Structure-activity and in vivo evaluation of a novel lipoprotein lipase (LPL) activator

Article abstract of DOI:10.1016/j.bmcl.2016.11.053

Elevated triglycerides (TG) contribute towards increased risk for cardiovascular disease. Lipoprotein lipase (LPL) is an enzyme that is responsible for the metabolism of core triglycerides of very-low density lipoproteins (VLDL) and chylomicrons in the vasculature. In this study, we explored the structure-activity relationships of our lead compound (C10d) that we have previously identified as an LPL agonist. We found that the cyclopropyl moiety of C10d is not absolutely necessary for LPL activity. Several substitutions were found to result in loss of LPL activity. The compound C10d was also tested in vivo for its lipid lowering activity. Mice were fed a high-fat diet (HFD) for four months, and treated for one week at 10?mg/kg. At this dose, C10d exhibited in vivo biological activity as indicated by lower TG and cholesterol levels as well as reduced body fat content as determined by ECHO-MRI. Furthermore, C10d also reduced the HFD induced fat accumulation in the liver. Our study has provided insights into the structural and functional characteristics of this novel LPL activator.

Full text of DOI:10.1016/j.bmcl.2016.11.053

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure-activity and in vivo evaluation of a novel lipoprotein lipase
(LPL) activator
b
b
c
d
d
Werner J. Geldenhuys a, , Joel Caporoso , Thomas C. Leeper , Yoon-Kwang Lee , Li Lin , Altaf S. Darvesh ,
⇑
Prabodh Sadana d,
⇑
a Department of Pharmaceutical Sciences, School of Pharmacy, West Virginia University, Morgantown, WV 26505, United States
b Department of Chemistry, University of Akron, Akron, OH 44323, United States
c Department of Integrative Medical Sciences, Northeast Ohio Medical University, Rootstown, OH 44272, United States
d Department of Pharmaceutical Sciences, Northeast Ohio Medical University, Rootstown, OH 44272, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Elevated triglycerides (TG) contribute towards increased risk for cardiovascular disease. Lipoprotein
lipase (LPL) is an enzyme that is responsible for the metabolism of core triglycerides of very-low density
lipoproteins (VLDL) and chylomicrons in the vasculature. In this study, we explored the structure-activity
relationships of our lead compound (C10d) that we have previously identified as an LPL agonist. We
found that the cyclopropyl moiety of C10d is not absolutely necessary for LPL activity. Several substitu-
tions were found to result in loss of LPL activity. The compound C10d was also tested in vivo for its lipid
lowering activity. Mice were fed a high-fat diet (HFD) for four months, and treated for one week at 10 mg/
kg. At this dose, C10d exhibited in vivo biological activity as indicated by lower TG and cholesterol levels
as well as reduced body fat content as determined by ECHO-MRI. Furthermore, C10d also reduced the
HFD induced fat accumulation in the liver. Our study has provided insights into the structural and func-
tional characteristics of this novel LPL activator.
Received 8 October 2016
Revised 18 November 2016
Accepted 18 November 2016
Available online xxxx
Keywords:
Lipoprotein lipase
Homology modeling
Hyperlipidemia
High-fat diet
Liver cirrhosis
Obesity
Ó 2016 Elsevier Ltd. All rights reserved.
Diabetes
Elevated triglycerides (TGs) are considered an independent risk
factor for coronary heart disease (CHD). In many cases, the contin-
uing residual CHD risk in spite of optimal low-density cholesterol
(LDL-C) after statin monotherapy can be attributed to elevated
apolipoprotein B (apo-B) and hypertriglyceridemia.1–3 TGs associ-
ated with remnant lipoproteins formed as a result of partial
hydrolysis by LPL, are atherogenic as well as subject to endothelial
accumulation and uptake by macrophages to form foam cells.4,5
Similarly type 2 diabetes mellitus (DM) and metabolic syndrome
patients commonly present with combined dyslipidemia, which
is characterized with fasting and postprandial hypertriglyc-
eridemia and low HDL-C. These patients are at risk for CHD even
if LDL-C levels are optimal. Hence, there is a significant need for
novel approaches to control plasma TG levels.
critical regulatory protein involved in TG metabolism.6,7 TG-rich
lipoproteins such as VLDL and chylomicrons are catabolized by
the action of LPL into free fatty acids and remnant lipoproteins.
Such activity is crucial for effective utilization of TGs and for the
resultant uptake of fatty acids by tissues. Furthermore, intact LPL
activity is crucial to guard against fluctuations resulting from
intake of high fat meals or production of TG rich lipoproteins from
the liver.
As such, a reduction in the expression and activity of LPL is asso-
ciated with hypertriglyceridemia. Both gain-of-function and loss-
of-function genetic mutations of LPL, resulting in dysregulated
plasma TG levels in patients, have been reported.7,8 Glybera (alipo-
gene tiparvovec), a gene therapy product that replaces the LPL gene
is available in Europe from uniQure (www.uniqure.com) for the
treatment of patients diagnosed with a genetic deficiency in famil-
ial lipoprotein lipase (LPLD). A large gap in the LPL field is the lack
of clinically used small molecule drugs to modulate LPL activity.
Previous attempts to modulate LPL activity using small molecules
produced highly significant effects in several animal models of
hyperlipidemia. Developed by Otsuka Pharmaceutical Factory
(Japan), NO-1886 (generic name: Ibrolipim) remains the best char-
acterized specific LPL activator. NO-1886 was discovered in early
Extracellular lipases have been shown to tightly regulate the
plasma TG and /or HDL-cholesterol levels. The lipoprotein lipase
enzyme (LPL) that is found lining the capillary endothelium is a
⇑
Corresponding authors at: 1 Medical Center Drive, Morgantown, WV 26506,
United States (W.J. Geldenhuys). 4209 State Route 44, Rootstown, OH 44272, United
States (P. Sadana).
E-mail address: werner.geldenhuys@hsc.wvu.edu (W.J. Geldenhuys).
http://dx.doi.org/10.1016/j.bmcl.2016.11.053
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.
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2
W.J. Geldenhuys et al. / Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
1990s and significantly stimulated LPL activity, lowered plasma
triglycerides, as well as elevated the levels of HDL-C.9,10 In addition
to affecting LPL activity, NO-1886 also increases LPL mRNA thereby
increasing post-heparin LPL mass.9 In streptozotocin (STZ) treated
diabetic rats, NO-1886 increased LPL activity 59% over the con-
trol.11 Finally, long term NO-1886 administration to rats with
experimental atherosclerosis caused by high-cholesterol feeding
significantly inhibited the development of atherosclerotic lesions
in coronary arteries.9 Similar results were observed in rabbit model
of atherosclerosis.12
Our group previously reported the identification of a novel
small organic compound with LPL activation property.13 This lead
compound named C10d which was identified from an in vitro
high-throughput (HTS) screening assay exhibited potent LPL acti-
vation twofold as compared to NO-1886. In the present study,
we have carried out additional structure-activity relationship stud-
ies to identify the key structural features of the C10d molecule that
is responsible for its LPL activation. Moreover, we present the first
in vivo studies to show lipid lowering properties of C10d in a high
fat diet model of hyperlipidemia.
To explore the structure-activity around C10d, we synthesized
benzoic acid derivatives of C10d through a condensation of 1-(3-
aminopropyl) imidazole with the appropriate aromatic moiety.
The carboxylic group of the aromatic side chain was first activated
using 10-carbonyldiimidazole (CDI) by stirring at room tempera-
ture for 24 h in tetrahydrofuran (THF), after which the imidazole
was added (Scheme 1). Analytical data of the compounds given
in the Supplementary data.
In this study, we evaluated the structure-activity relationship
surrounding the imidazole moiety of C10d. Compounds 2A–5E
were tested in comparison with C10, our originally identified hit
compound and C10d, the more potent activator identified in subse-
quent studies (Figs. 1 and 2). Compound 2A improved LPL activity,
although still much less than C10d (Fig. 2). Similar trend was
observed for the benzoic acid derivatives of C10d, including
C10d-Cl, C10d-H, and C10d-F as well. Compounds that acted as
substantial inhibitors of LPL enzyme activity were 2E, 2F, 4E,
C10d-SH, and C10d-OH.
To further explore the activity of C10d, we synthesized several
benzoic acid derivatives lacking the cyclopropyl group present in
C10d. We found that C10d-H and C10d-Cl have similar activity to
the control compound C10, but less than C10d (Fig. 2). Based on
previous docking studies, we expect that the aromatic ring of
C10d is oriented towards a pocket ideal for aromatic or hydropho-
bic interactions. The lack of the cyclopropyl group allows for more
flexibility and can potentially lead to decreased occupancy in this
pocket, explaining in part the loss of activity compared to C10d.
However, since the compounds lacking the cyclopropyl group also
show agonist activity, it suggests that the cyclopropyl moiety of
C10d is not essential for activity on LPL and can be removed for
alternative scaffold hopping efforts.
To understand how C10d acts as activator of LPL, we docked
C10d onto the homology model of LPL we had previously published
(Fig. 3).6,13 We found that upon docking of C10d to LPL, the cat-
alytic amino acids are pushed closer in space suggesting that the
induced docking of C10d may lead to more efficient enzyme kinet-
ics via a stabilization of the catalytic unit resulting in lowering of
the activation energy of TG catalysis. In comparison with C10d,
the compounds that act as inhibitors, for example 2E, seem to bind
to LPL but prevent the induced fit suggested from the C10d exper-
iments. As can be seen from our docking studies, when C10d binds
to LPL, hydrogen bonds are formed with Ser216 and an aromatic
interaction with Pro217 occurs. The induced fit docking also indi-
cated that the catalytic residues are moved slightly towards the
catalytic site via the amino acids Ile272-Phe275. In the case of
the antagonists such as 2E, an extra hydrogen bond is formed with
Lys294, effectively preventing the inductive effect seen from the
C10d binding.
We tested the enzyme kinetics of C10d and found that the Km
for the enzymatic reaction is significantly lowered (Km = 648 lM
in presence of C10d versus Km = 8.15 mM in presence of DMSO)
by using C10d, suggesting enhanced affinity of LPL for its substrate
in the presence of C10d (Fig. 4).
We furthermore explored the in vivo efficacy of C10d in its abil-
ity to lower serum triglycerides. Mice were fed a high fat diet (60%
kcal from fat) for four months. In the last week of the study, we
F
F
O
O
CDI/
N
N
H
THF
HO
N
+
C10d
N
N
NH2
R
R
O
O
CDI/
THF
HO
N
N
N
N
H
R=
+
4-F
4-OH
4-Cl
4-H
N
NH2
4-SH
Scheme 1. Synthesis of the C10d and its benzoic acid derivatives which are devoid of the cyclopropyl moiety. The carboxylic group of the aromatic side chain is activated
with CDI in THF for 24 h, after which the 1-(3-aminopropyl) imidazole is added and stirred for 48 h until workup.
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W.J. Geldenhuys et al. / Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
3
Fig. 1. Structures of C10d-derivatives tested in the LPL assay.
Fig. 2. Activity profile of the hit compound C10, the lead compound C10d and structural derivatives using an in vitro recombinant lipoprotein lipase (LPL) activity high-
throughput (HTS) screening assay. All compounds were screened at 100 M, in an assay set up in a 384 well plate. p-Nitrophenyl butyrate was used as a substrate in the assay
l
and absorbance detection at 405 nm was performed to detect product of cleavage, butyric acid. The fold change in absorbance as a measure of LPL activation is plotted. Bars
represent average S.D. with the error bars contained within the size of the bar where N = 2.
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W.J. Geldenhuys et al. / Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Fig. 3. Docking studies of the lead compound C10d (A and C) and compound 2E (B and D) in the homology model of LPL.13 C10d shows agonist behavior and 2E shows
antagonist behavior. Based on the docking studies, it appears that C10d allows for the amino acids in the catalytic site to be optimally place for a lower activation energy,
while the tight binding of 2D inhibits the flexibility of the protein to allow for efficient catalysis of the TG.
80
Control
HFD
10 mg/kg
60
40
20
0
fat (g)
Weight
lean (g)
F.water (g) T.water (g)
Fig. 5. ECHO-MRI analysis for different groups of animals in the high-fat diet (HFD)
study. At the conclusion of the four months of high fat diet feeding, body weight, as
well as total body fat and lean mass, were monitored by EchoMRI. Each bar
represents mean S.D., where n = 4.
Fig. 4. Enzyme kinetics for LPL stimulated hydrolysis was determined in the
presence or absence (DMSO vehicle control) of C10d. Using a dilution series for the
substrate, velocity of the reaction was determined. A Lineweaver-Burk plot of 1/V
vs. 1/S is shown. C10d significantly lowered the Km from 8.15 mM for the vehicle
treated to 0.648 mM in the presence of C10d. Symbols represent average S.D. with
the error bars contained within the size of the symbols where N = 3.
in body weight as compared to untreated HFD controls as well as
lower total body fat content (Fig. 5).
The lowering of total body fat content observed after C10d
treatment of mice only in the final week of the four-month
feeding suggests a potent in vivo efficacy of this LPL activator.
treated the mice with C10d at 10 mg/kg dose i.p. daily until the end
of the study. The dose of C10d used for the in vivo study was
selected based on a dose response study wherein a 10 mg/kg dose
of C10d exhibited an increase in LPL activity whereas the doses of
1 mg/kg and 5 mg/kg did not alter the LPL activity (data not
shown). Before sacrifice, ECHO-MRI was performed on the animals.
C10d treated animals showed a small but not significant decrease
A
decrease in serum triglycerides and cholesterol was also
observed (Fig. 6 A and B).
Additionally, mice treated with C01d were found to have livers
which morphologically resembled that of the mice fed a normal
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W.J. Geldenhuys et al. / Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
5
Fig. 8. Mice treated with C10d show reduction in liver injury from a high-fat diet
(HFD). Mice were fed a HFD for four months, and treated with C10d for seven days
at 10 mg/kg. The protein levels of SIRT1 was increased in mice treated with C10d.
*Statistical significance P < 0.05. n = 5 mice.
Fig. 6. Serum triglycerides (A; TG) and cholesterol (B; CHO) measurements in mice.
High-fat diet (HFD) feeding was performed in mice for 4 months. HFD + C10d group
received 10 mg/kg i.p. of C10d in the last week of the study. The mice were
sacrificed and lipids were measured in the serum using commercially available
assay kits (n = 3). Data show mean SE.
Fig. 7. Mice treated with C10d (10 mg/kg) show reduction in liver fat deposition
from a high-fat diet (HFD) as compared to mice on a HFD alone. The livers of mice
on HFD were enlarged as expected, and treatment with C10d showed a reduction in
liver size (Top panel). Histochemical staining with hematoxylin and eosin (H and E)
showed reduction in lipid accumulation in the livers treated with C10d (bottom
panel; 20ꢀ magnification).
Fig. 9. Mice treated with C10D show reduction in liver injury from a high-fat diet
(HFD). Mice were fed a HFD for four months, and treated with C10D for seven days
at 10 mg/kg. The protein level of LC3 was increased in mice treated with C10D.
*Statistical significance P < 0.05. n = 5 mice.
and the autophagy marker – microtubule associated protein 1 light
chain 3 alpha (LC3). These markers were increased in mice treated
with C10d (Figs. 8 and 9), suggesting a likely mechanism of action
of C10d in the liver might be via stimulation of liver protection
pathways.
diet (Fig. 7). In contrast, the livers of mice fed a high fat diet (HFD)
only, appeared enlarged and steatotic. Histochemical H & E stain-
ing indicated that the livers of mice treated with C10d showed less
lipid accumulation as compared to the HFD-only group.
Based on our morphological findings of the livers, we evaluated
the protein levels of two markers that are associated with liver
protection, the NAD dependent deacetylase – sirtuin 1 (SIRT1)
In conclusion, we explored the SAR for our lead compound C10d
as LPL agonist and found that the imidazole moiety plays an
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W.J. Geldenhuys et al. / Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
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tionally we tested the compound C10d in a high fat diet study to
evaluate its effect on lipid lowering activity. We found the com-
pound can reduce whole body fat, serum triglyceride levels, and
hepatic fat accumulation in mice fed high fat diet. Taken together
our data shows the feasibility of using LPL agonists in lowering
triglycerides. Lastly, the novel scaffolds here may lead to identifica-
tion of new classes of lipid lowering drugs which has therapeutic
potential in a variety of metabolic diseases such as dyslipidemia,
obesity, diabetes and fatty liver disease.
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We wish to thank The Goodyear Corporation, Akron OH for
donation of the NMR instrument used in this work. The project
described was supported by the National Institute of General Med-
ical Sciences, U54GM104942. The content is solely the responsibil-
ity of the authors and does not necessarily represent the official
views of the NIH.
A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2016.11.
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