 ꢀg human GAL4ꢀPPARγꢀHingeꢀLBD, 4.5 ꢀg 5x multimerized UASꢀ
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luciferase reporter, and 27ꢀL Xꢀtreme Gene 9 transfection reagent were cotransfected into HEK293T
cells (ATCC; cat. no. CRLꢀ3216) and grown in serumꢀfree OptiꢀMEM reduced serum media (Gibco).
The Gal4ꢀpBind vector and UASꢀLUC were coꢀtransfected into cells as a control. Cells were grown
for 18h at 37oC in a 5% CO2 incubator after which they were plated in quadruplicate in white 384ꢀ
well plates (PerkinElmer) at a density of 10,000 cells in each well. Cells were treated with either
DMSO only or the compound of interest (doses from 169pM to 10ꢀM) after replating. Cells were
treated with Brite Lite Plus (PerkinElmer) after 18 h incubation and read in a 384ꢀwell Luminescence
PerkinElmer EnVision Multilabel plate reader. Fold change of treated cells over DMSOꢀtreated
control cells were plotted to define EC50 values. GraphPad Prism was used for plotting and statistical
analysis including error bars. Each data point in the EC50 does response was repeated in triplicate and
standard Error of mean (S.E.M.) was derived from these values. In addition, two biological replicates
were performed to ensure reproducibility of the data
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Protein purification. The PPARγ ligand binding domain (residues 205ꢀ477) including an Nꢀterminal
hexaꢀhistidine tag were encoded in the pET11 expression vector. The expression vector was
transformed into E. coli BL21(DE3). Cells were grown at 37oC in LB media containing 50ꢀg/mL
ampicillin until an optical density of 0.5 was reached. Cells were induced at 16oC for 18 hours,
harvested by centrifugation, resuspended in buffer A (20mM Tris 8.0, 0.5M NaCl, 10mM imidazole,
and 2mM BME), and stored at ꢀ80oC. Cells were lysed by three passes through a French Press. Cell
lysate was clarified by centrifugation at 10,000g for 1 h and applied to a 5mL HisꢀTrap FF crude
column (GE Healthcare), washed with 100mL buffer A, and eluted with 25mL buffer B (20mM Tris
8.0, 0.5M NaCl, 250mM imidazole, and 2mM BME). The elution fractions were pooled and dialyzed
to buffer C (20mM Tris 8.0, 10mM NaCl, and 1mM DTT) using a 10,000 molecular weight cutꢀoff
dialysis bag (Spectrum Laboratories, Inc.) for 18 hours). The PPARγ sample was applied to a HiPrep
26/60 Sephacryl Sꢀ300 HR sizeꢀexclusion column and eluted at 1mL/minute over one column
volume. Fractions containing purified PPARγ were concentrated to 10mg/mL using a 10,000
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molecular weight cutꢀoff centripetal concentrator (Millipore). Protein used in crystallization trials
was used fresh and without freeze/thaw (within three days of preparation).
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Crystallization/Data Processing. Crystallization trials were carried out with all compounds,
but only compound 10 produced diffracting coꢀcrystals. For complex formation, a sample of
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10mg/mL PPARγ LBD was mixed with 10 (5mM final concentration 10) and incubated on ice for 30
minutes. Prior to crystallization the sample was clarified by centrifugation at 10,000 g at 4oC for 10
minutes and the supernatant was extracted for use in crystallization trials. Crystals were formed using
the vapor diffusion method by mixing 1ꢀL of PPARγꢀ10 complex with 1ꢀL well solution in an Intelliꢀ
plate (Art Robbins) using a sitting drop style plate. The well solution consisted of 75ꢀL 2M
ammonium sulfate. Crystallization trials were conducted at 289 K. Cubic crystals of approximately
150 microns in each dimension appeared after three days. The well solution containing 15% ethylene
glycol was used as a cryoꢀprotectant. Crystals were harvested using a cryoꢀloop (Hampton Research)
and flash cooled to 100 K. All data was collected at 100 K. Data was collected at APS beamline 22ꢀ
ID. 450 images were collected at 0.5o oscillations (225o data total) at 0.6 second exposure time per
image. Data was processed using iMosflm19 and scaled in aimless20 to a resolution of 2.2Å.
Resolution cutꢀoff was determined by use of the CC1/2 criteria21. PDB:3FUR stripped of ligands and
water molecules was used as a search model and phases were obtained by molecular replacement in
Phaser22. Initial difference Fourier maps revealed clear electron density for 10 and was modelled
manually. Refinement, including TLS refinement, was carried out in Phenix23 with multiple rounds of
manual rebuilding carried out in Coot24. Refinement was completed when Rꢀfactors converged.
Unsuccessful soaks of PPARγ apo crystals were diffracted at the Australian Synchrotron beamline
MX1 and MX225.
Docking. The ICM Molsoft suite26 was used to dock compound 1 analogs into the PPARγ LBD
structure. PDB 3FUR with ligands removed was used as the starting model for docking. The PPARγ
LBD structure was prepared for docking by protonation, deletion of water molecules, and energy
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minimization by means of the ICM force field and distance dependent dielectric potential with an
RMS gradient of 0.1. PocketFinder within ICM was used to define the ligand binding pocket and was
consistent with previously published Xꢀray structures. Default settings within the ICM docking
module were used with a rectangular box centered at the LBD with a grid spacing of 0.5Å. The top
ranked docking for each ligand was chosen for interpretation as the conformations were very
consistent with scaffold placement of 1 and 10 in the Xꢀray crystal structure.
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ASSOCIATED CONTENT
Additional figures (S1 and S2) illustrating sample electron density and comparison of the docking to
structures derived from experimental Xꢀray. Details of compounds synthesis including H1 NMR, MS
and HPLC analysis. This material is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: +61 (08) 8313 5218. Eꢀmail: john.bruning@adelaide.edu.au.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGEMENTS
We would like to thank the staff at SERꢀCAT beamline for assistance with data collection. Use of the
Advanced Photon Source was supported by the U. S. Department of Energy, Office of Science, Office
of Basic Energy Sciences, under Contract No. Wꢀ31ꢀ109ꢀEngꢀ38. We would like to thank the staff at
beamlines MX1 and MX2 of the Australian Synchrotron for help with data collection of the PPAR
soaks (This work was supported by CAP6919 and CAP8221 of the AS). This work was funded in
part by NIH NIDDK DK105825 PI: Griffin.
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The PDB code for PPARγ in complex with 10 is 5TTO. Authors will release the atomic coordinates
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and the experimental data upon article publication.
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ANCILLARY INFORMATION
Molecular Formula Strings. Strings are available in the supporting information.
Abbreviations. PPAR: peroxisome proliferator activated receptor; TZD: thiazolidinedione;
SAR: structure activity relationship; RXR: retinoid X receptor; T2DM: Type II Diabetes
Mellitus.
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43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Garman, E.; Gonzalez, A.; Sauter, N. K.; Phizackerley, R. P.; Soltis, S. M.; Kuhn, P. BluꢀIce and the
distributed control system: software for data acquisition and instrument control at macromolecular
crystallography beamlines. J. Synchrotron Radiat. 2002, 9, 401ꢀ406.
(26)
Abagyan, R.; Totrov, M. Biased probability Monte Carlo conformational searches and
electrostatic calculations for peptides and proteins. J. Mol. Biol. 1994, 235, 983ꢀ1002.
Table 1. Structure Activity of Compound 1 Analogs. Each data point in the EC50 does response
was repeated in triplicate and standard deviation was derived from these values. In addition, two
biological replicates were performed to ensure reproducibility of the data.
Formula
Compound
EC50 (nM)
n=3
Trans-
activation (%)*
n=2
Percent
Purity
(%)
1 (INT131)
>95
>99
170±10
600±61
24±4
29±6
2
4±1
21±9
3
>99
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4
85
97
93
5
6
7
8
134±13
27±8
18±7
9
10
11
12
13
14
15
16
17
18
19
20
21
22
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
5
957±499
94±10
ND
34±10
2±1
6
7
>99
88
2±1
13±7
8
9
94
169±14
131±6
9±5
10±6
10
> 98
11
12
96
90
151±20
11±6
4289±289
23±12
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4
13
5
6
7
8
5810±945
19±11
14±6
14±8
99
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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30
31
32
33
34
35
36
37
38
39
40
41
42
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49
50
51
52
53
54
55
56
57
58
59
60
14
>99
3314±2923
15
>99
ND
Table 2. Crystallographic Data.
Parameter
Compound (10)
C2
Space group
Cell dimensions a, b, c (Å)
Monoclinic angle β (deg)
Xꢀray source
93.1, 62.2, 118.9
102.2
Synchrotron: APS 22ꢀID
Wavelength (Å)
Resolution range (Å)
Last shell (Å)
1.0
50ꢀ2.24
2.32ꢀ2.25
0.051 (0.178)
Rmerge (%)
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9
Observations
146759 (13592)
31321 (2946)
15.3 (5.9)
Unique reflections
Mean (I)/σ(I)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
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43
44
45
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47
48
49
50
51
52
53
54
55
56
57
58
59
60
Completeness
Multiplicity
98.0 (99.7)
4.7 (4.6)
Structure refinement
Resolution range (Å)
45.5ꢀ2.24
0.2039
Rwork (%)
Rfree (%)
0.2555
Total number of
Nonꢀhydrogen atoms
Protein atoms
Ligand atoms
Water molecules
4139
64
159
RMSD
Bond length (Å)
Bond angle (deg)
0.007
0.878
Bꢀfactors (Å2)
Overall
61.2
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Average protein atoms
Average ligand atoms
Average solvent
61.3
63.2
58.8
10
11
12
13
14
15
16
17
18
19
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21
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59
60
Ramachandran statistics
Most favoured regions (%)
Allowed regions (%)
96.5
3.29
0.19
Disallowed regions (%)
aValues in parentheses are for the highest resolution shell.
FIGURE LEGENDS
Figure 1: Chemical composition of partial agonist 1. The compound is comprised of three
major moieties, denoted A, B, and C. The potential substitution positions on ring A are
numbered.
Figure 2: Crystal structure of 10 bound to PPARγ LBD. (A) Ribbons diagram of the
PPARγ LBD (green) in complex with 10 (blue sticks). (B) Comparison of 10 (blue sticks)
binding mode to 1 (yellow sticks), with the main scaffold in the same position and some
similar hydrogen bonds formed (1 PBD:3FUR)11. (C) Superimposition of 10 and 1
(PDB:3FUR) in the region contacting the betaꢀsheet and H3.
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Figure 3: Superimposition of docked 0 analogs. The ligands (colored sticks) that have been
shown to bind to PPARγ were docked in silico to the PPARγ LBD receptor (blue ribbons).
The scaffold of the ligands binds in a similar position, with slight variations at ring A due to
substitutions at that location.
3
4
5
6
7
8
9
10
11
12
13
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60
Figure 4: The substitution of a sulfonamide for an amide linker has detrimental effects
on the capability of 7 to bind to PPARγ. Shown is a superimposition of the 7 structure
(white) with the 1 structure (green). The carbon of the C=O in 7 (white sticks) is confined to a
planar conformation which prevents the A ring of 7 from making favorable pi-pi interactions
with Phe363 as well as hydrophobic interactions with residues of the binding pocket.
Figure 5: Superimposition of ligands containing a 4ꢀBr substitution in benzene ring A
reveal very similar positions of the bromine atom within the binding pocket. Shown is a
superimposition of the compounds containing a bromine atom at positon 4 (colored sticks)
bound to PPARγ (green ribbons). A bromine at position 4 of ring A results in higher affinity
of the ligand. The presence of a Br atom in this position enables a weak hydrogen bond with
the backbone nitrogen of Phe282 (shown as dashes).
Figure 6: Effective space filling within the PPARγ LBD binding pocket has been shown
to correlate with higher affinities. Displayed are superimpositions of the compound 1
analogs (colored sticks) bound to PPARγ (ribbons, green or blue), and a surface
representation of the ligand binding pocket (grey surface). Ligands with substitutions at (A)
position 2 of ring A have better packing within the pocket and higher affinities than (B) those
which have substitutions at other positions. Ligands with substitutions at position 2 include 3
(red), 4 (orange), 6 (green), and 8 (cyan). Ligands with additional substitutions at positions 3
or 5 have lower affinities and include 2 (yellow), 5 (pale orange), 9 (teal), 11 (purple), 2ꢀ
chloroꢀNꢀ(3,5ꢀdichloroꢀ4ꢀ(quinolinꢀ3ꢀyloxy)phenyl)benzenesulfonamide (13) (light blue), 14
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(turquoise) 15 (lilac), and 10 (dark blue). (C) The oxygen linker in the trifluro substitution of
3 enables it to reach further into the binding pocket than 6 and confer a better lockꢀandꢀkey fit
in addition to forming additional hydrogen bonds with Gln286. (D) the naphthalene moiety of
12 (pink) extends as far into the binding pocket as other ligands, as shown by comparison
with 9 (teal), 10 (dark blue), and 11 (purple).
3
4
5
6
7
8
9
10
11
12
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Figure 7: The naphthalene substitution at position A of 12 enables extensive
hydrophobic contacts contributed by helices 3 and 7. Shown is a superimposition of the 1
(yellow sticks) and 12 (purple sticks) compounds bound to the PPARγ LBD (green ribbons).
The naphthalene moiety of 12 (purple) is more hydrophobic than other ligands, enabling
unique hydrophobic interactions with the PPARγ binding pocket. Structure compared to 1
(yellow).
Table of Contents Graphic
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