U.S. patent application number 17/726994 was filed with the patent office on 2022-08-18 for methods and compositions for treating obesity and/or diabetes and for identifying candidate treatment agents.
The applicant listed for this patent is The J. David Gladstone Institutes, a testamentary trust established under the Will of J. David Glads. Invention is credited to Yadong Huang, Robert W. Mahley, Qin Xu.
Application Number | 20220257576 17/726994 |
Document ID | / |
Family ID | |
Filed Date | 2022-08-18 |
United States Patent
Application |
20220257576 |
Kind Code |
A1 |
Huang; Yadong ; et
al. |
August 18, 2022 |
METHODS AND COMPOSITIONS FOR TREATING OBESITY AND/OR DIABETES AND
FOR IDENTIFYING CANDIDATE TREATMENT AGENTS
Abstract
Provided are methods and compositions for identifying candidate
agents for treatment of obesity, liver disease, and/or diabetes.
Such methods include, e.g., contacting a mammalian cell or cell
population with a test agent, and measuring an expression level
and/or activity level of ClpP in the mammalian cell or in cells of
the cell population. Also provided are methods and compositions for
treating an individual (e.g., one who is obese and/or has
diabetes). Treatment methods include administering an inhibitor of
ClpP to the individual (e.g., to prevent or reduce weight gain, to
increase insulin sensitivity, and/or to increase glucose
tolerance).
Inventors: |
Huang; Yadong; (Daville,
CA) ; Xu; Qin; (Burlingame, CA) ; Mahley;
Robert W.; (San Francisco, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The J. David Gladstone Institutes, a testamentary trust established
under the Will of J. David Glads |
San Francisco |
CA |
US |
|
|
Appl. No.: |
17/726994 |
Filed: |
April 22, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16129296 |
Sep 12, 2018 |
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17726994 |
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PCT/US2017/022584 |
Mar 15, 2017 |
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16129296 |
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62309311 |
Mar 16, 2016 |
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International
Class: |
A61K 31/44 20060101
A61K031/44; A61K 31/365 20060101 A61K031/365; C12Q 1/37 20060101
C12Q001/37; A61P 1/16 20060101 A61P001/16; A61P 3/04 20060101
A61P003/04; A61P 3/10 20060101 A61P003/10; A61K 9/00 20060101
A61K009/00; G01N 33/50 20060101 G01N033/50 |
Claims
1. A method of treating an individual with obesity, liver disease,
and/or diabetes, the method comprising: administering an inhibitor
of ClpP to the individual in an amount effective for decreasing an
amount of fat tissue in the individual, preventing or reducing
weight gain of the individual, increasing insulin sensitivity of
the individual, and/or increasing glucose tolerance of the
individual.
2. The method according to claim 1, wherein the inhibitor of ClpP
is an RNAi agent or a gene editing agent that specifically reduces
expression of ClpP.
3. The method according to claim 1, wherein the inhibitor of ClpP
is administered such that reduction of ClpP expression is
substantially liver-specific, and the amount administered is
effective for increasing insulin sensitivity of the individual.
4. The method according to claim 1, wherein the inhibitor of ClpP
is a small molecule.
5. The method according to claim 4, wherein the small molecule is a
.beta.-Lactone.
6. The method according to claim 5, wherein the .beta.-Lactone is
(3RS,4RS)-3-(non-8-en-1-yl)-4-(2-(pyridin-3-yl)ethyl)oxetan-2-one
or a pharmaceutically acceptable salt thereof.
7. The method according to claim 1, wherein the inhibitor of ClpP
is delivered directly to the individual's liver, and the amount
administered is effective for increasing insulin sensitivity of the
individual.
8. The method according to claim 1, wherein the administering
comprises local injection.
9. The method according to claim 1, further comprising a step of
measuring insulin sensitivity of the individual.
Description
CROSS-REFERENCE
[0001] This application is a divisional of U.S. patent application
Ser. No. 16/129,296, filed Sep. 12, 2018, which application is a
continuation-in-part of International Application No.
PCT/US2017/022584, filed Mar. 15, 2017, which application claims
the benefit of priority to U.S. Provisional Patent Application No.
62/309,311, filed Mar. 16, 2016, which applications are
incorporated herein by reference in their entireties.
INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS A TEXT
FILE
[0002] A Sequence Listing is provided herewith as a text file,
"GLAD-408WO_SEQUENCE_LISTTNG_ST25.txt" created on Mar. 13, 2017 and
having a size of 9 KB. The contents of the text file are
incorporated by reference herein in their entirety.
INTRODUCTION
[0003] Mammalian ClpXP is a mitochondrial matrix protease complex
that requires ATP to unfold and hydrolyze protein substrates. The
ClpXP complex includes a catalytic subunit (ClpP) and a regulatory
subunit (ClpX). The Escherichia coli ClpP homolog forms a
barrel-shaped complex with hydrolytic active sites sequestered
inside, while the Escherichia coli ClpX homolog forms a hexamer
ring that attaches to each end of the barrel and is responsible for
substrate recognition and unfolding. Prokaryotic ClpXP protease
facilitates the degradation of damaged or unneeded polypeptides for
protein quality control. Like E. coli ClpP, human ClpP also forms a
chamber-like structure in the presence of ClpX. The physiological
functions of ClpP in mitochondria, the prime energy generators of
mammalian cells that play critical roles in energy homeostasis and
metabolic regulation, are largely unknown.
[0004] There is a need in the art for methods and compositions for
identifying candidate agents for treating diseases related to
energy homeostasis and metabolic regulation, such as obesity, liver
disease, and/or diabetes. For example, there is a need in the art
for methods and compositions for identifying candidate agents for
increasing insulin sensitivity, preventing and/or reducing weight
gain, preventing and/or reducing fat tissue, e.g., white adipose
tissue, and the like. There is also a need in the art for methods
and compositions for treating obesity, liver disease, and/or
diabetes (e.g., by increasing insulin sensitivity, preventing
and/or reducing weight gain, preventing and/or reducing fat tissue,
e.g., white adipose tissue, and the like).
SUMMARY
[0005] Provided are methods and compositions for identifying
candidate agents for treating obesity, liver disease, and/or
diabetes (e.g., candidate agents for decreasing an amount of fat
tissue in an individual, preventing or reducing weight gain of an
individual, increasing insulin sensitivity of an individual, and/or
increasing glucose tolerance of an individual). In some embodiments
of the present disclosure, such methods include (a) contacting a
mammalian cell or cell population (e.g., a rodent cell, a mouse
cell, a rat cell, a non-human primate cell, a monkey cell, a human
cell, or a cell population thereof) with a test agent, and (b)
measuring an expression level (e.g., protein and/or mRNA) and/or
activity level of ClpP in the mammalian cell (the agent-contacted
cell) or in cells of the agent-contacted cell population. It is
then determined whether the test agent caused a reduction of ClpP
expression and/or activity. Those test agents that reduce ClpP
expression (e.g., protein and/or mRNA) and/or activity, can be
identified as candidate agents for treatment. Thus, a compound is
considered to be a. "test agent" prior to contact with a cell or
cell population (e.g., in vitro, ex vivo, or in vivo), and if the
compound (the test agent) reduces ClpP expression (e.g., as can be
shown by measuring levels of ClpP protein and/or ClpP-encoding
mRNA) and/or activity, it is then considered to be a "candidate
agent" (e.g., a candidate agent for treatment, such as for treating
obesity, liver disease and/or diabetes). Thus, in some cases a
subject method further includes a step (c): determining whether the
test agent caused a reduction of ClpP expression (e.g., where a
reduction of ClpP expression is indicative that the test agent is a
candidate agent for treatment) and/or activity. In some cases, a
subject method includes either: (step c) determining that the test
agent caused a decrease in the expression level and/or activity
level of ClpP (e.g., relative to a reference value, e.g., an
expression level and/or activity level of ClpP prior to contact
with the test agent, an expression level and/or activity level of
ClpP after contact with a control agent that is known not to reduce
ClpP expression, and the like), and identifying the test agent as a
candidate agent for treating obesity, liver disease, and/or
diabetes, or (step d) determining that the test agent did not cause
a decrease in the expression level and/or activity level of ClpP
(e.g., relative to the reference value).
[0006] In some cases, the test agent is a small molecule or a
polypeptide. In some cases, the expression level (of ClpP) is an
RNA expression level and the measuring includes, e.g., the use of
quantitative RT-PCR, a microarray, or RNA sequencing. In some
cases, the expression level (of ClpP) is a protein expression level
and the measuring includes detecting ClpP protein (e.g., using an
anti-ClpP antibody, mass spectrometry, and/or an enzyme-linked
immunosorbent assay (ELISA) assay). In some cases, the method
includes screening a plurality of test agents to identify one or
more candidate agents for treating obesity, liver disease, and/or
diabetes.
[0007] In some cases, the mammalian cell (the target cell to be
contacted with the test agent) is a liver cell (a hepatocyte). In
some cases, the contacting is in vitro (e.g., the mammalian cell is
in vitro). In some cases, the contacting is ex vivo (e.g., the
mammalian cell is ex vivo). In some cases, the contacting is in
vivo (e.g., the mammalian cell is in vivo). In some cases, the
contacting includes administering the test agent to a mouse. In
some cases, the method includes a step of measuring an expression
level and/or activity level of ClpP in the mouse prior to the
contacting of step (a) in order to obtain the reference value. In
some cases, the method includes a step of generating a report that
the test agent is a candidate agent for treating obesity, liver
disease, and/or diabetes.
[0008] In some cases, a subject method includes, after determining
that the test agent is a candidate agent for treatment, a step of
administering the identified candidate agent to an individual that
has obesity, liver disease, and/or diabetes (e.g., as a treatment
or as a way to test whether the candidate agent causes a desired
outcome). In some cases, the individual is a mouse, a non-human
primate, or a human. In some cases, the method includes, after
administering the identified candidate agent to the individual,
measuring one or more features of the individual selected from:
insulin sensitivity; blood glucose level; glucose tolerance; body
fat mass; an amount of fat tissue; an amount of white adipose
tissue; percent fat mass; body weight; visceral adipose adipocyte
size; plasma leptin level; growth hormone level; basal energy
expenditure; a level of phosphorylated AKT (p-AKT) in muscles
and/or fibroblasts; percent lean mass; mitochondrial number in
hepatocytes; mitochondrial mass in hepatocytes; mitochondrial
morphology in hepatocytes; fibroblast respiratory capacity;
fibroblast maximal oxygen consumption rate (OCR); and fibroblast
resistance to H.sub.2O.sub.2-induced cytotoxicity.
[0009] Also provided are methods and compositions for treating an
individual (e.g., one who is obese and/or has diabetes or who has
been diagnosed as being obese and/or having diabetes). Treatment
methods include administering an inhibitor of ClpP (e.g., an agent
that reduces the amount and/or activity of ClpP protein) to the
individual in an amount effective for decreasing an amount of fat
tissue in the individual, preventing or reducing weight gain of the
individual, increasing insulin sensitivity of the individual,
and/or increasing glucose tolerance of the individual. In some
cases, the inhibitor of ClpP is a small molecule, e.g., any small
molecule ClpP inhibitor described herein, e.g., a .beta.-Lactone,
such as any .beta.-Lactone molecule described herein. In some
cases, the small molecule is
(3RS,4RS)-3-(non-8-en-1-yl)-4-(2-(pyridin-3-yl)ethyl)oxetan-2-one.
In some cases, the inhibitor of ClpP is an RNAi agent or a gene
editing agent that targets ClpP (e.g., specifically reduces the
expression level of the ClpP protein). In some cases, the inhibitor
of ClpP is administered such that reduction of ClpP expression is
substantially liver-specific, and the amount administered is
effective for increasing insulin sensitivity of the individual. In
some cases, the inhibitor of ClpP is delivered directly to the
individual's liver, and the amount administered is effective for
increasing insulin sensitivity of the individual. In some cases,
the administering includes local injection. In some cases, a
subject treatment method includes a step of measuring insulin
sensitivity of the individual.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] The invention is best understood from the following detailed
description when read in conjunction with the accompanying
drawings. It is emphasized that, according to common practice, the
various features of the drawings are not to-scale. On the contrary,
the dimensions of the various features are arbitrarily expanded or
reduced for clarity. Included in the drawings are the following
figures.
[0011] FIG. 1 (panels a-m) provides data showing that ClpP.sup.-/-
mice had less body fat and a resistance to high-fat diet-induced
obesity. FIG. 1 (panel a) provides images showing that ClpP.sup.-/-
mice were smaller and had lower body fat. FIG. 1 (panel b) provides
graphs showing that body weights of CdpP.sup.-/- mice were lower
than those of wild-type (WT) and ClpP.sup.+/- littermates (n=9 for
WT female, n=10 for ClpP.sup.+/- female, n=13 for ClpP.sup.-/-
female, n=13 for WT male, n=13 for ClpP.sup.-/- male, n=10 for
ClpP.sup.-/- male). FIG. 1 (panel c) provides a graph showing that
new-born ClpP.sup.-/- pups (postnatal day 1) had similar body
weights to WT and Clp.sup.-/- pups (n=5 for WT, n=7 for
ClpP.sup.+/-, n=6 for ClpP.sup.-/-). FIG. 1 (panel d), FIG. 1
(panel e) provide graphs depicting the fat mass and fat content of
5-month-old male mice (n=7), FIG. 1 (panel f), FIG. 1 (panel g)
provide graphs depicting lean mass and lean content of 5-month-old
male mice (n=7). FIG. 1 (panel h), FIG. 1 (panel i) provide data
depicting brown adipose tissue mass and content of 8 month-old male
mice (n=7 for WT and Clp.sup.+/-, n=4 for ClpP.sup.-/-. FIG. 1
(panel j) provides a graph depicting body weight gain of
8-month-old male mice on a high-fat diet (HFD) (n=8 for WT and
ClpP.sup.+/-, n=7 for ClpP.sup.-/-). FIG. 1 (panel k), FIG. 1
(panel l) provide graphs of fat mass and content of 8-month-old
male mice after 40 days on HFD (n=8 for WT, n=7 for ClpP.sup.-/-
and ClpP.sup.-/-). FIG. 1 (panel m) provides a histogram of
adipocyte size distribution of WT, ClpP.sup.+/-, and ClpP.sup.-/-
mice. Data are mean.+-.SD. *P<0.05, **P<0.01, ***P<0.005
versus WT.
[0012] FIG. 2 (panels a-i) provide data showing that ClpP.sup.-/-
mice had altered whole-body energy expenditure. FIG. 2 (panel a),
FIG. 2 (panel h) provide graphs depicting food intake of
7-8-month-old male mice (n=12 for WT and ClpP.sup.-/-, n=15 for
ClpP.sup.+/-). FIG. 2 (panel c) provides a timeline of
fasting-refeeding experiments. FIG. 2 (panel d), FIG. 2 (panel e)
provide graphs depicting body weight loss of 7-8-month-old male
mice after a 24-h fast (n=14 for WT and ClpP.sup.-/-, n=15 for
ClpP.sup.+/-). FIG. 2 (panel f) provides graphs depicting food
intake of 7-8-month-old male mice during 0-8 hour or 8-24 hour
refeeding periods (n=3 for WT and ClpP.sup.-/-, n=4 for
ClpP.sup.+/-). FIG. 2 (panel g) provides graphs depicting the
percentage body weight gain of 7-8-month-old male mice after 8 hour
or 24 hour refeeding (n=14 for WT and ClpP.sup.-/-, n=15 for
ClpP.sup.+/-). FIG. 2 (panel h), FIG. 2 (panel i) provide graphs
depicting body temperature of 8-month-old male mice after a 16-h
fast or during free feeding (n=13 for WT and ClpP.sup.+/-, n=14 for
ClpP.sup.-/-). Data are mean.+-.SD. *P<0.05, **P<0.01,
***P<0.005 versus WT.
[0013] FIG. 3 (panels a-o) provide data showing that ClpP.sup.-/-
mice had increased insulin sensitivity. FIG. 3 (panels a-c) provide
graphs depicting blood glucose and plasma insulin levels of
8-month-old male mice on chow diet (n=8 for each group). FIG. 3
(panel d), FIG. 3 (panel e) provide graphs depicting blood glucose
and plasma insulin levels of 8-month-old male mice on a high-fat
diet (n=5 for each group). FIG. 3 (panel 0 provides a graph
depicting glucose tolerance curve of 6-10-month-old male mice (n=9
for each group). FIG. 3 (panel g) provides graphs depicting plasma
insulin levels of 6-10-month-old male mice after glucose challenge
(n=5 for each group). FIG. 3 (panel h) provides a graph depicting
an insulin tolerance curve of 12-month-old male mice (n=7 for each
group). FIG. 3 (panel i) provides a graph depicting a pyruvate
tolerance curve of 14-month-old male mice (n=5 for WT and
ClpP.sup.-/-, n=7 for ClpP.sup.-/-). FIG. 3 (panel j), FIG. 3
(panel k) provide a representative image (j) and quantification (k)
of pAKT immunoblot of mouse fibroblasts from WT, ClpP.sup.-/-, or
ClpP.sup.-/- mice. FIG. 3 (panel l) provides a graph depicting pAKT
levels in gastrocnemius muscles of 10-month-old male mice (n=3 for
each group). FIG. 3 (panel m) provides a graph depicting pAKT
levels in mouse fibroblasts after IGF treatment (n=6 per dose for
each group). FIG. 3 (panel n) provides a graph depicting blood
glucose levels of 3-7-month-old db/db mice with different ClpP
genotypes after a 4-h fast (n=8 for WT, n=7 for ClpP.sup.+/-, n=6
for ClpP.sup.-/-). FIG. 3 (panel o) provides a glucose tolerance
curve of 3-7-month-cold db/db mice with different ClpP genotypes
(n=8 for WT, n=7 for ClpP.sup.+/-, n=6 for ClpP.sup.-/-). Data are
mean.+-.SD. *P<0.05, **P<0.01,***P<0.005 versus WT.
[0014] FIG. 4 (panels a-l) provide data showing that ClpP.sup.-/-
mice had increased mitochondrial chaperone levels. FIG. 4 (panels
a-h) provide representative western blots and quantification of
TRAP1, Grp75, LRPPRC, and ClpX in lysates from various organs of
mice (n=3 for each group). FIG. 4 (panel i), FIG. 4 (panel j)
provide representative western blots (i) and quantification (j) of
ClpX, TRAP1, LRPPRC, and OAT in lysates of WT or ClpP.sup.-/- mouse
fibroblasts. FIG. 4 (panel FIG. 4 (panel l) provide representative
western blots (k) and quantification (1) of ClpX, LRPPRC, and OAT
in ClpP.sup.-/- fibroblasts transfected with an empty vector
(control) or a mouse ClpP cDNA construct. Data are mean.+-.SD.
*P<0.05, **P<0.01, ***P<0005 versus WT.
[0015] FIG. 5 (panels a-j) provide data showing that ClpP.sup.-/-
mice had increased mitochondrial numbers, improved mitochondrial
function, and enhanced anti-oxidative stress capability. FIG. 5
(panel a) provides representative electron microscopic image of
mitochondria in WT and ClpP.sup.-/- mouse hepatocytes. Scale bars
are 2 .mu.m. FIG. 5 (panel b), FIG. 5 (panel c) provide graphs of
mitochondrial number (b) and mass (c, measured by mitochondrial
area) increased in ClpP.sup.-/- mouse hepatocytes. The
mitochondrial numbers were counted for each random field at
.times.13,600 magnification (n=17 for WT, n=12 for ClpP.sup.-/-).
The total mitochondrial area per field was measured by Image J at
.times.13,600 magnification (n=17 for WT, n=12 for ClpP.sup.-/-).
FIG. 5 (panel d) provides an oxygen consumption rate (OCR) curve of
fibroblasts from ClpP.sup.-/- and WT mice under basal and
uncoupling conditions (n=12 for each group), FIG. 5 (panel e)
provides a graph showing that fibroblasts from ClpP.sup.-/- mice
were resistant to H.sub.2O.sub.2-induced cell death compared with
fibroblasts from WT mice (n=8 for each group). FIG. 5 (panel l)
provides a graph showing that overexpression of mouse ClpP.sup.-/-
decreased the respiratory capacity of fibroblasts (n=12 for each
group). FIG. 5 (panel g) provides a graph showing that
overexpression of mouse ClpP abolished the H.sub.2O.sub.2
resistance of ClpP.sup.-/- fibroblasts (n=8 for each group). FIG. 5
(panel h), FIG. 5 (panel i) provide graphs showing that lentiviral
shRNA-mediated knockdown of TRAP1 or Grp75 lowered the resistance
of ClpP.sup.-/- fibroblasts to H.sub.2O.sub.2 cytotoxicity (n=8 for
each group). FIG. 5 (panel j) provides a graph showing the effects
of lentiviral shRNA-mediated knockdown of different mitochondrial
proteins on the maximum respiration capacity of ClpP.sup.-/-
fibroblasts (n=9 for each group). Data are mean.+-.SD. *P<0.01,
**P<0.01, ***P<0.005 versus WT (b-e) or ClpP.sup.-/-
(f-j).
[0016] FIG. 6 (panels a-i) provide data showing that
AAV-Cre-mediated knockdown of ClpP in livers increased insulin
sensitivity in ClpP-cKO mice. 3-5 month-old ClpP-cKO mice were
injected with AAV-CMV-Cre (Cre group, n=11) or control AAV (Con
group, n=10) through tail vein at a dose of 5.times.10.sup.9
gc/gram. FIG. 6 (panel a) provides images of ClpP immunostaining,
showing a significant reduction of ClpP in livers of
AAV-Cre-injected ClpP-cKO mice compared to control AAV-injected
mice. Scale bars are 50 .mu.m. FIG. 6 (panels b-d) provide graphs
of body weights (b and c) of ClpP-cKO mice before and 3 weeks after
AAV injection. The body weight gain of the AAV-Cre-injected mice
was significant lower than that of control AAV-injected mice (d).
FIG. 6 (panel e) provides a graph of plasma insulin levels of
ClpP-cKO mice before and 3 weeks after AAV injection. FIG. 6 (panel
0 provides a graph of blood glucose levels of ClpP-cKO mice before
and 3 weeks after AAV injection. FIG. 6 (panel g) provides a
glucose tolerance curve of ClpP-cKO mice 4 weeks after AAV
injection. FIG. 6 (panel h), provides a glucose tolerance curve of
ClpP-CKO mice injected with AAV-Cre or control AAV and on HFD for 2
weeks. FIG. 6 (panel i) provides a graph of data collected after
HFD was given to mice 6 weeks after AAV injection. No differences
of body weight gain were detected after 2 or 4 weeks on HFD. Data
are mean.+-.SD. *P<0.01, ***P<0.005 versus control.
[0017] FIG. 7 (panels a-i) provide data showing the generation of
ClpP.sup.-/- mice. FIG. 7 (panel a) provides a schematic of the
gene-trapping strategy used to generate ClpP.sup.-/- mice. FIG. 7
(panels b-i) provide western blots and quantification of ClpP
protein levels in lysates of the liver, adipose tissue, muscle, and
brain. Data are mean.+-.SD. ***P<0.005 versus WT.
[0018] FIG. 8 (panels a-b) provide data showing that ClpP.sup.-/-
mice have normal histology of the liver and muscle. FIG. 8 (panel
a), FIG. 8 (panel b) provide representative H&E-stained images
of the liver (a) and gastrocnemius muscle (b) WT, ClpP.sup.-/-, and
ClpP.sup.-/- mice. Scale bars are 50 .mu.m.
[0019] FIG. 9 (panels a-j) provide data showing that ClpP.sup.-/-
mice have normal neurological profile. FIG. 9 (panels a-e) provide
graphs of data collected from performing the grip test, incline
test, tail suspense test, and rotarod test of 8-month-old male mice
(n=8 for each group). FIG. 9 (panel f) provides a graph from the
elevated plus maze test performed with 8-9-month-old male mice (n=8
for each group). FIG. 9 (panels g-j) provide graphs from the Morris
water maze test performed with 8-9-month-old male and female mice
(n=8 for each group) (--.diamond-solid.-- WT, --.ident.--
ClpP.sup.-/-, --.tangle-solidup.-- ClpP.sup.-/-). H, hidden trial;
V, visible trial. Data are mean.+-.SEM.
[0020] FIG. 10 (panels a-d) provide data showing that ClpP.sup.-/-
mice have smaller adipocytes. FIG. 10 (panels a-b) provide
representative images of H&E-stained adipose tissues from WT,
ClpP.sup.+/-, and ClpP.sup.-/- mice.
[0021] FIG. 11 (panels a-b) provide data showing that ClpP.sup.-/-
mice have normal histology of brown adipose tissue and pancreas.
FIG. 11 (panel a), FIG. 11 (panel b) provide representative
H&E-stained images of the brown adipose tissue (a) and pancreas
(b) in WT, ClpP.sup.+/-, and ClpP.sup.-/- mice. Scale bars are 100
.mu.m,
[0022] FIG. 12 (panels a-d) provide data showing that ClpP.sup.-/-
mice are resistant to high-fat diet (HFD)-induced obesity. FIG. 12
(panel a), FIG. 12 (panel b) provide graphs of body weight gain of
8-month-old male mice after being on an HFD for 10 days and 20 days
(n 8 for WT and ClpP.sup.+/-, n=7 for ClpP). FIG. 12 (panel c),
FIG. 12 (panel d) provide graphs of the lean body mass of
8-month-old male mice after being on the HFD for 40 days (n=6 for
WT, n=7 for ClpP.sup.+/- and ClpP.sup.-/-). Data are mean.+-.SD.
**P<0.01, ***P<0.005 versus WT.
[0023] FIG. 13 (panels a-b) provide data showing that ClpP.sup.-/-
mice have normal locomotor activities. FIG. 13 (panel a), FIG. 13
(panel b) provide graphs depicting the total movement and the ratio
of center to total movement for 8-10-month-old male mice during an
open field test (n=10 for WT, n=12 for ClpP.sup.+/- and
ClpP.sup.-/-). Data are mean.+-.SEM.
[0024] FIG. 14 (panels a-c) provide data related to the effects of
knocking out ClpP on obesity and insulin resistance in db/db mice.
FIG. 14 (panel a) provides a graph of the body weight of
4-8-month-old db/db mice with different ClpP genotypes (n=10 for
WT, n=17 for ClpP.sup.+/-, n=8 for ClpP.sup.-/-), FIG. 14 (panel
b), FIG. 14 (panel c) provide graphs depicting the levels of blood
glucose and plasma insulin of 3-7-month-old db/db mice with
different ClpP genotypes after a 16-h fast (n=8 for WT, n=7 for
ClpP.sup.+/-, n=6 for ClpP.sup.-/-). Data are mean SD. *P<0.05
versus WT.
[0025] FIG. 15 (panels a-c) provide representative images from
comparative 2D fluorescence difference gel electrophoresis
(2D-DIGE) on different organs. FIG. 15 (panel a) provides an image
of 2D-DIGE profiles of WT versus ClpP.sup.-/- or WT versus
ClpP.sup.+/- livers. FIG. 15 (panel b) provides an image of 2D-DIGE
profiles of WT versus ClpP.sup.-/- or WT versus ClpP.sup.+/-
muscles. FIG. 15 (panel c) provides an image of 2D-DIGE profiles of
WT versus ClpP.sup.-/- or WT versus ClpP.sup.+/- hippocampi. Green,
WT; red, ClpP.sup.-/- or ClpP.sup.-/-.
[0026] FIG. 16 (panels a-b) provide data showing that the absence
of ClpP increases the levels of many mitochondrial proteins in
various organs. FIG. 16 (panel a), FIG. 16 (panel b) provide
Western blots and quantification of OAT, LonP, SDH2, ATP6V1A, VDAC,
Hsp60, CPS1, Hsp70, Grp78, and Hsp60 in lysates of different
oceans. Data are mean.+-.SD. *P<0.05, **P 0.01, ***P<0.005
versus WT.
[0027] FIG. 17 (panels a-d) provide data showing that knocking out
ClpP altered mitochondrial numbers and morphology in mouse
hepatocytes, FIG. 17 (panel a), FIG. 17 (panel b) provide
representative electron microscopic image of mitochondria in WT (a)
and ClpP.sup.-/- (b) mouse hepatocytes. Scale bars are 2 .mu.m.
FIG. 17 (panel c) provides a histogram of mitochondrial size
distribution in WT and ClpP.sup.-/- mouse hepatocytes (n=507 for
WT, n=529 for ClpP.sup.-/-). FIG. 17 (panel d) provides a histogram
of mitochondrial roundness distribution in WT and ClpP.sup.-/-
mouse hepatocytes (n=507 for WT, n=529 for ClpP.sup.-/-). The
roundness was defined as 4.times.(area)/(.pi..times.(major
axis).sup.2) and measured via Image J at .times.13,600
magnification.
[0028] FIG. 18 (panels a-f) provide data related to knocking down
ClpP targeting proteins in ClpP.sup.-/- fibroblasts and a cell
viability assay. FIG. 18 (panels a-d) provide representative
western blots and quantifications of LRPPRC, TRAP1, Grp75, and ClpX
protein levels in ClpP.sup.-/- fibroblasts treated by different
lentiviral shRNAs for 48 hours. FIG. 18 (panel e) provides a graph
depicting cell viability in response to H.sub.2O.sub.2 treatment of
ClpP.sup.-/- fibroblasts treated with LRPPRC lentiviral shRNAs (n=8
for each group) (--.diamond-solid.-- ClpP.sup.-/-+LRPPRC-shRNA).
FIG. 18 (panel f) provides a graph depicting cell viability in
response to H.sub.2O.sub.2 treatment of ClpP.sup.-/- fibroblasts
treated with ClpX lentiviral shRNAs (n=8 for each group)
(--.diamond-solid.-- ClpP.sup.+, --.box-solid.--
ClpP.sup.-/-+ClpX-shRNA). Data are mean.+-.SD. **P<0.01,
***P<0.005 versus ClpP.sup.-/-.
[0029] FIG. 19 (panels a-f) provide data showing that
adipocyte-specific knockout of ClpP did not affect body weight,
blood glucose levels, and insulin sensitivity in mice. FIG. 19
(panels a-b) provide a western blot of ClpP levels in adipose
tissues of adipocyte-specific ClpP-cKO mice, and a graph of the
data. FIG. 19 (panel e) provides a graph of body weights of
5-10-month-old adipocyte-specific ClpP-cKO mice compared to
controls (no Crc littermates) (n=30 for no Cre, n=24 for Ad-Cre),
FIG. 19 (panel d) provides a graph of the blood glucose levels of
5-10-month-old adipocyte-specific ClpP-cKO mice compared to
controls (no Cre littermates) (n=30 for no Cre, n=24 for Ad-Cre).
FIG. 19 (panel e) provides a glucose tolerance curve of
3-6-month-old adipocyte-specific ClpP-cKO mice compared to controls
(no Cre littermates) (n=13 for no Cre, n=11 for Ad-Cre)
(--.diamond-solid.-- no Crc, --.box-solid.-- Ad-Cre). FIG. 19
(panel f) provides a graph of body weight gain of 9-12-month-old
adipocyte-specific ClpP-cKO mice in response to FWD (n=9 for no
Cre, n=11 for Ad-Cre). FIG. 19 (panel g) provides a glucose
tolerance curve of 9-12-month-old adipocyte-specific ClpP-cKO mice
after 4 Week on HFD (n=9 for no Cre, n=11 for Ad-Cre).
***P<0.005 versus No Cre.
[0030] FIG. 20 provides a table of proteins differentially
expressed in ClpP-KO tissues.
[0031] FIG. 21 provides a table of potential substrates of
ClpXP.
[0032] FIG. 22 provides a table showing that the expression of
potential ClpXP substrates were not upregulated at transcription
levels, as determined by microarray assays.
[0033] FIG. 23 provides a western blot analysis of ClpP substrates
following treatment with the small molecule ClpP inhibitors
A2-32-01, AV167, and AV179.
[0034] FIG. 24 provides graphs showing protein levels for ClpP
substrates (normalized to GAPDH) following treatment with the small
molecule ClpP inhibitors A2-32-01-AV167, and AV179.
[0035] FIG. 25 provides graphs showing protein levels for ClpP
substrates (normalized to Actin) following treatment with the small
molecule ClpP inhibitors A2-32-01. AV167, and AV179.
[0036] FIG. 26 provides graphs showing mRNA levels for ClpP
substrates following treatment with A2-32-01.
[0037] FIG. 27 provides an overview of the in vivo study design of
Examples 9 and 10.
[0038] FIG. 28 provides a graph showing body weight data from the
first two days of in vivo 12-32-01 treatment from Example 9.
[0039] FIG. 29 provides graphs showing the results of a glucose
tolerance test following in vivo administration of a ClpP inhibitor
in a high fat diet (HFD)-induced obesity and diabetes mouse
model.
[0040] FIG. 30 provides a schematic showing an experimental scheme
of HFD, A2-32-01 treatment, and metabolic assays.
[0041] FIG. 31 provides a graph showing body weight change of
HFD-fed WT mice in response to A2-32-01 treatment (n=9 fir vehicle
group, n=10 for A2-32-01 group) (left panel). FIG. 31 also provides
a graph showing the results of a glucose tolerance test showing
increased insulin sensitivity in HFD-fed WT mice treated with
A2-32-01 compared to those treated with vehicle (n=9 for vehicle
group, n=10 for A2-32-01 group) (right panel).
[0042] FIG. 32 provides a graph showing ClpP activity was lower in
liver mitochondrion lysates from A2-32-01-treated mice compared to
those from vehicle-treated mice (n=8 for vehicle group, n=10 for
A2-32-01 group) (left panel). FIG. 32 also provides a graph showing
that the levels of tentative ClpP effectors, measured by western,
were higher in liver mitochondrion lysates from A2-32-01-treated
mice compared to those from vehicle-treated mice (n=8 for vehicle
group, n=10 for A2-32-01 group) (right panel).
[0043] FIG. 33 provides representative H&E and oil red staining
of liver sections from vehicle or A2-32-01 treated WT mice on HFD.
The micrographs showed that A2-32-01 treatment lowered the lipid
accumulation in liver cells and restored normal morphology of liver
cells in WT mice on HFD, as compared to vehicle-treated WT mice on
HFD.
[0044] FIG. 34, Panel A, provides a schematic showing an
experimental scheme of A2-32-01 treatment and metabolic assays in
db/db mice. FIG. 34, Panel B, provides a graph showing body weight
change of db/db mice in response to A2-32-01 treatment. (n=7 for
vehicle group, n=9 for A2-32-01 group).
[0045] FIG. 35 provides, Panel C, provides a graph showing results
of a glucose tolerance test showing increased insulin sensitivity
in db/db mice treated with A2-32-01 compared with those treated
with vehicle (n=7 for vehicle group, n=9 for A2-32-01 group). FIG.
35, Panel D, provides a graph showing that fasting blood glucose
levels in db/db mice treated with A2-32-01 are significantly lower
than those treated with vehicle (n=7 for vehicle group, n=9 for
A2-32-01 group). FIG. 35, Panel E, provides a graph showing that
ClpP activity was lower in liver mitochondria lysates from db/db
mice treated with A2-32-01 compared to those treated with vehicle
(n=7 for vehicle group, n=9 for A2-32-01 group).
[0046] FIG. 36, panels F-I, provide representative H&E (Panels
F, H) and oil red staining (Panels G, I) of liver sections from
vehicle or A2-32-01 treated db/dh mice. The micrographs showed that
A2-32-01 treatment lowered the lipid accumulation in liver cells
and restored normal morphology of liver cells in db/db mice, as
compared to vehicle-treated db/db mice.
DETAILED DESCRIPTION
[0047] Provided are methods and compositions for identifying
candidate agents for the treatment of obesity, liver disease,
and/or diabetes. In some embodiments, such methods include (a)
contacting a mammalian cell or cell population with a test agent,
and (h) measuring an expression level (e.g., protein and/or mRNA)
and/or activity level of ClpP in the mammalian cell (the
agent-contacted cell) or in cells of the agent-contacted cell
population. If the compound (the test agent) reduces ClpP
expression (e.g., as can be show by measuring levels of ClpP
protein and/or ClpP-encoding mRNA) and/or activity level, it is
then considered to be a "candidate agent" (e.g., a candidate agent
for treatment). Also provided are methods and compositions for
treating an individual (e.g., one who is obese and/or has diabetes
or who has been diagnosed as such). Treatment methods include
administering an inhibitor of ClpP (e.g., an RNAi agent or a gene
editing agent that targets ClpP) to the individual (e.g., to
prevent or reduce weight gain, to increase insulin sensitivity,
and/or to increase glucose tolerance).
[0048] Before the present methods and compositions are described,
it is to be understood that this invention is not limited to the
particular methods or compositions described, as such may, of
course, vary. It is also to be understood that the terminology used
herein is for the purpose of describing particular embodiments
only, and is not intended to be limiting, since the scope of the
present invention will be limited only by the appended claims.
[0049] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limits of that range is also specifically disclosed. Each
smaller range between any stated value or intervening value in a
stated range and any other stated or intervening value in that
stated range is encompassed within the invention. The upper and
lower limits of these smaller ranges may independently be included
or excluded in the range, and each range where either, neither or
both limits are included in the smaller ranges is also encompassed
within the invention, subject to any specifically excluded limit in
the stated range. Where the stated range includes one or both of
the limits, ranges excluding either or both of those included
limits are also included in the invention.
[0050] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, some potential and preferred methods and materials are
now described. All publications mentioned herein are incorporated
herein by reference to disclose and describe the methods and/or
materials in connection with which the publications are cited. It
is understood that the present disclosure supersedes any disclosure
of an incorporated publication to the extent there is a
contradiction.
[0051] As will be apparent to those of skill in the art upon
reading this disclosure, each of the individual embodiments
described and illustrated herein has discrete components and
features which may be readily separated from or combined with the
features of any of the other several embodiments without departing
from the scope or spirit of the present invention. Any recited
method can be carried out in the order of events recited or in any
other order that is logically possible.
[0052] It must be noted that as used herein and in the appended
claims, the singular forms "a", "an", and "the" include plural
referents unless the context clearly dictates otherwise. Thus, for
example, reference to "a cell" includes a plurality of such cells
and reference to "the peptide" includes reference to one or more
peptides and equivalents thereof, e.g., polypeptides, known to
those skilled in the art, and so forth.
[0053] The publications discussed herein are provided solely for
their disclosure prior to the filing date of the present
application, Nothing herein is to be construed as an admission that
the present invention is not entitled to antedate such publication.
Further, the dates of publication provided may be different from
the actual publication dates which may need to be independently
confirmed.
ClpP Protein
[0054] Mammalian ClpXP is a protein complex (a protease) that has a
catalytic subunit (the ClpP protein) and a regulatory subunit (the
ClpX protein). As described herein, it has been discovered that the
ClpP protein plays an important biological role in mitochondrial
function in mammalian cells, and that altering the
expression/function of ClpP has wide-ranging physiological
consequences.
[0055] The wild type mouse and human ClpP protein amino acid
sequences (and their encoding mRNAs) are depicted here.
[0056] Wild Type human ClpP (NP_006003.1)
[0057] *also known as "caseinolytic mitochondrial matrix peptidase
proteolytic subunit", DFNB81, and PRLTS3
TABLE-US-00001 (SEQ ID NO: 1)
MWPGILVGGARVASCRYPALGPRLAAHFPAQRPPQRTLQNGLALQRCLHAT
ATRALPLIPIVVEQTGRGERAYDIYSRLLRERIVCVMGPIDDSVASLVIAQ
LLFLQSESNKKPIHMYINSPGGVVTAGLAIYDTMQYILNPICTWCVGQAAS
MGSLLLAAGTPGMRHSLPNSRIMIHQPSGGARGQATDIAIQAEEIMKLKKQ
LYNIYAKHTKQSLQVIESAMERDRYMSPMEAQEFGILDKVLVHPPQDGEDE
PTLVQKEPVEAAPAAEPVPAST
[0058] DNA version of the mRNA Encoding Human ClpP (Above)
(NM_006012.2)
[0059] *ORF is underlined
TABLE-US-00002 (SEQ ID NO: 3)
CCTTAATGGCGCCCGCCCAGACTCCTGGAAGTGAGCGGCCTAGCGA
GCGAGCTCCCAGGCGCAAAGCACGCCGGAAGCTGTAGTTCCGCCAT
CGGACGGAAGCCGACCGGGGCGTGCGGAGGGATGTGGCCCGGAATA
TTGGTAGGGGGGGCCCGGGTGGCGTCATGCAGGTACCCCGCGCTGG
GGCCTCGCCTCGCCGCTCACTTTCCAGCGCAGCGGCCGCCGCAGCGG
ACACTCCAGAACGGCCTGGCCCTGCAGCGGTGCCTGCACGCGACGG
CGACCCGGGCTCTCCCGCTCATTCCCATCGTGGTGGAGCAGACGGGT
CGCGGCGAGCGCGCCTATGACATCTACTCGCGGCTGCTGCGGGAGC
GCATCGTGTGCGTCATGGGCCCGATCGATGACAGCGTTGCCAGCCTT
GTTATCGCACAGCTCCTCTTCCTGCAATCCGAGAGCAACAAGAAGCC
CATCCACATGTACATCAACAGCCCTGGTGGTGTGGTGACCGCGGGCC
TGGCCATCTACGACACGATGCAGTACATCCTCAACCCGATCTGCACC
TGGTGCGTGGGCCAGGCCGCCAGCATGGGCTCCCTGCTTCTCGCCGC
CGGCACCCCAGGCATGCGCCACTCGCTCCCCAACTCCCGTATCATGA
TCCACCAGCCCTCAGGAGGCGCCCGGGGCCAAGCCACAGACATTGC
CATCCAGGCAGAGGAGATCATGAAGCTCAAGAAGCAGCTCTATAAC
ATCTACGCCAAGCACACCAAACAGAGCCTGCAGGTGATCGAGTCCG
CCATGGAGAGGGACCGCTACATGAGCCCCATGGAGGCCCAGGAGTT
TGGCATCTTAGACAAGGTTCTGGTCCACCCTCCCCAGGACGGTGAGG
ATGAGCCCACGCTGGTGCAGAAGGAGCCTGTAGAAGCAGCGCCGGC
AGCAGAACCTGTCCCAGCTAGCACCTGAGAGCTGGGCCTCCTCTCCA
GAATCATGTGGAGGGGCCAGAGGCCTGCCAGACCCCCAGCTGGGCC
CTGCTCACCCCTTGTTGCTGGGCTTGGAGGGGCCTCTTGAGGAACTT
TTAATTTGCAGGGGTGCCCGCTATGGACGGGGCATTCCAGCTGAGAC
ACTGTGATTTTAAATTAAATCTTTGTGGTCTTTGCAAAAAAAAAAAA
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
[0060] Wild Type Mouse ClpP (NP_059089.1)
[0061] *also known as "caseinolytic mitochondrial matrix peptidase
proteolytic subunit", AU019820, and D17Wsu160e
TABLE-US-00003 (SEQ ID NO: 2)
MWPRVLLGEARVAVDGCRALLSRLAVHFSPPWTAVSCSPLRRSLHGTATRA
FPLIPIVVEQTGRGERAYDIYSRLLRERIVCVMGPIDDSVASLVIAQLLFL
QSESNKKPIHMYINSPGGVVTAGLAIYDTMQYILNPICTWCVGQAASMGSL
LLAAGSPGMRHSLPNSRIMIHQPSGGARGQATDIAIQAEEIMKLKKQLYNI
YAKHTKQSLQVIESAMERDRYMSPMEAQEFGILDKVLVHPPQDGEDEPELV
QKETATAPTDPPAPTST
[0062] DNA Version of the mRNA Encoding Mouse ClpP (Above)
(NM_017393.2)
[0063] *ORF is underlined
TABLE-US-00004 (SEQ ID NO: 4)
AGTGACTCCCGCAAAGCACGCCGGGTGTTGTAGTTCCGGAAGCCAA
GCCGGAGTGCGCGTCGTCATGTGGCCCAGAGTGCTGCTGGGGGAGG
CCCGGGTGGCTGTGGACGGATGTCGCGCTCTGTTGTCTCGCCTTGCC
GTGCATTTCTCCCCGCCATGGACTGCTGTGAGCTGCTCACCCCTGCG
GAGGAGCCTGCATGGAACTGCGACGCGAGCTTTCCCGCTCATCCCCA
TAGTGGTGGAGCAGACGGGTCGAGGCGAGCGCGCTTATGACATATA
CTCGAGGCTGTTGCGGGAACGCATCGTGTGCGTCATGGGCCCGATTG
ACGACAGTGTGGCCAGTCTGGTCATTGCCCAGCTGTTGTTCTTACAG
TCTGAAAGCAACAAGAAGCCCATTCATATGTATATCAACAGCCCAG
GTGGTGTGGTAACTGCGGGCCTGGCCATCTACGACACAATGCAGTAC
ATCCTGAACCCCATCTGCACGTGGTGTGTTGGACAGGCTGCCAGCAT
GGGCTCCCTGCTCCTCGCTGCTGGCAGCCCGGGCATGCGCCATTCAC
TGCCCAATTCCAGAATCATGATCCACCAGCCCTCTGGAGGAGCCAGG
GGCCAAGCCACAGACATCGCCATCCAGGCAGAGGAAATCATGAAGC
TGAAAAAGCAGCTATACAACATCTACGCCAAACACACCAAGCAGAG
CCTACAGGTGATCGAGTCAGCAATGGAGAGGGACCGCTACATGAGC
CCCATGGAGGCCCAAGAGTTTGGCATCTTGGACAAGGTCTTGGTCCA
CCCACCTCAGGACGGGGAGGATGAGCCAGAACTGGTACAGAAGGAG
ACTGCCACAGCGCCGACGGATCCTCCTGCCCCGACAAGCACCTAAG
GAGTGGAGACCAGACTGAAACTTCCTCTGCTGGGCCCAAGAACAAC
CCCTAGAGGAGATGTGGATTGAGGTTGCCCTCAGAGCAGGGCAGAC
TGCCTGAGACACTGTGATTTAAATTAAATCTTTGTAGTCTTTGTCCCA
TGTCTGAAGCACCTTCCATTACTTCTCCAAGACAGCAGGCCTCCTTC
ACCTTGACAAACCACTTCAGTAAGCAAACCCIGGCTCTCCIGGAACT
AAACCAATCTAGCCTCAGACTCAGGTACCCACCTGCCTCACCTCCTG
AGTGCTAGGATTAAAGGTGTACACCACCACACCTGACTTCAA
Screening Methods
[0064] Provided are screening methods for identifying candidate
agents for treating obesity, liver disease, and/or diabetes. By way
of example, such candidate agents may include candidate agents for
decreasing an amount of fat tissue in an individual, preventing or
reducing weight gain of an individual, increasing insulin
sensitivity of an individual, and/or increasing glucose tolerance
of an individual. In some embodiments, screening methods provided
herein include (a) contacting a mammalian cell or cell population
(e.g., a mouse cell, a rat cell, a non-human primate cell, a human
cell, or a cell population thereof) with a test agent, and (b)
measuring an expression level and/or activity level of ClpP (e.g.,
a protein and/or mRNA expression level or an activity level of
ClpP) in the mammalian cell (the agent-contacted cell) or in cells
of the agent-contacted cell population to determine whether the
test agent caused a reduction of ClpP expression and/or activity
level in the cell or cells of the cell population.
[0065] Test agents that reduce ClpP expression (e.g., protein
and/or mRNA) and/or activity level, can be identified as candidate
agents for use in the treatment of obesity, liver disease, and/or
diabetes. Thus, a compound is considered to be a "test agent" prior
to contact with a cell or cell population (e.g., in vitro, ex vivo,
or in vivo), and if the compound (the test agent) reduces ClpP
expression (e.g., as can be show by measuring levels of ClpP
protein and/or ClpP-encoding mRNA) and/or activity level, it is
then considered to be a "candidate agent" (e.g., a candidate agent
for the treatment of diseases in which ClpP reduction is
beneficial, such as obesity, liver disease, and/or diabetes). Thus,
the provided screening methods can also be referred to as methods
of identifying an inhibitor of ClpP, methods of identifying an
agent that reduces ClpP expression and/or activity level, methods
of identifying an inhibitor of ClpP expression and/or activity
level, and the like.
[0066] In some embodiments, a subject method (e.g., a method as
described above) includes (a) contacting a mammalian cell (e.g., a
mouse cell, a rat cell, a non-human primate cell, a human cell)
with a test agent; (b) measuring a decrease in the expression level
and/or activity level of ClpP caused by the contacting step (e.g.,
relative to a reference value, e.g., an expression level and/or
activity level of ClpP prior to contact with the test agent, an
expression level and/or activity level of ClpP after contact with a
control agent that is known not to reduce ClpP expression, and the
like); (c) determining that the test agent caused a decrease in the
expression level and/or activity level relative to the reference
value; and (d) identifying the test agent as a candidate agent for
treating obesity, liver disease, and/or diabetes.
[0067] In some cases, the contacting is in vitro (e.g., the cell is
in culture and is contacted in vitro). In some cases, the
contacting is ex vivo (e.g., the cell is in culture and is a
primary cell isolated from an individual).
[0068] A subject screening method includes a step of contacting a
cell (or cell population) with a test agent. When a test agent
reduces a ClpP expression level (e.g., at the level of ClpP protein
or mRNA encoding the ClpP protein) and/or activity level, that
agent is determined to be a candidate agent (e.g., for treating
obesity, liver disease, and/or diabetes). Thus, the contacting will
generally be for a period of time such that a change in an
expression level and/or activity level of ClpP (e.g., protein
and/or mRNA expression level) can potentially be detected. In other
words, the period of contact will be for a suitable period of time
after which one may reasonably expect that if a test agent is in
fact a candidate agent for treatment, a change in ClpP expression
and/or activity level will be detectable, in some cases, the
contacting is for a period of time of 2 or more minutes (e.g., 5 or
more minutes, 10 or more minutes, 15 or more minutes, 30 or more
minutes, 1 or more hours, 2 or more hours, 5 or more hours, 6 or
more hours, 12 or more hours or more hours, etc.). In some cases,
the contacting is for a period in a range of from 2 minutes to 48
hours (e.g., 5 minutes to 24 hours, 5 minutes to 6 hours, 5 minutes
to 2 hours, 15 minutes to 24 hours, 15 minutes to 6 hours, 15
minutes to 2 hours, 1 hour to 24 hours, 1 hour to 6 hours, or 1
hour to 2 hours).
[0069] In some cases, the contacting is in vivo. For example, in
some cases, a subject method (e.g., a screening method) includes a
step of administering an agent (e.g., a test agent or a candidate
agent) to an individual. A step of administering can serve a number
of different purposes. For example, in some cases a subject method
includes a step of administering a test agent to an individual
(e.g., a mouse or a rat), and then measuring an expression level
and/or activity level of ClpP in order to determine whether the
test agent is a candidate agent for treatment. Such administration
can be performed as part of the screen for identifying those test
agents that are candidate agents for treatment.
[0070] As another example, in some cases a subject method (e.g., a
screening method) includes a step of administering a candidate
agent (i.e., an agent that has already been determined to reduce an
expression level and/or activity level of ClpP, e.g., using a
subject method and contacting a cell in vitro, ex vivo, or in
vivo). Such administration can be performed as a treatment step, or
for example, in order to determine whether the candidate agent
causes a measurable change in features associated with obesity,
liver disease, and/or diabetes (e.g., decrease in an amount of fat
tissue, prevention or reduction of weight gain, increase in insulin
sensitivity, increase in glucose tolerance, and the like).
Target Cells
[0071] Any suitable mammalian cell or cell population can be used
in the provided screening methods as a target cell or cell
population (i.e., a cell or cell population that is contacted with
a test agent). For example, in some cases, the target cell or cell
population (the cell or cell population contacted with the test
agent) is a mammalian cell, a rodent cell (e.g., a mouse cell, a
rat cell), a rabbit cell, a non-human primate cell, a human cell,
etc. As noted above, the target cell or cell population can be in
vitro (e.g., from an establish cell line), ex vivo (e.g., primary
cells), or in vivo. The target cell can be any suitable type of
cell (e.g., a stem cell, a progenitor cell, an adipocyte, a neuron,
a cell of the pancreas, a fibroblast, an epithelial cells, a kidney
cell, an embryonic cell, and the like), and in some cases, the
contacted cell (the target cell) is a hepatocyte.
[0072] Examples of suitable mammalian cells for the subject
screening methods include, but are not limited to: monkey kidney
CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human
embryonic kidney line (293 or 293 cells subcloned for growth in
suspension culture, Graham et al., J. Gen Virol. 36:59 (1977));
baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary
cells/-DHFR(CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216
(1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod, 23:243-251
(1980)); monkey kidney cells (CV1 ATCC CCL 70); African green
monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical
carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC
CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human
lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB
8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TR1 cells
(Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1.982)); MRC 5
cells; FS4 cells; and a human hepatoma line (Hep G2).
Test Agents
[0073] A "test agent" (e.g., "test compound") to be used in the
provided screening methods can be any suitable agent (e.g.,
including, but not limited to, organic molecules, small molecules,
polynucleotides, RNA, DNA, proteins, antibodies, peptides, lipids,
carbohydrates, and the like). A test agent can also be a mixture of
chemical compounds. In some cases, an array of spatially localized
test agents can be used (e.g., a peptide array, polynucleotide
array, and/or combinatorial small molecule array; where "array"
refers to a collection of different molecular species immobilized
on a surface). In some cases, a small molecule library is screened
(i.e., a test agent can be a member of a small molecule library).
Test agents can be biological macromolecules, can be part of a
bacteriophage peptide display library, can be part of a
bacteriophage antibody (e.g., scFv) display library, a polysome
peptide display library, and the like. A test agent can be an
extract made from biological materials such as bacteria, plants,
fungi, or animal (e.g., mammalian) cells and/or tissues. As
provided in the subject screening methods, test agents are
evaluated for potential activity as agents for treating obesity,
liver disease, and/or diabetes (e.g., candidate agents for
decreasing an amount of fat tissue in an individual, preventing or
reducing weight gain of an individual, increasing insulin
sensitivity of an individual, and/or increasing glucose tolerance
of an individual). In some cases, a subject method is for screening
a plurality of test agents. Test agents can be evaluated (screened)
individually (sequentially), or in parallel.
[0074] In some cases, a test agent (e.g., a test compound) can have
a formula weight of less than 10,000 grams per mole (e.g., less
than 5,000 grams per mole, less than 1,000 grains per mole, or less
than 500 grams per mole). A test agent can be naturally occurring
(e.g., an herb or a natural product), synthetic; or can include
both natural and synthetic components. Examples of small molecules
include peptides, peptidomimetics (e.g., peptoids), amino acids,
amino acid analogs, polynucleotides, polynucleotide analogs,
nucleotides, nucleotide analogs, and small molecules, such as
organic or inorganic compounds, e.g., heterorganic or
organometallic compounds.
ClpP Expression Level and/or Activity Level
[0075] In some cases, a subject screening method includes a step of
measuring a ClpP expression level and/or activity level. Because
reduction of ClpP mRNA can result in reduced ClpP protein level,
either assay (one to measure ClpP-encoding mRNA or one to measure
ClpP protein) can be used for measuring an expression level. For
example, in some cases, a test agent that reduces the level of ClpP
protein in the target cell (or in cells of the target cell
population) will be identified as a candidate agent for use in
treating obesity, liver disease, and/or diabetes. In some cases, a
test agent that reduces the level of ClpP-encoding mRNA in the
target cell (or in cells of the target cell population) will be
identified as a candidate agent for use in treating obesity, liver
disease; and/or diabetes.
[0076] The terms "assaying" and "measuring" are used herein to
include the physical steps of manipulating a biological sample
(e.g., cell sample) to generate data related to the sample (e.g.,
measuring an expression level and/or activity level in a biological
sample). In practicing the subject methods, the expression level of
a ClpP expression product (e.g., mRNA or protein) and/or an
activity level of ClpP can be measured. The expression level may be
the expression level in a cell, in a population of cells, in a
biological sample from an individual, and the like. The expression
level(s) and/or activity level(s) can be measured by any suitable
method. For example, an RNA expression level can be measured by
measuring the levels/amounts of one or more nucleic acid
transcripts, e.g. mRNAs, of ClpP, Protein expression levels of ClpP
can be detected by measuring the levels/amounts of the ClpP
protein.
[0077] "Measuring" can be used to determine whether the measured
expression level and/or activity level is less than, greater than.
"less than or equal to", or "greater than or equal to" a particular
threshold, (the threshold can be pre-determined or can be
determined by assaying a control sample). On the other hand,
"measuring to determine the expression level" (and/or activity
level) or simply "measuring expression levels" (and/or activity
levels) can mean determining a quantitative value (using any
suitable metric) that represents the level of expression (e.g., the
amount of protein and/or RNA, e.g., mRNA) of a particular
expression product (e.g., a ClpP expression product) and/or the
activity level of ClpP. The level of expression and/or activity can
be expressed in arbitrary units associated with a particular assay
(e.g., fluorescence units, e.g., mean fluorescence intensity (WO,
threshold cycle (Ct), quantification cycle (CO, and the like), or
can be expressed as an absolute value with defined units (e.g.,
number of mRNA transcripts, number of protein molecules,
concentration of protein, amount of substrate cleaved, etc.).
[0078] An expression level and/or activity level can be a raw
measured value, or can be a normalized and/or weighted value
derived from the raw measured value. The terms "expression level"
and "measured expression level" are used herein to encompass raw
measured values as well as values that have been manipulated in
some way (e.g., normalized and/or weighted). In some cases, a
normalized expression level and/or activity level is a measured
expression level of an expression product and/or an activity level
from a sample where the raw measured value for the expression
product and/or activity level has been normalized. For example, the
expression level of an expression product (e.g., an RNA encoding
ClpP, a ClpP protein) can be compared to the expression level of
one or more other expression products (e.g., the expression level
of a housekeeping gene, the averaged expression levels of multiple
genes, etc.) to derive a normalized value that represents a
normalized expression level. Methods of normalization will be known
to one of ordinary skill in the art and any suitable normalization
method can be used. The specific metric (or units) chosen is not
crucial as long as the same units are used (or conversion to the
same units is performed) when evaluating multiple markers and/or
multiple biological samples (e.g., samples from multiple
individuals or multiple samples from the same individual).
[0079] A reduction of a ClpP expression level and/or activity level
can be determined in a number of different ways. For example, in
some cases, a ClpP expression level and/or activity level is
measured in a cell (or in an individual, e.g., in a biological
sample from the individual) prior to and after contact with a test
agent (or prior to and after administration of an agent to an
individual), and a determination is made as to whether a reduction
was caused by the contacting (or the administration). In some
cases, some cells of a cell population are not contacted with an
agent (e.g., a test agent) and other cells of the cell population
are contacted with an agent (e.g., a test agent) and a comparison
can be made among the contacted and non-contacted cells. In some
cases, some cells of a cell population are not contacted with an
agent (e.g., a test agent) and other cells of the cell population
are contacted with a mock agent (e.g., an agent known not to cause
a change) and a comparison can be made among the cells contacted
with the test agent and those (control cells) contacted with the
mock agent (control agent).
[0080] Such comparisons can generally be referred to as comparing a
measured value to a reference value, or determining that an agent
caused a reduction in a ClpP expression level and/or activity level
as compared to a reference value. A reference value can be a level
of ClpP (protein and/or mRNA) and/or activity level of ClpP
measured prior to contact with an agent, a level of ClpP (protein
and/or mRNA) and/or activity level of ClpP measured after contact
with a mock agent (control agent), a level of ClpP (protein and/or
mRNA) and/or activity level of ClpP measured in the absence of
contact with an agent, etc. A reference value can also be in some
cases a pre-determined (threshold) value.
[0081] In some cases, a subject method includes determining that a
test agent or a candidate agent caused a decrease in the expression
level and/or activity level of ClpP. In some cases, the expression
level and/or activity level is reduced by 10% or more (e.g., 20% or
more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or
more, 80% or more, or 90% or more) compared to a reference value
(e.g., an activity level and/or expression level, e.g., of ClpP
protein and/or mRNA, prior to contact with the agent; an activity
level and/or expression level, e.g., of ClpP protein and/or mRNA,
in mock-treated cells or in mock-treated control individuals;
etc.). In some cases, the measured expression level and/or activity
level is 95% of a reference value or less (e.g., 90% of the
reference value or less, 85% of the reference value or less, 80% of
the reference value or less, 75% of the reference value or less,
70% of the reference value or less, 65% of the reference value or
less, 60% of the reference value or less, 55% of the reference
value or less, 50% of the reference value or less, 45% of the
reference value or less, 40% of the reference value or less, 35% of
the reference value or less, 30% of the reference value or less,
25% of the reference value or less, 20% of the reference value or
less, 15% of the reference value or less, 10% of the reference
value or less, or 5% of the reference value or less). In some
cases, the reference value of the expression level and/or activity
level of ClpP is 1.1-fold or more (e.g., 1.2-fold or more, 1.3-fold
or more, 1.4-fold or more, 1.5-fold or more, 2-fold or more,
2.5-fold or more, 3-fold or more, 4-fold or more, 5-fold or more;
7.5-fold or more, or 10-fold or more) greater than the measured
value.
Measuring RNA
[0082] An expression level of an expression product of ClpP may be
measured by detecting the amount or level of one or more RNA
transcripts or a fragment thereof encoded by the gene of interest
(ClpP). Such detection may include detecting the level of one or
more RNA transcripts or a fragment thereof encoded by the ClpP gene
in a cell extract, in a fixed cell, in a living cell, in a
biological sample, etc. For measuring RNA levels, the amount or
level of an RNA in the sample is determined, e.g., the expression
level of an mRNA. In some instances, the expression level of one or
more additional RNAs may also be measured, and the level of ClpP
RNA expression compared to the level of the one or more additional
RNAs to provide a normalized value for the ClpP expression
level.
[0083] The expression level of nucleic acids in a sample (e.g., the
expression level of a ClpP mRNA) may be detected using any suitable
protocol. A number of exemplary methods for measuring RNA (e.g.,
mRNA) expression levels in a sample are known by one of ordinary
skill in the art, such as those methods employed in the field of
differential gene expression analysis, and any suitable method can
be used. Exemplary methods include, but are not limited to:
hybridization-based methods (e.g., Northern blotting, array
hybridization (e.g., microarray); in situ hybridization; in situ
hybridization followed by FACS; and the like) (Parker & Barnes,
Methods in Molecular Biology 106:247-283 (1999)); RNAse protection
assays (Hod, Biotechniques 13:852-854 (1992)); PCR-based methods
(e.g.; reverse transcription PCR (RT-PCR), quantitative RT-PCR
(qRT-FCR), real-time RT-PCR, etc.)(Weis et al., Trends in Genetics
8:263-264 (1992)); nucleic acid sequencing methods (e.g., Sanger
sequencing, Next Generation sequencing (i.e., massive parallel high
throughput sequencing, e.g., Illumina's reversible terminator
method, Roche's pyrosequencing method (454), Life Technologies'
sequencing by ligation (the SOLiD platform), Life Technologies' Ion
Torrent platform, single molecule sequencing, etc.); nanopore based
sequencing methods; and the like.
[0084] In some embodiments, the biological sample can be assayed
directly. In some embodiments, nucleic acid of the biological
sample is amplified (e.g., by PCR) prior to assaying. As such,
techniques such as PCR (Polymerase Chain Reaction), RT-PCR (reverse
transcriptase PCR), qRT-PCR (quantitative RT-PCR, real time
RT-PCR), etc. can be used prior to the hybridization methods and/or
the sequencing methods discussed above.
[0085] Examples of some of the methods listed above are described
in the following references: Margulies et al (Nature 2005 437:
376-80); Ronaghi et al (Analytical Biochemistry 1996 242: 84-9);
Shendure (Science 2005 309: 1728); Imelfort et al (Brief Bioinform.
2009 10:609-18); Fox et al (Methods Mol Biol. 2009; 553:79-108);
Appleby et al (Methods Mol Biol. 2009; 513:19-39); Soni et al Clin
Chem 53: 1996-2001 2007; and Morozova (Genomics. 2008 92:255-64),
which are herein incorporated by reference for the general
descriptions of the methods and the particular steps of the
methods, including starting products, reagents, and final products
for each of the steps.
Measuring Protein
[0086] An expression level of an expression product of ClpP may be
measured by detecting the amount or level of one or more proteins
(e.g., ClpP) or a fragment thereof. Such detection may include
detecting the level of a ClpP protein or a fragment thereof in a
cell extract, in a fixed cell, in a living cell, in a biological
sample, etc. For measuring a protein level, the amount or level of
protein the sample (e.g., in a cell, in a population of cells, in a
cell extract, etc.) is determined. In some instances, the
concentration of one or more additional proteins may also be
measured, and the measured expression level compared to the level
of the one or more additional proteins to provide a normalized
value for the measured expression level. In some embodiments, the
measured expression level is a relative value calculated by
comparing the level of one protein relative to another protein. In
other embodiments the concentration is an absolute measurement
(e.g., weight/volume or weight/weight).
[0087] The expression level of a protein (e.g., ClpP) may be
measured by detecting in a sample the amount or level of one or
more proteins/polypeptides or fragments thereof. The terms
"polypeptide," "peptide" and "protein" are used interchangeably
herein to refer to a polymer of amino acid residues. "Polypeptide"
refers to a polymer of amino acids (amino acid sequence) and does
not refer to a specific length of the molecule. Thus peptides and
oligopeptides are included within the definition of polypeptide. In
some cases, cells are removed from a biological sample (e.g., via
centrifugation; via adhering cells to a dish or to plastic, etc.)
prior to measuring the expression level. In some cases, the
intracellular protein level is measured by lysing cells of the
sample to measure the level of protein in the cellular contents. In
some cases, a level of protein can be measured without disrupting
cell morphology (e.g., a protein level can be visualized, e.g., via
fluorescent antibody staining, etc.) When protein levels are to be
detected, any suitable protocol for measuring protein levels may be
employed. Examples of methods for assaying protein levels include
but are not limited to antibody-based methods as well as methods
that are not antibody based. Examples of suitable methods include
but are not limited to: enzyme-linked immunosorbent assay (ELISA),
mass spectrometry, proteomic arrays, xMAP.TM. microsphere
technology, flow cytometry, western blotting, immunofluorescence,
and immunohistochemistry.
[0088] Some protein detection methods are antibody-based methods.
The term "antibody" is used in the broadest sense and specifically
covers monoclonal antibodies (including full length monoclonal
antibodies), polyclonal antibodies, multispecific antibodies (e.g.,
bispecific antibodies); and antibody fragments so long as they
exhibit the desired biological activity. Examples of antibody
fragments include Fab, Fab', Fab'-SH, F(ab').sub.2, and Fv
fragments; scFvs, diabodies; and multispecific or multivalent
structures formed from antibody fragments.
[0089] ClpP Activity Level
[0090] As noted above, ClpP functions as part of a complex (ClpXP)
that includes both ClpP and ClpX, and requires ATP to unfold and
hydrolyze protein substrates. As such, any agent that disrupts the
interaction between ClpP and ClpX, or that reduces the activity of
the ClpP/ClpX complex (e.g., reduces the hydrolysis of protein
substrates) can be considered an agent that reduces an activity
level of ClpP. An activity level of ClpP can be measured using any
suitable method. For example, an enzymatic assay can be used to
measure ClpP activity. Parameters such as the kinetics of
hydrolysis of ATP by ClpXP and/or degradation of substrate by ClpXP
can be measured using any suitable method. For examples of various
ways to measure ClpP activity level, see, e.g., Burton et al.,
Protein Sci. 2003 May; 12(5):893-902; Joshi et al., Nat Struct Mol
Biol. 2004 May; 11(5):404-11; Kang et al., J Biol Chem. 2005 Oct.
21; 280(42):35424-32; Baker et al., Biochim Biophys Acta. 2012
January; 0823(1):15-28, and Al-Furoukh et al, (2014) PUS ONE 9(7):
e103141, the disclosure of each of which are incorporated by
reference herein.
[0091] Furthermore, as demonstrated in the examples below, a
decrease in ClpP activity level (in this case caused by an absence
of ClpP in knockout mice) results in an increase in the amounts of
the following proteins: TNF receptor-associated protein 1 (TRAP1)
(mitochondrial Hsp90), heat shock protein family A (Hsp70) member 9
(Grp75) (mitochondrial Hsp70), leucine rich pentatricopeptide
repeat containing (LRPPRC), caseinolytic mitochondrial matrix
peptidase chaperone subunit (ClpX) (mitochondrial Hsp100),
ornithine aminotransferase (OAT), and Ion peptidase 1,
initochondrial (LonP1) (a mitochondrial protease), As such, in some
cases, measuring an activity level of ClpP can include measuring an
amount of one or more (e.g., two or more, three or more, four or
more, 5 or more, or all 6) of the following proteins: TRAP1, Grp75,
LRPPRC, ClpX, OAT, and LonP. For example, a decrease in ClpP
activity can be detected by measuring an increase in the levels of
one or more (e.g., two or more, three or more, four or more, 5 or
more, or all 6) of: TRAP1, Grp75, LRPPRC, ClpX, OAT, and LonP,
e.g., relative to a suitable control, such as a level of one or
more of TRAP1, Grp75, LRPPRC, ClpX, OAT, and LonP prior to
contacting with a test agent as described herein.
Evaluation Steps
[0092] As described in the examples below, the inventors have
discovered that mice with decreased ClpP expression (due to
knockout in the genome of the ClpP gene, designated ClpP
.sup.(-/-)) exhibit a number of phenotypes, including but not
limited to decreased: white adipose tissue, body fat (as measured
by total body fat mass or body fat content (% fat mass)), body
weight, visceral adipose adipocyte size, levels of plasma leptin,
and blood glucose level (e.g., after fasting); and increased:
insulin sensitivity, glucose tolerance, growth hormone levels,
energy consumption (e.g., increased basal energy expenditure),
levels of phosphorylated AKT (p-AKT) in muscles and/or fibroblasts,
and lean content (% lean mass). ClpP mice gained less weight and
generated less fat while consuming more food, and ClpP.sup.-/- mice
were resistant to high fat diet (HFD)-induced weight gain (e.g.,
the increase in fat mass and body weight due to HFD was less than
the increase seen in wild type controls).
[0093] In some cases, a subject method (e.g. a screening method or
a treatment method) includes, after administration of an agent to
an individual (e.g., a candidate agent for treatment as identified
with a subject screening method, an inhibitor of ClpP such as an
RNAi agent or a genome editing agent specific for ClpP, etc.), a
step of measuring one or more features of the individual (e.g., to
verify that the agent produces a desired outcome). Suitable
features that can be measured include, but are not limited to
insulin sensitivity, blood glucose level, glucose tolerance, body
fat mass, an amount of fat tissue, an amount of white adipose
tissue, percent fat mass, body weight, visceral adipose adipocyte
size, plasma leptin level, growth hormone level, basal energy
expenditure, a level of phosphorylated AKT (p-AKT) in muscles
and/or fibroblasts, percent lean mass, mitochondrial number in
hepatocytes, mitochondrial mass in hepatocytes, mitochondrial
morphology in hepatocytes, fibroblast respiratory capacity,
fibroblast maximal oxygen consumption rate (OCR), and fibroblast
resistance to H.sub.2O.sub.2-induced cytotoxicity. An evaluation
step (e.g., a step of measuring one or more of the above features),
can be included as part of a subject screening method (e.g., a
method of identifying a candidate agent). An evaluation step (e.g.,
a step of measuring one or more of the above features), can also be
included as part of a subject treatment method.
Generating a Report
[0094] In some cases, a subject method (e.g., any of the screening
methods described above) includes a step of generating a report
(e.g., a report that the test agent is a candidate agent for
treating obesity, liver disease, and/or diabetes). A "report," as
described herein, is an electronic or tangible document which
includes report elements that provide information of interest
relating to the results and/or assessments of such results of a
subject method (e.g., a screening method). In some embodiments, a
subject report includes a measured expression level and/or activity
level as discussed in greater detail above (e.g., a raw value, a
normalized value, a normalized and weighted value, etc.) (e.g., an
activity level of ClpP or an expression level of a ClpP expression
product, such as a ClpP protein and/or a ClpP-encoding mRNA). In
some embodiments, a subject report includes a ClpP expression level
and/or activity level. In some cases, a subject report includes an
assessment (e.g. a determination of whether one or more test agents
caused a reduction in a ClpP expression level and/or activity
level). For example, a report can state whether a given test agent
or list of test agents caused a reduction in a ClpP expression
level and/or activity level (e.g., at the level of protein and/or
mRNA), and/or whether a given test agent or list of test agents is
a candidate agent for treatment.
Treatment Methods
[0095] Provided are methods that include administering to an
individual an inhibitor of ClpP (such as an RNAi agent or gene
editing agent specific for ClpP), where the inhibitor of ClpP
reduces an activity level and/or expression level of ClpP (e.g., as
can be detected at the level of protein and/or mRNA). Such methods
can be referred to as methods of reducing an expression level
and/or activity level of ClpP, and/or methods of treating an
individual with obesity, liver disease, and/or diabetes. A
discussion of ClpP activity levels and expression levels (e.g., at
the level of protein and/or mRNA), and methods for measuring such
levels can be found elsewhere herein.
[0096] In some embodiments, such methods are methods of treating an
individual with obesity, liver disease, and/or diabetes, methods of
increasing energy output, methods of reducing an amount of adipose
tissue (e.g., white adipose tissue), methods of preventing or
reducing weight gain, methods of increasing insulin sensitivity,
and/or methods of increasing glucose tolerance. For example, in
some cases, a subject method is a method of increasing energy
output, reducing an amount of adipose tissue, increasing insulin
sensitivity, increasing glucose tolerance, and/or preventing or
reducing weight gain, and the method includes administering to the
individual a ClpP inhibitor (such as an RNAi agent or gene editing
agent specific for ClpP) that reduces the expression level and/or
activity level of ClpP. In some embodiments, a subject method is a
method of treating an individual with obesity, liver disease,
and/or diabetes, and the method includes administering to the
individual an inhibitor of ClpP (such as an RNAi agent or gene
editing agent specific for ClpP) that reduces the expression level
and/or activity level of ClpP.
[0097] In some embodiments, a subject method is a method of
administering an inhibitor of ClpP to an individual (e.g., an
individual who has obesity, liver disease, and/or diabetes) in an
amount effective for decreasing an amount of fat tissue in the
individual, preventing or reducing weight gain of the individual,
increasing insulin sensitivity of the individual, and/or increasing
glucose tolerance of the individual.
[0098] In some cases, any of the above subject treatment methods
can include a step of measuring a ClpP expression level and/or
activity level in a biological sample from the individual that is
being treated (e.g., in a hepatocyte of/from the individual to whom
a ClpP inhibitor was administered). In some cases, a subject
treatment method includes a step of obtaining a biological sample
from the individual and measuring a ClpP expression level and/or
activity level of the sample. In some cases, such a step is
performed before and after treatment (e.g., to verify that
administration had the desired outcome), In some cases, an
inhibitor of ClpP (e.g., an RNAi agent or a gene editing agent that
targets ClpP) is administered to an individual (e.g., an individual
who is obese and/or has diabetes), in an amount effective for
decreasing an amount of fat tissue in the individual, preventing or
reducing weight gain of the individual, increasing insulin
sensitivity of the individual, and; or increasing glucose tolerance
of the individual.
RNAi Agents and Genome Editing Agents
[0099] In some cases, an inhibitor of ClpP (e.g., an agent that
reduces an expression level and/or activity level of ClpP) is an
RNAi agent or a genome editing agent that targets ClpP (e.g., is
specific for ClpP). The term "RNAi agent" is used herein to mean
any agent that can be used to induce a gene specific RNA
interference (RNAi) response in a cell. Suitable examples of RNAi
agents include, but are not limited to short interfering RNAs
(siRNAs) and short hairpin RNAs (shRNAs), and micro RNAs (miRNA).
An RNAi agent (e.g., shRNA, siRNA, miRNA) specific for ClpP is an
agent that targets the LIANA encoding the ClpP protein. RNAi agents
can readily be designed to specifically target any desired mRNA
(e.g., one encoding ClpP) by choosing an appropriate nucleotide
sequence.
[0100] Various RNAi agent designs (RNAi agents with various
features) are known in the art and any suitable RNAi agent that
targets ClpP can be used. For example, various designs of RNAi
agents (as well as methods of their delivery) can be found in
numerous patents, including, but not limited to U.S. Pat. Nos.
7,022,828; 7,176,304; 7,592,324; 7,667,028; 7,718,625; 7,732,593;
7,772,203; 7,781,414; 7,807,650; 7,879,813; 7,892,793; 7,910,722;
7,947,658; 7,973,019; 7,973,155; 7,981,446; 7,993,925; 8,008,271;
8,008,468; 8,017,759; 8,034,922; 8,399,653; 8,415,319; 8,426,675;
8,466,274; 8,524,679; 8,524,679; 8,569,065; 8,569,256; 8,569,258;
9,233,102; 9,233,170; and 9,233,174; all of which are incorporated
herein by reference. Where appropriate, an RNAi agent may be
provided in the form of a DNA encoding the agent (e.g., an
expression vector encoding a shRNA). shRNAs targeting the ClpP
(accession no. NM_003321) coding sequence are provided for example
in Cole et al., 2015, Cancer Cell 27, 864-876, the disclosure of
which is incorporated by reference herein in its entirety and for
all purposes. These shRNAs include 5'-GCCCATCCACATGTACATCAA-3' (SEQ
ID NO:5); 5'-CACGATGCAGTACATCCTCAA-3' (SEQ NO:6); and
5'-GCTCAAGAAGCAGCTCTATAA-3' (SEQ ID NO:7).
[0101] The terms "genome editing agent" and "genome targeting
composition" are used interchangeably herein to mean a composition
that includes a genome editing nuclease. In some embodiments, the
genome editing nuclease binds a native or endogenous recognition
sequence. In some embodiments, the genome editing nuclease is a
modified endonuclease that binds a non-native or exogenous
recognition sequence and does not bind a native or endogenous
recognition sequence.
[0102] Examples of suitable genome editing nucleases include but
are not limited to zinc finger nucleases (ZFNs), TAL-effector DNA
binding domain-nuclease fusion proteins (transcription
activator-like effector nucleases (TALENs)), CRISPR/Cas
endonucleases (e.g., class 2 CRISPR/Cas endonucleases such as a
type II, type V, or type VI CRISPR/Cas endonucleases), and
recombinases (e.g., Cre recombinase, Hin recombinase, RecA, RAD51,
Tre, FLP, and the like). Thus, in some embodiments, a genome
editing agent is a composition that can include one or more genome
editing nucleases selected from: a ZFN, a TALEN, a recombinase
(e.g., Cre recombinase, Hin recombinase. RecA, RAD51, ire, FLP, and
the like), and a CRISPR/Cas endonuclease (e.g., a class 2
CRISPR/Cas endonuclease such as a type II, type V, or type VI
CRISPR/Cas endonuclease).
[0103] Recombinases include but are not limited to Cre recombinase,
Hin recombinase, RecA, RAD51, Tre, and FLP.
[0104] Information related to class 2 type II CRISPR/Cas
endonuclease Cas9 proteins and Cas9 guide RNAs (as well as methods
of their delivery) (as well as information regarding requirements
related to protospacer adjacent motif (PAM) sequences present in
targeted nucleic acids) can be found in the art, for example, see
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et. al., Proc Natl Acad Sci USA. 2013 Sep. 24; 110(39):15514-5; Xie
et. al., Mol Plant. 2013 Oct. 9; Yang et, al., Cell. 2013 Sep. 12;
154(6):1370-9; Briner et al., Mol Cell. 2014 Oct. 23; 56(2):333-9;
and U.S. patents and patent applications: U.S. Pat. Nos. 8,906,616;
8,895,308; 8,889,418; 8,889,356; 8,871,445; 8,865,406; 8,795,965;
8,771,945; 8,697,359; 20140068797; 20140170753; 20140179006;
20140179770; 20140186843; 20140186919; 20140186958; 20140189896;
20140227787; 20140234972; 20140242664; 20140242699; 20140242700;
20140242702; 20140248702; 20140256046; 20140273037; 20140273226;
20140273230; 20140273231; 20140273232; 20140273233; 20140273234;
20140273235; 20140287938; 20140295556; 20140295557; 20140298547;
20140304853; 20140309487; 20140310828; 20140310830; 20140315985;
20140335063; 20140335620; 20140342456; 20140342457; 20140342458;
20140349400; 20140349405; 20140356867; 20140356956; 20140356958;
20140356959; 20140357523; 20140357530; 20140364333; and
20140377868; all of which are hereby incorporated by reference in
their entirety. Examples and guidance related to type V or type VI
CRISPR/Cas endonucleases and guide RNAs (as well as information
regarding requirements related to protospacer adjacent motif (PAM)
sequences present in targeted nucleic acids) can be found in the
art, for example, see Zetsche et al, Cell. 2015 Oct. 22;
163(3):759-71; Makarova et al, Nat Rev Microbiol. 2015 November;
13(11):722-36; and Shmakov et al., Mol Cell. 2015 Nov. 5;
60(3):385-97.
[0105] Useful designer zinc finger modules include those that
recognize various GNN and ANN triplets (Dreier, et al., (2001) J
Biol Chem 276:29466-78; Dreier, et al., (2000) J Mol Biol
303:489-502; Liu, et al., (2002) J Biol Chem 277:3850-6), as well
as those that recognize various CNN or TNN triplets (Dreier, et
al., (2005) J Biol. Chem 280:35588-97; Jamieson, et al., (2003)
Nature Rev Drug Discov 2:361-8). See also, Durai, et al., (2005)
Nucleic Acids Res 33:5978-90; Segal, (2002) Methods 26:76-83;
Porteus and Carroll, (2005) Nat Biotechnol 23:967-73; Pabo, et al.,
(2001) Ann Rev Biochem 70:313-40; Wolfe, et al., (2000) Ann Rev
Biophys Biomol Struct 29:183-212; Segal and Barbas, (2001) Curr
Opin Biotechnol 12:632-7; Segal, et al., (2003) Biochemistry
42:2137-48; Beerli and Barbas, (2002) Nat Biotechnol 20:135-41;
Carroll, et al., (2006) Nature Protocols 1:1329; Ordiz, et al.,
(2002) Proc Natl Acad Sci USA 99:13290-5; Guan, et al., (2002) Proc
Natl Acad Sci USA 99:13296-301.
[0106] For more information on ZFNs and TALENs (as well as methods
of their delivers), refer to Sanjana et al., Nat Protoc. 2012 Jan.
5; 7(1):171-92 as well as international patent applications
WO2002099084; WO00/42219; WO02/42459; WO2003062455; WO03/080809;
WO05/014791; WO05/084190; WO08/021207; WO09/042186; WO09/054985;
WO10/079430; and WO10/065123; U.S. Pat. Nos. 8,685,737; 6,140,466;
6,511,808; and 6,453,242; and US Patent Application Nos.
2011/0145940, 2003/0059767, and 2003/0108880; all of which are
hereby incorporated by reference in their entirety.
Small Molecule ClpP Inhibitors
[0107] Small molecule inhibitors of ClpP are known Which may find
use in one or more of the screening or treatment methods described
herein. For example .beta.-Lactones, such as
(3RS,4RS)-3-(non-8-en-1-yl)-4-(2-(pyridin-3-yl)ethyl)oxetan-2-one
(also known as A2-32-01 and referred to herein interchangeably as
A2-32-01 and A2-32) have been identified as inhibitors of both
bacterial and mammalian ClpP. See, e.g., Cole et al., 2015, Cancer
Cell 27, 864-876, the disclosure of which is incorporated by
reference herein in its entirety and for all purposes.
[0108] Beta-lactones are described, for example, in U.S. Patent
Application Publication 2014/0243255, the disclosure of which is
incorporated by reference herein in its entirety and for all
purposes, and can include, but are not limited to,
trans-beta-lactones, beta-propiolactone, saturated aliphatic
beta-lactones, beta-butyrolactone, beta-isobutyrolactone,
beta-valerolactone, beta-isovalerolactone, beta-n-caprolactone,
alpha-ethylbeta-propiolactone, alpha-isopropyl-beta-propiolactone,
alpha-butyl-beta-propiolactone, alpha,
isopropyl-beta-propiolactone, beta isopropyl-beta-propiolactone,
alpha-methyl-beta-butyrolactone,
beta-ethyl-beta-butyrolactone-alpha-ethyl-beta-butyrolactone,
alpha-methyl beta-propiolactone, lactones of
betahydroxy-mono-carboxylic acids containing cycloalkyl, aryl and
aralkyl substituents such as betacyclohexyl-beta-propiolactone,
beta-phenyl-betapropiolactone, alpha-phenyl-beta-propiolactone,
beta-taenzyl-beta-propiolactone and derivatives thereof.
[0109] .beta.-Lactones inhibitors of ClpP, which may be utilized in
the context of the disclosed methods, include those described in
U.S. Patent Application Publication No. 2016/0221977, the
disclosure of which is incorporated by reference herein in its
entirely and for all purposes, Such .beta.-Lactones inhibitors of
ClpP include compounds of the following structures:
##STR00001##
wherein R.sub.2 is a single substitution of a hydrogen at any
position on the benzene ring where the substituted moiety is
selected from the group consisting of alkyl, substituted alkyl,
alkynyl, substituted alkyl, vinyl, nitro, halo (e.g., includes
bromine, chlorine, fluorine and iodine), cyano, amyl, hetero aryl,
alkoxy; and C.sub.n is carbon and n is a number of from 1 to 5.
##STR00002##
[0110] wherein R.sub.1 is selected from the group consisting of
alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,
substituted alkynyl, alkoxy, substituted alkoxy, alkoxyalkyl,
substituted alkoxyalkyl, NH2, NHR, NR2, mono- or
polyhydroxy-substituted alkyl, aryl, substituted heteroaryl, and
substituted heteroaryl; R.sub.2 is a single substitution of a
hydrogen at any position on the benzene ring where the substituted
moiety is selected from the group consisting of alkyl, substituted
alkyl, alkynyl, substituted alkyl, vinyl, nitro, halo, cyano, aryl,
heteroaryl, alkoxy; and, C.sub.n is carbon and n is a number of
from 1 to 5. Specific forms of the above structure are provided
below:
##STR00003##
[0111] Small molecule phenyl esters have also been shown to inhibit
bacterial ClpP. See, e.g., Hackl et al., J. Am. Chem. Soc., 137,
8475-8483 (2015), the disclosure of which is incorporated by
reference herein in its entirety and for all purposes. Such small
molecule inhibitors include, e.g.:
##STR00004##
[0112] In addition to the compounds described above,
pharmaceutically acceptable salts and pro-drugs thereof may be
utilized in the methods disclosed herein.
Agent Delivery
[0113] In some embodiments, a subject method (e.g., a screening
method or a treatment method as described herein) includes a step
of administering an agent to an individual (e.g., a test agent, a
candidate agent, a ClpP inhibitor that reduces an expression level
and/or activity level of ClpP, e.g., an RNAi agent or a gene
editing agent that specifically reduces expression of ClpP, and the
like). Depending on context, the individual can be of any suitable
species (e.g., a mammal, a rodent, a mouse, a rat, a non-human
primate, a human, etc.). For example, in some cases a test agent is
administered to a mouse. In some cases, a candidate agent (e.g., as
identified by a subject screening method) is administered to a
mouse, a rat, a non-human primate, or a human (e.g., an individual
with obesity, diabetes, reduced insulin sensitivity, reduced
glucose tolerance, etc. in some cases, a ClpP inhibitor that
reduces an expression level and/or activity level of ClpP as
described herein is administered to a mouse, a rat, a non-human
primate, or a human.
[0114] In some cases, an agent as described herein is administered
systemically. In some cases, an agent as described herein is
administered locally (e.g., directly to a desired tissue such as
the liver). In some cases, an agent as described herein is
administered by parenteral, topical, intravenous, intratumoral,
oral, subcutaneous, intraarterial, intracranial, intraperitoneal,
intranasal or intramuscular means. A typical route of
administration is intravenous or intratumoral, although other
routes can be equally effective. In some cases, an agent as
described herein is administered via injection. In some cases, an
agent as described herein is administered in a tissue-specific
manner (e.g., administration is directed to a specific tissue such
as the liver).
[0115] Those of skill in the art will readily appreciate that dose
levels can vary as a function of the specific compound, the
severity of the symptoms and the susceptibility of the subject to
side effects. Preferred dosages for a given compound are readily
determinable by those of skill in the art by a variety of
means.
[0116] In some embodiments, a single dose of a ClpP inhibitor is
administered. In other embodiments, multiple doses of a ClpP
inhibitor are administered. Where multiple doses are administered
over a period of time, a ClpP inhibitor is administered twice daily
(qid), daily (qd), every other day (god), every third day, three
times per week (tiw), or twice per week (biw) over a period of
time. For example, a ClpP inhibitor is administered qid, qd, god,
tiw, or biw over a period of from one day to about 2 years or more.
For example, a ClpP inhibitor is administered at any of the
aforementioned frequencies for one week, two weeks, one month, two
months, six months, one year, or two years, or more, depending on
various factors.
[0117] In some cases, a small molecule inhibitor of ClpP as
described herein, e.g., a .beta.-Lactone, such as
(3RS,4RS)-3-(non-8-en-1-yl)-4-(2-(pyridin-3-yl)ethyl)oxetan-2-one
or a pharmaceutically acceptable salt or pro-drug thereof is
administered to a subject in need thereof, e.g., a subject as
described herein, in an amount from about 50 mg/kg to about 1000
mg/kg per day, e.g., from about 100 mg/kg to about 900 mg/kg per
day, from about 200 mg/kg to about 800 mg/kg per day, from about
300 mg/kg to about 700 mg/kg per day, or from about 400 mg/kg to
about 600 mg/kg per day. In some cases, a small molecule inhibitor
of ClpP as described herein, e.g., a .beta.-Lactone, such as
(3RS,4RS)-3-(non-8-en-1-yl)-4-(2-(pyridin-3-yl)ethyl)oxetan-2-one
or a pharmaceutically acceptable salt or pro-drug thereof is
administered to a subject as a once daily dose or a twice daily
dose.
[0118] In some cases, a RNAi agent or genome editing agent is
configured such that the reduction of ClpP expression brought about
by the agent is limited to a particular tissue type (e.g., the
liver). For example, the components of an RNAi agent (e.g., shRNA,
siRNA) or genome editing agent (CRISPR/Cas protein plus guide RNA)
can be active only in a particular tissue, e.g., the components can
be delivered to the particular tissue, the components can be
operably linked to a tissue-specific promoter, etc. For example, as
described in the examples below, delivery of the Cre recombinase to
the liver in mice that harbor a foxed allele of the ClpP gene in
their genome, recapitulates the increase in insulin sensitivity
exhibited by ClpP knockout mice. Thus, reduction of ClpP expression
in the liver can result in an increase in insulin sensitivity
without the potential side effects of reducing ClpP expression
throughout the whole individual. In some cases, the inhibitor of
ClpP that reduces ClpP expression and/or activity (e.g., a small
molecule inhibitor, an RNAi agent or gene editing agent specific
for ClpP) is administered systemically, and in some cases locally
(e.g., to the liver, e.g., see U.S. Pat. Nos. 8,524,679; and
8,008,468, both of which are incorporated by reference in their
entirety).
[0119] In some cases, the effect of an agent as described herein is
substantially liver-specific (e.g., the genome editing nuclease
such as Cre recombinase, a CRISPR/Cas endonuclease, a ZFN, a TALEN,
etc., can be delivered in a tissue specific manner or can be under
the control of a tissue specific promoter). By "substantially
liver-specific" it is meant that the majority of the reduction of
ClpP expression is in the liver. Such can be achieved in a number
of different ways. It is understood that the reduction of ClpP
expression may not be limited only to the liver, but that one or
more other tissues may exhibit reduction of ClpP expression as
well. For example, if a promoter is used (e.g., as part of an
expression vector to drive expression of a ClpP inhibitor, e.g., a
genome editing nuclease, or a component of a ClpP inhibitor, e.g.,
a CRISPR/Cas guide RNA), that promoter need not be perfectly
limited only to the liver. Such a promoter may exhibit `leaky`
expression in other tissues or may be limited to tissues other than
just the liver (e.g., the liver plus one or more additional
tissues, but not all tissues, i.e., the promoter is not
constitutive). As another example, if an agent is locally delivered
(e.g., via direct injection), it is understood that some of the
agent may also affect tissues in addition to the liver (e.g.,
neighboring organs, the blood, etc.). Thus, when the term
"substantially liver-specific" is used, it is meant that global
ClpP expression (e.g., throughout the whole body) is not reduced,
while liver ClpP expression is reduced.
[0120] For example, when the agent is an RNAi agent,
liver-specificity can be accomplished by delivering the agent
directly to the liver (e.g., injection into the liver etc.). On the
other hand, liver-specificity can be accomplished by expressing the
RNAi agent from a DNA encoding the agent (e.g., an expression
vector encoding an shRNA) where the promoter driving expression of
the agent drives expression of the agent in the liver (e.g., a
liver-specific promoter). Likewise, genome editing agents can be
delivered directly to the liver, or can be functional in the liver
(e.g., by expressing one or more of the components from an
expression vector that has a promoter that drives expression in the
liver, e.g., a liver-specific promoter). In some cases, the
inhibitor of ClpP is administered such that reduction of ClpP
expression is substantially liver-specific, and the amount
administered is effective for increasing insulin sensitivity of the
individual.
[0121] Liver-specificity can also be accomplished when the
inhibitor of ClpP that reduces ClpP expression and/or activity is
in an inactive form unless converted to an active form by a
liver-specific enzyme. For example, the inhibitor of ClpP can be
delivered in the form of a prodrug, which is then converted to an
active form in the liver, for example by a carboxylesterase such as
human liver carboxylesterase 1 (hCE1), a paraoxonase such as PON3,
an alkaline phosphatase, human valacyclovirase (VACVase), a
purine-nucleosidephosphorylase (PNP), and the like). See, e.g.,
Yang, et. al., Enzyme-mediated hydrolytic activation of prodrugs,
Acta PharmaceuticaSinica B 2011; 1(3):143-159.
[0122] Agents can be prepared as injectables, either as liquid
solutions or suspensions; solid forms suitable for solution in, or
suspension in, liquid vehicles prior to injection can also be
prepared. The preparation also can be emulsified or encapsulated in
liposomes or micro particles such as polylactide, polyglycolide, or
copolymer for enhanced adjuvant effect, as discussed above, Langer,
Science 249: 1527, 1990 and Hanes, Advanced Drug Delivery Reviews
28: 97-119, 1997. Subject agents can be administered in the form of
a depot injection or implant preparation which can be formulated in
such a manner as to permit a sustained or pulsatile release of the
active ingredient. The agents can be formulated as sterile,
substantially isotonic and are in full compliance with all Good
Manufacturing Practice (GMP) regulations of the U.S. Food and Drug
Administration.
[0123] Also within the scope of the disclosure are kits. For
example, in some cases a subject kit can include (i) a detection
reagent for detecting a ClpP activity level and/or expression level
(e.g., protein, mRNA), e.g., an anti-ClpP antibody, and (ii) a
control agent (e.g. a positive control agent that is known to
reduce a ClpP activity level and/or expression level, and/or a
negative control agent that is known not to reduce a ClpP activity
level and/or expression level) In some cases, a subject kit can
include instructions for use. Kits typically include a label
indicating the intended use of the contents of the kit. The term
label includes any writing, or recorded material supplied on or
with the kit, or which otherwise accompanies the kit.
Exemplary Non-Limiting Aspects of the Disclosure
[0124] Aspects, including embodiments, of the present subject
matter described above may be beneficial alone or in combination,
with one or more other aspects or embodiments. Without limiting the
foregoing description, certain non-limiting aspects of the
disclosure numbered 1-26 are provided below. As will be apparent to
those of skill in the art upon reading this disclosure, each of the
individually numbered aspects may be used or combined with any of
the preceding or following individually numbered aspects. This is
intended to provide support for all such combinations of aspects
and is not limited to combinations of aspects explicitly provided
below:
1. A method of identifying a candidate agent for treating obesity,
liver disease, and/or diabetes, the method comprising: [0125] (a)
contacting a mammalian cell with a test agent; [0126] (b) measuring
the expression level and/or activity level of ClpP in the mammalian
cell relative to a reference value following the contacting; [0127]
(c) determining that the test agent caused a decrease in the
expression level and/or activity level relative to the reference
value; and [0128] (d) identifying the test agent as a candidate
agent for treating obesity, liver disease, and/or diabetes. 2. The
method according to 1, wherein the mammalian cell is a mouse cell.
3. The method according to 1, wherein the mammalian cell is a human
cell. 4. The method according to any of 1-3, wherein the mammalian
cell is in vitro. 5. The method according to any of 1-3, wherein
the mammalian cell is ex: vivo. 6. The method according to any of
1-3, wherein the mammalian cell is in vim. 7. The method according
to any of 1-6, wherein the mammalian cell is a hepatocyte. 8. The
method according to 2, wherein the contacting comprises
administering the test agent to a mouse. 9. The method according to
8, further comprising, measuring an expression level and/or
activity level of ClpP in the mouse prior to the contacting of step
(a) in order to obtain the reference value. 10. The method
according to any of 1-9, further comprising, after step (d),
administering the identified candidate agent to an individual that
has obesity, liver disease, and/or diabetes. 11. The method
according to 10, wherein the individual is a mouse, a non-human
primate, or a human. 12. The method according to 10 or 11, further
comprising, after administering the identified candidate agent to
the individual, measuring one or more features of the individual
selected from: insulin sensitivity, blood glucose level, glucose
tolerance, body fat mass, an amount of fat tissue, an amount of
white adipose tissue; percent fat mass, body weight, visceral
adipose adipocyte size, plasma leptin level, growth hormone level,
basal energy expenditure, a level of phosphorylated AKT (p-AKT) in
muscles and/or fibroblasts, percent lean mass, mitochondrial number
in hepatocytes, mitochondrial mass in hepatocytes, mitochondrial
morphology in hepatocytes, fibroblast respiratory capacity,
fibroblast maximal oxygen consumption rate (OCR), and fibroblast
resistance to H.sub.2O.sub.2-induced cytotoxicity. 13. The method
according to any of 1-12, wherein in the test agent is a small
molecule or a polypeptide. 14. The method according to any of 1-13,
comprising measuring an expression level of ClpP, wherein the
expression level is an RNA expression level and the measuring
comprises the use of quantitative RT-PCR, a microarray, or RNA
sequencing. 15. The method according to any of 1-13, comprising
measuring an expression level of ClpP, wherein the expression level
is a protein expression level and the measuring comprises detecting
ClpP protein using an anti-ClpP antibody, mass spectrometry, and/or
an enzyme-linked immunosorbent assay (ELISA) assay. 16. The method
according to any of 1-13, comprising measuring a decrease in an
activity level of ClpP by measuring an increase in an amount of one
or more proteins selected from: TNF receptor-associated protein 1
(TRAP1), heat shock protein family A (Hsp70) member 9 (Grp75),
leucine rich pentatricopeptide repeat containing (LRPPRC),
caseinolytic mitochondrial matrix peptidase chaperone subunit
(ClpX), ornithine aminotransferase (OAT), and Ion peptidase 1
(LonP1). 17. The method according to any of 1-16, wherein the
method comprises screening a plurality of test agents to identify
one or more candidate agents for treating obesity, liver disease,
and/or diabetes. 18. A method of treating an individual with
obesity, liver disease, and/or diabetes, the method comprising:
[0129] administering an inhibitor of ClpP to the individual in an
amount effective for decreasing an amount of fat tissue in the
individual, preventing or reducing weight gain of the individual,
increasing insulin sensitivity of the individual, and/or increasing
glucose tolerance of the individual. 19. The method according to
18, wherein the inhibitor of ClpP is an RNAi agent or a gene
editing agent that specifically reduces expression of ClpP. 20. The
method according to 18 or 19, wherein the inhibitor of ClpP is
administered such that reduction of ClpP expression is
substantially liver-specific, and the amount administered is
effective for increasing insulin sensitivity of the individual. 21.
The method according to 18, wherein the inhibitor of ClpP is a
small molecule. 22. The method according to 21, wherein the small
molecule is a 13-Lactone. 23. The method according to 22, wherein
the .beta.-Lactone is
(3RS,4RS)-3-(pon-8-en-1-yl)-4-(2-(pyridin-3-yl)ethyl)oxetan-2-one
or a pharmaceutically acceptable salt thereof. 24. The method
according to any of 18-23, wherein the inhibitor of ClpP is
delivered directly to the individual's liver, and the amount
administered is effective for increasing insulin sensitivity of the
individual. 25. The method according to any of 18-24, wherein the
administering comprises local injection. 26. The method according
to any of 18-25, further comprising a step of measuring insulin
sensitivity of the individual.
[0130] It will be apparent to one of ordinary skill in the art that
various changes and modifications can be made without departing
from the spirit or scope of the invention.
EXPERIMENTAL
[0131] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the present invention, and are
not intended to limit the scope of the invention nor are they
intended to represent that the experiments below are all or the
only experiments performed. Efforts have been made to ensure
accuracy with respect to numbers used amounts, temperature, etc)
but some experimental errors and deviations should be accounted
for. Unless indicated otherwise, parts are parts by weight,
molecular weight is weight average molecular weight, temperature is
in degrees Centigrade, and pressure is at or near atmospheric.
[0132] All publications and patent applications cited in this
specification are herein incorporated by reference as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference.
[0133] The present invention has been described in terms of
particular embodiments found or proposed to comprise preferred
modes for the practice of the invention. It will be appreciated by
those of skill in the art that, in light of the present disclosure,
numerous modifications and changes can be made in the particular
embodiments exemplified without departing from the intended scope
of the invention. For example, due to codon redundancy, changes can
be made in the underlying DNA sequence without affecting the
protein sequence. Moreover, due to biological functional
equivalency considerations, changes can be made in protein
structure without affecting the biological action in kind or
amount. All such modifications are intended to be included within
the scope of the appended claims.
Materials and Methods
[0134] The following methods were utilized in the examples
described herein:
[0135] Animals. All mice were maintained on a 12-h light/dark cycle
in a pathogen-free animal facility. Mice were separately housed by
their genotypes for all metabolic studies. For high-fat diet
(HFD)-induced obesity studies, animals were fed a FWD (21% by
weight, 42% kcal from fat, 0.2% total cholesterol; TD.01064,
Harlan-Teklad) for specified periods. Anesthesia was induced with
isoflurane (survival procedures) or with avertin (terminal
procedures). All procedures were approved by the UCSF Animal
Research Committee and followed NIH guidelines. Animal sample sizes
for different assays were chosen based on previous literature.
Animals were grouped according to genotype; no randomization was
used. For all behavior tests, all animal Ins were blinded to
tester, Testers were not blinded for other assays.
[0136] Generation and maintenance of ClpP knockout (ClpP.sup.-/-)
and ClpP conditional knockout (ClpP-cKO) mice. Frozen ClpP.sup.+/-
sperm was recovered and ClpP.sup.+/- mice were generated.
ClpP.sup.-/- mice were back-crossed into a C57BL/6 genetic
background for three or more generations. Since Clp.sup.-/- mice
were infertile, ClpP.sup.+/- mice were bred with each other to
generate littermates with three different genotypes (WT,
ClpP.sup.+/-, ClpP.sup.-/-) and used to maintain the line.
[0137] Generation and maintenance of ClpP conditional knockout
(Clp-cKO) mice. A ClpP-cKO construct was made by flanking ClpP gene
with a long arm of homology (5' terminal, about 6 Kb) and a short
arm of homology (3' terminal, about 3 Kb). LoxP sites were added to
both ends of the region containing ClpP exons 1-3 for future
removal. A puromycin resistant gene flanked by F3 sites were
inserted right before the 3' terminal loxP site. A mouse ES cell
clone carrying the targeted ClpP allele was selected and used for
microinjection. Resulting chimeras were bred with Flp mice to
generate offspring carrying the targeted ClpP allele. Heterozygous
ClpP-cKO mice were then bred to generate homozygous ClpP-cKO mice.
The ClpP-cKO mice were generated on a C57BL/6 genetic background.
Homozygous ClpP-cKO mice were used to maintain the line.
[0138] Body composition analysis. Body composition of mice after a
4-h fast was analyzed under isoflurane anesthesia by dual-energy
X-ray absorptiometry (DEXA) with a PIXImus2 scanner (GE Healthcare
Lunar).
[0139] Food intake study. Mice were separately housed by their
genotypes. For each cage, food was weighted every day at 10:00 a.m.
for 4 days. The daily food intake for each cage was calculated by
subtracting the food weight at the end of a 24-h period from the
food weight at the starting time. The daily food intake is
normalized either animal number or body weight.
[0140] Fasting-refeeding study. Mice fasted for 24 h and then
returned to free feeding. The body weights of mice were measured
before and after the fasting. The food intake and body weight gain
were monitored after refeeding periods of 8 and 24 h.
[0141] Body temperature measurement. The core body temperatures of
mice were taken with a rodent rectal thermometer.
[0142] Measurement of blood glucose and plasma insulin. Blood
glucose was measured with a glucometer and glucose test strips
(Free Style Lite). Plasma insulin was quantified with an insulin
Elisa kit (Crystal Chem).
[0143] Glucose, insulin, and pyruvate tolerance tests, Glucose,
insulin, and pyruvate tolerance tests were performed by
intraperitoneal injection of glucose (2 g/kg), insulin (0.5
units/kg) or pyruvate (2 g/kg) after an overnight fast for glucose
and pyruvate or a 4-h fast for insulin. Blood glucose levels were
measured before injection and at different time points after
injection. To assess glucose-stimulated insulin release, mice
fasted overnight (for 16 h) and were then injected
intraperitoneally with glucose (2 g/kg). Blood samples were
collected from tail veins before injection and at different time
points after injection.
[0144] Behavior tests. The general neurological behavior profiles
of ClpP.sup.-/- mice were assessed by the grip strength, incline,
and tail suspense tests. The motor function of these mice was
determined by the rotarod test. Anxiety levels were determined by
elevated plus maze. Activity levels were studied by an open field
test. The learning and memory of these mice were examined using the
Morris water maze. The auditory functions were tested by prepulse
inhibition test.
[0145] Histological analysis. Tissues (liver, visceral adipose,
gastrocnemius muscle, interscapular brown adipose, and pancreas)
were fixed in 4% paraformaldehyde (pH 7.4) overnight, embedded in
paraffin, and serially sectioned at 7-8 .mu.m. Standard
haematoxylin and eosin staining was performed.
[0146] Adipocyte size measurement, Haematoxylin and eosin staining
of adipose tissue sections was performed and images were analyzed
with image J.
[0147] Electron microscopic analysis of liver sections, WT or
ClpP.sup.-/- mice were perfused with EM fixative. The liver tissues
were collected and fixed in the EM fixative for 2 days. The tissues
were processed and electron microscopy images were collected using
a JEOL, JEM-1230 transmission electron microscope. The
mitochondrial number, area, and roundness were measured by Image J
software at 13,600 magnifications.
[0148] Generation of adipocyte-specific ClpP-cKO mice.
Adipocyte-specific ClpP knockout mice were generated through
breeding ClpP-cKO mice with ap2-Cre mice. The Cre.sup.+ mice had
greatly reduced ClpP protein levels in adipose tissues and the
Cre-littermates were served as controls in metabolic assays.
[0149] Tail vein injection of AAV-CMV-Cre. AAV8.2-CMV-Cre and
control AAV8.2 were utilized. These viruses were injected into 3-5
month-old ClpP-cKO mice through tail vein at a dose of
5.times.10.sup.9 genome copies/gram (gc/gram) body weight. The body
weights of the injected mice were measured once every week. The
blood glucose and plasma insulin levels were measured before and 3
weeks after injection. The glucose tolerance test was performed on
these mice 4 weeks after injection. HFD was given to these mice 6
weeks after injection. The body weight gain in response to HFD was
followed and the glucose tolerance test was performed after two
weeks on HFD.
[0150] Western blot, Tissues or cells were lysated in low-detergent
buffer (50 mM Tris/HCl, pH 8.0, 150 call NaCl, 0.1% SDS, 0.5%
Nonidet P-40, 0.5% sodium deoxycholate) with a mixture of protease
inhibitors. Phosphatase inhibitors were also included in the lysate
buffer. The lysates were centrifuged at 13,000 rpm for 10 min to
remove the pellets. SDS-PAGE was performed with a WAGE his-tris
system from Invitrogen, following the vendor's protocol. A standard
western blot protocol was used with IRDye-labeled secondary
antibodies (Li-cor). A Li-cor image system was used to scan the
western blot images.
[0151] 2D fluorescence difference gel electrophoresis. Organs from
ClpP.sup.-/-, ClpP.sup.+/-, or WT mice were submitted for 2D
fluorescence difference gel electrophoresis analysis. The most
dramatically changed spots were cut from the gel for mass
spectrometry analysis to identify the proteins, which was done by
the same company.
[0152] Mouse fibroblast preparation. Mouse fibroblasts were
prepared from skin tissue collected from new-born pups. The tissue
was cut into small pieces and placed on a 100-mm cell culture dish
with the skirt side up. After 5-10 min, 10 ml of fibroblast medium
(DMEM with 10% FBS) was slowly added to the dishes. The implants
were cultured in a humidified 37.degree. C., 5% CO.sub.2 incubator.
Fibroblasts eventually proliferated after 7-10 day in culture.
Mouse fibroblasts were maintained in the same medium for multiple
passages.
[0153] Overexpression of ClpP in ClpP.sup.-/- fibroblasts.
Lentiviral overexpression vector for human ClpP was constructed
with pLenti7.3TOPO TA Cloning Kit from Life Technologies. The
lentiviruses were packed with ViraPower.TM. lentiviral packaging
mix from Life Technologies. Lentivirus carrying either empty vector
(control) or human ClpP cDNA were added to ClpP-/- fibroblasts and
incubated for 48 hr before further experiments.
[0154] shRNA knockdown of different genes in ClpP.sup.-/-
fibroblasts. Lentiviral shRNA constructs were obtained. The
lentiviruses were packed with ViraPower.TM. lentiviral packaging
mix from Life Technologies. Lentivirus carrying either empty vector
(control) or different shRNAs were added to ClpP.sup.-/-
fibroblasts and incubated for 48 hr before further experiments.
[0155] Cell viability assay. Mouse fibroblasts were plated on black
clear-bottomed 96-well plates at 10,000 cells per well. After
incubating for 24 h, the cells were cultured in OPTI-MEM medium
overnight. The cells were treated with H.sub.2O.sub.2 at various
doses for 4 h. Alamar blue reagent (Invitrogen) was then added to
the wells (1:10 ratio). After a 2-h incubation, the fluorescence
intensity was measured at 590 nm emission (560 nm excitation).
[0156] Seahorse OCR assay. Mouse fibroblasts were maintained in
fibroblast growth medium until confluence. The day before the
assay, fibroblasts were trypsinized and plated on a 96-well
Seahorse culture plate at 40000 cells per well. Before the assay,
the culture medium was changed to a CO.sub.2-independent medium
supplied by Seahorse Bioscience. The cells were then incubated at
37.degree. C. without CO.sub.2 for 0.5-1 h. The oxygen consumption
rate (OCR) assay was performed with an NT Cell mito-stress test kit
and XF Extracellular Flux Analyzers (Seahorse Bioscience) following
the vendor's protocol.
[0157] Growth hormone (Gil) and leptin level determination. The GH
and leptin levels in mouse plasma were determine by a mouse GH
ELISA kit and ultra sensitive mouse insulin ELISA kit from Crystal
Chem, Inc.
[0158] Antibodies. The antibodies used were anti-ClpP (rabbit,
Novus Biologicals), anti-ClpX (SDI, custom made), anti-Akt (rabbit,
Cell Signaling), anti-phospho-Akt (rabbit, Cell Signaling),
anti-Grp75 (rabbit, Cell Signaling), anti-TRAP1 (moue mAb-AbCam),
anti-LRPPRC (rabbit, Proteintech Group), anti-LonP (rabbit,
Sigma-Aldrich), anti-OAT (mouse, Abeam), anti-SDH2 (rabbit, aric
antibodies), anti-ATP6V1A (rabbit, Proteintech Group), anti-CPS1
(mouse, Lifespan), anti-Hsp70 (rabbit, Cell Signaling), anti-GAPDH
(mouse mAb, Millipore), anti Hsp60 (rabbit, Abeam), anti-VDAC
(mouse mAb, EMD Chemical), and anti-actin (rabbit,
Sigma-Aldrich).
Statistical analyses. Data are presented as mean.+-.SD unless
otherwise specified. All statistical analyses were done using Prism
6 software (GraphPad). Differences between means were assessed by
t-test, one-way ANOVA, or repeated measures ANOVA, followed by
Bonferroni or Tukey-Kramer post hoc tests. In all cases, P<0.05
was considered statistically significant.
[0159] Mammalian ClpXP, an ATPase complex consisting of the
catalytic subunit ClpP and the regulatory subunit ClpX, is a
mitochondrial protease with unclear physiological functions. The
data presented in the following examples show that ClpP knockout
(ClpP.sup.-/-) mice expended more energy and had reduced adipose
tissue and enhanced insulin sensitivity compared to wild type
controls. Drastic increases in mitochondrial chaperones were
detected in various organs of ClpP.sup.-/- mice, accompanied with
increased mitochondrial numbers. Eliminating ClpP increased
mitochondrial function and anti-stress capacity of cells by
elevating LRPPRC and/or TRAP1 levels (both of which are proteins
with roles in mitochondrial function), which were reversed by
knocking down either protein. Hepatic ClpP was responsible for
regulating insulin sensitivity, while adipocytic ClpP was not,
Thus, ClpP is a master regulator of mitochondrial function and
stress response, which in turn modulate energy homeostasis, lipid
storage, and insulin sensitivity. These data highlight ClpP as a
therapeutic target for treating obesity and diabetes.
Example 1: Absence of ClpP in Mice Reduced Adipose Tissue on a Chow
or High Fat Diet
[0160] To understand the physiological roles of mammalian ClpXP
protease, a ClpP knockout (ClpP.sup.-/-) mouse model was generated
through a gene-trapping strategy (FIG. 7, panel a). Quantitative
polymerase chain reaction (qPCR) (not shown) and western blotting
confirmed that the Clpp gene was efficiently deleted in different
organs (FIG. 7, panels h-i). ClpP.sup.-/- pups were born viable and
at normal Mendelian ratios. Morphological assessment of liver and
muscle revealed no abnormalities in 6-month-old homozygous
(ClpP.sup.-/-) or heterozygous (ClpP.sup.+/-) knockout mice (FIG.
8). There were no differences between ClpP.sup.-/- and WT
littermates in general neurological behavior, motor function
(rotarod and swim speed tests), anxiety (elevated plus maze test),
or learning and memory (Morris water maze test) (FIG. 9),
indicating a normal neurological profile.
[0161] However, both male and female adult ClpP.sup.-/- mice were
significantly smaller in weight and size than WT and ClpP.sup.+/-
littermates (FIG. 1, panels a-b). In contrast, all new-born pups
had similar body weights regardless of their ClpP genotypes (FIG.
1, panel c). In addition, growth hormone levels in 5-6-month-old
ClpP.sup.-/- mice were higher than in other groups of mice
(ClpP.sup.-/-, 8.54.+-.5.41 ng/ml; ClpP.sup.+/-, 2.48.+-.0.67
ng/ml; WT, 0.91.+-.0.53 ng/ml; n=3-5 mice for each genotype; one
way ANOVA, p=0.0053), so body weight differences in adults did not
stem from early developmental defects or growth hormone
deficiencies.
[0162] Clp.sup.-/- mice had dramatically lower body fat, as
measured by total body fat mass or body fat content (% fat mass)
(FIG. 1, panels d-e), than WT mice. Although ClpP.sup.+/- mice had
similar body weight to WT mice, they tended to have lower body fat
(FIG. 1, panels d-e). In contrast, the lean content (% lean mass)
of ClpP.sup.-/- mice was significantly higher than that of
ClpP.sup.+/- and WT mice (FIG. 1, panel g), although the lean mass
was significantly lower (FIG. 1, panel f). Interestingly,
quantification of interscapular brown adipose tissue, which is
critical in thermogenesis and beneficial in metabolic regulation,
showed a similar profile as lean tissue among three ClpP genotypes
(FIG. 1, panels h-i). Consistent with their lower body fat content,
visceral adipose from ClpP.sup.-/- mice had smaller adipocytes
compared to those from ClpP.sup.+/- and WT mice (FIG. 1, panel m,
and FIG. 10), in contrast, the morphology of brown adipose tissue
in ClpP.sup.-/-, ClpP.sup.+/-, and WT mice were similar (FIG. 11,
panel a). Thus, knocking out ClpP greatly reduces white adipose
tissue, which likely contributes to the lower body weights seen in
adult ClpP.sup.-/- mice.
[0163] Next, mice were fed a high-fat diet (HFD) for 2 months to
determine the effect of eliminating ClpP on HFD-induced obesity.
ClpP.sup.-/- mice were resistant to HFD-induced weight gain, and
ClpP.sup.+/- mice showed delayed weight gain within the first 10
days (FIG. 1, panel j, and FIG. 12, panel a). At 40 days on the
HFD, when body weight changes in all three groups had plateaued,
ClpP.sup.-/- mice showed only a small increase in fat mass and body
weight, while ClpP.sup.+/- and WT mice gained significantly more
(FIG. 1, panel k, FIG. 1, panel l). Thus, ClpP.sup.-/- mice are
resistant to HFD-induced obesity, HFD did not change the
differences in lean mass and content among various groups of mice
(comparing FIG. 1, panels f-g, with FIG. 12, panels c-d).
[0164] Also consistent with lower body fat content, ClpP.sup.-/-
mice had much lower levels of plasma leptin than other groups of
mice (ClpP.sup.-/-, 0.409.+-.0.224 ng/ml; ClpP.sup.+/-,
1.927.+-.1.928 minis; WT, 2.056.+-.2.022; n=11-12 mice at 5-6
months of age, one way ANOVA, p<0.05). Interestingly,
ClpP.sup.-/- mice were infertile, as observed in leptin depleted
animal models. While not intending to be bound by any particular
theory, it is possible that low leptin levels are responsible for
the reproductive defect in ClpP.sup.-/- mice.
Example 2: Absence of ClpP in Mice Altered the Enemy Expenditure
Profiles
[0165] Low leptin levels lead to feeding behavior and adipose
deposition in WT mice. Consistent with lower serum leptin levels,
the food intake normalized to body weight was significantly higher
in ClpP.sup.-/- mice than in WT and Clp.sup.+/- mice (FIG. 2, panel
b), although mice in all groups had similar food intake per animal
per day (FIG. 2, panel a). Thus, ClpP.sup.-/- mice gained less
weight and generated less fat while consuming more food, suggesting
that they either expended more energy or did not utilize nutrition
properly.
[0166] A fasting-refeeding approach was used to dissect these two
possibilities (FIG. 2, panel c). After 24 h of fasting,
ClpP.sup.-/- mice lost the same amount of weight as WT and
ClpP.sup.+/- mice (FIG. 2, panel d). Since ClpP.sup.-/- mice had
lower body weight, their percentage of body weight loss was
significantly higher than that of WT and ClpP.sup.+/- formates
(FIG. 2, panel e), suggesting that ClpP.sup.-/- mice expend more
energy. During the refeeding period, ClpP.sup.-/- mice consumed
significantly more food and gained a significantly higher
percentage of weight compared with WT and ClpP.sup.+/- littermates
(FIG. 2, panels f-g).
[0167] Higher energy consumption may be attributable to
hyperactivity or high thermogenesis.
[0168] The open field test showed similar activity levels among all
groups (FIG. 13). Surprisingly, the core body temperature was
significantly lower in ClpP.sup.-/- mice than in WT and
ClpP.sup.+/- mice (FIG. 2, panels h-i), suggesting that
ClpP.sup.-/- mice did not use more energy for thermogenesis. Thus,
it is likely that the ClpP.sup.-/- mice had higher basal energy
expenditure.
Example 3: Absence of ClpP in Mice Improved Insulin Sensitivity
[0169] Blood glucose levels and plasma insulin levels in
ClpP.sup.-/- mice were analyzed. Blood glucose levels were
significantly lower in ClpP.sup.-/- mice than in ClpP.sup.+/- and
WT mice (FIG. 3, panel a). Plasma insulin levels were also
significantly lower in ClpP.sup.-/- mice than in WT controls, with
those in ClpP.sup.+/- mice being in the middle, under both
free-feeding and fasting conditions (FIG. 3, panels b-c). The low
insulin levels were unlikely due to a pancreas deficit, based on
the morphological data (FIG. 11, panel b). Thus, ClpP.sup.-/- mice
maintained normal blood glucose levels in spite of having very low
insulin levels. After 2 months on HFD, blood glucose levels in WT
and ClpP.sup.+/- mice increased greatly. In contrast, ClpP.sup.-/-
mice maintained much lower blood glucose and insulin levels on HFD
(FIG. 3, panels d-e). Glucose and insulin tolerance tests further
revealed enhanced insulin sensitivity in ClpP.sup.-/- mice (FIG. 3,
panels f-h). A trend toward increased pyruvate tolerance in
ClpP.sup.-/- mice was detected, although not reaching significance
(FIG. 3, panel i). In support of increased insulin sensitivity,
higher p-AKT levels, a main component of the insulin signaling
pathway, were found in muscles and fibroblasts of ClpP.sup.-/- mice
(FIG. 3, panels j-l). Additionally, ClpP.sup.-/- fibroblasts were
more sensitive than WT fibroblasts to IGF-induced AKT activation in
culture (FIG. 3, panel m). These data strongly support the
conclusion that eliminating ClpP improves insulin sensitivity in
mice.
[0170] ClpP.sup.-/- mice were cross-bred with db/db mice (a model
of obesity, diabetes, and dyslipidemia) to determine whether and
how depleting ClpP affects the obese and diabetic phenotypes of
db/db mice. Knocking out ClpP led to a small but significant
decrease in body weight of db/dh mice (FIG. 14, panel a). ClpP
knockout also significantly decreased blood glucose levels in db/db
mice after 4 h of fasting (FIG. 3, panel n), although this effect
disappeared after 16 h of fasting probably due to the fact that the
glucose levels in db/db mice with WT ClpP after 16 h fasting were
already dropped to normal levels (FIG. 14, panels b-c). More
importantly, eliminating ClpP greatly improved the glucose
tolerance of db/db mice (FIG. 3, panel o).
Example 4: Absence of ClpP in Mice Upregulates Mitochondrial
Chaperon Levels
[0171] To identify downstream effectors of ClpP, two dimensional
(2D) fluorescence difference gel electrophoresis (2D-DICE) was
employed to compare the protein profiles of various organs from
mice with different ClpP genotypes (FIG. 15). Mass spectrometry was
then used to identify the most dramatically changed proteins in
different organs of ClpP.sup.-/- mice (FIG. 20). Validation by
western blots confirmed six potential ClpXP downstream effectors
(TRAP1, Grp75, LRPPRC, ClpX, OAT, and LonP; FIG. 21); their protein
levels increased greatly in multiple organs (FIG. 4, panels a-h and
FIG. 16), but their mRNA levels remained the same in most cases
(FIG. 22). Thus, ClpP likely controls the levels of these proteins
post-transcriptionally. Significantly increased protein levels were
also detected in embryonic day-19 pups (not shown), without a
decrease in body weight, suggesting that the accumulation of these
proteins is likely the cause, rather than a consequence, of the
observed phenotypes. Increased levels of the same proteins were
also detected in mouse fibroblasts (FIG. 4, panels i-j), Which were
subsequently reduced by overexpressing mouse ClpP (FIG. 4, panel
k-l), suggesting that ClpP specifically regulates these protein
levels in a cell-autonomous manner.
[0172] Strikingly, many of the potential ClpP downstream effectors
are related to mitochondrial protein homeostasis, including the
molecular chaperones ClpX (mitochondrial Hsp100), TRAP1
(mitochondrial Hsp90), and Grp75 (mitochondrial Hsp70) as well as
mitochondrial protease LonP (FIG. 4, panels a-h, FIG. 16). ClpP
knockout had no significant effect on the levels of mitochondrial
proteins VDAC and Hsp60 (FIG. 16). Furthermore, there were no
significant differences in many cytosol and endoplasmic reticulum
(ER) heat shock proteins, such as Bip and Hsp90 (FIG. 16). Thus,
either directly or indirectly, ClpP controls a specific group of
mitochondrial proteins, especially chaperons.
Example 5: Absence of ClpP Increased Mitochondrial Numbers and
Enhanced Mitochondrial Function and Anti-Stress Capacity
[0173] Mitochondrial number and function were analyzed in
ClpP.sup.-/- mice. Electron microscopic (EM) study of ClpP.sup.-/-
liver sections showed that depletion of ClpP increased
mitochondrial numbers and altered mitochondrial morphology in
hepatocytes (FIG. 5, panels a-b and FIG. 17, panels a-b),
Mitochondrial mass (measured by total mitochondrial area per field)
was also significantly increased in ClpP.sup.-/- hepatocytes (FIG.
5, panel c, and FIG. 17, panels a-b). The ClpP.sup.-/- mitochondria
had denser matrixes compared to those of WT (FIG. 5, panel a, and
FIG. 17, panels a-b), probably due to increased protein content in
ClpP.sup.-/- mitochondria. Although the size distribution of
mitochondria was similar between ClpP.sup.-/- and WT hepatocytes
(FIG. 17, panel c), there was a higher percentage of ClpP.sup.-/-
mitochondria with lower roundness (FIG. 17, panel d), suggesting
that more ClpP.sup.-/- mitochondria had elongated shapes.
[0174] To determine whether ClpP regulates mitochondrial function,
the respiratory capacity of fibroblasts from ClpP.sup.-/- and WT
mice were compared using a Seahorse XF analyzer. ClpP.sup.-/-
fibroblasts had a similar basal oxygen consumption rate (OCR) but a
significantly higher maximal OCR compared with WT fibroblasts (FIG.
5, panel d), indicating enhanced mitochondrial function in the
absence of ClpP. ClpP.sup.-/- fibroblasts also showed resistance to
H.sub.2O.sub.2-induced cytotoxicity compared with WT fibroblasts
(FIG. 5, panel e), suggesting increased anti-oxidative stress
capacity in the absence of ClpP. Overexpression of mouse ClpP in
ClpP.sup.-/- fibroblasts not only led to decreased levels of the
ClpP downstream effectors (FIG. 4, panels k-l) but also abolished
the enhancement of mitochondrial function and resistance to
H.sub.2O.sub.2-induced cytotoxicity observed in ClpP.sup.-/- cells
(FIG. 5, panels f-g).
Example 6: Mitochondrial Effectors TRAP1, Grp75, and LRPPRC
Mediated the Effects of ClpP Absence on Mitochondrial Function and
Anti-Stress Capacity
[0175] To determine whether and which of the mitochondrial
effectors regulated by ClpP mediated the enhanced mitochondrial
function seen in ClpP.sup.-/- fibroblasts, lentiviral shRNAs were
used to knock down each of them in ClpP.sup.-/- fibroblasts. This
was followed by functional assays (FIG. 18, panels a-d). Knocking
down TRAP1 or Grp75, but not LRPPRC and ClpX, abolished or lowered
the resistance of ClpP.sup.-/- fibroblasts to H.sub.2O.sub.2
cytotoxicity (FIG. 5, panels h-i and FIG. 18, panels e-f). Knocking
down either LRPPRC or TRAP1 abolished the enhanced respiratory
capacity of ClpP.sup.-/- cells, while lowering the level of ClpX or
Grp75 had no such an effect (FIG. 5, panel j). These data suggest
that ClpP regulates mitochondrial function by controlling its
effector levels, specifically TRAP1, Grp75, and LRPPRC. These
proteins may affect mitochondrial function via distinct pathways or
have synergistic effects on the same pathway.
Example 7: Hepatic ClpP was Responsible for Regulating Insulin
Sensitivity
[0176] Multiple tissues can affect insulin sensitivity through
different mechanisms. ClpP conditional knockout mice (ClpP-cKO)
were employed to further dissect the tissue specific mechanism(s)
underlying the phenotypes of ClpP.sup.-/- mice. In this mouse
model, LoxP sites were inserted to flank the mouse genomic region
containing ClpP exons 1-3 for future removal. To delete ClpP
specifically in the liver, AAV-CMV-Cre was injected through the
tail vein into ClpP-cKO mice. The ClpP levels in livers, detected
by immunostaining, were dramatically reduced in Cre-injected mice
compared to control AAV-injected mice (FIG. 6, panel a). At three
weeks after injection, the Cre-injected mice had a significantly
lower body weight gain than the control mice (FIG. 6, panel d),
although the total body weights were not significantly different
between the two groups of mice (FIG. 6, panels b-c). The blood
glucose levels of the Cre-injected mice were also significantly
lower than those of the control mice (FIG. 6, panel f), while no
difference in plasma insulin levels was detected (FIG. 6, panel e),
The enhanced insulin sensitivity of Cre-injected mice was revealed
by glucose tolerance test in mice on either chow diet or HFD for
two weeks (FIG. 6, panels g-h). Unlike ClpP.sup.-/- mice, liver
specific knockdown of ClpP did not affect FWD-induced body weight
gain (FIG. 6, panel i), These data suggest that hepatic ClpP levels
are responsible for regulating the insulin sensitivity observed in
ClpP.sup.-/- mice, without affecting the adipose content.
[0177] ClpP-cKO mice were also crossed with ap2-Cre mice to
establish adipocyte-specific ClpP-KO mice. The ClpP levels in
adipose tissues of ClpP-cKO/ap2-Cre mice were about a quarter of
those in control mice (FIG. 19, panels a-b). It was not clear
whether the residue ClpP levels were due to uncompleted deletion of
Clpp by apt-Cry; or contamination of other tissues or cells (such
as vascular or immune cells) in the collected adipose tissues. No
significant changes in body weights, blood glucose levels, and
glucose tolerance were detected in ClpP-ckO/ap2-Cre mice (FIG. 19,
panels c-e). The body weight gain and glucose tolerance of
ClpP-cKO/ap2-Cre mice on HFD were also similar to those of control
mice on the same diet (FIG. 19, panels f-g). Therefore, adipocytic
ClpP is unlikely attributable to the reduced adipose deposition and
enhanced insulin sensitivity seen in ClpP.sup.-/- mice.
Example 8: In-Vitro Analysis of Small Molecule ClpP Inhibitors in
Mouse Embryonic Fibroblast (MEF) Cells
[0178] Wild type mouse MEF cells were seeded at 200,000 cells per
well at 6 well plates the day before treatment. Cells were treated
with the small molecule ClpP inhibitors A2-32-01, AV167, and AV179
(described previously herein) at 10 .mu.M in OPTI-MEM twice a day
for 2 days (48 hr). For each treatment, the old medium will be
removed and fresh medium added to the wells. After 48 hr treatment,
the cells were harvested for western blot analysis or gPCR assay of
ClpP substrates.
[0179] As shown in FIGS. 24 and 25, treatment with ClpP inhibitor
A2-32-01 increased ClpP substrate protein levels in mouse WEF
cells, mimicking the phenotypes observed in ClpP knock out MEF and
in ClpP knock out mouse organs. As shown in FIG. 26, the mRNA level
of these genes in mouse MEF cells remained unchanged after A2-32-01
treatment, which excluded potential off-target effects of A2-32-01
through transcription regulation.
[0180] Based on these in vitro data, further in vivo testing of
A2-32-01 in high fat diet (HFD)-induced obesity and diabetes mouse
models was performed as described in Example 9 below.
Example 9: In-Vivo Analysis of Small Molecule ClpP Inhibitor
A2-32-01 in High Fat Diet (HFD)-Induced Obesity and Diabetes Mouse
Models
[0181] 7-9 month old WT female (20 mice), were fed with high fat
diet for 3 weeks. Body weight change and blood glucose/plasma
insulin levels in response to HFD were measured. At the beginning
of the 4.sup.th week, for treatment group, A2-32-01 was injected
peritoneally twice daily at 300 mg/kg for 8 days. For the control
group, corn oil (vehicle) was injected. The mice were maintained on
HFD. The body weight change was followed every day. At the 8.sup.th
day of A2-32-01 injection, blood glucose levels were measured, and
a glucose tolerance test was applied to measure insulin
sensitivity. Body weight data from the first seven days of in vivo
A2-32-01 treatment is provided in FIG. 28, which show a significant
decrease in body weight on day 2 through day 7 as compared to the
vehicle control group. A glucose tolerance test, which reflects
insulin sensitivity, was performed on day 8 and the data are
provided in FIG. 29, which show a significant improvement of
insulin sensitivity in A2-32-01-treated mice as compared to the
vehicle control mice.
Example 10: Continued In-Vivo Analysis of Small Molecule ClpP
Inhibitor A2-32-01 in High Fat Diet (HFD)-Induced Obesity and
Diabetes Mouse Models (Prophetic)
[0182] The in-vivo analysis of Example 9 is continued, with
collection of the blood and multiple organs (liver/muscle/brain) at
day 9 of A2-32-01 injection. Mitochondria are isolated from mouse
liver and ClpP activity is tested using peptide substrates, e.g.,
as described herein. Western blot analysis is performed to detect
ClpP substrate levels.
Example 11: Continued In-Vivo Analysis of Small Molecule ClpP
Inhibitor A2-32-01 in High Fat Diet (HEM-Induced Obesity and
Diabetes Mouse Models
[0183] The in-vivo analysis described in Examples 9 and 10 was
continued in accordance with the experimental protocol shown in
FIG. 30. As shown in FIG. 31 (left panel), the A2-32-01-treated
mice on HFD had significantly decreased body weight compared to
vehicle-treated mice on HFD. As shown in FIG. 31 (right panel),
glucose tolerance tests showed increased insulin sensitivity in
HFD-fed WT mice treated with A2-32-01 compared to those treated
with vehicle. ClpP activity was lower in liver mitochondrion
lysates from A2-32-01-treated mice compared to those from
vehicle-treated mice as shown in FIG. 32, left panel. As shown in
FIG. 32, right panel, the levels of tentative ClpP effectors Trap1,
ClpX, LRPPRC, LonP, and OAT), measured by western blot, were higher
in liver mitochondrion lysates from A2-32-01-treated mice compared
to those from vehicle-treated mice. Finally, FIG. 33 shows liver
morphology from vehicle or A2-32-01-treated WT mice on HFD,
indicating that A2-32-01 treatment lowered the lipid accumulation
in liver cells and restored normal morphology of liver cells in WT
mice on HID, as compared to vehicle-treated WT mice on HID.
Overall, these data demonstrate that treatment with the ClpP
inhibitor A2-32-01 in high fat diet (HFD)-fed wildtype (WT) mice
significantly lowers body weight, increases insulin sensitivity,
and restores normal liver morphology.
Example 12: In-Vivo Analysis of Small Molecule ClpP Inhibitor
A2-32-01 in db/db Mice
[0184] The following in-vivo analysis of db/db mice (a model of
obesity, diabetes, and dyslipidemia--see, e.g., Kobayashi et al.
Metabolism, 2000 January; 49(1):22-31.) was conducted according to
the experimental scheme set forth in FIG. 34, panel A. As shown in
FIG. 34, panel B, the A2-32-01-treated db/db mice had significantly
decreased body weight compared to vehicle-treated dh/db mice at the
end of the treatment. In addition, glucose tolerance tests showed
increased insulin sensitivity in db/db mice treated with A2-32-01
compared with those treated with vehicle (FIG. 35, panel C).
Fasting blood glucose levels in db; db mice treated with A2-32-01
were significantly lower than those treated with vehicle as shown
in FIG. 35, panel D. ClpP activity was lower in liver mitochondria
lysates from db/db mice treated with A2-32-01 compared to those
treated with vehicle as shown in FIG. 35, panel E. FIG. 36, panels
F-I, show liver morphology from vehicle or A2-32-01-treated db/db
mice, indicating that A2-32-01 treatment lowered the lipid
accumulation in liver cells and restored normal morphology of liver
cells in db/db mice, as compared to vehicle-treated db/db mice.
Overall, these data demonstrate that treatment with ClpP inhibitor
A2-32-01 in db/db mice significantly lowers body weight, increases
insulin sensitivity, and restores normal liver morphology.
Sequence CWU 1
1
71277PRTHomo Sapiens 1Met Trp Pro Gly Ile Leu Val Gly Gly Ala Arg
Val Ala Ser Cys Arg1 5 10 15Tyr Pro Ala Leu Gly Pro Arg Leu Ala Ala
His Phe Pro Ala Gln Arg 20 25 30Pro Pro Gln Arg Thr Leu Gln Asn Gly
Leu Ala Leu Gln Arg Cys Leu 35 40 45His Ala Thr Ala Thr Arg Ala Leu
Pro Leu Ile Pro Ile Val Val Glu 50 55 60Gln Thr Gly Arg Gly Glu Arg
Ala Tyr Asp Ile Tyr Ser Arg Leu Leu65 70 75 80Arg Glu Arg Ile Val
Cys Val Met Gly Pro Ile Asp Asp Ser Val Ala 85 90 95Ser Leu Val Ile
Ala Gln Leu Leu Phe Leu Gln Ser Glu Ser Asn Lys 100 105 110Lys Pro
Ile His Met Tyr Ile Asn Ser Pro Gly Gly Val Val Thr Ala 115 120
125Gly Leu Ala Ile Tyr Asp Thr Met Gln Tyr Ile Leu Asn Pro Ile Cys
130 135 140Thr Trp Cys Val Gly Gln Ala Ala Ser Met Gly Ser Leu Leu
Leu Ala145 150 155 160Ala Gly Thr Pro Gly Met Arg His Ser Leu Pro
Asn Ser Arg Ile Met 165 170 175Ile His Gln Pro Ser Gly Gly Ala Arg
Gly Gln Ala Thr Asp Ile Ala 180 185 190Ile Gln Ala Glu Glu Ile Met
Lys Leu Lys Lys Gln Leu Tyr Asn Ile 195 200 205Tyr Ala Lys His Thr
Lys Gln Ser Leu Gln Val Ile Glu Ser Ala Met 210 215 220Glu Arg Asp
Arg Tyr Met Ser Pro Met Glu Ala Gln Glu Phe Gly Ile225 230 235
240Leu Asp Lys Val Leu Val His Pro Pro Gln Asp Gly Glu Asp Glu Pro
245 250 255Thr Leu Val Gln Lys Glu Pro Val Glu Ala Ala Pro Ala Ala
Glu Pro 260 265 270Val Pro Ala Ser Thr 2752272PRTMus musculus 2Met
Trp Pro Arg Val Leu Leu Gly Glu Ala Arg Val Ala Val Asp Gly1 5 10
15Cys Arg Ala Leu Leu Ser Arg Leu Ala Val His Phe Ser Pro Pro Trp
20 25 30Thr Ala Val Ser Cys Ser Pro Leu Arg Arg Ser Leu His Gly Thr
Ala 35 40 45Thr Arg Ala Phe Pro Leu Ile Pro Ile Val Val Glu Gln Thr
Gly Arg 50 55 60Gly Glu Arg Ala Tyr Asp Ile Tyr Ser Arg Leu Leu Arg
Glu Arg Ile65 70 75 80Val Cys Val Met Gly Pro Ile Asp Asp Ser Val
Ala Ser Leu Val Ile 85 90 95Ala Gln Leu Leu Phe Leu Gln Ser Glu Ser
Asn Lys Lys Pro Ile His 100 105 110Met Tyr Ile Asn Ser Pro Gly Gly
Val Val Thr Ala Gly Leu Ala Ile 115 120 125Tyr Asp Thr Met Gln Tyr
Ile Leu Asn Pro Ile Cys Thr Trp Cys Val 130 135 140Gly Gln Ala Ala
Ser Met Gly Ser Leu Leu Leu Ala Ala Gly Ser Pro145 150 155 160Gly
Met Arg His Ser Leu Pro Asn Ser Arg Ile Met Ile His Gln Pro 165 170
175Ser Gly Gly Ala Arg Gly Gln Ala Thr Asp Ile Ala Ile Gln Ala Glu
180 185 190Glu Ile Met Lys Leu Lys Lys Gln Leu Tyr Asn Ile Tyr Ala
Lys His 195 200 205Thr Lys Gln Ser Leu Gln Val Ile Glu Ser Ala Met
Glu Arg Asp Arg 210 215 220Tyr Met Ser Pro Met Glu Ala Gln Glu Phe
Gly Ile Leu Asp Lys Val225 230 235 240Leu Val His Pro Pro Gln Asp
Gly Glu Asp Glu Pro Glu Leu Val Gln 245 250 255Lys Glu Thr Ala Thr
Ala Pro Thr Asp Pro Pro Ala Pro Thr Ser Thr 260 265 27031194DNAHomo
Sapiens 3ccttaatggc gcccgcccag actcctggaa gtgagcggcc tagcgagcga
gctcccaggc 60gcaaagcacg ccggaagctg tagttccgcc atcggacgga agccgaccgg
ggcgtgcgga 120gggatgtggc ccggaatatt ggtagggggg gcccgggtgg
cgtcatgcag gtaccccgcg 180ctggggcctc gcctcgccgc tcactttcca
gcgcagcggc cgccgcagcg gacactccag 240aacggcctgg ccctgcagcg
gtgcctgcac gcgacggcga cccgggctct cccgctcatt 300cccatcgtgg
tggagcagac gggtcgcggc gagcgcgcct atgacatcta ctcgcggctg
360ctgcgggagc gcatcgtgtg cgtcatgggc ccgatcgatg acagcgttgc
cagccttgtt 420atcgcacagc tcctcttcct gcaatccgag agcaacaaga
agcccatcca catgtacatc 480aacagccctg gtggtgtggt gaccgcgggc
ctggccatct acgacacgat gcagtacatc 540ctcaacccga tctgcacctg
gtgcgtgggc caggccgcca gcatgggctc cctgcttctc 600gccgccggca
ccccaggcat gcgccactcg ctccccaact cccgtatcat gatccaccag
660ccctcaggag gcgcccgggg ccaagccaca gacattgcca tccaggcaga
ggagatcatg 720aagctcaaga agcagctcta taacatctac gccaagcaca
ccaaacagag cctgcaggtg 780atcgagtccg ccatggagag ggaccgctac
atgagcccca tggaggccca ggagtttggc 840atcttagaca aggttctggt
ccaccctccc caggacggtg aggatgagcc cacgctggtg 900cagaaggagc
ctgtagaagc agcgccggca gcagaacctg tcccagctag cacctgagag
960ctgggcctcc tctccagaat catgtggagg ggccagaggc ctgccagacc
cccagctggg 1020ccctgctcac cccttgttgc tgggcttgga ggggcctctt
gaggaacttt taatttgcag 1080gggtgcccgc tatggacggg gcattccagc
tgagacactg tgattttaaa ttaaatcttt 1140gtggtctttg caaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaa 119441207DNAMus musculus
4agtgactccc gcaaagcacg ccgggtgttg tagttccgga agccaagccg gagtgcgcgt
60cgtcatgtgg cccagagtgc tgctggggga ggcccgggtg gctgtggacg gatgtcgcgc
120tctgttgtct cgccttgccg tgcatttctc cccgccatgg actgctgtga
gctgctcacc 180cctgcggagg agcctgcatg gaactgcgac gcgagctttc
ccgctcatcc ccatagtggt 240ggagcagacg ggtcgaggcg agcgcgctta
tgacatatac tcgaggctgt tgcgggaacg 300catcgtgtgc gtcatgggcc
cgattgacga cagtgtggcc agtctggtca ttgcccagct 360gttgttctta
cagtctgaaa gcaacaagaa gcccattcat atgtatatca acagcccagg
420tggtgtggta actgcgggcc tggccatcta cgacacaatg cagtacatcc
tgaaccccat 480ctgcacgtgg tgtgttggac aggctgccag catgggctcc
ctgctcctcg ctgctggcag 540cccgggcatg cgccattcac tgcccaattc
cagaatcatg atccaccagc cctctggagg 600agccaggggc caagccacag
acatcgccat ccaggcagag gaaatcatga agctgaaaaa 660gcagctatac
aacatctacg ccaaacacac caagcagagc ctacaggtga tcgagtcagc
720aatggagagg gaccgctaca tgagccccat ggaggcccaa gagtttggca
tcttggacaa 780ggtcttggtc cacccacctc aggacgggga ggatgagcca
gaactggtac agaaggagac 840tgccacagcg ccgacggatc ctcctgcccc
gacaagcacc taaggagtgg agaccagact 900gaaacttcct ctgctgggcc
caagaacaac ccctagagga gatgtggatt gaggttgccc 960tcagagcagg
gcagactgcc tgagacactg tgatttaaat taaatctttg tagtctttgt
1020cccatgtctg aagcaccttc cattacttct ccaagacagc aggcctcctt
caccttgaca 1080aaccacttca gtaagcaaac cctggctctc ctggaactaa
accaatctag cctcagactc 1140aggtacccac ctgcctcacc tcctgagtgc
taggattaaa ggtgtacacc accacacctg 1200acttcaa 1207521DNAArtificial
sequenceSynthetic primer 5gcccatccac atgtacatca a
21621DNAArtificial sequenceSynthetic primer 6cacgatgcag tacatcctca
a 21721DNAArtificial sequenceSynthetic primer 7gctcaagaag
cagctctata a 21
* * * * *