U.S. patent application number 13/841530 was filed with the patent office on 2013-08-15 for lipase inhibitors.
The applicant listed for this patent is Rolf Breinbauer, Klaus Fortschegger, Johannes Grillari, Regina Grillari, Nicole Mayer, Matthias Romauch, Elisabeth Schraml, Martina Schweiger, Robert Zimmermann. Invention is credited to Rolf Breinbauer, Klaus Fortschegger, Johannes Grillari, Regina Grillari, Nicole Mayer, Matthias Romauch, Elisabeth Schraml, Martina Schweiger, Robert Zimmermann.
Application Number | 20130210883 13/841530 |
Document ID | / |
Family ID | 48946113 |
Filed Date | 2013-08-15 |
United States Patent
Application |
20130210883 |
Kind Code |
A1 |
Grillari; Johannes ; et
al. |
August 15, 2013 |
LIPASE INHIBITORS
Abstract
The invention provides a compound of formula (I) as defined
herein ##STR00001## that is useful in the treatment and prevention
of a disorder such as cachexia, stroke, atherosclerosis, coronary
artery disease, or diabetes and disorders and conditions associated
therewith. The invention also provides a method of screening for
lipase inhibitors using a compound of formula (I) and determining
its lipase inhibitory activity. activity.
Inventors: |
Grillari; Johannes;
(Bisamberg, AT) ; Breinbauer; Rolf; (Graz, AT)
; Schweiger; Martina; (Graz, AT) ; Romauch;
Matthias; (Graz, AT) ; Zimmermann; Robert;
(Graz, AT) ; Mayer; Nicole; (Graz, AT) ;
Schraml; Elisabeth; (Wien, AT) ; Fortschegger;
Klaus; (Maria Anzbach, AT) ; Grillari; Regina;
(Bisamberg, AT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Grillari; Johannes
Breinbauer; Rolf
Schweiger; Martina
Romauch; Matthias
Zimmermann; Robert
Mayer; Nicole
Schraml; Elisabeth
Fortschegger; Klaus
Grillari; Regina |
Bisamberg
Graz
Graz
Graz
Graz
Graz
Wien
Maria Anzbach
Bisamberg |
|
AT
AT
AT
AT
AT
AT
AT
AT
AT |
|
|
Family ID: |
48946113 |
Appl. No.: |
13/841530 |
Filed: |
March 15, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13699272 |
Nov 20, 2012 |
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PCT/EP11/58379 |
May 23, 2011 |
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13841530 |
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61755332 |
Jan 22, 2013 |
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Current U.S.
Class: |
514/407 ;
435/184; 435/19; 514/406; 514/539; 514/543; 548/369.7; 548/374.1;
560/48; 560/59 |
Current CPC
Class: |
C07C 69/94 20130101;
C07D 231/14 20130101; C12Q 1/44 20130101; C07C 69/78 20130101; C07C
65/24 20130101; C07D 231/18 20130101; C07C 229/52 20130101 |
Class at
Publication: |
514/407 ;
548/369.7; 548/374.1; 560/59; 560/48; 514/406; 514/543; 514/539;
435/184; 435/19 |
International
Class: |
C07C 229/52 20060101
C07C229/52; C07D 231/14 20060101 C07D231/14; C07C 65/24 20060101
C07C065/24; C07D 231/18 20060101 C07D231/18 |
Foreign Application Data
Date |
Code |
Application Number |
May 21, 2010 |
EP |
10163604.1 |
Claims
1. A compound of formula (I) ##STR00009## wherein: L.sup.1 is a
bond, C.sub.1-10 alkylene, C.sub.2-10 alkenylene, or C.sub.2-10
alkynylene, wherein said alkylene, said alkenylene or said
alkynylene is optionally substituted with one or more groups
independently selected from --C.sub.1-4 alkyl, --CF.sub.3, --CN,
--OH, --O(C.sub.1-4 alkyl), --NH.sub.2, --NH(C.sub.1-4 alkyl), or
--N(C.sub.1-4 alkyl)(C.sub.1-4 alkyl), and further wherein one or
more --CH.sub.2-- units comprised in said alkylene, said alkenylene
or said alkynylene are each optionally replaced by a group
independently selected from --O--, --NH--, --N(C.sub.1-4 alkyl)-,
--CO--, --CO--NH--, --NH--CO--, --S--, --SO--, or --SO.sub.2--,
R.sup.1 is independently selected from C.sub.1-4 alkyl, halogen,
--CF.sub.3, --CN, --OH, --O(C.sub.1-4 alkyl), --NH.sub.2,
--NH(C.sub.1-4 alkyl), or --N(C.sub.1-4 alkyl)(C.sub.1-4 alkyl), a
5 to 7-membered, saturated or unsaturated carbon ring structure
wherein optionally one to three of the carbon atoms are replaced by
N, O, or S, said ring structure being optionally substituted with
C.sub.1-4 alkyl, halogen, --CF.sub.3, --CN, --OH, --O(C.sub.1-4
alkyl), --NH.sub.2, --NH(C.sub.1-4 alkyl), or --N(C.sub.1-4
alkyl)(C.sub.1-4 alkyl), or a aryl or heteroaryl optionally
substituted with C.sub.1-4 alkyl, halogen, --CF.sub.3, --CN, --OH,
--O(C.sub.1-4 alkyl), --NH.sub.2, --NH(C.sub.1-41 alkyl), or
--N(C.sub.1-4 alkyl)(C.sub.1-4 alkyl), R.sup.2 is optionally
substituted aryl or optionally substituted heteroaryl but not
pyrazolyl, wherein said aryl or said heteroaryl may be substituted
with one or more groups independently selected from C.sub.1-4
alkyl, halogen, --CF.sub.3, --CN, --OH, --O(C.sub.1-4 alkyl),
--NH.sub.2, --NH(C.sub.1-4 alkyl), or --N(C.sub.1-4
alkyl)(C.sub.1-4 alkyl), or R.sup.2 is pyrazolyl optionally
substituted with one or more groups independently selected from
C.sub.1-4 alkyl, halogen, --CF.sub.3, --CN, --O(C.sub.1-4 alkyl),
--NH.sub.2, --NH(C.sub.1-4 alkyl), or --N(C.sub.1-4
alkyl)(C.sub.1-4 alkyl); R.sup.3 is C.sub.1-10 alkylene, C.sub.2-10
alkenylene, or C.sub.2-10 alkynylene, wherein said alkylene, said
alkenylene or said alkynylene is optionally substituted with one or
more groups independently selected from halogen, --CF.sub.3, --CN,
--OH, --O(C.sub.1-4 alkyl), --NH.sub.2, --NH(C.sub.1-4 alkyl), or
--N(C.sub.1-4 alkyl)(C.sub.1-4 alkyl), and further wherein one or
more --CH.sub.2-- units comprised in said alkylene, said alkenylene
or said alkynylene are each optionally replaced by a group
independently selected from --O--, --NH--, --N(C.sub.1-4 alkyl)-,
--CO--, --CO--NH--, --NH--CO--, --S--, --SO--, or --SO.sub.2--; m
is an integer of 0 to 8; and each R.sup.4 is independently selected
from C.sub.1-4 alkyl, halogen, --CF.sub.3, --CN, --OH,
--O(C.sub.1-4 alkyl), --NH.sub.2, --NH(C.sub.1-4 alkyl), or
--N(C.sub.1-4 alkyl)(C.sub.1-4 alkyl); R.sup.5 is aryl or
heteroaryl; or a pharmaceutically acceptable salt, solvate or
prodrug thereof.
2. A compound of formula (I) ##STR00010## wherein: L.sup.1 is a
bond, C.sub.1-10 alkylene, C.sub.2-10 alkenylene, or C.sub.2-10
alkynylene, wherein said alkylene, said alkenylene or said
alkynylene is optionally substituted with one or more groups
independently selected from --C.sub.1-4 alkyl, --CF.sub.3, --CN,
--OH, --O(C.sub.1-4 alkyl), --NH.sub.2, --NH(C.sub.1-4 alkyl), or
--N(C.sub.1-4 alkyl)(C.sub.1-4 alkyl), and further wherein one or
more --CH.sub.2-- units comprised in said alkylene, said alkenylene
or said alkynylene are each optionally replaced by a group
independently selected from --O--, --NH--, --N(C.sub.1-4 alkyl)-,
--CO--, --CO--NH--, --NH--CO--, --S--, --SO--, or --SO.sub.2--;
R.sup.1 is independently selected from C.sub.1-4 alkyl, halogen,
--CF.sub.3, --CN, --OH, --O(C.sub.1-4 alkyl), --NH.sub.2,
--NH(C.sub.1-4 alkyl), or --N(C.sub.1-4 alkyl)(C.sub.1-4 alkyl), a
5 to 7-membered, saturated or unsaturated carbon ring structure
wherein optionally one to three of the carbon atoms are replaced by
N, O, or S, said ring structure being optionally substituted with
C.sub.1-4 alkyl, halogen, --CF.sub.3, --CN, --OH, --O(C.sub.1-4
alkyl), --NH.sub.2, --NH(C.sub.1-4 alkyl), or --N(C.sub.1-4
alkyl)(C.sub.1-4 alkyl), or a aryl or heteroaryl optionally
substituted with C.sub.1-4 alkyl, halogen, --CF.sub.3, --CN, --OH,
--O(C.sub.1-4 alkyl), --NH.sub.2, --NH(C.sub.1-4 alkyl), or
--N(C.sub.1-4 alkyl)(C.sub.1-4 alkyl); R.sup.2 is optionally
substituted aryl or optionally substituted heteroaryl but not
pyrazolyl, wherein said aryl or said heteroaryl may be substituted
with one or more groups independently selected from C.sub.1-4
alkyl, halogen, --CF.sub.3, --CN, --OH, --O(C.sub.1-4 alkyl),
--NH.sub.2, --NH(C.sub.1-4 alkyl), or --N(C.sub.1-4
alkyl)(C.sub.1-4 alkyl); or R.sup.2 is pyrazolyl optionally
substituted with one or more groups independently selected from
C.sub.1-4 alkyl, halogen, --CF.sub.3, --CN, --O(C.sub.1-4 alkyl),
--NH.sub.2, --NH(C.sub.1-4 alkyl), or --N(C.sub.1-4
alkyl)(C.sub.1-4 alkyl) R.sup.3 is C.sub.1-10 alkylene, C.sub.2-10
alkenylene, or C.sub.2-10 alkynylene, wherein said alkylene, said
alkenylene or said alkynylene is optionally substituted with one or
more groups independently selected from halogen, --CF.sub.3, --CN,
--OH, --O(C.sub.1-4 alkyl), --NH.sub.2, --NH(C.sub.1-4 alkyl), or
--N(C.sub.1-4 alkyl)(C.sub.1-4 alkyl), and further wherein one or
more --CH.sub.2-- units comprised in said alkylene, said alkenylene
or said alkynylene are each optionally replaced by a group
independently selected from --O--, --NH--, --N(C.sub.1-4 alkyl)-,
--CO--, --CO--NH--, --NH--CO--, --S--, --SO--, or --SO.sub.2--; m
is an integer of 0 to 8; each R.sup.4 is independently selected
from C.sub.1-4 alkyl, halogen, --CF.sub.3, --CN, --OH,
--O(C.sub.1-4 alkyl), --NH.sub.2, --NH(C.sub.1-4 alkyl), or
--N(C.sub.1-4 alkyl)(C.sub.1-4 alkyl); R.sup.5 is aryl or
heteroaryl optionally substituted as shown in formula (I), selected
from phenyl, benzyl, pyridinyl, pyrazinyl, pyrimidinyl,
pyridazinyl, pyrazolyl, imidazolyl, pyrrolyl, triazinyl,
tetrazolyl, thiophenyl (thienyl), thiazolyl, furanyl (furyl),
furazanyl, oxazolyl, isoxazolyl, benzthienyl, benzfuryl,
benzimidazolyl, benzthiazolyl, isothiazolyl, benzisothiazolyl,
benzoxazolyl, benzisoxazolyl, isothiazolyl, benztriazolyl,
thiadiazolyl, oxadiazolyl, indolyl indazolyl, chinolinyl, naphthyl,
anthracenyl; or a pharmaceutically acceptable salt, solvate or
prodrug thereof.
3. A compound of claim 1 wherein R.sup.5 is thiophenyl,
piperidinyl, indolyl or methylindolyl.
4. A compound of claim 1 wherein R.sup.2 is selected from phenyl,
benzyl, pyridyl, furanyl, thiophenyl, pyrrolyl.
5. A compound of claim 1 wherein R.sup.2 is selected from
pyrrazolyl, pyridinyl, thienyl, and furanyl.
6. A compound of claim 1 wherein R.sup.2 is selected from among
##STR00011## wherein R signifies the point of attachment to the
structure of formula (I)
7. A compound of claim 1 wherein R.sup.2 is phenyl.
8. A compound of claim 7 having an IC.sub.50 for lipase activity of
less than 40 uM.
9. A compound of claim 8 wherein the lipase is ATGL.
10. A compound of claim 1, for use in the treatment or prevention
of a disorder.
11. A compound of claim 10, for use in the treatment or prevention
of diabetes type II, stroke, atherosclerosis, coronary artery
disease, high triglyceride levels, cachexia, or its associated or
related disorders or conditions, including weight loss, muscle
atrophy or wasting, fat loss, reduced WAT.
12. A method of inhibiting the activity of a lipase in vitro, by
contacting said lipase with a compound of formula (I) as per claim
1.
13. A method of claim 12 wherein the lipase is ATGL.
14. A pharmaceutical composition comprising the compound of formula
(I) as per claim 1 and a pharmaceutically acceptable excipient.
15. A method for treating or preventing diabetes type II, coronary
artery disease, atherosclerosis, stroke, cachexia, or its
associated or related disorders or conditions, including weight
loss, muscle atrophy or wasting, fat loss, and/or reduced WAT, the
method comprising the administration of the compound of formula (I)
as per claim 1, optionally in a pharmaceutical composition that
includes a pharmaceutically acceptable excipient, to a subject in
need of such a treatment or prevention.
16. A method for identifying a compound that inhibits the activity
of a lipase, comprising: (a) contacting a compound with a cell
expressing lipase polypeptide; and (b) measuring the activity of
said lipase in the presence or absence of said compound, wherein a
compound that blocks or reduces the activity of said lipase is
identified as a compound that inhibits the activity of the lipase,
and wherein said compound of step (a) is a compound of formula (I)
as per claim 1.
17. The method of claim 16 wherein said lipase is ATGL.
18. A method for identifying a compound useful in the prevention,
amelioration, inhibition or treatment of cachexia, atherosclerosis,
stroke, coronary artery disease, type II diabetes, and/or disorders
and/or conditions associated or related thereto, comprising: a.
contacting a compound with a cell expressing ATGL polypeptide; and
b. measuring the expression and/or activity of ATGL in the presence
or absence of said compound, wherein a compound that blocks or
reduces the activity of ATGL is identified as a compound useful in
the prevention, amelioration, inhibition or treatment of said
disorders, diseases or the disorders or conditions associated
thereto, and wherein the compound of step (a) is a compound of
formula (I) as per claim 1.
19. The method of claim 18 wherein the compound is one that is able
to reduce blood FFA levels.
20. The method of claim 18 wherein the compound is one that does
not lead to reduced blood glucose levels.
21. The method of claim 18 wherein the compound is substantially
non-toxic.
22. A method for identifying a compound that prevents, ameliorates
and/or inhibits cachexia, stroke, type II diabetes,
atherosclerosis, or coronary artery disease, and/or the disorders
or conditions associated or related thereto, comprising: a)
contacting a compound with an ATGL polypeptide or fragments
thereof; and b) measuring a compound-polypeptide property.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to lipase inhibitors, methods
of screening therefor, and to methods of prevention, treatment or
alleviation of a disorder that can be influenced by lowering lipase
activity.
BACKGROUND OF THE INVENTION
[0002] In mammals, triglycerides are stored in adipose tissue
providing the primary source of energy during periods of food
deprivation. Whole body energy homeostasis depends on the precisely
regulated balance of lipid storage and mobilization. White adipose
tissue (WAT) functions as buffer for dietary lipids and stores
excess energy in the form of triacylglycerol (TG). Mobilization of
fatty acids from TG stores in adipose tissue critically depends on
the activation of lipolytic enzymes. Upon demand, TG stores are
hydrolyzed by lipolytic enzymes and the body is provided with free
fatty acids (FA) for energy conversion or for the synthesis of
complex lipids. Efficient lipolysis requires a three-step process
involving three enzymes: Adipose triglyceride lipase (ATGL, also
annotated as patatin-like phospholipase domain containing 2,
desnutrin, phospholipase A2zeta, and transport secretion protein
2.2), hormone-sensitive lipase (HSL), and monoglyceride lipase
(MGL). ATGL removes the first fatty acid from the TG molecule and
generates diacylglycerol (DG). HSL is the rate-limiting enzyme for
the hydrolysis of DG and MGL performs the last step in this
reaction leading to the liberation of FA and glycerol.
[0003] FA mobilization in WAT and non-adipose tissues is strongly
dependent on ATGL and its co-activator protein CGI-58 (comparative
gene expression 58, also known as .alpha./.beta. hydrolase
fold-containing 5). In humans, loss-of-function mutations in either
of these genes are associated with Neutral Lipid Storage Disease
(NLSD), a rare autosomal recessive disorders characterized by the
excessive accumulation of neutral lipids in multiple tissues.
Similarly as observed in humans, a complete absence of ATGL
function in mice is associated with severely reduced lipolysis,
obesity, and fat deposition in virtually all tissues of the body.
Under fasting conditions, ATGL-deficient animals are unable to
mobilize sufficient energy in the form of FA to maintain normal
energy homeostasis. Prolonged starvation induces a torpor-like
metabolic state characterized by decreased plasma FA
concentrations, hypoglycemia, reduced oxygen consumption, and
hypothermia. It has been observed that increased circulating FA
concentrations, as seen in obesity, can promote fat deposition,
insulin resistance, and inflammation in non-adipose tissues. These
adverse effects of ectopic lipid overload are known under the term
"lipotoxicity" and central in the pathogenesis of metabolic
disorders.
[0004] Dysfunctional lipolysis affects energy homeostasis and may
contribute to the pathogenesis of obesity and insulin resistance.
Dysregulation of TG-lipolysis in man has been linked to variations
in the concentration of circulating FA, an established risk factor
for the development of insulin resistance (Bergman, R. N. et al
(2001) J Investig Med 49: 119-26; Blaak, E. E. (2003) Proc Nutr Soc
62: 753-60; Boden, G. and G. I. Shulman (2002) Eur J Clin Invest 32
(Suppl 3):14-23; Arner, P. (2002) Diabetes Metab Res Rev 18 (Suppl
2): S5-9).
[0005] During periods of increased energy demand, lipolysis in
adipocytes is activated by hormones, such as catecholamines.
Hormone interaction with G-protein coupled receptors is followed by
increased adenylate cyclase activity, increased cAMP levels, and
the activation of cAMP-dependent protein kinase (protein kinase A,
PKA) (Collins, S. and R. S. Surwit (2001) Recent Prog Horm Res
56:309-28). PKA then phosphorylates targets with established
function in lipolysis including hormone-sensitive lipase (HSL),
resulting in the translocation of HSL from the cytoplasm to the
lipid droplet where efficient TG hydrolysis occurs (Sztalryd, C. et
al (2003) J Cell Biol 161:1093-103).
[0006] The mobilization of free fatty acids from adipose
triacylglycerol (TG) stores requires the activities of
triacylglycerol hydrolases. Adipose triglyceride lipase (ATGL) and
hormone-sensitive lipase (HSL) are the major enzymes contributing
to TG breakdown. ATGL (also named PNPLA 2 (patatin-like
phospholipase domain containing protein-2, desnutrin, phospholipase
A2.delta., and transport-secretion protein)) is highly expressed in
adipose tissue and specifically removes the first fatty acid from
the TG molecule, generating FFA and DG (Zimmermann, R. et al (2004)
Science 306:1383-1386; Wang, S P et al (2001) Obes Res 9:119-128;
Villena, J A et al (2004) J Biol Chem 279:47066-47075; Jenkins, C M
et al (2004) J Biol Chem 279:48968-48975). An essential role of
ATGL in lipolypsis has been demonstrated in studies of
ATGL-deficient (ATGL-ko) mice (Haemmerle, G. et al (2006) Science
312:734-737). ATGL-deficient mice accumulated large amounts of
lipid in the heart, causing cardiac dysfunction and premature
death. The relative contribution of these hydrolases to the
lipolytic catabolism of fat has been determined, in mutant mouse
models lacking ATGL or HSL (Schweiger, M. et al (2006) J Biol Chem
281(52):40236-40241). Both HSL and ATGL enzymes contribute to
hydrolysis of TG, however, ATGL deficient mice studies indicate
that ATGL is rate limiting in the catabolism of cellular fat
deposits and plays an important role in energy homeostasis
(Haemmerle, G. et al (2006) Science 312(5774):734-737).
[0007] Cachexia is a life-threatening syndrome characterized by the
unattended loss of body weight, muscle atrophy, fatigue, weakness
and significant loss of appetite in someone who is not actively
trying to lose weight. It can be a sign of various underlying
disorders. It occurs in about 50% of cancer patients but is also
observed in other diseases including certain infectious diseases
(e.g. tuberculosis, AIDS), in or alcoholchronic obstructive
pulmonary disease, and advanced organ failure (liver, heart,
kidney). Cachexia physically weakens patients to a state of
immobility stemming from loss of appetite, asthenia, and anemia,
and response to standard treatment is usually poor (Lainscak M, et
al (2007) Curr Opin Support Palliat Care 1(4): 299-305; Bossola M
et al (2007) Expert Opin Investig Drugs 16 (8): 1241-53). Recently,
is has been shown that lipolysis is also increased in cancer
associated cachexia, leading to a loss of adipose tissue (Thompson
et al). Another study provided evidence that ATGL deficiency
protects from cancer cachexia associated loss of adipose tissue and
skeletal muscle (Das, 2011). Thus, inhibiting ATGL might provide a
novel medical intervention technique to prevent the loss of adipose
and skeletal muscle mass in cancer cachexia. This could prevent
uncontrolled weight loss and increase life expectancy of cancer
patients.
[0008] Cachexia is also prevalent in HIV patients before the advent
of highly active anti-retroviral therapy (HAART) and in patients
that have any of the range of illnesses classified as "COPD"
(chronic obstructive pulmonary disease), particularly emphysema.
Some severe cases of schizophrenia can present this condition where
it is named vesanic cachexia (from vesania, a Latin term for
insanity). Metabolic syndrome is a name for a group of risk factors
that occur together and increase the risk for coronary artery
disease, stroke, and type 2 diabetes. All of the risks for the
syndrome are related to obesity. The two most important risk
factors for metabolic syndrome are: Extra weight around the middle
and upper parts of the body (central obesity), and insulin
resistance. As a result, blood glucose and fat levels rise. Other
risk factors include lack of exercise and age. Metabolic syndrome
is associated with dyslipidemia and especially increased plasma
levels of FA may have a causal role in the development of the
syndrome.
[0009] Moreover, increased blood levels of FA are also a risk
factor for the development of atherosclerosis, stroke, and coronary
artery disease. To prevent or treat disorders caused by
dyslipidemia, an effective tool represents may be the effective
reduction of excessive blood FA levels.
SUMMARY OF THE INVENTION
[0010] The invention provides a compound of formula (I) as defined
herein
##STR00002##
that is useful in the treatment and prevention of a disorder. The
disorder comprises cachexia, stroke, atherosclerosis, coronary
artery disease, diabetes and disorders and conditions associated
therewith. The invention also provides a method of screening for
lipase inhibitors using a compound of formula (I) and determining
its lipase inhibitory activity.
[0011] Aspects of the present method include the in vitro assay of
compounds using ATGL and/or HSL, and cellular assays wherein
inhibition is followed by observing indicators of efficacy,
including alteration of the release of free fatty acid, TG
hydrolysis, binding affinity, lowering blood FFA values etc.
Another aspect of the invention is a method of treatment or
prevention of a condition involving cachexia, stroke,
artherosclerosis, coronary artery disease, diabetes, preferably
diabetes type II, and its associated or related disorders and
conditions, including weight loss, muscle atrophy or wasting, in a
subject suffering or susceptible thereto, by administering a
pharmaceutical composition comprising an agent which is able to
inhibit ATGL. Also contemplated herein, are compositions comprising
one or more ATGL-inhibiting agents of the invention, alone, or in
combination with each other or in combinations with one or more
lipase inhibitors, such as HSL or MGL inhibitors, and/or inhibitors
of inflammatory cytokines such as tumor necrosis factor-alpha
(TNF-.alpha.), interleukins 1 and 6 (IL-1 and IL-6), interferon
gamma (IFN-.gamma.), and leukemia-inhibitory factor (LAF).
[0012] Other objects and advantages will become apparent from a
consideration of the ensuing description taken in conjunction with
the following illustrative drawings.
BRIEF DESCRIPTION OF THE FIGURES
[0013] FIG. 1. Structure of compounds 1-4 and effect on ATGL
activity. For determination of lipase activity, lysates of E. coli
overexpressing recombinant strep-tagged ATGL and CGI-58 were
incubated with a substrate containing radiolabeled
[9,10-3H(N)]-triolein. Subsequently, FA were extracted and
quantified by liquid scintillation counting. Lysates of cells
expressing empty vector were set as blank. Inhibitors were
solubilized in DMSO and added at the indicated concentrations. DMSO
alone was used as negative control. (A) Structure and IC50 values
of compound 1-4. (B) Dose-dependent inhibition of ATGL in the
presence of compound 3 and 4. The insert in (B) shows a Western
blot confirming the expression of Strep-tagged proteins at their
correct molecular weight. (C) Lineweaver-Burk plots for kinetic
analysis of ATGL inhibition. Experiments were performed at varying
concentrations of substrate (0.05-3 mM) in presence and absence of
compound 4 (Atglistatin). The insert shows the intersection with
the y- and x-axis representing 1/Vmax and -1/Km, respectively. Data
are presented as mean+/-S.D. of triplicate determinations. Data for
compound 4 are representative for at least three independent
experiments.
[0014] FIG. 2. Selectivity of Atglistatin. Lipase activity of ATGL,
HSL, lipoprotein lipase, and pancreatic lipase was determined using
triolein as substrate. MGL activity was detected in the presence of
rac-(1,3)-monooleoylglycerol as substrate. Inhibitors were
solubilized in DMSO and added at the indicated concentrations. DMSO
alone was used as negative control. (A) Dose-dependent inhibition
of TG hydrolase activity in WAT lysates obtained from wild-type and
ATGL-ko mice. (B) Inhibition of TG hydrolase activity in wild-type
WAT lysates by Atglistatin (40 .mu.M) and by the HSL inhibitor Hi
76-0079 (20 .mu.M). (C) Effect of Atglistatin on murine MGL
(purified from E. coli), and murine HSL (overexpressed in COS-7
lysates) activity. (D) Effect of Atglistatin on purified bovine LPL
(Sigma) and pancreatic lipase (from porcine pancreas, Sigma). Data
are presented as mean+/-S.D. of triplicate determinations and are
representative for at least three independent experiments
(p<0.05**, p<0.01; ***, p<0.001).
[0015] FIG. 3 Inhibition of lipolysis in 3T3-L1 adipocytes and WAT
organ cultures. (A,B) Differentiated 3T3 cells were preincubated
with the indicated concentrations of Atglistatin. Thereafter, the
medium was replaced by DMEM containing 2% BSA (fatty acid free), 10
.mu.M forskolin, in the presence or absence of inhibitors. The
release of FA (A) and glycerol (B) in the media was determined
using commercial kits. Basal FA and glycerol release (not shown)
was barely detectable under the applied conditions. Data are
presented as mean+/-SD and are representative for 2 independent
experiments. (C-F) Effect of Atglistatin on NAT basal (C, D) and
forskolin-stimulated (E, F) lipolysis. Adipose tissue pieces
(.about.15 mg, n=5 for each concentration) of wild-type mice were
cultured for 8 h in DMEM containing 2% FA-free BSA and the
indicated concentrations of Atglistatin. Subsequently, the medium
was replaced by identical fresh medium and samples were collected
after incubation for another hour in the presence or in the absence
of 10 .mu.M forskolin. Atglistatin was solubilized in DMSO and DMSO
alone was used as negative control. Data are presented as
mean+/-S.D. (*, p<0.05**, p<0.01; ***, p<0.001).
[0016] FIG. 4 Inhibition of lipolysis in vivo. (A,B) Time dependent
effect of Atglistatin on plasma free fatty acids (FFA) and glycerol
levels of mice. C57Bl6 mice were fasted overnight (ON) and received
an oral gavage containing 100 .mu.mol/kg Atglistatin dissolved in
olive oil, or olive oil as control. At the indicated timepoints
blood was taken retroorbitally and plasma parameters were measured
using commercial kits. (C,D) Dose dependent effect of Atglistatin
on plasma FFA and glycerol levels of mice. C57Bl6 mice were fasted
ON and received an oral gavage containing 0, 50, 100, or 200
.mu.mol/kg Atglistatin dissolved in olive oil, or olive oil as
control. 8 h after gavage blood was taken retroorbitally and plasma
parameters were measured using commercial kits. Data are presented
as mean+S.D. (*, p<0.05**, p<0.01; ***, p<0.001). n=5
DETAILED DESCRIPTION OF THE INVENTION
[0017] The following terms are intended to have the meanings
presented therewith below and are useful in understanding the
description and intended scope of the present invention.
[0018] The term `agent` means any molecule, including polypeptides,
antibodies, polynucleotides, chemical compounds and small
molecules. In particular the term agent includes compounds such as
test compounds or drug candidate compounds.
[0019] The term `agonist` refers to a ligand that stimulates the
receptor the ligand binds to in the broadest sense.
[0020] As used herein, the term `antagonist` is used to describe a
compound that does not provoke a biological response itself upon
binding to a receptor, but blocks or dampens agonist-mediated
responses, or prevents or reduces agonist binding and, thereby,
agonist-mediated responses.
[0021] The term `assay` means any process used to measure a
specific property of an agent. A `screening assay` means a process
used to characterize or select agents based upon their activity
from a collection of agents.
[0022] The term `binding affinity` is a property that describes how
strongly two or more compounds associate with each other in a
non-covalent relationship. Binding affinities can be characterized
qualitatively (such as `strong`, `weak`, `high`, or `low`) or
quantitatively (such as measuring the K.sub.D).
[0023] The term `carrier` means a non-toxic material used in the
formulation of pharmaceutical compositions to provide a medium,
bulk and/or useable form to a pharmaceutical composition. A carrier
may comprise one or more of such materials such as an excipient,
stabilizer, or an aqueous pH buffered solution. Examples of
physiologically acceptable carriers include aqueous or solid buffer
ingredients including phosphate, citrate, and other inorganic and
organic acids; antioxidants including ascorbic acid; low molecular
weight (less than about 10 residues) polypeptide; proteins, such as
serum albumin, gelatin, or immunoglobulins; hydrophilic polymers
such as polyvinylpyrrolidone; amino acids such as glycine,
glutamine, asparagine, arginine or lysine; monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose,
or dextrins; chelating agents such as EDTA; sugar alcohols such as
mannitol or sorbitol; salt-forming counterions such as sodium;
and/or nonionic surfactants such as TWEEN.RTM., polyethylene glycol
(PEG), and PLURONICS.RTM..
[0024] The term `complex` means the entity created when two or more
compounds bind to, contact, or associate with each other.
[0025] The term `compound` is used herein in the context of a `test
compound` or a `drug candidate compound` described in connection
with the assays of the present invention.
[0026] As such, these compounds comprise organic or inorganic
compounds, derived synthetically, recombinantly, or from natural
sources.
[0027] The compounds include inorganic or organic compounds such as
polynucleotides, lipids, hormone analogs, or other small molecules.
Other biopolymeric organic test compounds include peptides
comprising from about 2 to about 40 amino acids and larger
polypeptides comprising from about 40 to about 500 amino acids,
including polypeptide ligands, enzymes, receptors, channels,
antibodies or antibody conjugates. The term `condition` or
`disease` means the overt presentation of symptoms (i.e., illness)
or the manifestation of abnormal clinical indicators (for example,
biochemical indicators or diagnostic indicators). Alternatively,
the term `disease` refers to a genetic or environmental risk of or
propensity for developing such symptoms or abnormal clinical
indicators.
[0028] The term `contact` or `contacting` means bringing at least
two moieties together, whether in an in vitro system or an in vivo
system.
[0029] The term `derivatives of a polypeptide` relates to those
peptides, oligopeptides, polypeptides, proteins and enzymes that
comprise a stretch of contiguous amino acid residues of the
polypeptide and that retain a biological activity of the protein,
for example, polypeptides that have amino acid mutations compared
to the amino acid sequence of a naturally-occurring form of the
polypeptide. A derivative may further comprise additional naturally
occurring, altered, glycosylated, acylated or non-naturally
occurring amino acid residues compared to the amino acid sequence
of a naturally occurring form of the polypeptide. It may also
contain one or more non-amino acid substituents, or heterologous
amino acid substituents, compared to the amino acid sequence of a
naturally occurring form of the polypeptide, for example a reporter
molecule or other ligand, covalently or non-covalently bound to the
amino acid sequence.
[0030] The term `derivatives of a polynucleotide` relates to
DNA-molecules, RNA-molecules, and oligonucleotides that comprise a
stretch of nucleic acid residues of the polynucleotide, for
example, polynucleotides that may have nucleic acid mutations as
compared to the nucleic acid sequence of a naturally occurring form
of the polynucleotide. A derivative may further comprise nucleic
acids with modified backbones such as PNA, polysiloxane, and
2'-O-(2-methoxy)ethyl-phosphorothioate, non-naturally occurring
nucleic acid residues, or one or more nucleic acid substituents,
such as methyl-, thio-, sulphate, benzoyl-, phenyl-, amino-,
propyl-, chloro-, and methanocarbanucleosides, or a reporter
molecule to facilitate its detection.
[0031] The term `endogenous` shall mean a material that a mammal
naturally produces. Endogenous in reference to the term `protease`,
`kinase`, `factor`, or `receptor` shall mean that which is
naturally produced by a mammal (for example, and not limitation, a
human). In contrast, the term non-endogenous in this context shall
mean that which is not naturally produced by a mammal (for example,
and not limitation, a human). Both terms can be utilized to
describe both in vivo and in vitro systems. For example, and
without limitation, in a screening approach, the endogenous or
non-endogenous target (e.g., ATGL and/or HSL, alternative species
forms, isoforms, and variants) may be in reference to an in vitro
screening system. As a further example and not limitation, where
the genome of a mammal has been manipulated to include a
non-endogenous target, screening of a candidate compound by means
of an in vivo system is viable. The term `expressible nucleic acid`
means a nucleic acid coding for a proteinaceous molecule, an RNA
molecule, or a DNA molecule.
[0032] The term `expression` comprises both endogenous expression
and overexpression by transduction.
[0033] The term `expression inhibitory agent` means a
polynucleotide designed to interfere selectively with the
transcription, translation and/or expression of a specific
polypeptide or protein normally expressed within a cell. More
particularly, `expression inhibitory agent` comprises a DNA or RNA
molecule that contains a nucleotide sequence identical to or
complementary to at least about 15-30, particularly at least 17,
sequential nucleotides within the polyribonucleotide sequence
coding for a specific polypeptide or protein. Exemplary expression
inhibitory molecules include ribozymes, double stranded siRNA
molecules, self-complementary single-stranded siRNA molecules
(shRNA), genetic antisense constructs, and synthetic RNA antisense
molecules with modified stabilized backbones.
[0034] The term `inhibit` or `inhibiting`, in relationship to the
term `response` means that a response is decreased or prevented in
the presence of a compound as opposed to in the absence of the
compound.
[0035] The term `inhibition` refers to the reduction, down
regulation of a process or the elimination of a stimulus for a
process, which results in the absence or minimization of the
expression or activity of a protein or polypeptide.
[0036] The term `pharmaceutically acceptable salts` refers to the
non-toxic, inorganic and organic acid addition salts, and base
addition salts, of compounds which inhibit the expression or
activity of targets as disclosed herein. These salts can be
prepared in situ during the final isolation and purification of
compounds useful in the present invention.
[0037] The term `preventing` or `prevention` refers to a reduction
in risk of acquiring or developing a disease or disorder (i.e.,
causing at least one of the clinical symptoms of the disease not to
develop) in a subject that may be exposed to a disease-causing
agent, or predisposed to the disease in advance of disease
onset.
[0038] The term `prophylaxis` is related to and encompassed in the
term `prevention`, and refers to a measure or procedure the purpose
of which is to prevent, rather than to treat or cure a disease.
Non-limiting examples of prophylactic measures may include the
administration of vaccines; the administration of low molecular
weight heparin to hospital patients at risk for thrombosis due, for
example, to immobilization; and the administration of an
anti-malarial agent such as chloroquine, in advance of a visit to a
geographical region where malaria is endemic or the risk of
contracting malaria is high.
[0039] The term `solvate` means a physical association of a
compound useful in this invention with one or more solvent
molecules. This physical association includes hydrogen bonding. In
certain instances the solvate will be capable of isolation, for
example when one or more solvent molecules are incorporated in the
crystal lattice of the crystalline solid. "Solvate" encompasses
both solution-phase and isolable solvates. Representative solvates
include hydrates, ethanolates and methanolates.
[0040] The term `subject` includes humans and other mammals. In the
context of this invention, it is particularly envisaged that
mammals are to be treated which are economically, agronomically or
scientifically important. Scientifically important organisms
include, but are not limited to, mice, rats, and rabbits.
Non-limiting examples of agronomically important animals are sheep,
cattle and pigs, while, for example, cats and dogs may be
considered as economically important animals. Preferably, the
subject is a human.
[0041] `Therapeutically effective amount` means that amount of a
drug, compound, expression inhibitory agent, or pharmaceutical
agent that will elicit the biological or medical response of a
subject that is being sought by a medical doctor or other
clinician. The term `treating` or `treatment` of any disease or
disorder refers, in one embodiment, to ameliorating the disease or
disorder (i.e., arresting the disease or reducing the
manifestation, extent or severity of at least one of the clinical
symptoms thereof). In another embodiment `treating` or `treatment`
refers to ameliorating at least one physical parameter, which may
not be discernible by the subject. In yet another embodiment,
`treating` or `treatment` refers to modulating the disease or
disorder, either physically, (e.g., stabilization of a discernible
symptom), physiologically, (e.g., stabilization of a physical
parameter), or both. In a further embodiment, `treating` or
`treatment` relates to slowing the progression of the disease.
[0042] The term "vectors" also relates to plasmids as well as to
viral vectors, such as recombinant viruses, or the nucleic acid
encoding the recombinant virus.
[0043] The term "vertebrate cells" means cells derived from animals
having vertera structure, including fish, avian, reptilian,
amphibian, marsupial, and mammalian species. Preferred cells are
derived from mammalian species, and most preferred cells are human
cells. Mammalian cells include feline, canine, bovine, equine,
caprine, ovine, porcine murine, such as mice and rats, and
rabbits.
[0044] Lipolytic activity may be detected by assay methods known to
those of skill in the art. Preferably, methods such as described
hereinunder in Examples 1 and 7 are used. In a preferred embodiment
of the invention, the method as described herein in paragraphs 1-3
of example 7 herein, including references made therein, is used. A
preferred embodiment of the invention includes measurement of
lipase activity according to Jenkins et al. and Chung et al. as
described in Example 7 herein. A further preferred embodiment of
the invention includes measurement of lipase activity as described
Example 7 using HIS-tagged ATGL. Any such assays and tests are
described for the purpose of illustration and guidance; variations,
alterations, adaptations and modifications will be obvious and
possible to the person of skill in the art. For instance, the test
described in example 7 may be used to test the lipolytic activity
and if desired the inhibition thereof by a given inhibitor, among
others, of variants, fragments, variants due to premature
termination or the like, of a lipase, preferably of ATGL lipase,
without deviating from the scope of the invention.
[0045] The target is the structure used to detect the desired
activity of a compound of the invention. It is preferably a lipase,
more preferably ATGL, and particularly human ATGL. The amino acid
and nucleic acid sequences for ATGL are known to the skilled
artisan. For example, the ATGL sequence is set out in FIG. 7 (human
ATGL) and FIG. 8 (mouse ATGL) of WO2010115825.
[0046] The term "cachexia and its associated or related disorders
and conditions" or "cachexia and its related conditions or
physiology" or variants thereof, refers to a disease or condition
which involves, results at least in part from, or includes loss of
weight, muscle atrophy, fatigue, weakness and significant loss of
appetite in someone who is not actively trying to lose weight. It
can be associated with or result from (directly or indirectly)
various underlying disorders including cancer, metabolic acidosis
(from decreased protein synthesis and increased protein
catabolism), certain infectious diseases (e.g. tuberculosis, AIDS),
some autoimmune disorders, addiction to drugs such as amphetamines
or cocaine, chronic alcoholism and cirrhosis of the liver, chronic
inflammatory disorders, anorexia, and neurodegenerative disease. In
a particular aspect, cachexia is cancer cachexia. In other such
aspects, muscle wasting and/or unintended body weight loss
associated with neurological conditions, immobility or impaired
mobility due to various diseases such as neurodegenerative disease,
MS, spinal cord injury, is included in the term.
[0047] The term "alkyl" relates to a monovalent saturated aliphatic
(i.e. non-aromatic) acyclic hydrocarbon group (i.e. a group
consisting of carbon atoms and hydrogen atoms) which may be linear
or branched and does not comprise any carbon-to-carbon double bond
or any carbon-to-carbon triple bond.
[0048] The term "alkenyl" refers to a monovalent unsaturated
aliphatic acyclic hydrocarbon group which may be linear or branched
and comprises at least one carbon-to-carbon double bond while it
does not comprise any carbon-to-carbon triple bond.
[0049] The term "alkynyl" refers to a monovalent unsaturated
aliphatic acyclic hydrocarbon group which may be linear or branched
and comprises at least one carbon-to-carbon triple bond and
optionally one or more carbon-to-carbon double bonds.
[0050] The term "alkylene" refers to an alkanediyl group including
straight chain and/or branched chain groups.
[0051] The term "alkenylene" refers to an alkenediyl group
including straight chain and/or branched chain groups, and
comprising at least one carbon-to-carbon double bond, while it does
not comprise any carbon-to-carbon triple bond.
[0052] The term "alkynylene" refers to an alkynediyl group
including straight chain and/or branched chain groups, and
comprising at least one carbon-to-carbon triple bond and optionally
one or more carbon-to-carbon double bonds.
[0053] The term "aryl" refers to a monovalent aromatic hydrocarbon
group, including bridged ring and/or fused ring systems, containing
at least one aromatic ring. "Aryl" may, for example, refer to
phenyl, naphthyl or anthracenyl.
[0054] The term "heteroaryl" refers to a monocyclic or fused-ring
polycyclic group having 5 to 14 ring atoms, having 6, 10 or 14 pi
electrons shared in a cyclic array, and containing carbon ring
atoms and 1, 2 or 3 hetero ring atom independently selected from O,
N, or S. The term "heteroaryl" may, for example, relate to
thiophenyl (thienyl), furanyl (furyl), pyrrolyl, imidazolyl,
pyrazolyl, pyridinyl (pyridyl; including, e.g., 2-pyridyl,
3-pyridyl, and 4 pyridyl), pyrazinyl, pyrimidinyl, pyridazinyl,
oxazolyl, isoxazolyl, or furazanyl.
[0055] The term "heteroaryl having 5 or 6 ring atoms, wherein 1, 2
or 3 ring atoms are each independently selected from oxygen,
sulfur, or nitrogen and the other ring atoms are carbon atoms"
refers to a monocyclic group having 5 or 6 ring atoms (i.e., ring
members), having 6 pi electrons shared in a cyclic array, and
containing carbon atoms and 1, 2 or 3 heteroatoms independently
selected from O, N, or S, Non-limiting examples of heteroaryl
groups include thiophenyl (thienyl), furanyl (furyl), pyrrolyl,
imidazolyl, pyrazolyl, pyridinyl (pyridyl; including, e.g.,
2-pyridyl, 3-pyridyl, and 4 pyridyl), pyrazinyl, pyrimidinyl,
pyridazinyl, oxazolyl, isoxazolyl, and furazanyl. The term
"halogen" refers to fluoro, chloro, bromo, or iodo, and in
particular to fluoro, chloro, or bromo.
[0056] The present invention provides the first small molecule
inhibitor for ATGL having IC.sub.50 concentrations in the
submicromolar range. The inhibitor is preferably a competitive
inhibitor and does not affect the activity of other known
acylglycerol hydrolases. Further, the inhibitor is preferably
orally bioavailable and capable of inhibiting lipolysis in vivo. It
is therefore capable of serving as lead structure for the
identification of further inhibitors.
[0057] FA metabolism is closely linked to the development of
metabolic disorders. Increased lipolysis can promote lipid overload
of non-adipose tissues such as liver, skeletal and cardiac muscle,
and pancreas, which causes lipotoxicity impairing the metabolic
functions of these tissues (Boden, 2011). Mice lacking ATGL exhibit
improved glucose tolerance and insulin sensitivity and are
resistant to high-fat diet-induced insulin resistance implicating
that inhibition of ATGL represents a strategy to improve insulin
resistance (Haemmerle, 2006; Kienesberger, 2009). Apparently,
ATGL-deficiency causes a shift from fatty acid to glucose usage in
insulin-sensitive tissues which is opposite to that observed in
type 2 diabetes. Notably, ATGL-deficient animals show improved
insulin sensitivity despite severe TG accumulation in non-adipose
tissues indicating that increased ectopic lipid storage per se does
not cause lipotoxicity. In fact, ATGL activity generates FA
required for the synthesis of lipotoxic metabolites such as
acyl-CoA (Li, 2010), ceramides (Summers, 2006), and diglycerides
(Samuel, 2010) which cause insulin resistance.
[0058] ATGL deficiency is associated with a severe metabolic
phenotype characterized by TG accumulation in multiple tissues
which is most pronounced in the heart causing cardiomyopathy and
premature death in ATGL-ko mice (Haemmerle, 2006). TG accumulation
seems to be the only cause for premature death since ATGL-ko
animals overexpressing ATGL exclusively in cardiac muscle exhibit
normal life expectancy (Haemmerle, 2011). Similarly, humans with
defective ATGL function develop cardiac myopathy which is lethal or
necessitates cardiac transplantation (Hirano, 2008). It is
reasonable to assume that also inhibitor-mediated ablation of ATGL
activity can cause cardiac TG accumulation limiting the potential
value of ATGL inhibitor as therapeutic target. However, TG
accumulation in the mouse heart is slowly progressive and humans
with defective ATGL function reach adulthood. Severe cardiac
myopathy appears to develop at .about.30 years of age (Hirano,
2009) suggesting that transient inhibition of ATGL must not
necessarily result in cardiac dysfunction since tissue TG stores
can be rapidly mobilized for energy production or membrane lipid
synthesis.
[0059] Importantly, the compounds of the present invention do not
result in TG accumulation in heart and other tissues in treated
mice although lipolytic parameters were significantly reduced. This
implicates that a more severe and continuous inhibition of ATGL
activity is required for ectopic TG accumulation. Reduced
circulating glycerol and FA levels suggest that the compounds of
the present invention efficiently inhibits WAT lipolysis. Thus, the
compounds of formula (I), preferably compound 4, represent a
suitable tool to advance the development of drugs for the treatment
of the metabolic syndrome.
[0060] Another devastating disease which is strongly linked with
deregulated lipolysis is cachexia, a complex metabolic disorder
characterized by general physical wasting and frequently associated
with cancer (Tisdale, 2010, Ryden, 2008). Cancer-associated
cachexia leads to depletion of adipose and muscle tissue mass and
is considered as important adverse prognostic factor responsible
for the immediate cause of death in an estimated 15% of all cancer
patients (Deans, 2005). Importantly, ATGL-deficiency in mice
protects from cancer-induced WAT loss. Moreover, these mice are
also resistant to muscle loss although the protective mechanism
remains unexplained (Das, 2011). In this respect, it is important
to note that insulin resistance is present in many cancer patients
and may be one mechanism through which muscle wasting occurs
(Honors, 2012). Ectopic lipid accumulation induced by
muscle-specific overexpression of LPL in mice has been shown to
result in increased proteasomal activity, apoptosis and skeletal
muscle damage (Tamilarasan, 2012). Similarly, cancer-mediated loss
of adipose tissue due to elevated lipolysis could have lipotoxic
effects in non-adipose tissues promoting insulin resistance and
muscle loss. Thus, pharmacological inhibition of ATGL may also help
to prevent cachexia.
[0061] In a further aspect of the invention, the compounds of the
invention are provided as tool compounds to study the
pathophysiology of FA metabolism and offer new opportunities to
understand the chemical biology of ATGL.
[0062] In one aspect, the invention relates to a compound of
formula (I),
##STR00003##
[0063] wherein R1-R5, L1, m have the meanings defined herein, or a
pharmaceutically acceptable salt, solvate or prodrug thereof.
[0064] In particular:
L.sup.1 is a bond, C.sub.1-10 alkylene, C.sub.2-10 alkenylene, or
C.sub.2-10 alkynylene, wherein said alkylene, said alkenylene or
said alkynylene is optionally substituted with one or more groups
independently selected from --C.sub.1-4 alkyl, --CF.sub.3, --CN,
--OH, --O(C.sub.1-4 alkyl), --NH.sub.2, --NH(C.sub.1-4 alkyl), or
--N(C.sub.1-4 alkyl)(C.sub.1-4 alkyl), and further wherein one or
more --CH.sub.2-- units comprised in said alkylene, said alkenylene
or said alkynylene are each optionally replaced by a group
independently selected from --O--, --NH--, --N(C.sub.1-4 alkyl)-,
--CO--, --CO--NH--, --NH--CO--, --S--, --SO--, or --SO.sub.2--;
R.sup.1 is independently selected from C.sub.1-4 alkyl, halogen,
--CF.sub.3, --CN, --OH, --O(C.sub.1-4 alkyl), --NH.sub.2,
--NH(C.sub.1-4 alkyl), or --N(C.sub.1-4 alkyl)(C.sub.1-4 alkyl), a
5 to 7-membered, saturated or unsaturated carbon ring structure
wherein optionally one to three of the carbon atoms are replaced by
N, O, or S, said ring structure being optionally substituted with
C.sub.1-4 alkyl, halogen, --CF.sub.3, --CN, --OH, --O(C.sub.1-4
alkyl), --NH.sub.2, --NH(C.sub.--.sub.4 alkyl), or --N(C.sub.1-4
alkyl)(C.sub.1-4 alkyl), or a aryl or heteroaryl optionally
substituted with C.sub.1-4 alkyl, halogen, --CF.sub.3, --CN, --OH,
--O(C.sub.1-4 alkyl), --NH.sub.2, --NH(C.sub.1-4 alkyl), or
--N(C.sub.1-4 alkyl)(C.sub.1-4 alkyl), R.sup.2 is optionally
substituted aryl or optionally substituted heteroaryl but not
pyrazolyl, wherein said aryl or said heteroaryl may be substituted
with one or more groups independently selected from C.sub.1-4
alkyl, halogen, --CF.sub.3, --CN, --OH, --O(C.sub.1-4 alkyl),
--NH.sub.2, --NH(C.sub.1-4 alkyl), or --N(C.sub.1-4
alkyl)(C.sub.1-4 alkyl), or R.sup.2 is pyrazolyl optionally
substituted with one or more groups independently selected from
C.sub.1-4 alkyl, halogen, --CF.sub.3, --CN, --O(C.sub.1-4 alkyl),
--NH.sub.2, --NH(C.sub.1-4 alkyl), or --N(C.sub.1-4
alkyl)(C.sub.1-4 alkyl); R.sup.3 is C.sub.1-10 alkylene, C.sub.2-10
alkenylene, or C.sub.2-10 alkynylene, wherein said alkylene, said
alkenylene or said alkynylene is optionally substituted with one or
more groups independently selected from halogen, --CF.sub.3, --CN,
--OH, --O(C.sub.1-4 alkyl), --NH.sub.2, --NH(C.sub.1-4 alkyl), or
--N(C.sub.1-4 alkyl)(C.sub.1-4 alkyl), and further wherein one or
more --CH.sub.2-- units comprised in said alkylene, said alkenylene
or said alkynylene are each optionally replaced by a group
independently selected from --O--, --NH--, --N(C.sub.1-4 alkyl)-,
--CO--, --CO--NH--, --NH--CO--, --S--, --SO--, or --SO.sub.2--; m
is an integer of 0 to 8; and each R.sup.4 is independently selected
from C.sub.1-4 alkyl, halogen, --CF.sub.3, --CN, --OH,
--O(C.sub.1-4 alkyl), --NH.sub.2, --NH(C.sub.1-4 alkyl), or
--N(C.sub.1-4 alkyl)(C.sub.1-4 alkyl); and R.sup.5 is aryl or
heteroaryl; R.sup.5 is preferably selected from optionally
substituted phenyl, benzyl, pyridinyl, pyrazinyl, pyrimidinyl,
pyridazinyl, pyrazolyl, imidazolyl, pyrrolyl, triazinyl,
tetrazolyl, thiophenyl (thienyl), thiazolyl, furanyl (furyl),
furazanyl, oxazolyl, isoxazolyl, benzthienyl, benzfuryl,
benzimidazolyl, benzthiazolyl, isothiazolyl, benzisothiazolyl,
benzoxazolyl, benzisoxazolyl, isothiazolyl, benztriazolyl,
thiadiazolyl, oxadiazolyl, indolyl indazolyl, chinolinyl, naphthyl,
anthracenyl. Further preferably, R5 is selected from optionally
substituted phenyl, thienyl, indolyl, indolinyl, methylindolyl,
piperidinyl. Most preferably, R5 is optionally substituted
phenyl.
[0065] Further preferably, R.sup.2 is selected from optionally
substituted phenyl, benzyl, pyridyl, furanyl, thiophenyl, pyrrolyl.
Also preferably, R.sup.2 is selected from optionally substituted
pyrrazolyl, pyridinyl, thienyl, and furanyl. More preferably,
R.sup.2 is selected from optionally substituted phenyl. The term
"optionally substituted" has the meaning given for the structure to
which it relates as defined in the claims. For instance, when
referring to optionally substituted R.sup.2, the optional
substituents as defined in the claims, particularly claim 1, for
R.sup.2 are intended. Further preferably, R.sup.2 is selected from
among
##STR00004##
wherein R signifies the point of attachment to the structure of
formula (I). More preferably, R2 and R5 are optionally substituted
phenyl. Further preferably, R2 and R5 are both unsubstituted
phenyl. R3 is preferably --NH--CO--N(CH3)2, preferably attached to
the R.sup.2 ring structure at the meta position relative to the
benzyl ring in formula (I). Preferably, L.sup.1 is a bond and
R.sup.1 is --N(CH3)2. Also preferably, R2 is optionally substituted
phenyl, L1 is a bond and R.sup.5 is optionally substituted
piperidine. R1 is preferably --O--CH2-CH3. More preferably, R1 is
--N(CH3)2. R3 is preferably --CO--O--CH2-CH3. More preferably, R3
is --NH--CO--N(CH3)2.
[0066] Also contemplated within the invention is a pharmaceutical
composition comprising a compound as described herein above.
Preferably, the compound is one wherein R2 and/or R5 are optionally
substituted phenyl. Further preferably, L.sup.1 is a bond and
R.sup.1 is --N(CH3)2. Also preferably, R.sup.2 is optionally
substituted phenyl, L1 is a bond and R.sup.5 is optionally
substituted piperidine. R1 is preferably --O--Ch2-Ch3. More
preferably, R1 is --N(CH3)2. R3 is preferably --CO--O--CH2-CH3.
More preferably, R3 is --NH--CO--N(CH3)2. The composition is
preferably useful in the treatment, prevention, amelioration, or
inhibition of cachexia, atherosclerosis, stroke, coronary artery
disease, type II diabetes, and/or disorders and/or conditions
associated or related thereto.
[0067] The compound preferably has lipase inhibitory activity. Such
activity may be measured by methods described hereinabove and
below. For example, the method described in example 3, 4 or 5
and/or the Figures referred to therein (e.g., FIG. 1) may be used.
The compound preferably has an IC50 of less than 200 uM, less than
100 uM, 40, or 14 micromolar. Further preferably, the IC 50 of the
compound is less than 1 micromolar.
[0068] The compound preferably inhibits ATGL or an enzymatically
active portion thereof. The inhibition is preferably selective.
Selectivity may be measured by methods known to the skilled person.
For example, the methods described herein in example 4 and/or FIG.
2 may be used. The inhibition of another lipase (e.g. of HSL) is
preferably at least 10%, 15%, 30%, 50%, 70% or at least 90% weaker
than that observed for ATGL. The compound preferably leads to
lowering of blood FFA levels in vivo. Methods of measuring the
effect of a compound on blood FFA levels are known to the skilled
artisan. For example, the method described herein in example 1
and/or FIG. 4 may be used. Also preferably, the compound does not
lead to substantially altered blood glucose levels in vivo. For
instance, blood glucose levels may be changed less than 80%, 65%,
50%, 40%, 30%, 20% or 10%. Blood glucose levels may be measured as
known to the skilled person; for example, the method described
herein in example 1 may be used. The compound preferably does not
lead to a reduction in muscle mass in vivo. Methods of measuring
muscle mass are known to the skilled artisan. For example, the
methods described in Das, 2011, may be used. Preferably,
administration of the compound leads to accumulation of muscle mass
or inhibition of loss of muscle mass. This is especially preferred
when the compound is used in the treatment or prevention of
cachexia or an associated disorder.
[0069] Further preferably, the compound has a low or absent
toxicity. Methods of measuring toxicity are known to the skilled
person. Preferably, the method described herein in example 1
"toxicity test" is used. The viability of the cells, measured in OD
units, preferably does not change or rises when administering the
compound. Also preferably, the viability is reduced by less than
80%, 70%, 50%, 40%, 30%, 20%, 15%, 10% or 5%. Also preferably, the
compound exhibits binding affinity for a lipase, which is
preferably, MGL, HSL or ATGL, more preferably ATGL or an
enzymatically active portion thereof. The binding affinity may be
measured as known in the art. Preferably, affinity is measured as
described hereinbelow. Preferably, binding affinity is in the
micromolar range, more preferably below 100 micromolar, below 70
micromolar, below 40 micromolar, below 25 micromolar, below 14
micromolar, below 10 micromolar, below 5 micromolar, or below 1
micromolar.
[0070] Using a compound of formula (I), or a preferred embodiment
thereof, the skilled artisan may according to the invention conduct
a screening for compounds with desirable and/or improved
properties. For instance, the method described in example 7 may be
used. Also, further methods used as known in the art may be
used.
[0071] The invention further provides a method for inhibiting the
activity of a lipase in vitro, by contacting the lipase with a
compound as defined hereinabove and below. The lipase is preferably
HSL, MGL, or ATGL, more preferably ATGL. The inhibition may be
advantageously used in in vitro assays, for instance when activity
of ATGL is not desired or in order to find out what activity of
ATGL contributes to an observed phenomenon.
[0072] The invention further provides a method for binding of a
compound of the invention to a lipase in vitro, by contacting the
lipase with a compound as defined hereinabove and below. The lipase
is preferably HSL, MGL, or ATGL, more preferably ATGL. The binding
may be advantageously used in in vitro assays, for instance, where
the compound comprises a label or is labeled and it is desired to
locate ATGL within a cellular, tissue, or organ structure. Thus,
also variants of the compound as defined herein, which are labeled,
are contemplated within the invention. Using a compound of the
invention, such labeled compounds may be prepared by methods known
to the skilled person. For instance, the methods described
hereinbelow may be used.
[0073] In one aspect, the present invention relates to a method for
providing further compounds useful in inhibiting lipases, in
particular ATGL.
[0074] More particularly, the invention relates to a method for
identifying a compound that inhibits ATGL comprising: [0075] (a)
contacting a population of mammalian cells with one or more
compound of formula (I), and [0076] (b) measuring the inhibitory
effect of the compound on TG hydrolysis by ATGL.
[0077] In particular the inhibition of the release of free fatty
acid from TG may be assessed. Preferably, inhibition of ATGL
activity is assessed as described in example 7. More preferably,
the inhibition of ATGL is specific and the compound identified does
not inhibit other lipases to the same degree. Specificity may be
assessed by methods known to the skilled artisan. Preferably, the
method described in example 7 and FIG. 4 herein is used. Further
preferably, the compound inhibits other lipases, such as for
example HSL, to a substantially lesser degree than it inhibit ATGL.
The inhibition of said other lipase may be at least 15%, 30%, 50%,
70% or 90% weaker as that observed for ATGL inhibition.
[0078] Further, the invention relates to a method for identifying
an agent or compound that alleviates cachexia, stroke,
atherosclerosis, coronary artery disease, diabetes or its
associated physiology whereby said agent or compound inhibits ATGL
said method comprising: [0079] (a) contacting a population of
mammalian cells with one or more compound of formula (I), and
[0080] (b) measuring the inhibitory effect of the compound on TG
hydrolysis.
[0081] In particular the inhibition of the release of free fatty
acid from TG may be assessed. Preferably, inhibition of ATGL
activity is assessed as described in example 7. More preferably,
the inhibition of ATGL is specific and the compound identified does
not inhibit other lipases to the same degree. Further preferably,
the compound inhibits other lipases to a substantially lesser
degree. The inhibition of said other lipases may be at least 15%,
30%, 50%, 70% or 90% weaker as that observed for ATGL.
[0082] In a further aspect of the present invention said method is
used to identify a compound that inhibits lipolysis, particularly
inhibits ATGL activity or expression. In particular the inhibition
of the release of free fatty acid from TG may be assessed.
[0083] The present assay method may be practiced in vitro, using
ATGL or an enzymatically active portion thereof.
[0084] The binding affinity of the compound with ATGL can be
measured by methods known in the art, such as using surface plasmon
resonance biosensors (Biacore), by saturation binding analysis with
a labeled compound (e.g. Scatchard and Lindmo analysis), by
differential UV spectrophotometer, fluorescence polarization assay,
Fluorometric imaging Plate Reader (FLIPR.RTM.) system, Fluorescence
resonance energy transfer, and Bioluminescence resonance energy
transfer. The binding affinity of compounds can also be expressed
in dissociation constant (Kd) or as IC.sub.50 or EC.sub.50. The
IC.sub.50 represents the concentration of a compound that is
required for 50% inhibition of binding of another ligand to the
polypeptide. The EC.sub.50 represents the concentration required
for obtaining 50% of the maximum effect in any assay that measures
ATGL function. The dissociation constant, Kd, is a measure of how
well a ligand binds to the polypeptide, it is equivalent to the
ligand concentration required to saturate exactly half of the
binding-sites on the polypeptide. Compounds with a high affinity
binding have low Kd, IC.sub.50 and EC.sub.50 values, i.e. in the
range of 100 nM to 1 pM; a moderate to low affinity binding relates
to a high Kd, IC.sub.50 and EC.sub.50 values, i.e. in the
micromolar range.
[0085] One embodiment of the present method for identifying a
compound that inhibits cachexia, and its associated or related
disorders and conditions, comprises culturing a population of
mammalian cells expressing ATGL, determining a first level of FA
release from TG or of ATGL activity or expression in said
population of cells; exposing said population of cells to a
compound, or a mixture of compounds; determining a second level of
FA release from TG or of ATGL activity or expression in said
population of cells under the same or commensurate conditions,
during or after exposure of said population of cells to said
compound, or the mixture of said compounds; and identifying the
compound(s) that suppress TG hydrolysis and/or ATGL activity or
expression. In a specific embodiment, the cells are adipose cells.
In a specific embodiment the cells are human cells.
[0086] The release of FA from TG or lipolysis or lipase activity or
ATGL activity can be determined by methods known in the art such as
the methods as described herein. The assay method may be based on
the particular expression or activity of the ATGL polypeptide,
including but not limited to an enzyme activity. Thus, assays for
the enzyme targets may be based on enzymatic activity or enzyme
expression. The measurable phenomenon, activity or property may be
selected or chosen by the skilled artisan. The person of ordinary
skill in the art may select from any of a number of assay formats,
systems or design one using his knowledge and expertise in the art.
Specific methods to determine the inhibition by a compound by
measuring the cleavage of the substrate by the polypeptide ATGL,
which is a lipase, are well known in the art.
[0087] In one particular embodiment the methods of the present
invention further comprise the step of contacting the population of
cells with an agonist of the polypeptide. For instance, the
activity of the ATGL enzyme can be enhanced by adding the
endogenous activator cgi-58. This improves assay quality in cases
where extracts lacking sufficient cgi-58 or purified enzyme are
used. By using an agonist the polypeptide may be triggered,
enabling a proper read-out if the compound inhibits the
polypeptide. Similar considerations apply to the measurement of the
release of FA from TG. In a particular embodiment, the cells used
in the present method are mammalian adipocytes.
[0088] In a particular aspect of the present invention the methods
include the additional step of comparing the compound to be tested
to a control, where the control is a population of cells that have
not been contacted with the test compound. In a particular aspect
of the present invention the methods described above include the
additional step of comparing the compound to be tested to a
control, where the control is a population of cells that do not
express said polypeptide.
[0089] In a particular aspect of the present invention the methods
described above include the additional step of comparing the
compound to be tested to a control, where the control may be a
general lipase inhibitor such as a pancreatic lipase inhibitor, or
preferably, a lipase inhibitor such as orlistat.
[0090] According to another preferred embodiment, the assay method
uses a compound of formula (I) identified as having a binding
affinity for the ATGL, and/or has already been identified as having
down-regulating activity such as antagonist activity for the
target. In vivo animal models of cachexia or wasting conditions or
infections or other disorders wherein body weight loss or muscle
atrophy is seen may be utilized by the skilled artisan to further
or additionally screen, assess, and/or verify the agents or
compounds identified in the present invention, including further
assessing target/ATGL modulation in vivo. Such animal models
include, but are not limited to cachexia models, such as tumor
models or AIDS models.
[0091] The present invention also provides biologically compatible,
inhibiting or modulating compositions comprising an effective
amount of one or more compounds identified as target inhibitors,
and/or the expression-inhibiting agents as described
hereinabove.
[0092] A biologically compatible composition is a composition, that
may be solid, liquid, gel, or other form, in which the compound, of
the invention is maintained in an active form, e.g., in a form able
to effect a biological activity. For example, a compound of the
invention would have antagonist activity on the ATGL.
[0093] A particular biologically compatible composition is an
aqueous solution that is buffered using, e.g., Tris, phosphate, or
HEPES buffer, containing salt ions. Usually the concentration of
salt ions will be similar to physiological levels. Biologically
compatible solutions may include stabilizing agents and
preservatives. In a more preferred embodiment, the biocompatible
composition is a pharmaceutically acceptable composition. Such
compositions can be formulated for administration by topical, oral,
parenteral, intranasal, subcutaneous, intraperitoneal, and
intraocular, routes. Parenteral administration is meant to include
intravenous injection, intramuscular injection, intraarterial
injection or infusion techniques. The composition may be
administered parenterally in dosage unit formulations containing
standard, well-known non-toxic physiologically acceptable carriers,
adjuvants and vehicles as desired.
[0094] A particular embodiment of the present composition invention
is a pharmaceutical composition comprising a therapeutically
effective amount of an expression-inhibiting agent as described
hereinabove, in admixture with a pharmaceutically acceptable
carrier. Another particular embodiment is a pharmaceutical
composition for the treatment or prevention of a disease or
condition involving cachexia, coronary artery disease, stroke,
atherosclerosis, or type 2 diabetes, and its associated or related
disorders and conditions, or a susceptibility to such conditions,
comprising an effective amount of the ATGL antagonist or inverse
agonist of formula (I), its pharmaceutically acceptable salts,
hydrates, solvates, or prodrugs thereof in admixture with a
pharmaceutically acceptable carrier. Pharmaceutical compositions
for oral administration can be formulated using pharmaceutically
acceptable carriers well known in the art in dosages suitable for
oral administration. Such carriers enable the pharmaceutical
compositions to be formulated as tablets, pills, dragees, capsules,
liquids, gels, syrups, slurries, suspensions, and the like, for
ingestion by the patient. Pharmaceutical compositions for oral use
can be prepared by combining active compounds with solid excipient,
optionally grinding a resulting mixture, and processing the mixture
of granules, after adding suitable auxiliaries, if desired, to
obtain tablets or dragee cores. Suitable excipients are
carbohydrate or protein fillers, such as sugars, including lactose,
sucrose, mannitol, or sorbitol; starch from corn, wheat, rice,
potato, or other plants; cellulose, such as methyl cellulose,
hydroxypropylmethyl-cellulose, or sodium carboxymethyl-cellulose;
gums including arabic and tragacanth; and proteins such as gelatin
and collagen. If desired, disintegrating or solubilizing agents may
be added, such as the cross-linked polyvinyl pyrrolidone, agar,
alginic acid, or a salt thereof, such as sodium alginate. Dragee
cores may be used in conjunction with suitable coatings, such as
concentrated sugar solutions, which may also contain gum arabic,
talc, polyvinyl-pyrrolidone, carbopol gel, polyethylene glycol,
and/or titanium dioxide, lacquer solutions, and suitable organic
solvents or solvent mixtures. Dyestuffs or pigments may be added to
the tablets or dragee coatings for product identification or to
characterize the quantity of active compound, i.e., dosage.
[0095] The invention also relates to the use of the compounds
disclosed herein for the preparation of pharmaceutical compositions
for the treatment of prevention of various conditions.
[0096] Pharmaceutical preparations that can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a coating, such as glycerol or sorbitol.
Push-fit capsules can contain active ingredients mixed with filler
or binders, such as lactose or starches, lubricants, such as talc
or magnesium stearate, and, optionally, stabilizers. In soft
capsules, the active compounds may be dissolved or suspended in
suitable liquids, such as fatty oils, liquid, or liquid
polyethylene glycol with or without stabilizers.
[0097] Preferred sterile injectable preparations can be a solution
or suspension in a non-toxic parenterally acceptable solvent or
diluent. Examples of pharmaceutically acceptable carriers are
saline, buffered saline, isotonic saline (e.g. monosodium or
disodium phosphate, sodium, potassium; calcium or magnesium
chloride, or mixtures of such salts), Ringer's solution, dextrose,
water, sterile water, glycerol, ethanol, and combinations thereof
1,3-butanediol and sterile fixed oils are conveniently employed as
solvents or suspending media. Any bland fixed oil can be employed
including synthetic mono- or di-glycerides. Fatty acids such as
oleic acid also find use in the preparation of injectables.
[0098] The agents or compositions of the invention may be combined
for administration with or embedded in polymeric carrier(s),
biodegradable or biomimetic matrices or in a scaffold. The carrier,
matrix or scaffold may be of any material that will allow
composition to be incorporated and expressed and will be compatible
with the addition of cells or in the presence of cells.
Particularly, the carrier matrix or scaffold is predominantly
non-immunogenic and is biodegradable. Examples of biodegradable
materials include, but are not limited to, polyglycolic acid (PGA),
polylactic acid (PLA), hyaluronic acid, catgut suture material,
gelatin, cellulose, nitrocellulose, collagen, albumin, fibrin,
alginate, cotton, or other naturally-occurring biodegradable
materials. It may be preferable to sterilize the matrix or scaffold
material prior to administration or implantation, e.g., by
treatment with ethylene oxide or by gamma irradiation or
irradiation with an electron beam. In addition, a number of other
materials may be used to form the scaffold or framework structure,
including but not limited to: nylon (polyamides), dacron
(polyesters), polystyrene, polypropylene, polyacrylates, polyvinyl
compounds (e.g., polyvinylchloride), polycarbonate (PVC),
polytetrafluorethylene (PTFE, teflon), thermanox (TPX), polymers of
hydroxy acids such as polylactic acid (PLA), polyglycolic acid
(PGA), and polylactic acid-glycolic acid (PLGA), polyorthoesters,
polyanhydrides, polyphosphazenes, and a variety of
polyhydroxyalkanoates, and combinations thereof. Matrices suitable
include a polymeric mesh or sponge and a polymeric hydrogel. In the
particular embodiment, the matrix is biodegradable over a time
period of less than a year, more particularly less than six months,
most particularly over two to ten weeks. The polymer composition,
as well as method of manufacture, can be used to determine the rate
of degradation. For example, mixing increasing amounts of
polylactic acid with polyglycolic acid decreases the degradation
time. Meshes of polyglycolic acid that can be used can be obtained
commercially, for instance, from surgical supply companies (e.g.,
Ethicon, N.J). In general, these polymers are at least partially
soluble in aqueous solutions, such as water, buffered salt
solutions, or aqueous alcohol solutions that have charged side
groups, or a monovalent ionic salt thereof.
[0099] The composition medium can also be a hydrogel, which is
prepared from any biocompatible or non-cytotoxic homo- or
hetero-polymer, such as a hydrophilic polyacrylic acid polymer that
can act as a drug absorbing sponge. Certain of them, such as, in
particular, those obtained from ethylene and/or propylene oxide are
commercially available. A hydrogel can be deposited directly onto
the surface of the tissue to be treated, for example during
surgical intervention.
[0100] Embodiments of pharmaceutical compositions of the present
invention comprise a vector encoding an agent of the present
invention, particularly a recombinant replication defective vector,
and a transfection enhancer, such as poloxamer. An example of a
poloxamer is Poloxamer 407, which is commercially available (BASF,
Parsippany, N.J.) and is a non-toxic, biocompatible polyol. A
poloxamer impregnated with recombinant viruses may be deposited
directly on the surface of the tissue to be treated, for example
during a surgical intervention. Poloxamer possesses essentially the
same advantages as hydrogel while having a lower viscosity.
[0101] The active expression-inhibiting agents may also be
entrapped in microcapsules prepared, for example, by interfacial
polymerization, for example, hydroxymethylcellulose or
gelatin-microcapsules and poly-(methylmethacylate) microcapsules,
respectively, in colloidal drug delivery systems (for example,
liposomes, albumin microspheres, microemulsions, nano-particles and
nanocapsules) or in macroemulsions. Such techniques are disclosed
in Remington's Pharmaceutical Sciences (1980) 16th edition, Osol,
A. Ed.
[0102] Sustained-release preparations may be prepared. Suitable
examples of sustained-release preparations include semi-permeable
matrices of solid hydrophobic polymers containing the antibody,
which matrices are in the form of shaped articles, e.g. films, or
microcapsules. Examples of sustained-release matrices include
polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and gamma-ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT.TM.. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid. While polymers such as
ethylene-vinyl acetate and lactic acid-glycolic acid enable release
of molecules for over 100 days, certain hydrogels release proteins
for shorter time periods. When encapsulated antibodies remain in
the body for a long time, they may denature or aggregate as a
result of exposure to moisture at 37.degree. C., resulting in a
loss of biological activity and possible changes in immunogenicity.
Rational strategies can be devised for stabilization depending on
the mechanism involved. For example, if the aggregation mechanism
is discovered to be intermolecular S--S bond formation through
thio-disulfide interchange, stabilization may be achieved by
modifying sulfhydryl residues, lyophilizing from acidic solutions,
controlling moisture content, using appropriate additives, and
developing specific polymer matrix compositions.
[0103] As defined above, therapeutically effective dose means that
amount of a compound of formula (I) which ameliorate the symptoms
or condition. Therapeutic efficacy and toxicity of such compounds
can be determined by standard pharmaceutical procedures in cell
cultures or experimental animals, e.g., ED.sub.50 (the dose
therapeutically effective in 50% of the population) and LD.sub.50
(the dose lethal to 50% of the population). The dose ratio of toxic
to therapeutic effects is the therapeutic index, and it can be
expressed as the ratio, LD.sub.50/ED.sub.50. Pharmaceutical
compositions that exhibit large therapeutic indices are preferred.
The data obtained from cell culture assays and animal studies are
used in formulating a range of dosage for human use. The dosage of
such compounds lies preferably within a range of circulating
concentrations that include the ED.sub.50 with little or no
toxicity. The dosage varies within this range depending upon the
dosage form employed, sensitivity of the patient, and the route of
administration.
[0104] For any compound, the therapeutically effective dose can be
estimated initially either in cell culture assays or in animal
models, usually mice, rabbits, dogs, or pigs. The animal model is
also used to achieve a desirable concentration range and route of
administration. Such information can then be used to determine
useful doses and routes for administration in humans. The exact
dosage is chosen by the individual physician in view of the patient
to be treated. Dosage and administration are adjusted to provide
sufficient levels of the active moiety or to maintain the desired
effect. Additional factors which may be taken into account include
the severity of the disease state, age, weight and gender of the
patient; diet, desired duration of treatment, method of
administration, time and frequency of administration, drug
combination(s), reaction sensitivities, and tolerance/response to
therapy. Long acting pharmaceutical compositions might be
administered every 3 to 4 days, every week, or once every two weeks
depending on half-life and clearance rate of the particular
formulation.
[0105] The pharmaceutical compositions according to this invention
may be administered to a subject by a variety of methods. They may
be added directly to target tissues, complexed with cationic
lipids, packaged within liposomes, or delivered to target cells by
other methods known in the art. Localized administration to the
desired tissues may be done by direct injection, transdermal
absorption, catheter, infusion pump or stent. The DNA, DNA/vehicle
complexes, or the recombinant virus particles are locally
administered to the site of treatment. Alternative routes of
delivery include, but are not limited to, intravenous injection,
intramuscular injection, subcutaneous injection, aerosol
inhalation, oral (tablet or pill form), topical, systemic, ocular,
intraperitoneal and/or intrathecal delivery. Examples of ribozyme
delivery and administration are provided in Sullivan et al. WO
94/02595.
[0106] The present invention also provides a method of treating
and/or preventing cachexia, and its associated or related disorders
and conditions, including weight loss, muscle atrophy or wasting,
and reduction of WAT, a pharmaceutical composition or compound as
described herein, particularly a therapeutically effective amount
of an agent which inhibits the expression or activity of ATGL. In a
particular embodiment, the disease is cancer cachexia, wasting
disease associated with AIDS or other infectious disease or
condition, chronic substance abuse, alcoholism, cirrhosis of the
liver, or low body weight associated with anorexia or other
disorders. Further, the invention provides said method for the
treatment of stroke, atherosclerosis, coronary artery disease, and
diabetes. Preferably, the diabetes is diabetes type II.
[0107] Administration of the agent or pharmaceutical composition of
the present invention to the subject patient includes both
self-administration and administration by another person. The
patient may be in need of treatment for an existing disease or
medical condition, or may desire prophylactic treatment to prevent
or reduce the risk for diseases and medical conditions
characterized by cachexia, and its associated or related disorders
and conditions. The agent of the present invention may be delivered
to the subject patient orally, transdermally, via inhalation,
injection, nasally, rectally or via a sustained release
formulation.
[0108] The polypeptides or the polynucleotides of the present
invention employed in the methods described herein may be free in
solution, affixed to a solid support, borne on a cell surface, or
located intracellularly. To perform the methods it is feasible to
immobilize either the polypeptide of the present invention or the
compound to facilitate separation of complexes from uncomplexed
forms of the polypeptide, as well as to accommodate automation of
the assay. Interaction (e.g., binding) of the polypeptide of the
present invention with a compound can be accomplished in any vessel
suitable for containing the reactants. Examples of such vessels
include microtitre plates, test tubes, and microcentrifuge tubes.
In one embodiment, a fusion protein can be provided which adds a
domain that allows the polypeptide to be bound to a matrix. For
example, the polypeptide of the present invention can be "His"
tagged, and subsequently adsorbed onto Ni-NTA microtitre plates, or
ProtA fusions with the polypeptides of the present invention can be
adsorbed to IgG, which are then combined with the cell lysates
(e.g., .sup.(35)S-labelled) and the candidate compound, and the
mixture incubated under conditions favorable for complex formation
(e.g., at physiological conditions for salt and pH). Following
incubation, the plates are washed to remove any unbound label, and
the matrix is immobilized. The amount of radioactivity can be
determined directly, or in the supernatant after dissociation of
the complexes. Alternatively, the complexes can be dissociated from
the matrix, separated by SDS-PAGE, and the level of the protein
binding to the protein of the present invention quantitated from
the gel using standard electrophoretic techniques.
[0109] Other techniques for immobilizing protein on matrices can
also be used in the method of identifying compounds. For example,
either the polypeptide of the present invention or the compound can
be immobilized utilizing conjugation of biotin and streptavidin.
Biotinylated protein molecules of the present invention can be
prepared from biotin-NHS (N-hydroxy-succinimide) using techniques
well known in the art (e.g., biotinylation kit, Pierce Chemicals,
Rockford, Ill.), and immobilized in the wells of
streptavidin-coated 96 well plates (Pierce Chemical).
Alternatively, antibodies reactive with the polypeptides of the
present invention but which do not interfere with binding of the
polypeptide to the compound can be derivatized to the wells of the
plate, and the polypeptide of the present invention can be trapped
in the wells by antibody conjugation. As described above,
preparations of a labeled candidate compound are incubated in the
wells of the plate presenting the polypeptide of the present
invention, and the amount of complex trapped in the well can be
quantitated.
[0110] The polynucleotides encoding ATGL and HSL encoding
polynucleotides, particularly ATGL encoding nucleotides, such as
human or mouse ATGL are available to the skilled person. For
instance, WO2010115825 describes, in FIG. 7 therein, the amino acid
and nucleic acid sequence of human ATGL and in FIG. 8 therein the
amino acid and nucleic acid sequence of mouse ATGL. WO2010115825
further describes methods of expressing said lipases and conducting
assays to test for lipase activity which may be advantageously used
in the practice of the current invention.
EXAMPLES
Example 1
Methods
[0111] cDNA cloning and transient expression of recombinant
His-tagged proteins in COS-7 cells and 3T3-L1 adipocytes.
[0112] The coding sequences of mouse ATGL and HSL are amplified by
PCR from cDNA prepared from mRNA of mouse white adipose tissue by
reverse transcription. The open reading frame, flanked by KpnI/XhoI
sites for ATGL and HSL were cloned into the eucaryotic expression
vector pcDNA4/HisMax (Invitrogen). Transfection of COS-7 cells was
performed with Metafectene.TM. (Biontex) according to the
manufacturer's description. The PCR primers used to generate these
probes were as follows.
TABLE-US-00001 ATGL forward 5'-TGGTACCGTTCCCGAGGGAGACCAAGTGGA-3',
ATGL revers 5'-CCTCGAGCGCAAGGCGGGAGGCCAGGT-3'. HSL forward
5'-TGGTAGGT-ATGGATTTACGCACGATGACAGA-3', HSL revers
5'-CCTCGAGCGTTCAGTGGTGCAGCAGGCG-3'.
Construction of the Recombinant Adenovirus for ATGL Expression
(ATGL-Ad) and Infection of 3T3-L1 Cells:
[0113] The recombinant adenovirus coding for mouse ATGL is prepared
by cotransfection of the shuttle plasmid pAvCvSv containing the
ATGL cDNA and pJM 17 into HEK-293 cells. The 1.65 kb Mlu I-Cla I
flanked mouse ATGL cDNA fragment (His-tag included) is amplified by
PCR from the eucaryotic expression vector pcDNA4/HisMax containing
mouse ATGL cDNA and subcloned into Mlu I-Cla I digested pAvCvSv.
The resulting shuttle plasmid is cotransfected with pJM 17 into
HEK-293 cells using the calcium phosphate coprecipitation method.
Large scale production of high titer recombinant ATGL-Ad is
performed as described elsewhere. 3T3-L1 fibroblasts were cultured
in DMEM containing 10% FCS and differentiated using a standard
protocol (Bernlohr, D. A. et al (1985) J Biol Chem 260: 5563-7).
Adipocytes are infected on day 8 of differentiation with a
multiplicity of infection (moi) of 400 plaque forming units/cell.
For that purpose appropriate pfu are preactivated in DMEM
containing 0.5 .mu.g/ml of polylysin for 100 min and afterwards the
cells are incubated with this virus suspension for 24 hours. After
24 h the medium is removed and the cells are incubated for further
24 h with complete medium. For most of the experiments, recombinant
adenovirus expressing .beta.-galactosidase was used as a control
(LacZ-Ad).
Western Analysis.
[0114] Cellular proteins are separated by SDS-polyacrylamide gel
electrophoresis and transferred to a nitrocellulose membrane
(Schleicher & Schuell, Germany). For detection of His-tagged
proteins, blots were incubated with 1/10000 diluted Anti-His
monoclonal antibody (6.times.His, Clonetech). Bound immunoglobulins
are detected with a HRP-labeled IgG conjugates (Vector Inc.) and
visualized by ECL detection (ECL plus, Amersham Pharmacia Biotech,
Germany) on a Storm Image Analysis system. Quantitation is
performed using ImageQuant Software.
[0115] Reaction of ATGL and HSL with the fluorescent lipase
inhibitor NBD-HEHP. Transfected COS-7 cells are washed twice with
PBS, scraped into lysis buffer (0.25 M sucrose, 1 mM EDTA, 1 mM
dithioerythritol, 20 .mu.g/ml leupeptin, 2 .mu.g/ml antipain, 1
.mu.g/ml pepstatin) and disrupted on ice by sonication. Nuclei and
unbroken materials are removed by centrifugation at 1.000 g at
4.degree. C. for 15 min to obtain cytoplasmatic extracts. 50 .mu.g
of protein is incubated with 1 nmol fluorescently labelled lipase
inhibitor
O-((6-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl)aminoethyl-O-(n-h-
exyl)phosphonic acid p-nitrophenyl ester (NBD-HEHP) (Oskolkova, O.
V. et al (2003) Chem Phys Lipids 125:103-14.) and 1 mM Triton X-100
(especially purified for membrane research, Hofmann LaRoche) at
37.degree. C. for 2 hours under shaking. Protein is precipitated
with 10% TCA for 1 h on ice, washed with acetone and separated by
10% SDS-PAGE. Gels are fixed in 10% ethanol and 7% acetic acid.
Fluorescence is detected with a BioRad FX Pro Laserscanner
(excitation 488 nm, emission 530 nm).
Northern Analysis.
[0116] The cDNA probe for northern blot analysis of mouse ATGL is
prepared by RT-PCR by use of first-strand cDNA from mouse fat mRNA.
PCR primers used to generate this probe are as follows: forward
5'-TGGAACATCTCATTCGCTGG-3', reverse 5'-AATGCCGCCATCCACATAG-3'.
Total RNA was isolated from various mouse tissues using the TRI
Reagent procedure according to manufacturer's protocol (Molecular
Research Center, Karlsruhe, Germany). Specific mRNAs were detected
using standard Northern blotting techniques with 10 .mu.g total
RNA. 32P-labeled probes for hybridization were generated using
random priming. Northern blots are visualized by exposure to a
PhosphorImager Screen (Apbiotech, Freiburg, Germany) and analyzed
using ImageQuant Software.
Compound Synthesis
[0117] Compounds 1-4 were prepared using organic synthesis methods.
Unless otherwise stated, all experiments were carried out under
inert atmosphere by using standard Schlenk-techniques.
Acetonitrile, DMF, Ethanol and Dimethoxyethane were purchased as
absolute solvents from Acros Organics, Fisher Scientific and Sigma
Aldrich. All applied starting materials were commercially available
from Alfa Aesar and Sigma Aldrich and were used as received. Silica
gel chromatography was performed with Acros Organics silica gel 60
(35-70 .mu.M). .sup.1H and .sup.13C NMR spectra were recorded on
Bruker AVANCE III 300 spectrometer (.sup.1H, 300.36 MHz; .sup.13C,
75.53 MHz) and chemical shifts are referenced to residual
protonated solvent signals as internal standard. Electron impact
(El, 70 eV) HRMS spectra were recorded on Waters GCT Premier
equipped with direct insertion (DI) and GC (HP GC7890A). GC/MS
analyses were made on an Agilent Technologies 5975 C inert MSD with
Triple Axis Detector GC system with a mass sensitive detector.
Ethyl 1-(4-ethoxyphenyl)-4-hydroxy-1H-pyrazole-3-carboxylate
(1)
##STR00005##
[0118] Ethyl-4-chloro-2-((4-ethoxyphenyl)diazenyl)-3-oxobutanoate
(5)
[0119] A 10 mL one-neck round-bottom flask was charged with 250.0
mg (0.24 mL, 1.825 mmol, 1.04 eq) 4-ethoxyaniline which was
dissolved in 3.1 mL acetic acid at 10.degree. C. (ice bath). To
this cooled solution 128.0 mg (1.861 mmol, 1.06 eq) NaNO.sub.2 in
0.5 mL conc. H.sub.2SO.sub.4 were added and the reaction mixture
was stirred at 10.degree. C. for 1 h. In a second 25 ml one-neck
round-bottom flask 288.6 mg (0.24 mL, 1.755 mmol, 1.0 eq)
ethyl-4-chloro-3-oxobutanoate were dissolved in a mixture of 1.3 mL
acetic acid and 2.6 mL water and cooled to 0.degree. C. (ice bath
+NaCl). After one hour stirring at 10.degree. C. the generated
solution of the diazonium salt was given to the second solution at
0.degree. C. and stirred for further 15 min at this temperature. An
aqueous solution of 1.58 g (19.305 mmol, 11 eq) NaOAc in 3.0 mL
water was added to the reaction mixture at 0.degree. C. and the
product precipitated. Stirring at rt over night, filtration,
washing with a small amount of water and drying under high pressure
yielded the crude product which was used in the next reaction step
without further purification.
[0120] yield: 453.7 mg (83%); yellow-brown solid; M.p.:
106-110.degree. C.; .sup.1H-NMR (300 MHz, DMSO-d.sub.6): Isomer I
(25%): .delta. (ppm)=14.39 (s, 1H, NH), 7.57-7.35 (m, 2H, Ar--H),
7.98-7.95 (m, 2H, Ar--H), 4.95-4.92 (m, 2H, Cl--CH.sub.2),
4.31-4.27 (m, 2H, CH.sub.2), 4.03-3.98 (m, 4H, 2CH.sub.2),
1.34-1.27 (m, 6H, 2CH.sub.3), Isomer II (75%): .delta. (ppm)=12.35
(s, 1H, NH), 7.57-7.35 (m, 2H, Ar--H), 6.98-6.95 (m, 2H, Ar--H),
4.95-4.92 (m, 2H, Cl--CH.sub.2), 4.31-4.27 (m, 2H, CH.sub.2),
4.03-3.98 (m, 4H, 2CH.sub.2), 1.34-1.27 (m, 6H, 2CH.sub.3);
.sup.13C-NMR (75.5 MHz, DMSO-d.sub.6): Isomer I: .delta.
(ppm)=187.6 (C.dbd.O), 164.0 (C.dbd.O), 156.9 (C.sub.q), 135.2
(C.dbd.N), 125.1 (C.sub.q), 118.1 (2CH.sub.Ar), 115.2 (2CH.sub.Ar),
63.2 (OCH.sub.2), 60.3 (Cl--CH.sub.2), 49.6 (CH.sub.2), 14.1
(CH.sub.3), 13.9 (CH.sub.3), Isomer II: .delta. (ppm)=185.7
(C.dbd.O), 162.0 (C.dbd.O), 156.0 (C.sub.q), 135.2 (C.dbd.N), 125.1
(C.sub.q), 117.4 (2CH.sub.Ar), 115.1 (2CH.sub.Ar), 63.2
(OCH.sub.2), 60.8 (Cl--CH.sub.2), 46.9 (CH.sub.2), 14.5 (CH.sub.3),
13.9 (CH.sub.3).
Ethyl 1-(4-ethoxyphenyl)-4-hydroxy-1H-pyrazole-3-carboxylate
(1)
[0121] A 25 mL Schlenk tube was charged with 270.6 mg (0.866 mmol,
1.0 eq) ethyl-4-chloro-2-((4-ethoxyphenyl)diazenyl)-3-oxobutanoate
(5) which was suspended in 4 mL EtOH under a gentle stream of
nitrogen. After adding 102.0 mg (1.039 mmol, 1.2 eq) KOAc the
suspension was stirred under reflux for 1.5 h, during which the
suspension dissolved. TLC analysis (CH/EtOAc 3:1) indicated full
conversion of the starting material. After cooling to rt the
mixture was transferred to a flask to remove the EtOH with a rotary
evaporator. The residue was dissolved in 20 mL EtOAc and washed
with water (2.times.15 mL) and 15 mL brine, dried over MgSO.sub.4
and concentrated at the rotary evaporator. Drying at high vacuum
yielded the pure product without further purification.
[0122] yield: 232.4 mg (97%); dark red-brown solid; M.p.:
96-98.degree. C.; R.sub.f (CH/EtOAc 3:1): 0.35; .sup.1H-NMR (300
MHz, DMSO-d.sub.6): .delta. (ppm)=9.07 (s, 1H, OH), 7.96 (s, 1H,
Ar--H), 7.70 (d, J=9.0 Hz, 2H, Ar--H), 7.03 (d, .sup.3J=9.0 Hz, 2H,
Ar--H), 4.28 (q, .sup.3J=6.9 Hz, 2H, CH.sub.2), 4.06 (q,
.sup.3J=6.9 Hz, 2H, CH.sub.2), 1.36-1.27 (m, 6H, 2CH.sub.3);
.sup.13C-NMR (75.5 MHz, DMSO-d.sub.6): .delta. (ppm)=161.6
(C.dbd.O), 157.3 (C.sub.q), 145.0 (C.sub.q--OH), 132.8 (C.sub.q),
131.1 (C.sub.q), 120.0 (2CH.sub.Ar), 114.9 (2 CH.sub.Ar), 114.8
(CH.sub.Ar), 63.3 (CH.sub.2), 59.7 (CH.sub.2), 14.5 (CH), 14.2
(CH.sub.3); GC-MS (NM.sub.--50_S2): t.sub.R=8.071 min (m/z=276.1,
99.0% M.sup.+, BP).
Ethyl 1-(4-ethoxyphenyl)-1H-pyrazole-3-carboxylate (2)
##STR00006##
[0123] Ethyl 1H-pyrazole-3-carboxylate (6)
[0124] A 100 mL Schlenk tube was dried under vacuum, filled with
nitrogen and charged with 0.5 g (4.461 mmol, 1.0 eq)
1H-pyrazol-3-carboxylic acid which was dissolved in 20 mL EtOH.
After adding 1.31 g (0.72 mL, 13.382 mmol, 3.0 eq) conc.
H.sub.2SO.sub.4 the colorless reaction mixture was heated to reflux
(100.degree. C.) and stirred at this temperature for 4 h. TLC
analysis (DCM/MeOH 95:5) indicated full conversion of the starting
material. After cooling to rt the mixture was transferred to a
flask to remove the solvent at a rotary evaporator. The colorless
residue was diluted in 20 mL water and neutralized with 17 mL
saturated aqueous NaHCO.sub.3 solution. Thereby a white solid
precipitated. The aqueous layer was extracted with EtOAc
(4.times.50 mL), dried over MgSO.sub.4 and the solvent was
evaporated under reduced pressure to yield the pure title
compound.
[0125] yield: 582.5 mg (93%); colorless solid; M.p.:
158-161.degree. C.; R.sub.f, (DCM/MeOH 95:5): 0.42; .sup.1H-NMR
(300 MHz, CDCl.sub.3): .delta. (ppm)=10.69 (bs, 1H, NH), 7.84 (d,
.sup.4J=2.1 Hz, 1H, Ar--H), 6.86 (d, .sup.4J=2.1 Hz, 1H, Ar--H),
4.43 (q, .sup.3J=7.2 Hz, 2H, CH.sub.2), 1.41 (t, .sup.3J=7.2 Hz,
3H, CH.sub.3); .sup.13C-NMR (75.5 MHz, CDCl.sub.3): .delta.
(ppm)=161.8 (C.dbd.O), 141.6 (C.sub.q), 132.3 (CH.sub.Ar), 107.8
(CH.sub.Ar), 61.1 (CH.sub.2), 14.3 (CH.sub.3); GC-MS
(NM.sub.--50_S2): t.sub.R=4.655 min (m/z=140.1, 98.0% M.sup.+, BP:
95.0).
Ethyl 1-(4-ethoxyphenyl)-1H-pyrazole-3-carboxylate (2)
[0126] A 10 mL Schlenk tube was dried under vacuum, filled with
nitrogen and consecutively charged with 19.4 mg (0.102 mmol, 0.2
eq) CuI, 332.3 mg (1.020 mmol, 2.0 eq) Cs.sub.2CO.sub.3, 100.0 mg
(0.714 mmol, 1.4 eq) ethyl 1H-pyrazole-3-carboxylate (6), 102.4 mg
(73.0 .mu.L, 0.510 mmol, 1.0 eq) p-bromophenetol and 1 mL dry ACN.
The light brown suspension was degassed by vacuum/N.sub.2 cycles
and stirred first at 82.degree. C. for 7 h and than after adding
0.5 mL dry DMF (solubility issue) at 120.degree. C. for further 65
h. The GC-MS analysis showed full conversion. ACN and DMF were
removed under high pressure and the brown residue was suspended in
10 mL EtOAc. After filtration of the brown suspension through a pad
of silica and flushing with 150 mL EtOAc the colorless filtrate was
concentrated under reduced pressure leading to 52.1 mg (39%) crude
product as a green-brown oil. Final purification by column
chromatography (CH/EtOAc 3:1, size: 15.5.times.2.0 cm, 20 g silica
gel) yielded the pure title compound.
[0127] yield: 10.2 mg (8%); orange solid; M.p.: 88-90.degree. C.;
R.sub.f (CH/EtOAc 3:1): 0.40; .sup.1H-NMR (300 MHz, DMSO-d.sub.6):
.delta. (ppm)=8.51 (d, .sup.4J=2.4 Hz, 1H, Ar--H), 7.78 (d,
.sup.3J=9.0 Hz, 2H, Ar--H), 7.07 (d, .sup.3J=9.0 Hz, 2H, Ar--H),
6.97 (d, .sup.4J=2.4 Hz, 1H, Ar--H), 4.31 (q, .sup.3J=6.9 Hz, 2H,
CH.sub.2), 4.08 (q, .sup.3=7.2 Hz, 2H, CH.sub.2), 1.37-1.29 (m, 6H,
2CH.sub.3); .sup.13C-NMR (75.5 MHz, DMSO-d.sub.6):
[0128] .delta. (ppm)=161.4 (C.dbd.O), 157.6 (C.sub.q), 143.6
(C.sub.q), 132.5 (C.sub.q), 129.5 (CH.sub.Ar), 120.7 (2CH.sub.Ar),
115.0 (2CH.sub.Ar), 119.8 (CH.sub.Ar), 63.4 (CH.sub.2), 60.3
(CH.sub.2), 14.5 (CH.sub.3), 14.1 (CH.sub.3); GC-MS
(NM.sub.--50_S2): t.sub.R=7.722 min (m/z=260.1, 98.0% M.sup.+,
BP).
Ethyl 4-ethoxybiphenyl-3-carboxylate (3)
##STR00007##
[0130] A 20 mL Schlenk tube was dried under vacuum, filled with
nitrogen and charged consecutively with 100.0 mg (70 .mu.L, 0.437
mmol, 1.0 eq) ethyl-3-bromobenzoate, 72.5 mg (0.437 mmol, 1.0 eq)
4-ethoxyphenylboronic acid, 139.3 mg (0.917 mmol, 2.1 eq) CsF, 17.8
mg (0.022 mmol, 0.05 eq) PdCl.sub.2(dppf)*DCM and 5.0 mL DME. The
orange suspension was degassed by vacuum/N.sub.2 cycles and stirred
at 80.degree. C. for 7 h. GC-MS analysis indicated full conversion
(98% product) of the starting material. The reaction mixture was
filtered through a pad of celite which was rinsed with EtOAc. The
solvent from the filtrate was removed under reduced pressure and
final purification by column chromatography (CH/EtOAc 15:1, size:
12.5.times.2.0 cm, 15 g silica gel) yielded the pure product.
[0131] yield: 109.5 mg (93%); colorless solid; M.p.: 46-47.degree.
C.; R.sub.f (CH/EtOAc 15:1): 0.40; .sup.1H-NMR (300 MHz,
CDCl.sub.3): .delta. (ppm)=.delta. (ppm)=8.25 (t, .sup.4J=1.5 Hz,
1H, Ar--H), 7.98 (d, J=7.8 Hz, 1H, Ar--H), 7.74 (d, .sup.3J=7.8 Hz,
1H, Ar--H), 7.56 (d, J=9.0 Hz, 2H, Ar--H), 7.48 (t, .sup.3J=7.8 Hz,
1H, Ar--H), 6.98 (d, .sup.3J=9.0 Hz, 2H, Ar--H), 4.41 (q,
.sup.3J=7.2 Hz, 2H, OH.sub.2), 4.09 (q, .sup.3J=6.9 Hz, 2H,
CH.sub.2), 1.47-1.39 (m, 6H, 2CH.sub.3); .sup.13C-NMR (75.5 MHz,
CDCl.sub.3): .delta. (ppm)=166.6 (C.dbd.O), 158.8 (C.sub.q), 141.0
(C.sub.q), 132.5 (C.sub.q), 131.0 (C.sub.q), 130.9 (CH.sub.Ar),
128.7 (CH.sub.Ar), 128.2 (2CH.sub.Ar), 127.7 (CH.sub.Ar), 127.6
(CH.sub.Ar), 114.8 (2CH.sub.Ar), 63.8 (CH.sub.2), 61.0 (CH.sub.2),
14.8 (CH.sub.3), 14.3 (CH.sub.3); GC-MS (NM.sub.--50S2):
t.sub.R=7.796 min (m/z=270.1, 99% M.sup.+, BP); HRMS (El.sup.+):
m/z: calcd for C.sub.17H.sub.18O.sub.3[M].sup.+: 270.1256. found
270.1260.
Ethyl 4'-(dimethylamino)biphenyl-3-carboxylate (4)
##STR00008##
[0133] A 25 mL Schlenk tube was dried under vacuum, filled with
nitrogen and charged consecutively with 150.0 mg (0.1 mL, 0.655
mmol, 1.0 eq) ethyl-3-bromobenzoate, 107.9 mg (0.655 mmol, 1.0 eq)
4-(dimethylamino)phenylboronic acid, 208.9 mg (1.375 mmol, 2.1 eq)
CsF, 26.7 mg (0.033 mmol, 0.05 eq) PdCl.sub.2(dppf)*DCM and 7.5 mL
DME. The orange suspension was degassed by vacuum/N.sub.2 cycles
and stirred at 80.degree. C. for 26 h. GC-MS analysis indicated
full conversion (86% product) of the starting material. The
reaction mixture was filtered through a pad of celite which was
rinsed with EtOAc. The solvent from the filtrate was removed under
reduced pressure and final purification by column chromatography
(CH/EtOAc 19:1, size: 14.0.times.2.0 cm, 25 g silica gel) yielded
the pure product.
[0134] yield: 135.9 mg (77%); colorless solid; M.p.: 79-81.degree.
C.; R.sub.f (CH/EtOAc 19:1): 0.24; .sup.1H-NMR (300 MHz,
CDCl.sub.3): .delta. (ppm)=8.26 (s, 1H, Ar--H), 7.94 (d,
.sup.3J=7.8 Hz, 1H, Ar--H), 7.74 (d, .sup.3J=7.8 Hz, 1H, Ar--H),
7.55 (d, .sup.3J=9.0 Hz, 2H, Ar--H), 7.46 (t, .sup.3J=7.8 Hz, 1H,
Ar--H), 6.82 (d, .sup.3J=9.0 Hz, 2H, Ar--H), 4.41 (q, .sup.3J=7.2
Hz, 2H, CH.sub.2), 3.01 (s, 6H, 2CH.sub.3), 1.42 (t, .sup.3J=7.2
Hz, 3H, CH.sub.3); .sup.13C-NMR (75.5 MHz, CDCl.sub.3): .delta.
(ppm)=166.8 (C.dbd.O), 150.2 (C.sub.q--N(CH.sub.3).sub.2), 141.4
(C.sub.q), 130.8 (C.sub.q), 130.4 (CH.sub.Ar), 128.6 (CH.sub.Ar),
128.0 (C.sub.q), 127.7 (2CH.sub.Ar), 127.2 (CH.sub.Ar), 127.0
(CH.sub.Ar), 112.7 (2CH.sub.Ar), 60.9 (CH.sub.2), 40.5
(N(CH.sub.3).sub.2), 14.4 (CH.sub.3); GC-MS (NM.sub.--50_S2):
t.sub.R=8.288 min (m/z=269.1, 99% M.sup.+, BP).
[0135] Expression of Recombinant Proteins and Preparation of Cell
Lysates.
[0136] Monkey embryonic kidney cells (Cos-7; ATCC CRL-1651) were
cultivated in DMEM (GIBCO, Invitrogen Corp., Carlsbad, Calif.),
containing 10% fetal calf serum (FCS, Sigma-Aldrich) and
antibiotics (100 IU/ml penicillin and 100 .mu.g/ml streptomycin) at
standard conditions (37.degree. C., 5% CO.sub.2, 95% humidified
atmosphere). Cells were transfected with 1 .mu.g DNA complexed to
Metafectene (Biontex GmbH, Munich, Germany) in serum free DMEM.
After 4 h the medium was replaced by DMEM supplemented with 10%
FCS. For the preparation of cell lysates, cells were washed with
1.times.PBS, collected using a cell scraper, and disrupted in
buffer A (0.25 M sucrose, 1 mM EDTA, 1 mM dithiothreitol, 20
.mu.g/ml leupeptine, 2 .mu.g/ml antipain, 1 .mu.g/ml pepstatin, pH
7.0) by sonication (Virsonic 475, Virtis, Gardiner, NJ). Nuclei and
unbroken cells were removed by centrifugation (1,000.times.g,
4.degree. C., for 10 min).
[0137] For over-expression of ATGL and CGI-58 in E. coli (XL-1)
cDNA was cloned into the vector pASK-IBA5+ (IBA-BioTagnology).
Cells were transformed and cultured over night at 30.degree. C.
Thereafter cells were transferred into a fresh medium and grown
until OD600 reached 0.7-0.8. Expression was induced using 0.2
.mu.g/ml anhydrotetracycline. After 3 hours incubation at
37.degree. C. cells where harvested, resuspended in lysis buffer
(0.25M Sucrose, 1 mM DTT, 1 mM EDTA) and disrupted by sonication.
Lysates were centrifuged at 15,000 g for 20 min at 4.degree. C. and
the supernatant was collected.
[0138] Determination of protein concentrations of cell lysates and
detection of His-tagged proteins by Western blotting analysis were
performed as described below.
[0139] Preparation of Tissue Homogenates.
[0140] Murine adipose tissue samples were washed in PBS containing
1 mM EDTA and homogenized on ice in buffer A using an Ultra Turrax
(IKA, Staufen, Germany). Homogenates were centrifuged for 30 min at
20,000.times.g and 4.degree. C. to obtain tissue extracts. Protein
content was determined as described below.
[0141] Determination of TG Hydrolase Activity.
[0142] For the determination of TG hydrolase activity of cell
lysates containing various recombinant proteins, tissue extracts,
or purified proteins, samples in a total volume of 100 .mu.l buffer
A were incubated with 100 .mu.l substrate in a water bath at
37.degree. C. for 60 min. As a control, incubations under identical
conditions were performed in buffer A alone. After incubation, the
reaction was terminated by adding 3.25 ml of
methanol/chloroform/heptane (10:9:7) and 1 ml of 0.1 M potassium
carbonate, 0.1 M boric acid, (pH 10.5). After centrifugation
(800.times.g, 15 min), the radioactivity in 1 ml of the upper phase
was determined by liquid scintillation counting. Counts from
control incubations were subtracted and the rate of FA hydrolysis
calculated using .sup.3H radiolabelling of triolein substrate. TG
substrate was prepared by emulsifying 330 .mu.M triolein (40 000
cpm/nmol) and 45 .mu.M phosphatidylcholine/phosphatidylinositol
(3:1) in 100 mM potassium phosphate buffer, (pH 7.0) by sonication
and adjusted to 5% essentially FA-free BSA (Sigma, St. Louis,
Mo.).
[0143] Determination of MG Hydrolase Activity.
[0144] Monoacylglycerol hydrolase activities were determined using
recombinant, purified mMGL and rac-1-(3)-oleoylglycerol as
substrate as described (Taschler, 2011).
[0145] Lipolysis of 3T3-L1 Cells.
[0146] 3T3-L1 fibroblasts (CL-173) were obtained from ATCC
(Teddington, UK) and cultivated in DMEM containing 4.5 g/liter
glucose and L-glutamine (Invitrogen) supplemented with 10% FCS and
antibiotics under standard conditions. Cells were seeded in 12 well
plates and two days after confluence, medium was changed to DMEM
supplemented with 10% FCS containing 10 .mu.g/ml insulin
(Sigma-Aldrich), 0.25 .mu.M (0.4 .mu.g/ml) dexamethasone
(Sigma-Aldrich), and 500 .mu.M isobutylmethylxanthine
(Sigma-Aldrich). After 3 and 5 days, medium was changed to DMEM
supplemented with 10% FCS containing 10 .mu.g/ml and 0.5 .mu.g/ml
insulin, respectively. On day 7 of differentiation the cells were
incubated ON in the absence of insulin. Cells were used at day 8 of
differentiation. Therefore cells were preincubated with 0, 0.1, 1,
10, or 50 .mu.M of Atglistatin in the presence or absence of 10
.mu.M Hi 76-0079 (NNC 0076-0000-0079, provided by Novo Nordisk,
Denmark) for 2 h. Then, the medium was replaced by DMEM containing
2% BSA (fatty acid free, Sigma, St. Louis, Mo.), 10 .mu.M
forskolin, and 0, 0.1, 1, 10, or 50 .mu.M of Atglistatin in the
presence or absence of 10 .mu.M Hi 76-0079 for 1 h. The release of
FFA and glycerol in the media was determined using commercial kits
(NEFA C, WAKO, free glycerol reagent, Sigma). Protein concentration
was determined using BCA reagent (Pierce) after extracting total
lipids using hexane:isopropanol (3:2), and lysing the cells using
SDS:NaOH (0.3%:0.1N).
[0147] Lipolysis of Isolated WAT Organ Cultures--
[0148] Gonadal fat pads of C57Bl6 mice were surgically removed and
washed several times with PBS. Tissue pieces (.about.15 mg) were
preincubated in DMEM containing 0, 0.1, 1, 10, and 50 .mu.M
Atglistatin for 8 h at C37.degree. C., 5% CO.sub.2, 95% humidified
atmosphere. Thereafter, the medium was replaced by DMEM containing
2% BSA (fatty acid-free) either in the presence or in the absence
of 10 .mu.M forskolin and 0, 0.1, 1, 10, and 50 .mu.M, and
incubated for another 60 min at 37.degree. C. Then, aliquots of the
medium were removed and analyzed for FFA and glycerol content using
commercial kits (HR Series NEFA-HR(2), WAKO Diagnostics, Neuss,
Germany; Sigma, St. Louis, Mo.). For protein determinations, fat
pads were washed extensively with PBS and lysed in 0.3 N NaOH/0.1%
SDS. Protein measurements were performed using the BCA reagent
(Pierce Rockfort, Ill.).
[0149] Toxicity Test for Atglistatin in AML-12 Mouse
Hepatocytes.
[0150] For MTT-based in vitro viability assays, cells were seeded
at an initial density of 1.times.10.sup.4 cells per well in 96-well
plates and cultured under standard conditions for twenty-four
hours. The next day, cells were pretreated with different
concentrations of Atglistatin dissolved in DMSO or Cisplatin
dissolved in dimethylformamide (DMF) (Sigma-Aldrich) as positive
control for two hours. Medium was replaced by an identical fresh
medium and incubated again for the indicated timepoints. Thereafter
cells were incubated for 3 hours with 100 .mu.l Thiazolyl Blue
Tetrazolium Bromide (MTT). The resulting violet formazan crystals
were dissolved by adding 100 .mu.l of MTT Solubilization Solution
(0.1% NP-40, 4 mM HCl and anhydrous isopropanol). After complete
dissolution of the formazan product, absorbance was measured at 595
nm and 690 nm. The values obtained at 690 nm were subtracted from
the 595 nm measurements.
[0151] Animals.
[0152] Mice (C57Bl/6J) were maintained on a regular light-dark
cycle (14 hours light, hours dark) and kept on a standard
laboratory chow diet (4.5% wt/wt fat, Sniff GMBH Germany).
Maintenance, handling, and tissue collection from mice has been
approved by the Austrian Federal Ministry of Science and Research
Education and by the ethics committee of the University of Graz.
Atglistatin was orally administrated by gavage of 200 .mu.l olive
oil containing Atglistatin.
[0153] Determination of Tissue TG Content.
[0154] For tissue collection mice were euthanized by cervical
dislocation. Subsequently, tissues were excised and snap frozen.
After homogenisation in buffer A without DTT, TG was measured using
TG reagent (Thermo Electron Corp., Victoria, Australia).
[0155] Plasma Parameters.
[0156] Blood samples of fed or fasted mice were collected by
retro-orbital puncture. Plasma levels of TG, glycerol, FFA, and
total cholesterol were determined using commercial kits (Thermo
Electron Corp., Victoria, Australia; Sigma, St. Louis, Mo.; Wako
Chemicals, Wako Chemicals, Neuss, Germany; Roche Diagnostics,
Vienna, Austria). Blood glucose was determined using Accu-Check
glucometer (Roche, Diagnostics).
[0157] Determination of Protein Concentrations.
[0158] Protein concentrations were determined using Bio-Rad protein
assay according to the manufacturer's protocol (Bio-Rad 785,
Bio-Rad Laboratories, Munich, Germany), using BSA as standard.
Alternatively, protein measurements were performed using the BCA
reagent (Pierce, Rockford, Ill.).
[0159] Statistical Analysis.
[0160] Statistical analyses were determined by Student's unpaired
t-test (two tailed). Following levels of statistical significance
were used: * . . . p<0.05, ** . . . p<0.01, and *** . . .
p<0.001.
Example 2
Compound Chemistry
[0161] Compound 1 (FIG. 1A) inhibits the activity of recombinant
ATGL (IC.sub.50=50 .mu.M). The HO-group in 4-position could be an
anchor point for path II metabolism. In order to test the effect of
the HO-group in the 4-position on the inhibitory effect and
toxicity, compound 2 was prepared by Ullmann-arylation of ethyl
pyrazol-3-carboxylate. This compound inhibited ATGL with an
IC.sub.50 of =40 .mu.M. Both compounds showed some limited toxicity
in in vitro tests. The pyrazole moiety was therefore targeted for
replacement. Next, a series of compounds were prepared in which the
pyrazole was replaced by other aromatic and heteroaromatic rings
keeping the 1,3-arrangement of the 4-ethoxyphenyl- and
ethoxycarbonyl-substituents constant.
[0162] Using this strategy, biphenyl compound 3 (IC.sub.50=12
.mu.M, FIG. 1) emerged as a suitable scaffold. Next, the role of
the ethoxy substituent in the bottom ring was investigated. A
series of compounds prepared by Pd-catalyzed Suzuki-coupling of
different 4-substituted phenylboronic acids with
ethyl-3-bromobenzoate indicated that electron donating groups at
the 4'-phenyl position promote ATGL inhibition. Additionally
possibilities were surveyed to replace the ester moiety in the
3-position by other functional groups. This resulted in urea
compound 4 which showed improved inhibition activity. The
dose-dependent inhibition of ATGL activity by compounds 3 and 4 is
shown in FIG. 1B. Compound 4 was named Atglistatin and inhibited
the activity of recombinant ATGL with an IC.sub.50 of 0.7 .mu.M.
Cytotoxicity assays revealed for 4 only a very moderate decrease in
cell viability (tested as described in example 1/methods), at
concentrations .gtoreq.100 .mu.M.
Example 3
Kinetic Studies of Inhibitor Action
[0163] To investigate the mechanism of Atglistatin-mediated ATGL
inhibition, kinetic studies were performed by varying substrate and
inhibitor concentrations. Kinetic analysis revealed an increase in
the apparent K.sub.m values and unchanged V.sub.max (FIG. 1C)
indicating that Atglistatin acts as a competitive inhibitor. Based
on the increase of K.sub.m values determined in three independent
experiments and using non-linear regression analysis (SigmaPlot
12.0), a K.sub.i value of 0.5+/-0.2 .mu.M was calculated. However,
it has to be considered that this is a relative value, since the TG
substrate is not water-soluble and consequently only partially
available for the reaction.
Example 4
Inhibition of ATGL Activity in Adipose Tissue Lysates and
Selectivity of Atglistatin
[0164] To evaluate whether Atglistatin inhibits other lipases
present in adipose tissue, white adipose tissue (WAT) lysates of
wild-type and ATGL deficient mice were analysed for TG hydrolase
activity in the presence and absence of Atglistatin. As shown in
FIG. 2, Atglistatin inhibited TG hydrolase activity of wild-type
WAT in a dose dependent manner (FIG. 2A) up to 78% at a
concentration of 40 .mu.M. Compared to the wild-type control, TG
hydrolase activity in lysates obtained from ATGL-ko animals was
reduced by approximately 70%. Notably, a moderate but significant
effect of Atglistatin on the residual activity in these lysates was
observed at concentrations .gtoreq.20 .mu.M. This could indicate
that Atglistatin inhibits other minor lipases that are structurally
related to ATGL such as adiponutrin (PNPLA3). However, most of the
remaining activity present in ATGL-ko lysates can be ascribed to
HSL. As shown in FIG. 2B, the HSL inhibitor Hi 76-0079 decreased TG
hydrolase activity in wild-type lysates by 72% which is primarily
caused by defective DG degradation (Schweiger, 2006). In accordance
with previous data (Schweiger, 2006), the combined use of Hi
76-0079 and Atglistatin led to an almost complete depletion of TG
hydrolase activity (FIG. 2B).
[0165] Next, the effect of the inhibitor on various other
intracellular and secreted lipases was determined. Atglistatin had
only minor effects on HSL and MGL activity (FIG. 2C), the
downstream lipases of ATGL, as well as on pancreatic lipase and
lipoprotein lipase representing major secreted TG lipases in
intestine and plasma, respectively (FIG. 2D). Together, these
observations suggest that Atglistatin exhibits high selectivity for
ATGL and does not interfere with other known secreted or
intracellular acylglycerol lipases.
Example 5
Inhibition of Lipolysis in 3T3-L1 Adipocytes and Organ Cultures of
Murine White Adipose Tissue
[0166] To test the effect of Atglistatin on cellular lipolysis,
differentiated 3T3-L1 adipocytes were preincubated for 4 hours in
the presence or in the absence of the inhibitor and subsequently
stimulated with forskolin to induce lipolysis. Atglistatin reduced
forskolin-stimulated lipolysis in a dose dependent manner resulting
in almost complete inhibition of FA release (FIG. 3A) and
.about.90% inhibition of glycerol release (FIG. 3B). The inhibitor
was also highly effective in WAT organ cultures of wild-type mice.
Incubation of WAT organ cultures for 8 h reduced basal lipolysis
(FIG. 3 C, D) and hormone-stimulated lipolysis in a dose dependent
manner (FIG. 3 E, F). Under the applied conditions,
forskolin-stimulated FA and glycerol release were reduced up to 72%
and 66%, respectively. These results demonstrate that Atglistatin
is able to inhibit lipolysis on the cellular and organ level.
Example 6
Inhibition of Lipolysis In Vivo
[0167] The in vivo effect of Atglistatin on lipolysis was tested in
fasted wild-type C57Bl/6J mice. Fasted animals received an oral
gavage of olive oil containing the inhibitor or olive oil alone.
Before gavage, as well as 4 h, 8 h, 12 h, and 16 h after gavage
blood was collected by retro-orbital puncture and plasma FA and
glycerol concentrations were determined. Atglistatin lead to a time
dependent decrease in plasma levels for FA and for glycerol between
4 h and 12 h, and between 4 h and 8 h after gavage, respectively
(FIG. 4A,B). Moreover, Atglistatin produced a dose-dependent
decrease in plasma FA and glycerol levels up to 50% and 70%,
respectively (FIG. 4C,D).
[0168] Detailed analyses of plasma parameters revealed that the
Atglistatin group exhibited significantly reduced plasma FA,
glycerol, and TG levels. Plasma glucose, total cholesterol, and
insulin levels remained unchanged (Table 1A).
[0169] ATGL-deficiency in mice has been shown to cause TG
accumulation in many tissues which is most pronounced in the heart.
Importantly, Atglistatin did not promote the accumulation of TG in
cardiac muscle and other tissues including skeletal muscle, adipose
tissue, testis, pancreas, liver, and kidney (Table 1B).
TABLE-US-00002 TABLE 1 Inhibition of lipolysis in vivo. C57BI6 mice
were fasted overnight and received an oral gavage containing 200
.mu.mol/kg Atglistatin dissolved in olive oil, or olive oil as
control. After 8 h blood was taken retroorbitally and mice were
sacrificed. (A) Control Atglistatin FFA (mmol/L) 0.77 .+-. 0.13
0.53 .+-. 0.05 *** Glycerol (mmol/L) 0.41 .+-. 0.05 0.31 .+-. 0.08*
Triglycerides (mmol/L) 0.58 .+-. 0.08 0.33 .+-. 0.14** Glucose
(mg/dL) 78.85 .+-. 7.99 86.85 .+-. 12.19 CE (mmol/L) 3.25 .+-. 0.44
3.12 .+-. 0.25 Insulin (ng/ml) 0.17 .+-. 0.06 0.18 .+-. 0.07 (B)
Control Atglistatin (nmol TG/mg protein) (nmol TG/mg protein)
skeletal muscle 74 .+-. 17 99 .+-. 13 cardiac muscle 76 .+-. 17 71
.+-. 19 adipose tissue 6092 .+-. 630 6218 .+-. 742 testis 146 .+-.
50 168 .+-. 36 pancreas 115 .+-. 32 131 .+-. 39 liver 132 .+-. 30
112 .+-. 14 kidney 84 .+-. 16 72 .+-. 15 (A) Plasma parameters were
measured using commercial kits. (B) Tissue triglyceride (TG) and
protein levels were determined in whole tissue lysates and are
presented as TG (nmol) per protein (mg). Data are presented as mean
+ S.D. ( *p < 0.05 **p < 0.01; *** p < 0.001). n = 7
Example 7
Screening
Lipase Assay (Triacylgycerol Hydrolase Assay):
[0170] Mesenteric, retroperitoneal, omental and, gonadal white
adipose tissues (WAT) of mice were removed surgically and, washed
in phosphate-buffered saline (PBS) containing 1 mM
ethylenediaminetetraacetic-acid (EDTA). The tissue was homogenized
in lysis buffer (0.25 M sucrose, 1 mM EDTA, 1 mM dithiothreitol, 20
.mu.g/ml leupeptin, 2 .mu.g/ml antipain, 1 .mu.g/ml pepstatin, pH
7.0) using a Magna Lyser (Roche diagnostics GmbH, Mannheim,
Germany). The WAT lysate was centrifuged at 100,000 g for 1 hour
(h) at 4.degree. C. The lipid-free infranatant (cytosolic fraction)
was collected and, used for triacyl-glycerol (TG) hydrolase assays.
The substrate for the measurement of lipase activity containing
triolein and [9, 10-3H(N)-triolein](NEN Life Science Products,
Boston, Mass.) as radioactive tracer was emulsified with
phosphatidylcholine/phosphatidylinositol using a conventional
ultrasound sonicator. The cytosolic fractions supplemented with or,
without a specific inhibitor for HSL were incubated at 37.degree.
C. for 60 min under constant shaking. The reaction was terminated
by addition of 3.25 ml methanol/chloroform/heptane (10:9:7) and, 1
ml of 0.1 M potassium carbonate and, 0.1 M boric acid (pH 10.5).
After centrifugation at 800 g for 20 minutes (min) the
radioactivity in 1 ml of the upper phase was determined by liquid
scintillation counting in a LS 6500 Multi-Purpose Scintillation
Counter from Beckman Coulter Inc. (Fullerton, Calif.). The activity
of ATGL can primarily be measured by cleavage of triglycerides
(exemplary assay provided directly above or in example 1 above;
Lipase Assay), because the enzyme does not recognize water-soluble
substrates. Using this method, about 300 measurements can be
carried out per day by one person in a high throughput assay.
[0171] ATGL activity may also be measured using cell culture.
Preadipocyte-lines, e.g. 3T3-L1 cells, are differentiated to
adipocytes. Then after beta-adrenergic stimulation, these cells
secrete large amounts of glycerol and fatty acids into the culture
medium, which can be measured easily using commercial enzymatic
kits. By incubation with inhibitors, this secretion can be
inhibited. In the absence of ATGL, the secretion is inhibited by
about 70%. In the absence of HSL and ATGL, there is almost no
beta-adrenergic stimulation of lipolysis in adipocytes (Schweiger M
et al (2006) J Biol Chem 281(52):40236-40241). Therefore, in the
presence of an HSL inhibitor (such as 76-0079, Novo Nordisk),
specific ATGL activity can be determined in living adipocytes
(Schweiger M et al (2006) Biol Chem 281(52):40236-40241).
[0172] ATGL inhibitory activity of a phospholipase-inhibitor
(R)-Bromoenol lactone (Cayman Chemicals, Cat. No. 10006800, CAS No.
478288-90-3) has been demonstrated with expressed ATGL (Jenkins C M
et al (2004) J Biol Chem 279(47):48968-48975) or native ATGL in
hepatocytes (Chung C et al (2008) J Hepatol 48:471-41-478). This
lipase inhibitor also inhibits other lipases including iPLA2.beta.
and iPLA2.gamma. (Mancuso D J et al (2000) J boil Chem
275:9937-9945; Hazen S et al (1991) J Biol Chem 266:7227-7232;
Zupan L A et al (1993) J med Chem 36:95-100). This provides an ATGL
inhibiting compound for studies, assessment, and as a control in
further screening.
[0173] To further assess ATGL hydrolysis of neutral lipids,
His-tagged ATGL can be transiently expressed in COS-7 cells using a
eukaryotic expression vector. For comparison, COS-7 cells are also
transfected with a similar construction expressing His-tagged HSL.
In an alternative embodiment, strep-tagged ATGL and CGI-58 are
expressed in E. coli. Extracts from transfected cells are
preincubated with an inhibitor or candidate compound or compound
library. When extracts are preincubated with the fluorescent lipase
inhibitor (NBD-HEHP) and subsequently subjected to SDS-PAGE
analysis and fluorography, fluorescent signals can be observed in
positions corresponding to the expected molecular weight of ATGL
and HSL providing evidence that ATGL is enzymatically active in
transfected COS cells. To confirm ATGL activity, TG-hydrolase
activity assays can be performed using a radioactively labelled [9,
10-3H(N))]-triolein substrate. As controls, no enzymatic activities
should be observed when radioactively labeled retinyl palmitate,
cholesteryl oleate or phosphatidylcholine are used as lipid
substrates.
[0174] To determine ATGL function in adipocytes, including in
assays in the presence or absence of one or more candidate
compounds, modulators, or inhibitors, a recombinant adenovirus
encoding His-tagged full length mouse or human ATGL cDNA is
constructed and used to infect mouse 3T3-L1 adipocytes at day 6 of
differentiation. Western blotting analysis of cell-extracts of
infected adipocytes reveals expression of His-tagged ATGL at the
appropriate molecular weight. Overexpression of ATGL in adipocytes
can markedly augment both basal and isoproterenol-stimulated
lipolysis, indicative of a functional ATGL lipase in adipose
tissue.
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