T-3364366 Targets the Desaturase Domain of Delta-5 Desaturase with Nanomolar Potency and a Multihour Residence Time

Article abstract of DOI:10.1021/acsmedchemlett.6b00241

Delta-5 desaturase (D5D) catalyzes the conversion from dihomo-gamma linoleic acid (DGLA) to arachidonic acid (AA). DGLA and AA are common precursors of anti- and pro-inflammatory eicosanoids, respectively, making D5D an attractive drug target for inflammatory-related diseases. Despite several reports on D5D inhibitors, their biochemical mechanisms of action (MOAs) remain poorly understood, primarily due to the difficulty in performing quantitative enzymatic analysis. Herein, we report a radioligand binding assay to overcome this challenge and characterized T-3364366, a thienopyrimidinone D5D inhibitor, by use of the assay. T-3364366 is a reversible, slow-binding inhibitor with a dissociation half-life in excess of 2.0 h. The long residence time was confirmed in cellular washout assays. Domain swapping experiments between D5D and D6D support [³H]T-3364366 binding to the desaturase domain of D5D. The present study is the first to demonstrate biochemical MOA of desaturase inhibitors, providing important insight into drug discovery of desaturase enzymes.

Full text of DOI:10.1021/acsmedchemlett.6b00241

Letter
pubs.acs.org/acsmedchemlett
T‑3364366 Targets the Desaturase Domain of Delta‑5 Desaturase
with Nanomolar Potency and a Multihour Residence Time
Ikuo Miyahisa,*,† Hideo Suzuki,† Atsushi Mizukami,† Yukiya Tanaka,† Midori Ono,† Mark S. Hixon,‡
and Junji Matsui*,†
†Pharmaceutical Research Division, Takeda Pharmaceutical Company Ltd., 26-1, Muraoka-Higashi 2-Chome, Fujisawa, Kanagawa
251-8555, Japan
‡Takeda California Inc., 10410 Science Center Drive, San Diego, California 92121, United States
S
* Supporting Information
ABSTRACT: Delta-5 desaturase (D5D) catalyzes the con-
version from dihomo-gamma linoleic acid (DGLA) to
arachidonic acid (AA). DGLA and AA are common precursors
of anti- and pro-inflammatory eicosanoids, respectively,
making D5D an attractive drug target for inflammatory-related
diseases. Despite several reports on D5D inhibitors, their
biochemical mechanisms of action (MOAs) remain poorly
understood, primarily due to the difficulty in performing
quantitative enzymatic analysis. Herein, we report a radio-
ligand binding assay to overcome this challenge and characterized T-3364366, a thienopyrimidinone D5D inhibitor, by use of the
assay. T-3364366 is a reversible, slow-binding inhibitor with a dissociation half-life in excess of 2.0 h. The long residence time was
confirmed in cellular washout assays. Domain swapping experiments between D5D and D6D support [3H]T-3364366 binding to
the desaturase domain of D5D. The present study is the first to demonstrate biochemical MOA of desaturase inhibitors,
providing important insight into drug discovery of desaturase enzymes.
KEYWORDS: Delta-5 desaturase, T-3364366, slow-binding inhibition, mechanism of action (MOA)
Scheme 1. Major Metabolic Pathway of Long-Chain
Polyunsaturated Fatty Acids in Vertebrates
icosanoids are lipid signaling molecules synthesized by the
oxidation of 20-carbon fatty acids and have numerous
E
important biological functions as key regulators of inflamma-
tion.1 The eicosanoids derived from arachidonic acid (AA,
C20:4 n-6) are generally pro-inflammatory substances, and
their excess or uncontrolled production is implicated in a wide
range of inflammation-related diseases from atherosclerosis to
cancer.2,3 Thus, proteins involved in the biosynthesis and signal
transduction of these eicosanoids have been intensively
investigated as drug targets with significant attention of the
pharmaceutical industry.4
brain, and lung, although also found in other organs at low but
detectable levels.7 D5D is a dual domain protein comprising a
desaturase domain and a cytochrome b5 domain. The
desaturase domain catalyzes a desaturation reaction using a
di-iron active center, while the cytochrome b5 domain transfers
electrons from NADH cytochrome b5 reductase to the active
center of the desaturase domain.
DGLA is a precursor of anti-inflammatory eicosanoids such
as prostaglandin E1 (PGE1) and 15-hydroxytrienoic acid.8
Thus, inhibition of D5D is expected to exert dual anti-
inflammatory action, by decreasing AA-derived pro-inflamma-
tory eicosanoids and simultaneously by increasing DGLA-
In vertebrates, AA is synthesized via a sequential enzymatic
conversion from linoleic acid (LA, C18:2 n-6), which is an
essential fatty acid. Desaturation of LA by delta-6 desaturase
(D6D) and subsequent C2 elongation by fatty acid elongase
(Elovl5) yield dihomo-gamma linoleic acid (DGLA; 20:3, n-6).
Next, delta-5 desaturase (D5D) introduces another double
bond between C5 and C6 of DGLA, to produce AA (Scheme
1).5 The final oxidation catalyzed by D5D is one of the rate-
contributing steps in AA biosynthesis.
D5D is a member of the fatty acid desaturase family
including D6D, delta-9 desaturase (stearoyl-CoA desaturase,
SCD), and dihydroceramide desaturase 1 and 2. D5D catalyzes
the O2-dependent dehydrogenation of dihomo-gamma lino-
leoyl-CoA to yield arachidonyl-CoA.6 D5D is an integral
membrane protein localized in the endoplasmic reticulum and
is expressed primarily in the liver and to a lesser degree in heart,
Received: June 23, 2016
Accepted: August 7, 2016
© XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.6b00241
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ACS Medicinal Chemistry Letters
Letter
derived anti-inflammatory eicosanoids. In fact, D5D-deficient
mice exhibited significant increase in PGE1 levels and decrease
in PGE2 levels compared to wild-type mice, leading to anti-
inflammatory and antiproliferative cellular phenotypes.9 Taken
together, inhibition of D5D by small molecules may attenuate
inflammatory responses, potentially creating a next-generation
anti-inflammatory agent. For example, Obukowicz et al.
reported the discovery of several selective D5D inhibitors
from compound screening, including CP-74006 (Chart 1) and
other chemical classes of inhibitors (Supporting Information,
Figure S1B).10 CP-74006 was further optimized to indazole
series, displaying in vivo activity.11
separation of D5D substrate and product, making the assays
insensitive or low-throughput (or both).14,15 Second, D5D is an
integral membrane protein and has yet to be purified to
homogeneity, preventing crystallographic analysis and bio-
chemical characterization with purified proteins, both of which
have widely been utilized for the analysis of the soluble
desaturases.16 Finally, strict control of active concentrations of
substrates is difficult since one substrate is molecular oxygen
and the other, a long-chain fatty acyl-CoA, forms micelles at
concentrations around its Km.17
To overcome the activity assay challenges, we directly
analyzed the binding of T-3364366 by means of a radioligand
binding assay using tritiated T-3364366 ([3H]T-3364366) as a
probe. Ligand binding assays using test compounds as probes
offer an alternative strategy for direct observations of the
kinetics and mechanisms of ligand binding in the absence of
substrates. A filter binding assay was developed using rat liver
microsomes D5D as a surrogate enzyme for the lower protein
stability observed with recombinant human D5D (hD5D).
Since T-3364366 exhibited similar inhibitory potencies in
human and rat cellular assays (Table 1), the results obtained
with rat liver microsomes should well reflect those with hD5D.
As an assay validation step, we performed a saturation
binding experiment by varying the tritiated ligand concen-
trations. [3H]T-3364366 displayed specific and saturating
binding to rat liver microsomes and exhibited single class of
binding sites in the concentration range examined, consistent
with 1:1 binding to a protein (Figure 1A). The specific binding
Chart 1. Chemical Structures of CP-74006 and T-3364366
Compound library screening using D5D enzymatic assay
provided hits, which were then optimized leading to
compounds such as T-3364366, a thienopyrimidinone D5D
inhibitor (Chart 1).12 T-3364366 exhibited potent D5D
inhibitory activity and excellent selectivity away from D6D
and SCD in the enzymatic activity assay. In addition, T-
3364366 potently inhibits AA production in liver cell lines
derived from human (HepG2) and rat (RLN-10) (Table 1).
Curiously, the cellular IC50 values are more potent than that of
enzymatic assay.
Table 1. IC50 Values for Desaturase Activities of CP-74006
a
and T-3364366
Figure 1. Development of a ligand binding assay using [3H]T-
3364366. (A) Saturation binding of [3H]T-3364366; specific binding
CP-74006
T-3364366
b
enzymatic assay (nM)
■
○
( ) was calculated by subtracting nonspecific ( ) from total binding
D5D
160 (140−190)
>10000
19 (16−22)
6200 (4100−9300)
>10000
●
( ). Data are presented as mean standard errors of the mean (SEM;
D6D
n = 3). (B) Displacement of [3H]T-3364366 binding by known D5D
inhibitors; DMSO and 10 μM nonlabeled T-3364366 were used as
100% and 0% controls, respectively. Data are presented as mean
SEM (n = 3).
SCD
>10000
cellular D5D assay (nM)
HepG2
29 (25−34)
26 (22−30)
1.9 (1.7−2.0)
2.1 (1.8−2.4)
RLN-10
a
Numbers in parentheses represent 95% confidence intervals.
Evaluated using rat liver microsomes.
b
was fully displaced by CP-74006 and other selective D5D
inhibitors (Figure 1B and Supporting Information, Figure S1A),
but only partially displaced by the D6D inhibitor SC-26196 at
elevated concentrations (Supporting Information, Figure
S1B).10 The above study is consistent with the signal of
[3H]T-3364366 reporting engagement of D5D rather than
other proteins within the microsomal fraction including D6D.
The apparent 2.7 nM (2.5−2.9, 95% confidence interval (CI))
Kd value after 150 min incubation agrees with the cellular assay
IC50s listed in Table 1, providing further validation of the ligand
binding assay. The high sensitivity of this ligand displacement
assay permits a 6-fold reduction in microsome proteins over the
enzymatic activity assay.
To elucidate the cause of this discrepancy, we initially
performed a kinetic analysis of the inhibitor using an enzymatic
assay of D5D. Unfortunately, the low sensitivity and assay-
limited substrate concentration range failed to develop
enzymatic assays for quantitative evaluations (Supporting
Information, Supplementary Note S1).
For the lead generation effort in drug discovery, under-
standing the mechanism of biochemical interaction of a lead
compound with its target is useful since it provides the basis for
quantitative evaluation of the lead series.13 Although several
D5D inhibitors have been reported, detailed biochemical and
kinetic characterizations are lacking, resulting in poor under-
standing of the mechanisms of action (MOAs) of the inhibitors.
Several challenges arise when examining D5D inhibitor
potency. First, reported activity assays use HPLC or TLC for
There are several possibilities that could cause the
discrepancy in potency between enzymatic and cellular assay
results, including compound activation and compound
accumulation in cells in addition to overlooking time-
dependent inhibition.18 We first evaluated the slow onset of
B
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ACS Medicinal Chemistry Letters
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binding of T-3364366 using the binding assay. Various
concentrations of [3H]T-3364366 were incubated with D5D,
and specific binding at several time points was determined.
Increase in the specific binding of [3H]T-3364366 was
observed up to 2.5 h, indicating time-dependent D5D binding
of T-3364366 on the scale of our measurement (Figure 2A). In
incubated for an additional 4 h and [14C]AA production was
monitored. While the washout produced nearly a thousand-fold
shift inhibitory activity of AA production by CP-74006, it
shifted the inhibitory activity by T-3364366 by only several fold
(Figure 3). From the result, T-3364366 is expected to exert
extended duration of action in vivo.
Figure 2. Slow-binding D5D inhibition of T-3364366. (A) Slow onset
of binding kinetics of [3H]T-3364366; a single exponential association
curve was fitted with data. Data are presented as mean SEM (n = 6).
(B) A dilution experiment of [3H]T-3364366; a single exponential
decay curve was fitted to data. Data are presented as mean SEM (n
= 8).
Figure 3. Duration of inhibition by CP-74006 (A) and T-3364366 (B)
after washout in cellular D5D assays; normalized production of
[14C]AA from [14C]DGLA is plotted as a function of compound
concentrations; DMSO and 10 μM T-3364366 under no washout
conditions were used as 100 and 0% controls, respectively. Data are
presented as mean SEM (n = 4).
contrast, after 2.5 h incubation, a much slower decrease in the
specific binding was detected, which could be explained by
D5D protein denaturation (Supporting Information, Figure
S2). The slow decrease in specific binding could not be
described by a simple mathematical function. Despite several
attempts to build a mathematical model encompassing inhibitor
association, dissociation, and loss of native D5D, we were
unsuccessful in quantitatively determining the equilibrium and
kinetic constants including the Kd value obtained above.
Therefore, we report the upper limit of the true value (binding
is tighter and dissociation slower than our model fit).
To consider the contributions of the slow off-rate of T-
3364336 to in vivo efficacy, pharmacokinetic (PK) parameters
of T-3364366 were determined (Table 2). T-3364366 exhibited
a
Table 2. Pharmacokinetic Parameters of T-3364366
dose
AUC0−8h
CLtotal
(L/h/kg)
1.5
Vdss
(L/kg)
1.1
MRT
(h)
F
route
(mg/kg)
(ng·h/mL)
(%)
iv
0.10
1.0
65
0.65
1.8
oral
260
41
a
Mouse cassette dosing (n = 3); F indicates bioavailability.
Time-dependent inhibition can arise from reversible
inhibition with slow-binding kinetics or irreversible inhibition
by covalent-bond formation. A reversibility test of T-3364366
was conducted by use of a dilution experiment. The binary
complex between [3H]T-3364366 (6.0 nM) and D5D was
produced by preincubation for 150 min. Next, the mixture was
diluted 2-fold with an excess amount of nonlabeled T-3364366
(10 μM) to prevent rebinding of the dissociated ligand. The
binding signal of [3H]T-3364366 slowly decreased after the
dilution and was less than 10% of the control signal in 21 h,
suggesting reversible and slow-release interaction of T-3364366
(Supporting Information, Figure S3). As shown in Figure 2B,
the dissociation half-life was determined from a dilution
experiment as 2.0 h (1.9−2.1, 95% CI) without considering
the effect of the time-dependent loss of the specific binding.
To further exclude the possibility of covalent bond formation
of T-3364366 with D5D, binding of [3H]T-3364366 after the
addition of a protein denaturant was evaluated. Irreversible
inhibitors through a covalent bond formation should remain
attached to their targets even after the loss of the binding
pocket by protein denaturation. Addition of final 2%
trichloroacetate solution to D5D preincubated with [3H]T-
3364366 immediately abolished the specific binding of [3H]T-
3364366, consistent with a noncovalent and reversible binding
mechanism of T-3364366 (Supporting Information, Figure S4).
The dissociation half-life of T-3364366 is slow enough to
expect duration of action in HepG2 assay after washout of the
compounds in the medium. After treating CP-74006 and T-
3364366 with HepG2 cells for 3 h, the inhibitors were removed
from medium by washing cells three times and then the cells
were pulsed with D5D substrate [14C]DGLA. Cells were then
acceptable PK properties for oral administration in the mouse
with its mean plasma residence time after intravenous
administration (MRTiv) as 0.65 h, which is shorter than the
dissociation half-life of T-3364366.
Inhibitors with slow off-rates generally lead to extended
durations of enzyme occupancy in vivo, even after drug
concentrations decrease.19 The long residence time of T-
3364366 could reduce potential off-target risks by decreasing
systemic inhibitor exposure required for comparable in vivo
efficacy.
Since D5D is a dual domain protein, several possibilities on
MOA of D5D inhibition can be considered including inhibition
of the desaturase reaction or perturbation of electron
transduction. Thus, to determine the binding domain the
inhibitor engages, we conducted a domain swapping experi-
ment between D5D and D6D. These desaturases comprise
desaturase and b5 domains and share 76% amino acid sequence
similarity.20 Since T-3364366 possesses excellent selectivity
toward D6D, ligand binding assays against the domain-swapped
mutants of desaturase and b5 domain between D5D and D6D
could reveal the binding domain of T-3364366. Based on the
amino acid sequence alignment of the subtypes, we generated
the domain-swapped mutants, D6D-b5(1−100)/D5D-des(100−444)
and D5D-b5(1−99)/D6D-des(101−444), and performed a [3H]T-
3364366 binding experiment against these recombinant
proteins (Figure 4). [3H]T-3364366 only binds to the proteins
with the D5D desaturase domain (wild-type D5D and D6D-
b5(1−100)/D5D-des(100−444)), consistent with T-3364366 inhib-
ition of D5D through the interaction with the desaturase
C
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ACS Medicinal Chemistry Letters
Letter
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.6b00241.
Materials, synthetic procedure and analytical data for T-
3364366, full experimental procedures, and supporting
figures (PDF)
Figure 4. Identification of the [3H]T-3364366 binding site. Saturation
AUTHOR INFORMATION
Corresponding Authors
*(I.M.) E-mail: ikuo.miyahisa@takeda.com. Phone: +81-466-
32-2783.
*(J.M.) E-mail: junji.matsui@takeda.com. Phone: +81-466-32-
2780.
binding experiments of [3H]T-3364366 were performed against mock
■
■
●
( ), wild-type D5D ( ), and the domain-swapped mutants, D6D-b5/
□
○
D5D-des ( ) and D5D-b5/D6D-des ( ). Specific binding was
determined as described in the materials and methods, Supporting
Information. Data are presented as mean SEM (n = 3).
Notes
The authors declare no competing financial interest.
domain of the enzyme, but not with the electron transduction
system.
ACKNOWLEDGMENTS
■
To summarize, the MOA of T-3364366 was elucidated by
developing a radioligand binding assay using the test compound
as a probe. The advantages of the ligand binding assay over the
reported enzymatic assays of D5D include sensitivity,
throughput, and direct measurement of the binding, enabling
the quantitative evaluation of T-3364366. Making the best use
of these advantages, we demonstrate that T-3364366 exhibits
slow-binding kinetics with its dissociation half-life of >2.0 h,
which may in part explain the discrepancy between IC50 values
in enzyme activity and cell-based assay. The difficulty in
performing quantitative enzymatic assay for fatty acid
desaturases may also lead to poor information on the MOA
of inhibitors for enzymes of this family including D6D and
SCD. Recently, a ligand displacement assay for SCD1 inhibitors
using an azetidine compound ([3H]AZE) was reported, but a
detailed characterization of MOA of the compound has not yet
reported.21 The biochemical analysis of [3H]AZE using a ligand
binding method should be informative for further under-
standing of MOAs of inhibitors of desaturase family enzymes.
One of the important questions to be addressed on the
biochemical MOA of T-3364366 is inhibition modality against
a substrate, DGL-CoA. Since [3H]T-3364366 potently binds at
least to a free form of the enzyme, we subsequently tried to
investigate whether [3H]T-3364366 binds to the enzyme in the
presence of DGL-CoA. Despite our attempt to perform the
ligand binding assay, we failed to produce consistent results,
presumably due to the micelle formation of the substrate (data
not shown). In such cases, structural analysis of a target protein
followed by mutational experiments can be one of the powerful
solutions to determine the binding sites of substrates and
inhibitors. Unfortunately, since no crystal structures of D5D
have been reported to date, further chimeric or mutational
experiments based on topological information on desaturase
proteins, for example, may enable the speculation of binding
sites and thus inhibition modality against the substrate.22
In conclusion, by use of the ligand binding assay, we
demonstrate that T-3364366 is a potent, selective, and orally
available D5D inhibitor, exhibiting a reversible, slow-binding
interaction to the desaturase domain of D5D. T-3364366 is a
promising lead compound with potent affinity and prolonged
efficacy. The present study is the first to demonstrate MOA of a
fatty acid desaturase inhibitor, providing important insight into
drug discovery research on desaturase inhibitors.
We acknowledge all of the D5D project team members at
Takeda, especially Shuichi Takagahara for preparation of rat
liver microsomes, Nobuyuki Matsunaga for chemical synthesis
of D5D inhibitors, and Masanori Nakakariya for analyzing PK
data. We thank Tomoya Sameshima for helpful discussion on
slow-binding kinetics.
ABBREVIATIONS
■
D5D, delta-5 desaturase; D6D, delta-6 desaturase; SCD,
stearoyl-CoA desaturase; DGLA, dihomo-gamma linoleic acid;
AA, arachidonic acid; LA, linoleic acid; MOA, mechanism of
action; NADH, nicotinamide adenine dinucleotide; PGE,
prostaglandin E; DMSO, dimethyl sulfoxide; HPLC, high-
performance liquid chromatography; TLC, thin-layer chroma-
tography; PK, pharmacokinetics
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