Inhibition of long chain fatty acyl-CoA synthetase (ACSL) and ischemia reperfusion injury

Article abstract of DOI:10.1016/j.bmcl.2014.01.016

Various triacsin C analogs, containing different alkenyl chains and carboxylic acid bioisoteres including 4-aminobenzoic acid, isothiazolidine dioxide, hydroxylamine, hydroxytriazene, and oxadiazolidine dione, were synthesized and their inhibitions of long chain fatty acyl-CoA synthetase (ACSL) were examined. Two methods, a cell-based assay of ACSL activity and an in situ [¹⁴C]-palmitate incorporation into extractable lipids were used to study the inhibition. Using an in vivo leukocyte recruitment inhibition protocol, the translocation of one or more cell adhesion molecules from the cytoplasm to the plasma membrane on either the endothelium or leukocyte or both was inhibited by inhibitors 1, 9, and triacsin C. The results suggest that inhibition of ACSL may attenuate the vascular inflammatory component associated with ischemia reperfusion injury and lead to a decrease of infarct expansion.

Full text of DOI:10.1016/j.bmcl.2014.01.016

Bioorganic & Medicinal Chemistry Letters 24 (2014) 1057–1061
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Inhibition of long chain fatty acyl-CoA synthetase (ACSL)
and ischemia reperfusion injury
Allan M. Prior a, Man Zhang a, Nina Blakeman b, Palika Datta b, Hung Pham c, Qian Chen c, Lindon H. Young c,
Margaret T. Weis b, , Duy H. Hua a,
⇑
⇑
a Department of Chemistry, 213 CBC Building, Kansas State University, Manhattan, KS 66506, United States
b Department of Biomedical Sciences, Vascular Drug Research Center, School of Pharmacy, Texas Tech University, Health Sciences Center, Amarillo, TX 79106, United States
c Department of Pathology, Microbiology, Immunology and Forensic Medicine, Philadelphia College of Osteopathic Medicine, Philadelphia, PA 19131, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Various triacsin C analogs, containing different alkenyl chains and carboxylic acid bioisoteres including 4-
aminobenzoic acid, isothiazolidine dioxide, hydroxylamine, hydroxytriazene, and oxadiazolidine dione,
were synthesized and their inhibitions of long chain fatty acyl-CoA synthetase (ACSL) were examined.
Two methods, a cell-based assay of ACSL activity and an in situ [14C]-palmitate incorporation into extract-
able lipids were used to study the inhibition. Using an in vivo leukocyte recruitment inhibition protocol,
the translocation of one or more cell adhesion molecules from the cytoplasm to the plasma membrane on
either the endothelium or leukocyte or both was inhibited by inhibitors 1, 9, and triacsin C. The results
suggest that inhibition of ACSL may attenuate the vascular inflammatory component associated with
ischemia reperfusion injury and lead to a decrease of infarct expansion.
Received 23 December 2013
Accepted 6 January 2014
Available online 13 January 2014
Keywords:
Hydroxytriazene
Ischemia reperfusion injury
Long chain fatty acyl-CoA synthetase (ACSL)
inhibitors
Oxadiazolidine dione
Triacsin C
Ó 2014 Elsevier Ltd. All rights reserved.
Ischemia reperfusion (I/R) injury is a cascade of various cellular
and molecular events, including impaired nitric oxide (NO) release
and rapid translocation of cell adhesion molecules from the cyto-
plasmic compartment to the plasma membrane.1 Together, these
events initiate leukocyte recruitment and subsequent inflamma-
tory injury. Long chain fatty acyl CoA synthetase (ACSL or LC-FACS)
catalyzes the formation of a thioester between a coenzyme A (CoA)
and a fatty acid (FA) by a two-step process as shown in Eq. (1).2
molecule (ICAM), and platelet endothelial cell adhesion molecule
(PECAM), is dependent on a rapid depalmitoylaton/repalmitoyla-
tion cycle,5–8 suggesting that the I/R injury cascade is dependent,
at least in part, on protein palmitoylation. Hence, interrupting that
cascade would be expected to mitigate I/R injury. Triacsin C (Fig. 1)
blocks eNOS palmitoylation,9 increases post-ischemic intravascu-
lar NO generation, and inhibits neutrophil recruitment and infiltra-
tion in a model of ischemia.10 Triacsin C is a natural product
isolated from Streptomyces aureofaciens,11,12 which inhibits ACSL,
including those expressed by the vascular endothelium.13 To-
gether, the data suggests that these processes are dependent on
palmitoyl CoA formation and imply that protein palmitoylation is
dependent on palmitoyl CoA availability.
ð1Þ
Our earlier work14 described several triacsin C analogs, includ-
ing compounds 1 and 2 (Fig. 1), with high anti-viral activities. How-
ever, the efficacy of these compounds to inhibit ACSL activity was
not tested directly. Here, we expand the library of triacsin C analogs,
examine their activities as ACSL inhibitors both in vitro and in vivo,
and evaluate their potential as inhibitors of neutrophil infiltration
in an in vivo model of ischemia. The hydroxytriazene function of
triacsin C remains an elusive functionality to be synthesized. In
the reported synthesis of triacsin C,15 the final three steps for
the construction of hydroxyazoimine or hydroxytriazene moiety
involved the hydrazone formation of (E,E,E)-2,4,7-undecatrien-1-
al with hydrazine followed by silylation with trimethylsilyl chloride
and addition to dinitrogen trioxide, resulting in only 0.4% yield. The
Protein palmitoylation occurs when the fatty acid moiety is
transferred to a free SH group on a cysteine residue of CoA. While
fatty acyl CoA (FACoA) transferase has been described, the process
is also thought to occur spontaneously requiring seconds to com-
plete.3 Endothelial nitric oxide synthase (eNOS) generates endo-
thelial NO. Its activity is controlled by palmitoylation as well as
by protein–protein interaction and phosphorylation.4 The function
of cell adhesion molecules necessary for neutrophil infiltration of
ischemic tissue, such as p-selectin, integrins, intercellular adhesion
⇑
Corresponding authors. Tel.: +1 (806) 414 9125; fax: +1 (806) 356 4643
(M.T.W.); tel.: +1 (785) 532 6699; fax: +1 (785) 532 6666 (D.H.H.).
E-mail addresses: Margaret.Weis@ttuhsc.edu (M.T. Weis), duy@ksu.edu
(D.H. Hua)..
0960-894X/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2014.01.016

1058
A. M. Prior et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1057–1061
O2
S
H
N
N
N
CO2H
CO2H
2
1
3
H
N
H
N
OH
CO2H
4
OH
N
OH
N
N
N
N
N
CO2H
5
CO2H
6
OH
N
O2
S
H
N
H
N
N
N
OH
O
N
CO2H
7
9
8
H
N
H
N
H
N
O
O
O
OH
N
N
N
O
O
10
11
12
N
N
OH
Triacsin C
Figure 1. Synthesized and bioevaluated ACSL inhibitors 1–12 and triacsin C.
scarcity of triacsin C hinders various in vivo biological studies.
Moreover, the hydroxyazoimine function of triacsin C is a reactive
moiety. Hence, analogs 3–12 with varying alkenyl chains, possess-
ing different functionalities, and mimicking hydroxyazoimine moi-
ety of triacsin C were synthesized and bioevaluated (Fig. 1).
Following the previously reported method,14 compounds 3 and
4 were readily synthesized in moderate yields from reductive ami-
nation reactions of 10-undecenal (13) and 14 and 15, separately,
with sodium cyanoborohydride in ethanol at 25 °C (Scheme 1).
Hydroxyamine 9 was obtained in 76% yield from the coupling of
aldehyde 13 and hydroxylamine in ethanol followed by reduction
with NaCNBH3. Similar treatment of aldehydes14 16 and 17 affor-
ded hydroxyamines 10 and 18, respectively. Addition reactions of
hydroxyamine 9, 10, and 18, separately, with 4-(hydroxycarbon-
yl)phenyldiazonium chloride16 in methanol provided hydroxytria-
zene analogs 5, 6, and 7 in 83%, 48%, and 42% yield, respectively.
Notably, these three compounds are stable molecules. Because
the imino function of 214 hydrolyzes with water slowly, it was re-
duced with NaCNBH3 and HCl in methanol to furnish stable sulfo-
nylhydrazine 8 in a 53% yield. Under other reaction conditions
including the use of acetic acid as a catalyst in the reduction pro-
cedure resulted in a mixture of unidentifiable byproducts. 1,2,4-
Oxadiazolidine-3,5-dione is a carboxylic acid bioisostere,17 hence
we synthesized compounds 11 and 12 from 9 and 10, respectively,
by the addition reactions with ethyl isocyanoformate followed by
ring closure with NaOH.17
replication,14 suggesting that the effect on rotavirus replication
may not attribute only by the inhibition of ACSL. Significantly,
compound 3, having EC50 value of 4.95
undecenyl chain, has similar or better inhibitory activity as the
trienyl analog, 1 with EC50 value of 5.86 M. Among the head
lM and containing a 10-
l
groups examined, the 4-hydroxycarbonylphenylamino moiety (in
compounds 1 and 3) possesses the greatest activity. The carboxylic
acid bioisosteres, 1,2,4-oxadiazolidine-3,5-dione derivatives 11
and 12 do not appear to improve the inhibitory activity. Also, the
isothiazolidine dioxides 2 and 8 are >30 folds less active compare
with compound 3.
ACSL inhibition in situ: compounds 1, 2, 3, 4, 7, and 9 were
compared to triacsin C as inhibitors of [14C]-palmitate incorpora-
tion into extractable lipids of bEND 3 cells, and results are shown
in Figure 3A. Each of the analogs, with the exception of
compound 2, showed significant inhibition of ACSL, with
a
potency ratio of triacsin C > 3 > 1 > 4 > 9 = 7 > 2 and mirroring that
of in vitro ACSL inhibition. [14C]-Palmitoylated lipids were also
measured in aqueous fraction (Fig. 3B) and media (Fig. 3C), and
the total recovered radioactivity (lipid+aqueous+media) was
calculated (Fig. 3D). The control cultures incorporated 40.2 2.6
fmoles [14C]-palmitate/
lg of lipid. Incorporation of radioactive
fatty acids into lipid is a two-step process. First, the FACoA deriv-
ative is formed, a reaction mediated by ACSL and second, the fatty
acid moiety is transferred to an available site during de novo lipid
synthesis or during remodeling of existing lipids. These reactions
are mediated by various acyl transferases, so that simply demon-
strating that the lesser radioactivity in the lipid fraction is insuf-
ficient to distinguish between inhibition of ACSL and the relevant
acyl transferase.
ACSL inhibition in vitro: with the exception of Compounds 2 and
8, all of the triacsin C analogs inhibit ACSL activity in solubilized
brain endothelial bEND3 cells, and results and inhibitory curves
are shown in Table 1 and Figure 2, respectively. The median effec-
tive concentration (EC50) values ranged from ꢀ5 to ꢀ170
l
M. In
Free palmitic acid is insoluble in aqueous systems at neutral pH,
while the palmitoyl CoA derivative has appreciable water solubility
(about 1.74 g/L). Thus, free palmitic acid is more likely to partition
into the CHCl3 layer of the lipid extraction, while the CoA
derivative is more likely to remain in the aqueous layer. As shown
contrast, triacsin C is about 10 times more potent than compound
3, the most active analog of the series. Compound 1 is only
one-tenth as potent as triacsin C in the ACSL assay, yet it was at
least twice as potent as triacsin C as an inhibitor of rotavirus

A. M. Prior et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1057–1061
1059
NaCNBH3
R1
H2N
EtOH, r.t.
3
4
(66% yield)
(50% yield)
+
CHO
CO2H
13
14 : R1 = H
15 : R1 = OH
O
1) H2NOH•HCl
9 (76% yield)
R2
H
10% NaOH, EtOH
10 (92% yield)
13 : R2 = CH2-(CH2)7-CH=CH2
2) NaCNBH3, MeOH
HC
18 : R2
=
C
H
n-Pr
HC
E
16 : R2
=
C
H
n-Pr
(30% yield)
17 : R2
=
HC
E
C
n-Pr
H
5
(83% yield)
N2
CO2H
Cl
6 (48% yield)
7 (42% yield)
MeOH, 0oC
O
C
N
CO2Et
1)
11 (31% yield)
12 (48% yield)
9
NaCNBH3
CH2Cl2
8
2
HCl, MeOH
2) 3 N NaOH
dioxane
10
(53% yield)
Scheme 1. Synthesis of compounds 3–12.
Table 1
Inhibition of ACSL activity in solubilized brain endothelial bEND3 cells
duced total lipid mass, or loss of radioactivity to b–oxidation. The
mass of lipids recovered was slightly greater than control for all
compounds tested, with the exception of compound 2. In cells trea-
ted with triacsin C or compounds 1, 3, 4, 7 and 9, the mean lipid
extracted did not differ with treatment, and was 127 6% of con-
trol. The unincorporated radioactivity in the media (with the
exception of compound 2) was greater in treated cells as compared
to control cells, as shown in Figure 3C. Finally, the total recovery of
radioactive material (media+lipid+aqueous) did not differ among
the compounds tested, and was not different from control
(Fig. 3D). The uniform recovery of radioactivity in both the control
and treated cells strongly suggests that treatment did not increase
b–oxidation. Thus, the reduced incorporation into lipids is not
likely to be the consequence of reduced overall lipid content or
increased b–oxidation of fatty acid. The possible remaining
interpretation of the data is that the reduced incorporation of
Compound
EC50
(
lM)
Compound
EC50 (lM)
1
2
3
4
5
6
5.86 0.54
171 79.0
4.95 0.74
6.88 0.66
73.1 9.6
7
8
9
10
11
12
26.9 3.6
164 6 0.8
70.4 14.0
14.8 1.6
9.04 1.45
53.5 33.2
74.8 34.8
0.358 0.027
Triacsin C
The EC50 values were calculated by fitting the velocity versus inhibitor concentra-
tion to the constrained three-parameter log (inhibitor) versus response function of
the Prism 6.0 statistical analysis package.
in Figure 3B, there was significantly less radioactivity in the
aqueous layer for each of the compounds, with the exception of
compound 2. Furthermore, the potency ratio for inhibition of
radioactivity in the aqueous layer is similar to that for inhibition
of incorporation into extractable lipids. Together, these data are
consistent with the inhibition of ACSL and not with inhibition of
an acyl transferase. Alternative explanations might include re-
[
14C]-palmitate into extractable lipids is the consequence of
inhibition of ACSL.
Inhibition of leukocyte recruitment: Previously, we
demonstrated that 5
lM triacsin C virtually abolished neutrophil
recruitment in a model of ischemia.10 Figure 4 compares the effects
Figure 2. Concentration effect curves of the inhibition of ACSL activity in solubilized brain endothelial bEND3 cells for each of the analogs, as compared to Triacsin C.

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A. M. Prior et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1057–1061
Figure 3. In situ inhibitions of ACSL by triacsin C and analogs in bEND3 brain endothelial cells, as evaluated by the incorporation of [14C]-palmitic acid into extractable lipid
(A), aqueous (B), media (C), and recovery (D). Triacsin C was used at 1.5 lM, while all other compounds were evaluated at 15 lM. Differences between the treatment groups
and control were evaluated by one-way ANOVA, followed by Dunnett’s t test for significance. ⁄ = p <0.05; ⁄⁄ = p <0.01; ⁄⁄⁄ = p <0.001; ⁄⁄⁄⁄ = p <0.0001. n = 3–9 independent
experiments.
of compounds 1 and 9 (as representative analogs) to that of triacsin
C. Both compounds 1 and 9 significantly inhibited rolling, adhe-
sion, and extravasation. The magnitudes of inhibition for both
strongly suggest that all three compounds inhibit translocation of
one or more cell adhesion molecules from the cytoplasm to the
plasma membrane on either the endothelium or leukocyte or both.
Furthermore, the potency ratio of these three compounds to inhibit
leukocyte recruitment is the same as the potency ratio for inhibition
of ACSL activity. Taken together, the data strongly suggest that inhib-
iting ACSL may attenuate the vascular inflammatory component
associated with I/R injury,1 consequently limiting infarct expansion.
In conclusion, various triacsin C analogs were synthesized and
their ACSL inhibitory activities and amelioration of the vascular
inflammatory component associated with ischemia/reperfusion
were studied. 4-Akenylaminobenzoic acid 1 and 3 showed the
greatest activities. Further optimization of this class of ACSL inhib-
itors is possible, which may lead to the understanding of the
consequence of ACSL inhibition effect in ischemia and treatment
of ischemia.
compounds were less than that of 5 lM triacsin C, but mirrored
the potencies of compounds 1 and 9 to inhibit ACSL activity. Triac-
sin C inhibits palmitoylation of multiple proteins including eNOS.9
This suggests that inhibitors of ACSL exert their effects, at least in
part, by inhibiting protein palmitoylation, consequently changing
their function and/or sub-cellular location. Protein palmitoylation
is a very rapid (half-time of seconds) common pathway facilitating
protein trafficking.3,18 Indeed, proteins that are rapidly re-parti-
tioned between cytoplasm and membrane are suspects for palmi-
toylation.3 Cell adhesion proteins such as PECAM5 and P-selectin6
require palmitoylation for translocation to the plasma membrane
early in the ischemic injury cascade. Our observation that triacsin
C and both compounds 1 and 9 inhibit leukocyte recruitment
Figure 4. The L-NAME induced leukocyte recruitment at 120 min in the presence of compounds 1 and 9. Rolling, adhesion and extravasation were measured in post-capillary
venules of the mesentery, a model of ischemia, and in the presence or absence of 5 or 50 M compound 1 or 50 M compound 9. The rolling, adhesion and extravasation rates
for 50 M compound 1 and 5 M triacsin C were not different from each other nor were they different from vehicle alone. When evaluated at 5 M, compound 1 inhibited the
M. Each bar represents the mean SEM of 3–7
l
l
l
l
l
recruitment to about half the extent as at 50
lm. Compound 9 at 50 lM was about as effective as compound 1 at 5 l
independent experiments.

A. M. Prior et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1057–1061
4. Liu, J.; Garcia-Cardena, G.; Sessa, W. C. Biochemistry 1996, 35, 13277.
1061
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This work was generously supported by the National Institutes
of Health, National Institutes of Allergy and Infectious Disease (U01
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Supplementary data
Supplementary data (synthetic procedure and biological exper-
iments) associated with this article can be found, in the online ver-
sion, at http://dx.doi.org/10.1016/j.bmcl.2014.01.016.
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