NZ792168A - Mitoketoscins: mitochondrial-based therapeutics targeting ketone metabolism in cancer cells - Google Patents
Mitoketoscins: mitochondrial-based therapeutics targeting ketone metabolism in cancer cellsInfo
- Publication number
- NZ792168A NZ792168A NZ792168A NZ79216818A NZ792168A NZ 792168 A NZ792168 A NZ 792168A NZ 792168 A NZ792168 A NZ 792168A NZ 79216818 A NZ79216818 A NZ 79216818A NZ 792168 A NZ792168 A NZ 792168A
- Authority
- NZ
- New Zealand
- Prior art keywords
- mitoketoscin
- alkenes
- cyclic
- alkanes
- compounds
- Prior art date
Links
- 201000011510 cancer Diseases 0.000 title claims abstract 11
- 230000002438 mitochondrial Effects 0.000 title claims abstract 6
- 150000002576 ketones Chemical class 0.000 title claims 10
- 239000003814 drug Substances 0.000 title claims 5
- 230000036740 Metabolism Effects 0.000 title 1
- 230000004060 metabolic process Effects 0.000 title 1
- 230000035786 metabolism Effects 0.000 title 1
- 230000001225 therapeutic Effects 0.000 title 1
- 150000001875 compounds Chemical class 0.000 claims abstract 19
- 101710037523 ACAT1 Proteins 0.000 claims abstract 5
- 101710031075 OXCT1 Proteins 0.000 claims abstract 5
- 102100001061 OXCT1 Human genes 0.000 claims abstract 5
- 101700025022 SOAT1 Proteins 0.000 claims abstract 5
- 230000003712 anti-aging Effects 0.000 claims abstract 4
- 230000001093 anti-cancer Effects 0.000 claims abstract 4
- 230000002401 inhibitory effect Effects 0.000 claims abstract 2
- -1 cyclic alkanes Chemical class 0.000 claims 21
- WPYMKLBDIGXBTP-UHFFFAOYSA-N Benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 claims 14
- 229910052731 fluorine Inorganic materials 0.000 claims 11
- 239000011737 fluorine Substances 0.000 claims 11
- 241001120493 Arene Species 0.000 claims 8
- 150000001336 alkenes Chemical class 0.000 claims 8
- 150000001412 amines Chemical class 0.000 claims 8
- 229910052801 chlorine Inorganic materials 0.000 claims 8
- 239000000460 chlorine Substances 0.000 claims 8
- ZAMOUSCENKQFHK-UHFFFAOYSA-N chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 claims 8
- 150000002390 heteroarenes Chemical class 0.000 claims 8
- PNDPGZBMCMUPRI-UHFFFAOYSA-N iodine Chemical compound II PNDPGZBMCMUPRI-UHFFFAOYSA-N 0.000 claims 8
- 229910052740 iodine Inorganic materials 0.000 claims 8
- 239000011630 iodine Substances 0.000 claims 8
- 239000005711 Benzoic acid Substances 0.000 claims 7
- 150000001299 aldehydes Chemical class 0.000 claims 7
- 150000001335 aliphatic alkanes Chemical class 0.000 claims 7
- 150000001408 amides Chemical class 0.000 claims 7
- 235000010233 benzoic acid Nutrition 0.000 claims 7
- 238000004166 bioassay Methods 0.000 claims 7
- GDTBXPJZTBHREO-UHFFFAOYSA-N bromine Substances BrBr GDTBXPJZTBHREO-UHFFFAOYSA-N 0.000 claims 7
- WKBOTKDWSSQWDR-UHFFFAOYSA-N bromine atom Chemical compound [Br] WKBOTKDWSSQWDR-UHFFFAOYSA-N 0.000 claims 7
- 238000007877 drug screening Methods 0.000 claims 7
- 150000002148 esters Chemical class 0.000 claims 7
- 150000002170 ethers Chemical class 0.000 claims 7
- 239000001257 hydrogen Substances 0.000 claims 7
- 229910052739 hydrogen Inorganic materials 0.000 claims 7
- 125000002950 monocyclic group Chemical group 0.000 claims 7
- 230000035812 respiration Effects 0.000 claims 7
- 230000029058 respiratory gaseous exchange Effects 0.000 claims 7
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Natural products OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 claims 6
- 150000001345 alkine derivatives Chemical class 0.000 claims 6
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims 6
- 150000001735 carboxylic acids Chemical class 0.000 claims 6
- YCKRFDGAMUMZLT-UHFFFAOYSA-N fluorine atom Chemical compound [F] YCKRFDGAMUMZLT-UHFFFAOYSA-N 0.000 claims 6
- 150000002431 hydrogen Chemical class 0.000 claims 6
- 150000002989 phenols Chemical class 0.000 claims 6
- 210000000130 stem cell Anatomy 0.000 claims 6
- 125000001153 fluoro group Chemical group F* 0.000 claims 5
- 102100019388 SOAT1 Human genes 0.000 claims 4
- 238000004519 manufacturing process Methods 0.000 claims 4
- 239000008194 pharmaceutical composition Substances 0.000 claims 4
- 239000002253 acid Substances 0.000 claims 3
- 125000004122 cyclic group Chemical group 0.000 claims 3
- 101710037511 ACAT2 Proteins 0.000 claims 2
- 102100001062 OXCT2 Human genes 0.000 claims 2
- 101710031074 OXCT2 Proteins 0.000 claims 2
- 102100019390 SOAT2 Human genes 0.000 claims 2
- 101700032213 SOAT2 Proteins 0.000 claims 2
- 150000007513 acids Chemical class 0.000 claims 2
- 239000004480 active ingredient Substances 0.000 claims 2
- 230000000845 anti-microbial Effects 0.000 claims 2
- 239000002246 antineoplastic agent Substances 0.000 claims 2
- 239000005445 natural product Substances 0.000 claims 2
- 230000002165 photosensitisation Effects 0.000 claims 2
- 230000000637 radiosensitizating Effects 0.000 claims 2
- 230000000241 respiratory Effects 0.000 claims 2
- 238000010200 validation analysis Methods 0.000 claims 2
- RDHQFKQIGNGIED-MRVPVSSYSA-N Acetylcarnitine Chemical compound CC(=O)O[C@H](CC([O-])=O)C[N+](C)(C)C RDHQFKQIGNGIED-MRVPVSSYSA-N 0.000 claims 1
- 229960001009 Acetylcarnitine Drugs 0.000 claims 1
- 206010006187 Breast cancer Diseases 0.000 claims 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N D-Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims 1
- 102000004190 Enzymes Human genes 0.000 claims 1
- 108090000790 Enzymes Proteins 0.000 claims 1
- 229940076788 Pyruvate Drugs 0.000 claims 1
- 229940035295 Ting Drugs 0.000 claims 1
- 238000004458 analytical method Methods 0.000 claims 1
- 150000004945 aromatic hydrocarbons Chemical class 0.000 claims 1
- 235000020934 caloric restriction Nutrition 0.000 claims 1
- 239000000969 carrier Substances 0.000 claims 1
- 230000012292 cell migration Effects 0.000 claims 1
- 210000004027 cells Anatomy 0.000 claims 1
- 235000014113 dietary fatty acids Nutrition 0.000 claims 1
- 201000009910 diseases by infectious agent Diseases 0.000 claims 1
- 239000003937 drug carrier Substances 0.000 claims 1
- 229940079593 drugs Drugs 0.000 claims 1
- 230000000694 effects Effects 0.000 claims 1
- 239000000194 fatty acid Substances 0.000 claims 1
- 150000004665 fatty acids Chemical class 0.000 claims 1
- 230000004907 flux Effects 0.000 claims 1
- 239000008103 glucose Substances 0.000 claims 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims 1
- 238000000338 in vitro Methods 0.000 claims 1
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 claims 1
- 230000002503 metabolic Effects 0.000 claims 1
- 230000000813 microbial Effects 0.000 claims 1
- 230000036284 oxygen consumption Effects 0.000 claims 1
- 230000020477 pH reduction Effects 0.000 claims 1
- LCTONWCANYUPML-UHFFFAOYSA-M pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 claims 1
- 239000000758 substrate Substances 0.000 claims 1
- 238000011179 visual inspection Methods 0.000 claims 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N β-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims 1
- 206010060945 Bacterial infection Diseases 0.000 abstract 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 abstract 2
- 230000001717 pathogenic Effects 0.000 abstract 2
- 102100008820 ACAT1 Human genes 0.000 abstract 1
- 230000002407 ATP formation Effects 0.000 abstract 1
Abstract
The present disclosure relates to compounds that bind to at least one of ACAT1/2 and OXCT1/2 and inhibit mitochondrial ATP production, referred to herein as mitoketoscins. Methods of screening compounds for mitochondrial inhibition and anti-cancer properties are disclosed. Also described are methods of using mitoketoscins to prevent or treat cancer, bacterial infections, and pathogenic yeast, as well as methods of using mitoketoscins to provide anti-aging benefits. Specific mitoketoscin compounds are also disclosed. of using mitoketoscins to prevent or treat cancer, bacterial infections, and pathogenic yeast, as well as methods of using mitoketoscins to provide anti-aging benefits. Specific mitoketoscin compounds are also disclosed.
Description
TOSCINS: MITOCHONDRIAL-BASED THERAPEUTICS TARGETING
KETONE METABOLISM IN CANCER CELLS
FIELD
The present disclosure relates to mitoketoscins – non-carcinogenic compounds that
bind to at least one of ACAT1/2 and OXCT1/2 and inhibit ondrial ATP production, as well
as methods for identifying mitoketoscins, methods of using the inhibitors to target cancer stem
cells, to target ia and pathogenic yeast, and to provide ging ts, and
pharmaceutical compositions for treating cancer, bacterial infections, yeast infections, and aging,
containing one or more mitoketoscins as the active ingredient.
BACKGROUND
Researchers have struggled to develop new anti-cancer treatments. Conventional
cancer therapies (e.g. irradiation, ting agents such as cyclophosphamide, and antimetabolites
such as rouracil) have attempted to selectively detect and eradicate fast-growing
cancer cells by interfering with cellular mechanisms involved in cell growth and DNA replication.
Other cancer therapies have used immunotherapies that selectively bind mutant tumor antigens on
fast-growing cancer cells (e.g., monoclonal antibodies). Unfortunately, tumors often recur
following these therapies at the same or ent site(s), indicating that not all cancer cells have
been eradicated. Relapse may be due to insufficient chemotherapeutic dosage and/or emergence
of cancer clones resistant to therapy. Hence, novel cancer treatment strategies are needed.
Ketones (3-hydroxybutyrate, acetoacetate and acetone) are high-energy
mitochondrial fuels; they are naturally generated by hepatocytes during periods of caloric
restriction, fasting, and/or starvation. During nutrient ation, ketone bodies secreted into the
blood are then directed towards the brain, where neurons convert them back into -CoA so
they may be effectively lized as an energy source. The two most critical neuronal enzymes
for this ketone re-utilization process are OXCT1/2 and ACAT1/2, as they are directly involved in
the conversion of ketone bodies into Acetyl-CoA. Martinez-Outschoorn et al., Nat Rev Clin Oncol
2017; 14(1):11-31.
The inventors showed that a r “ketone-shuttle” also exists in human tumors,
y nic cancer-associated fibroblasts (CAFs) locally e ketone bodies for reutilization
by mitochondria in adjacent human breast cancer cells. Martinez-Outschoorn, et al.,
Cell Cycle 2012; 11(21):3956-63. In further support of this “metabolic-coupling” hypothesis, the
inventors found that recombinant over-expression of ACAT1/2 or OXCT1/2 in MDA-MB-231
breast cancer cells was indeed sufficient to promote tumor growth and lung metastasis. These data
provide genetic ce that ketone body re-utilization may help drive tumor progression and
metastasis.
SUMMARY
In view of the foregoing background, it appears that the enzymes ACAT1/2 and
OXCT1/2 may be bona-fide metabolic oncogenes. It is therefore an object of this disclosure to
demonstrate that ketone re-utilization plays a critical role in the ation and maintenance of
many cancers. It is also an object of this disclosure to present methods for identifying
mitoketoscins, non-carcinogenic compounds that bind to at least one of ACAT1/2 and OXCT1/2
and inhibit mitochondrial ATP production. It is also an object of this disclosure to identify
mitoketoscins having anti-cancer and antibiotic properties. It is also an object of this disclosure to
identify mitoketoscins having anti-aging properties. It is also an object of this disclosure to
mitoketoscins that function as radiosensitizers and photosensitizers. The term "mitoketoscin"
broadly refers to non-carcinogenic compounds that bind to at least one of ACATI/2 and OXCTI/2
and inhibit mitochondrial ATP production. These compounds therefore are ed to target the
ondrial enzymes sible for ketone re-utilization and that have anti-cancer and
antibiotic properties. These compounds bind to either or both active tic sites of OXCTI/2
and ACATI/2 to inhibit mitochondrial function. The t disclosure further relates to methods
of identifying mitoketoscins, methods of making such mitoketoscins, and methods of using
mitoketoscins for therapeutic purposes.
Given their mitochondrial inhibition properties, mitoketoscins may similarly be
used to target bacteria and enic yeast, provide anti-aging benefits, function as
ensitizers and/or photo-sensitizers, sensitize bulk cancer cells and cancer stem cells to
chemotherapeutic agents, pharmaceuticals, and/or other natural substances.
Mitoketoscins may be identified through a convergent approach of virtual highthroughput
in silico screening followed by in vitro validation for mitochondrial inhibition.
Mitoketoscins may be rapidly developed by combining in silico drug design with phenotypic drug
screening.
In a first aspect of the invention, there is provided a method of identifying a mitoketoscin
using virtual high-throughput screening and phenotypic drug screening, the method comprising;
identifying a compound that targets a mitochondrial ketone re-utilization enzyme using
virtual hroughput screening; and
testing the compound for mitochondrial inhibition activity.
In a second aspect of the invention, there is provided a mitoketoscin sing the
general formula:
, wherein R may be selected from the group consisting of chlorine,
bromine, , carboxyl, alkanes, cyclic alkanes, alkenes, cyclic alkenes, alkynes, ketones,
aldehydes, carboxylic acids, ethers, esters, amines, amides, clic or clic arene,
arenes, phenols and benzoic acid.
In a third aspect of the invention, there is provided the use of a mitoketoscin in the
manufacture of a medicament for treating cancer, wherein the mitoketoscin comprises the
general formula:
, wherein R may be selected from the group consisting of
hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkanes, cyclic alkanes, alkenes,
cyclic alkenes, alkynes, ketones, aldehydes, carboxylic acids, ethers, esters, amines, ,
monocyclic or polycyclic arene, heteroarenes, phenols and benzoic acid.
In a fourth aspect of the invention, there is provided the use of a mitoketoscin in
the manufacture of a ment for treating a microbial infection, wherein the toscin
comprises the general formula:
, wherein R may be selected from the group consisting of
hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkanes, cyclic alkanes, alkenes,
cyclic alkenes, alkynes, ketones, aldehydes, carboxylic acids, ethers, esters, amines, amides,
monocyclic or polycyclic arene, heteroarenes, phenols and benzoic acid.
In a fifth aspect of the invention, there is provided the use of a mitoketoscin in the
manufacture of a medicament for treating an age-related ion, wherein the mitoketoscin
comprises the general formula:
, wherein R may be selected from the group consisting of
hydrogen, ne, ne, e, iodine, carboxyl, alkanes, cyclic s, s,
cyclic alkenes, alkynes, ketones, aldehydes, carboxylic acids, ethers, esters, amines, amides,
monocyclic or polycyclic arene, heteroarenes, phenols and benzoic acid.
In a sixth aspect of the invention, there is provided a ceutical composition
comprising, as an active ingredient, a mitoketoscin comprising the general formula:
, wherein R may be selected from the group consisting of
hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkanes, cyclic alkanes, alkenes,
cyclic alkenes, alkynes, ketones, aldehydes, carboxylic acids, ethers, esters, amines, ,
monocyclic or polycyclic arene, heteroarenes, phenols, benzoic acid; and
a pharmaceutically acceptable carrier.
In a seventh aspect of the ion, there is provided a mitoketoscin comprising
the general formula:
, wherein R may be selected from the group consisting of hydrogen,
, chlorine, bromine, iodine, carboxyl, alkanes, cyclic alkanes, alkenes, cyclic alkenes,
alkynes, ketones, aldehydes, carboxylic acids, ethers, esters, , amides, monocyclic or
polycyclic arene, heteroarenes, phenols and c acid.
In an eighth aspect of the ion, there is provided the use of a mitoketoscin in
the manufacture of a medicament for treating cancer, wherein the mitoketoscin comprises the
general formula:
, n R may be selected from the group consisting of hydrogen,
fluorine, chlorine, bromine, iodine, carboxyl, alkanes, cyclic alkanes, s, cyclic alkenes,
alkynes, ketones, aldehydes, carboxylic acids, ethers, esters, , amides, clic or
polycyclic arene, heteroarenes, s and benzoic acid.
In a ninth aspect of the invention, there is provided a pharmaceutical composition
comprising, as an active ingredient, a mitoketoscin comprising the general formula:
, wherein R may be selected from the group consisting of hydrogen,
fluorine, chlorine, bromine, iodine, carboxyl, alkanes, cyclic alkanes, alkenes, cyclic alkenes,
alkynes, ketones, aldehydes, carboxylic acids, ethers, esters, amines, amides, monocyclic or
polycyclic arene, heteroarenes, phenols, benzoic acid; and
a pharmaceutically acceptable carrier.
In one embodiment of the present invention, the mitoketoscin comprises the
l formula:
or a ceutically acceptable salt thereof.
In another embodiment, wherein the mitoketoscin comprises the general formula:
or a pharmaceutically acceptable salt thereof, wherein Z is defined as
ethylpiperidine or ethylpyrrolidine
In a n embodiment, the mitoketoscin comprises the general formula:
or a ceutically acceptable salt thereof, wherein Z is d as
ethylpiperidine or ethylpyrrolidine.
In another embodiment, the mitoketoscin comprises the general formula:
or a pharmaceutically acceptable salt thereof.
In one embodiment, the toscin comprises the general formula:
or a pharmaceutically acceptable salt thereof.
In an embodiment, the mitoketoscin comprises the general pharmacophore:
or a pharmaceutically acceptable salt thereof.
In one embodiment, the mitoketoscin ses the general pharmacophore:
or a pharmaceutically acceptable salt thereof.
In another embodiment, the mitoketoscin comprises the general formula:
or a pharmaceutically acceptable salt thereof.
In yet another embodiment, the mitoketoscin comprises the l formula:
or a ceutically acceptable salt thereof.
In a n embodiment, the mitoketoscin ses the general formula:
or a pharmaceutically able salt thereof.
In one embodiment, the mitoketoscin comprises the general formula:
or a pharmaceutically acceptable salt thereof.
In another embodiment, the mitoketoscin comprises the general formula:
or a pharmaceutically acceptable salt thereof.
In one embodiment, the mitoketoscin comprises the general formula:
or a pharmaceutically acceptable salt f.
In another ment, the mitoketoscin comprises the general formula:
or a pharmaceutically acceptable salt thereof.
In yet another embodiment, the mitoketoscin comprises the general formula:
or a pharmaceutically acceptable salt thereof.
In one embodiment, the mitoketoscin possesses anti-aging activity.
In r embodiment, the mitoketoscin possesses ensitizing activity.
In a certain embodiment, the toscin possesses photosensitizing activity.
In one embodiment, the mitoketoscin sensitizes cancer cells to chemotherapeutic
agents.
In an embodiment, the mitoketoscin sensitizes cancer cells to natural nces.
In another embodiment, the mitoketoscin sensitizes cancer cells to caloric
restriction.
In yet another embodiment, the mitoketoscin binds to OXCT1.
In one ment, the mitoketoscin binds to ACAT1.
In an embodiment, the mitoketoscin binds to at least one of OXCT1/2 and
ACAT1/2.
In another certain embodiment of the present invention, there is provided a
method of ng cancer comprising administering to a patient in need thereof of a
pharmaceutically effective amount of the mitoketoscin disclosed herein and a pharmaceutically
acceptable carrier.
In one embodiment, the method of treating a microbial infection comprising
administering to a patient in need thereof of a pharmaceutically effective amount of the
mitoketoscin disclosed herein and a pharmaceutically acceptable carrier.
In another embodiment, the method of treating an lated condition
sing administering to a patient in need thereof of a pharmaceutically effective amount of
the mitoketoscin disclosed herein and a pharmaceutically able carrier.
In yet another embodiment, a pharmaceutical composition for treating cancer is
ed, containing, as the active ingredient, at least one toscin.
In one embodiment, the pharmaceutical composition for treating a bacterial
infection containing, as the active ient, at least one mitoketoscin.
In another embodiment, the pharmaceutical composition for treating a pathogenic
yeast infection containing, as the active ingredient, at least one mitoketoscin.
In an embodiment, the pharmaceutical composition for treating aging, as the
active ingredient, at least one mitoketoscin.
In one embodiment, the present invention provides a method of identifying a
mitoketoscin using virtual high-throughput screening and phenotypic drug ing, the method
sing;
identifying a mitoketoscin that targets a mitochondrial ketone re-utilization enzyme using
virtual high-throughput screening;
synthesizing the toscin; and
testing the mitoketoscin for mitochondrial inhibition activity.
One embodiment r comprising stering a mitochondrial support
substrate.
In yet another embodiment, the mitochondrial support substrate comprises at least
one of glucose, pyruvate, lactate, fatty acids and acetyl-carnitine.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. I shows a schematic diagram outlining a drug discovery strategy according to
embodiments of the present approach.
illustrates the chemical structures of eight candidate mitoketoscin
compounds 1-8 fied ing phenotypic drug screening.
FIGs. 3A-F shows the s of six candidate mitoketoscin compounds on
mammosphere formation in MCF7 cells.
FIGs. 4A-D shows the effects of four candidate mitoketoscin compounds on ATP-
ion in MCF7 cells.
FIGs. 5A-B and 6A-B each show the s of two candidate mitoketoscin
compounds on basal respiration, proton leak, ATP-linked respiration, maximal respiration, and
spare respiratory capacity in MCF7 cells.
shows the s of five candidate mitoketoscin compounds on aerobic
glycolysis in MCF7 cells. shows the effects of five candidate mitoketoscin compounds
on extracellular acidification rate (ECAR) over time in MCF7 cells.
FIGs. 8A-D illustrate docking images of four candidate mitoketoscin nds.
shows a schematic diagram outlining how OXCT1 and ACAT1 function to
drive ATP production.
A shows four candidate mitoketoscin compounds and their respective IC-
50s for ting CSC propagation. B shows the chemical structure of arecoline, a
naturally occurring ACAT1 inhibitor.
A compares the structures of two candidate mitoketoscin compounds. B shows the pharmacophores for two candidate mitoketoscin compounds.
A shows the structure of Coenzyme A (CoA). B compares the
structure of CoA with two candidate mitoketoscin compounds.
shows a schematic diagram outlining a follow-up treatment strategy with
mitochondrial substrates to ameliorate potential side effects of mitoketoscins, according to
embodiments of the t approach.
DESCRIPTION
The ing description illustrates embodiments of the present approach in
sufficient detail to enable practice of the present approach. gh the t approach is
described with reference to these specific embodiments, it should be iated that the present
approach may be embodied in different forms, and this description should not be construed as
limiting any ed claims to the specific embodiments set forth herein. Rather, these
embodiments are provided so that this sure will be thorough and complete, and will fully
convey the scope of the t approach to those skilled in the art.
Mitochondrial metabolism is an untapped gateway for treating a number of
afflictions, ranging from cancer to bacterial and fungal infections to aging. Functional
mitochondria are required for the propagation of cancer stem cells. Inhibiting mitochondrial
metabolism in cancer cells impedes the propagation of those cells. Mitochondrial inhibitors
targeting the re-utilization of ketone bodies as mitochondrial fuels therefore represent a new class
of anti-cancer therapeutics. These compounds may also inhibit mitochondrial protein translation,
and ore may function as spectrum antibiotics that target both ia and pathogenic
yeast. Research has also shown that mitochondrial inhibitors have anti-aging properties; hence
mitoketoscins may also impart anti-aging benefits.
Novel inhibitors of mitochondrial ATP production that bind to at least one of
OXCT1/2 and ACAT1/2 – mitoketoscins – may be identified through a convergent approach of
virtual high-throughput screening followed by in vivo validation for mitochondrial inhibition. is an overview of methods for identifying mitoketoscins by using in silico drug screening and
ypic drug screening disclosed herein. All or a portion of the three-dimensional structure of
the porcine OXCT1 and human ACAT1 proteins may be used in step S101 to fy novel
compounds that bind to these ns through virtual high-throughput screening (vHTS) (i.e., in
silico drug screening). The screening may be performed across a library of molecules. For instance,
during initial igations the inventors screened a collection of 30,000 small molecule
compounds for compounds expected to bind anywhere to the yl-CoA: 3-ketoacid CoA
transferase from pig heart covalently bound to CoA (PDB code 3OXO) or to the CoA binding site
of human mitochondrial cetyl-CoA thiolase (PDB code 2F2S). Initial vHTS may use various
screening programs, such as the eHiTS screening program, to identify a subset of compounds
having a strong binding affinity to either protein. For example, the ors used eHiTS to identify
the top 1,000 ranked compounds from an initial library, based on predicted binding affinity. eHiTS
is a screening method that systematically covers the part of the conformational and positional
search space that avoids severe steric clashes, producing highly accurate docking poses at a speed
that is well-suited for virtual high-throughput screening.
It should be appreciated that those skilled in the art may select or develop methods
for identifying a subset of compounds having a desired binding affinity. To efficiently m the
docking, a series of clip files may be prepared corresponding to the entire protein structure and
each compound docked sequentially at each of the clip files. Consensus scoring of the top
nds may be carried out using AutoDock 4.2, based on the same l binding site for
each compound ted from the eHiTS screen. Further analysis of predicted binding affinity
and visual inspection may be carried out using a number of methods, including for example a de
novo design program such as SPROUT. See Law et al., J Mol Struct. 666: 651-657 (2003), which
is incorporated by reference in its entirety, for information about SPROUT. Depending on the
l y size and results, a number of compounds may be selected for phenotypic drug
screening. For example, the inventors selected 227 compounds that performed well in these
analysis steps for ypic drug screening at step S103. 84 compounds were ed for the
OXCT1-based phenotypic screen and 143 compounds were selected for the ACAT1-based
phenotypic screen.
Phenotypic drug screening S103 may be accomplished by testing the mitochondrial
inhibition of selected compounds in a selected cell line. For example, ATP depletion assays may
be used. The inventors tested the selected 227 compounds on their ability to functionally induce
ATP-depletion in MCF7 human breast cancer cells. Approximately 85% of cellular ATP is
normally generated by OXPHOS in mitochondria, so ATP-depletion is a surrogate marker for
mitochondrial inhibition. It should be appreciated that those skilled in the art may employ other
surrogates for mitochondrial inhibition. However, for the ATP-depletion assay inventors
employed, MCF7 cells (6,000 cells/well) were plated into black clear-bottom 96-well plates and
incubated overnight before treatment. The 227 compounds identified by vHTS were applied to the
plated MCF7 cells at a concentration of 20 µM and were screened for ATP ion. Compounds
showing ATP-depletion effects were subsequently re-screened at a lower concentration (10 µM)
to identify the top eight compounds that most potently induce ATP-depletion. Compounds were
tested after 72 hours of incubation and experiments were performed in duplicate. After treatment,
media was ted from the wells and plates were washed with warm phosphate-buffered saline
(PBS) mented with Ca2+ and Mg2+. Then, cells were incubated with a t 33342
) staining solution (10 µg/ml) for 30 min and washed with PBS to te cell viability.
Fluorescence was read with a plate reader using tion/emission wavelengths at 355/460-nm.
Then, a CellTiter-Glo luminescent assay (Promega) was performed to measure metabolic activity
(ATP content) in the very same wells that were treated with a given compound. Assays were
performed according to the manufacturer’s protocol. Fluorescence intensity (Hoechst staining) and
luminescence intensity (ATP content) were normalized to vehicle-alone d controls and were
displayed as percent control for ison. All eight test compounds significantly depleted ATP
levels in viable cells. It should be appreciated that those of skill in the art may choose to employ
the same or similar ATP-depletion assays, modify such assays, or may replace the ATP-depletion
assay with another methodology for screening ed compounds for mitochondrial inhibition
(e.g., oxygen ption assays).
The present approach includes methods of confirming cell viability. Persons of skill
in the art may select one or more methods for ming cell viability suitable for the particular
embodiment. The inventors initially used the Sulphorhodamine (SRB) assay, which is based on
the measurement of cellular protein content. After treatment for 72 hours in 96-well plates, cells
were fixed with 10% trichloroacetic acid (TCA) for 1 hour in the cold room, and were dried
overnight at room temperature. Then, cells were incubated with SRB for 15 min, washed twice
with 1% acetic acid, and air dried for at least 1 hour. y, the protein-bound dye was dissolved
in a 10 mM Tris, pH 8.8 solution and read using the plate reader at 540-nm. Using the SRB assay,
the inventors ed only the compounds depleting ATP levels without prominent cytotoxicity
for further analysis. Prominent cytotoxicity was defined as fewer than 30% of cells still on the
plate. Of , embodiments employing other cell viability confirmation methodology may
select nds for further analysis based on other considerations as may be known in the art.
The present approach further es methods of functional validation at step
S105, during which a compound’s function as a ondrial inhibitor may be confirmed. A
number of methods may be used for functional validation, including, for example, lic flux
analysis, mammosphere assays, ity assays, and antibiotic (anti-bacterial and/or anti-fungal)
activity. For example, the inventors determined extracellular acidification rates (ECAR) and realtime
oxygen ption rates (OCR) for MCF7 cells using the Seahorse Extracellular Flux
(XF96) analyzer (Seahorse Bioscience, MA, USA). MCF7 cells were maintained in DMEM
supplemented with 10% FBS (fetal bovine , 2 mM GlutaMAX, and 1% Pen- Strep. 5,000
cells per well were seeded into XF96-well cell culture plates, and incubated overnight at 37°C in
a 5% CO2 humidified atmosphere. After 24 hours, cells were treated with selected compounds
showing ATP-depletion without prominent cytotoxicity at various concentrations (or vehicle
alone). After 72 hours of ent, cells were washed in pre-warmed XF assay media (for OCR
measurement, XF assay media was supplemented with 10mM glucose, 1mM Pyruvate, 2mM L-
glutamine and adjusted at pH 7.4). Cells were maintained in 175 μL/well of XF assay media at
37°C in a non-CO2 incubator for 1 hour. During incubation, 25 μL of 80mM e, 9μM
oligomycin, 1M 2-deoxyglucose (for ECAR measurement) and 25 μL of 10μM oligomycin, 9μM
FCCP, 10μM rotenone, 10μM antimycin A (for OCR measurement) in XF assay media was loaded
into the ion ports of the XFe-96 sensor cartridge. During the experiment, the instrument
ed these tors into the wells at a given time point, while ECAR/OCR was measured
continuously. ECAR and OCR measurements were normalized by protein content (using the
Sulphorhodamine B assay). Data sets were analyzed by XFe-96 software, using one-way ANOVA
and Student’s t-test calculations. All experiments were performed in triplicate, and results
validated the mitochondrial inhibition effects of the mitoketoscin compounds described herein. It
should be appreciated that numerous methods are known for functional validation, and that s
of skill in the art may select one or more depending on the tion needs (e.g., other assays that
measure or approximate mitochondrial function).
In y, the present approach may include methods of identifying potential
mitoketoscins using in silico drug screening and phenotypic drug screening. Novel compounds
identified using this ology may be tested for anti-cancer activity (e.g., the ability to inhibit
mammosphere ion and cell migration) and may be further tested on distinct bacterial and/or
yeast strains to investigate anti-microbial activity. summarizes the general methods for drug
screening and validation according to an embodiment of the t approach, but it should be
appreciated that those of skill in the art may deviate from the specific examples disclosed herein
without departing from the present approach.
The present approach has led to the identification of mitoketoscins that have anticancer
properties, and embodiments of the present approach may take the form of one or more of
these compounds, as well as pharmaceutical compositions including effective amounts of one or
more of these nds, and various methods of treatment using one or more of these
compounds. Based on the inventors’ initial screening and validation, the nds identified in
have anti-cancer properties and are therefore mitoketoscins. In view of the inventors’
research, these mitoketoscins are ore candidates for clinical trial. It should be appreciated
that the mitoketoscins identified in are not exhaustive, but are merely those compounds that
have been identified thus far using the novel methodology set forth herein. It should be appreciated
by those skilled in the art that the therapeutically-effective amount of each compound, for a
particular y can be determined h the application of straightforward procedures as are
known in the art.
Some embodiments may take the form of one or more mitoketoscins. The
embodiment may be included in a pharmaceutical composition for treating cancer, bacterial
infection, and/or pathogenic yeast infection. For e, a mitoketoscin may be a general
pharmacophore having the ing structure (or a salt thereof):
, , wherein Z is defined as ethylpiperidine or ethylpyrrolidine,
, or .
As another example, a mitoketoscin may be a l pharmacophore having the
ing structure (or a salt thereof):
wherein each R may be the same or different and is selected from the group
consisting of hydrogen, carbon, nitrogen, sulfur, oxygen, flourine, chlorine, bromine, iodine,
carboxyl, alkanes, cyclic alkanes, alkane-based derivatives, alkenes, cyclic alkenes, -based
derivatives, alkynes, alkyne-based derivative, ketones, ketone-based derivatives, aldehydes,
aldehyde-based tives, carboxylic acids, carboxylic acid-based derivatives, ethers, ether-
based derivatives, esters and ester-based derivatives, amines, amino-based derivatives, amides,
amide-based derivatives, monocyclic or polycyclic arene, heteroarenes, arene-based derivatives,
heteroarene-based derivatives, phenols, phenol-based derivatives, benzoic acid, and c sed
derivatives.
As another example, a mitoketoscin may be a general pharmacophore having the
following structure (or a salt thereof):
As a further example, a toscin may be a general pharmacophore having the
following structure (or a salt thereof):
n each R may be the same or different and is selected from the group
consisting of hydrogen, carbon, nitrogen, sulfur, oxygen, flourine, chlorine, bromine, iodine,
yl, alkanes, cyclic s, alkane-based derivatives, alkenes, cyclic alkenes, alkene-based
derivatives, alkynes, alkyne-based derivative, ketones, ketone-based derivatives, aldehydes,
aldehyde-based derivatives, carboxylic acids, carboxylic acid-based derivatives, ethers, etherbased
derivatives, esters and ester-based derivatives, amines, amino-based tives, amides,
based derivatives, monocyclic or polycyclic arene, heteroarenes, arene-based derivatives,
heteroarene-based derivatives, phenols, phenol-based derivatives, c acid, and benzoic acidbased
derivatives.
As another example, a mitoketoscin may be a general pharmacophore having the
following structure (or a salt f):
wherein each R may be the same or different and is selected from the group
consisting of hydrogen, carbon, en, sulfur, oxygen, flourine, chlorine, bromine, iodine,
carboxyl, alkanes, cyclic alkanes, alkane-based derivatives, alkenes, cyclic alkenes, alkene-based
derivatives, alkynes, alkyne-based tive, s, ketone-based tives, aldehydes,
aldehyde-based derivatives, carboxylic acids, carboxylic acid-based derivatives, , etherbased
derivatives, esters and ester-based derivatives, amines, amino-based derivatives, amides,
based derivatives, monocyclic or polycyclic arene, heteroarenes, arene-based derivatives,
heteroarene-based derivatives, phenols, phenol-based derivatives, benzoic acid, and benzoic acidbased
derivatives.
Another example of a mitoketoscin is a general pharmacophore having the
following structure (or a salt thereof):
A further example of a mitoketoscin is a general pharmacophore having the
following structure (or a salt thereof):
An additional example of a mitoketoscin is a general pharmacophore having the
following structure (or a salt thereof):
wherein each R may be the same or different and is selected
from the group consisting of hydrogen, carbon, nitrogen, sulfur, oxygen, flourine, chlorine,
e, iodine, carboxyl, alkanes, cyclic alkanes, alkane-based tives, alkenes, cyclic
alkenes, alkene-based derivatives, alkynes, alkyne-based derivative, ketones, ketone-based
derivatives, aldehydes, aldehyde-based derivatives, carboxylic acids, carboxylic acid-based
derivatives, ethers, ether-based derivatives, esters and ester-based tives, amines, aminobased
derivatives, amides, based derivatives, monocyclic or polycyclic arene, heteroarenes,
arene-based derivatives, heteroarene-based derivatives, phenols, phenol-based tives,
benzoic acid, and benzoic acid-based tives.
Another example of a mitoketoscin is a general pharmacophore having the
following structure (or a salt thereof):
wherein each R may be the same or different and is ed from the group
consisting of hydrogen, carbon, nitrogen, sulfur, oxygen, flourine, chlorine, bromine, iodine,
carboxyl, alkanes, cyclic s, -based derivatives, alkenes, cyclic alkenes, alkene-based
derivatives, alkynes, alkyne-based derivative, ketones, ketone-based derivatives, aldehydes,
aldehyde-based derivatives, carboxylic acids, carboxylic ased derivatives, ethers, etherbased
derivatives, esters and ester-based derivatives, amines, amino-based derivatives, amides,
amide-based tives, monocyclic or polycyclic arene, arenes, arene-based derivatives,
heteroarene-based derivatives, phenols, phenol-based tives, benzoic acid, and benzoic acidbased
derivatives.
A further example of a mitoketoscin is a general pharmacophore having the
following structure (or a salt f):
Another example of a mitoketoscin is a general pharmacophore having the
following structure (or a salt thereof):
wherein each R may be the same or different and is selected from the group
consisting of hydrogen, carbon, nitrogen, sulfur, oxygen, flourine, chlorine, bromine, iodine,
carboxyl, alkanes, cyclic alkanes, alkane-based derivatives, alkenes, cyclic alkenes, alkene-based
derivatives, alkynes, alkyne-based derivative, ketones, ketone-based tives, aldehydes,
aldehyde-based derivatives, carboxylic acids, carboxylic acid-based derivatives, , etherbased
tives, esters and ester-based derivatives, amines, amino-based derivatives, amides,
amide-based derivatives, clic or polycyclic arene, heteroarenes, arene-based derivatives,
heteroarene-based derivatives, phenols, phenol-based derivatives, benzoic acid, and c acidbased
derivatives.It should be appreciated that the mitoketoscins may be selected for therapeutic
use individually, or in combination with more than one specific toscin, and/or with other
substances to enhance the efficacy of other therapeutics. The therapeutics may be used in the form
of usual pharmaceutical compositions which may be prepared using one or more known methods.
For example, a pharmaceutical composition may be prepared by using diluents or excipients such
as, for example, one or more fillers, g agents, binders, wetting agents, disintegrating agents,
surface active agents, lubricants, and the like as are known in the art. Various types of
administration unit forms may be selected depending on the therapeutic e(s). Examples of
forms for pharmaceutical compositions include, but are not limited to, tablets, pills, powders,
liquids, suspensions, emulsions, granules, capsules, suppositories, injection preparations (solutions
and suspensions), topical creams, and other forms as may be known in the art. For the purpose of
shaping a pharmaceutical composition in the form of tablets, any excipients which are known may
be used, for example carriers such as lactose, white sugar, sodium de, glucose, urea, starch,
calcium ate, kaolin, cyclodextrins, crystalline cellulose, silicic acid and the like; binders
such as water, ethanol, propanol, simple syrup, glucose solutions, starch solutions, gelatin
solutions, carboxymethyl cellulose, shelac, methyl cellulose, potassium phosphate,
nylpyrrolidone, etc. Additionally, egrating agents such as dried starch, sodium
alginate, agar powder, laminalia powder, sodium en carbonate, calcium ate, fatty
acid esters of polyoxyethylene an, sodium laurylsulfate, yceride of stearic acid,
starch, lactose, etc., may be used. Disintegration inhibitors such as white sugar, stearin, coconut
butter, hydrogenated oils; absorption accelerators such as quaternary ammonium base, sodium
laurylsulfate, etc., may be used. Wetting agents such as glycerin, starch, and others known in the
art may be used. Adsorbing agents such as, for example, starch, lactose, kaolin, bentonite, colloidal
silicic acid, etc., may be used. Lubricants such as purified talc, stearates, boric acid powder,
polyethylene glycol, etc., may be used. If tablets are d, they may be further coated with the
usual coating materials to make the tablets as sugar coated s, gelatin film coated tablets,
s coated with enteric coatings, tablets coated with films, double layered tablets and multi-
layered tablets. ceutical compositions adapted for l administration may be
formulated as ointments, , suspensions, lotions, powders, solutions, pastes, gels, foams,
sprays, ls, or oils. Such pharmaceutical compositions may include conventional additives
which include, but are not limited to, preservatives, solvents to assist drug penetration, co-solvents,
emollients, propellants, viscosity modifying agents (gelling ), surfactants and carriers.
The present approach may, in some embodiments, involve methods of testing
compounds, and in particular mitoketoscins, for anti-cancer properties. As discussed above, vHTS
and computational chemistry may be used to identify candidate mitochondrial inhibitors. Those
candidates may be tested for specific anti-cancer properties. For example, the inventors compared
seven candidate compounds in parallel for their ability to inhibit mammosphere ion in
MCF7 cells. demonstrates that six compounds tested inhibited mammosphere formation.
Compound 2 and 8 (FIGs. 3A and 3B, tively) were the two most potent candidates in
decreasing the number of mammospheres, a measure of cancer stem cell activity, at a concentration
of 25 µM. Compound 6 and 3 were also ive (FIGs. 3C and 3D, respectively), while
compound 5 and 1 (FIGs. 3E and 3F, respectively) were less potent inhibitors of mammosphere
Compound ID IC-50 (µM)
OXCT1 Hits
1 ALB-H01004577 160.4
2 ALB-H09465625 11.3
3 ALB-H15358970 46.7
4 ALB-H15354504 166.8
ACAT1 Hits
ALB-H04367562 66.7
6 LEG19576081 22.9
8 ALB-H01005022 10.1
Table 1. Mitoketoscin inhibition of mammosphere formation in MCF7 cells
Table 1 summarizes the mammosphere formation inhibition results for seven
candidate compounds. Table 1 shows that seven compounds inhibited mammosphere formation
with half-maximal inhibitory concentrations (IC-50s) between 10 and 170 µM. nds 1 to 4
were identified from the OXCT1 screen, and Compounds 5, 6, and 8 were identified from the
ACAT1 screen.
The present ch may, in some embodiments, involve methods of on
validation of toscin compounds. For example, the inventors assessed functional validation
of four candidates using the Seahorse Analyzer, which quantitatively measures oxygen
consumption rate (OCR) and extracellular acidification rate (ECAR). OCR is a surrogate marker
for OXPHOS and ECAR is a surrogate marker for glycolysis and L-lactate production.
The inventors’ results demonstrated that Compounds 2, 3, 6, and 8 all dosedependently
inhibited mitochondrial oxygen-consumption in MCF7 cells. FIGs. 4A and 4B show
that Compounds 2 and 8 reduced mitochondrial respiration icantly, even at doses as low as
µM. Compounds 6 and 3 were also potent tors (FIGs. 4C and 4D). As shown in FIGs. 5
and 6, the compounds dosed dependently reduced basal respiration, proton leak, ATP-linked
respiration, and maximal respiration. shows how four compounds significantly inhibited
glycolysis compared to the control.
Some embodiments of the present approach may include testing compounds for
anti-cancer properties by ering compound effects on mammosphere formation. It should be
appreciated that those skilled in the art may use other methods known in the art for ing a
candidate mitochondrial tor’s effects on a particular cell line without departing from the
present approach. It should also be appreciated that those skilled in the art may assess a candidate
mitochondrial inhibitor’s effects on other cancer types, as the inhibitors target cancer stem cells
(CSCs). CSCs show conserved or similar features across most cancer types.
FIGs. 8A-8D illustrate the molecular ng of Compounds 2, 8, 3, and 6. The
goal of molecular modeling is to predict the predominant binding mode of a compound to a known
three dimensional structure. and 8C show the molecular docking of Compounds 2 and 3,
respectively, at the yl-CoA g site within the 3D crystal structure of OXCT1. A
comparison of and 8C shows that at least one amino acid, ASN-373, is predicted to
directly bind to both Compounds 2 and 3. FIGs. 8B and 8D shows the molecular docking of
Compounds 8 and 6, respectively, at the CoA binding site within the 3D crystal structure of
ACAT1. A comparison of and 8D shows that at least two amino acids, LEU-184 and HIS-
192, are predicted to directly bind to both Compounds 8 and 6. These predicted dominant binding
modes may be invaluable to r lead optimization.
Ketone bodies functionally behave as mitochondrial fuels, which may actively
drive tumor growth and metastasis. In this context, OXCT1 and ACAT1 are two mitochondrial
proteins that participate in ketone re-utilization, as is summarized in The inventors
molecularly targeted OXCT1 and ACAT1 to prevent cancer cells from recycling ketone bodies
into Acetyl-CoA, which normally enters the TCA cycle, driving mitochondrial ATP production.
A top hit for the OXCT1 screen und 2) and a hit for the ACAT1 screen
(Compound 8) have similar al structures, with the exception of minor functional side
groups, as shown in A. The underlying “chemical scaffold” or pharmacophore is the same
for both small les, as shown in FIG 11B. The structures of Compounds 2 and 8 were also
compared to the molecular structure of Coenzyme A in to trate structural
similarities.
Two compounds from the OXCT1 screen (Compounds 3 and 4) are structurally
similar to each other, as is shown in A. However, based on their observed IC-50 values,
Compound 3 is nearly 4 times more potent than Compound 4 in its ability to target CSC
propagation. The unique chemical groups that distinguish these two les structurally
(highlighted by arrows in A), may be responsible for the observed ences in their IC-
50s observed for their inhibition of CSC propagation.
Recent studies provide additional evidence of a role for ACAT1 as an oncogene, as
these studies identified arecoline as a potential ACAT1 inhibitor. Garcia-Bermudez et al, Mol Cell
2016, 64(5):856-857. Arecoline is a nicotinic ased alkaloid found within the areca nut,
which is the fruit of the areca palm tree (Areca catechu). Arecoline has shown anti-tumor activity,
r validating that drugs targeting ACAT1 might be valuable as anti-cancer agents. However,
the inventors did not assess its capacity to target CSCs. As arecoline is very small molecule (shown
in B for ison to Compounds 6 and 3), it may need to be modified significantly by
medicinal chemistry to increase its potency. ine is not a mitoketoscin, because the
compound is known to be carcinogenic.
While normal ketone metabolism occurs under conditions of organismal starvation
and/or severe nutrient deprivation, this regulation is lost in human tumors, and ketone metabolism
appears to occur constitutively in cancer cells. Targeting ketone lism in human tumors,
under normal dietary conditions, would be predicted to have minimal lic side effects.
Nevertheless, the ial side-effects of ketone inhibitors could be significantly ameliorated or
“controlled” by including a “rescue” step, consisting of a follow-up treatment with other
mitochondrial support ates, such as glucose, pyruvate, lactate, fatty acids and/or acetylcarnitine
, as is shown in FIG 13. Sterile ose and L-lactate intravenous solutions (D5W,
D5NS, Lactated Ringer’s) are already used routinely in hospitals for other clinical and eutic
indications; hence, a follow-up treatment is clinically feasible.
The inventors have shown that compounds inducing acute ATP depletion in cancer
cells may sensitize those cells to radiation, ultraviolet light, chemotherapeutic agents, natural
substances, and/or caloric restriction. Mitoketoscins, as discussed herein, have demonstrated ATP-
ion effects. Based on these preliminary s, mitoketoscins may also be used as
radiosensitizers and/or photo-sensitizers. Use as radiosensitizers and/or photo-sensitizers may be
in combination with other treatment vectors, including but not limited to other cancer treatment
s as may be known in the art, and cancer treatment through inhibiting mitochondrial
biogenesis as disclosed herein. Similarly, mitoketoscins may be used to functionally sensitize bulk
cancer cells and cancer stem cells to chemotherapeutic agents, pharmaceuticals, and/or other
natural substances, such as dietary ments and caloric restriction.
In addition to anti-cancer and anti-biotic behavior, the mitochondrial inhibitors that
may be identified by the present ch have the potential to slow the mammalian aging process.
c inhibition of ondrial protein translation has been shown to have beneficial sideeffects
, and in particular the side effect of slowing the aging process and increasing lifespan in
model organisms. Lower steady-state levels of Mrps5 (a mitoribosomal n) are strongly
functionally correlated with longer murine lifespan, resulting in a significant lifespan increase of
~250 days. In addition, selective knock-down of Mrps5 in C. elegans dramatically increases
lifespan. Mrps5 knock-down worms show significant decreases in ondrial respiration and
ATP production. Similarly, down of the worm homologs of mitochondrial complex I, III,
IV and V, as well as several TCA cycle enzymes, all robustly extended lifespan, further ating
d OXPHOS activity and lower ATP levels as the mechanism. Finally, pharmacological
inhibition of mitochondrial biogenesis (using the off-target effects of doxycycline) also
significantly increases an in C. elegans. Thus, mitoketoscins may be used to therapeutically
target the aging process and to extend an.
Mitoketoscins may also be used to ze and/or reverse drug resistance in
cancer cells. Drug resistance is thought to be based, at least in part, on sed mitochondrial
function in cancer cells. In ular, cancer cells demonstrating resistance to endocrine therapies,
such as tamoxifen, are expected to have increased mitochondrial function. Mitoketoscins inhibit
mitochondrial function, and therefore may be useful in reducing and, in some cases reversing, drug
resistance in cancer cells.
The terminology used in the description of the invention herein is for the purpose
of describing particular embodiments only and is not intended to be limiting of the ion. As
used in the description of the invention and the appended claims, the singular forms “a,” “an” and
“the” are intended to include the plural forms as well, unless the t clearly indicates
otherwise. The invention includes numerous alternatives, modifications, and equivalents as will
become apparent from consideration of the following ed description.
It will be understood that gh the terms “first,” d,” “third,” “a),” “b),”
and “c),” etc. may be used herein to describe s elements of the invention should not be
limited by these terms. These terms are only used to distinguish one element of the invention from
another. Thus, a first element discussed below could be termed a element aspect, and similarly, a
third without departing from the teachings of the present invention. Thus, the terms “first,”
“second,” “third,” “a),” “b),” and “c),” etc. are not intended to necessarily convey a sequence or
other hierarchy to the associated elements but are used for identification purposes only. The
sequence of operations (or steps) is not limited to the order presented in the claims.
Unless otherwise d, all terms (including 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. It will be r understood that terms, such as those defined in commonly
used dictionaries, should be interpreted as having a meaning that is consistent with their meaning
in the t of the present application and relevant art and should not be reted in an
idealized or overly formal sense unless expressly so defined . The terminology used in the
ption of the invention herein is for the purpose of describing particular embodiments only
and is not intended to be limiting of the invention. All publications, patent applications, patents
and other references mentioned herein are incorporated by reference in their entirety. In case of a
conflict in terminology, the present specification is controlling.
] Also as used herein, “and/or” refers to and encompasses any and all le
combinations of one or more of the associated listed items, as well as the lack of combinations
when interpreted in the alternative (“or”).
Unless the context indicates otherwise, it is specifically intended that the various
features of the invention bed herein can be used in any combination. Moreover, the present
invention also contemplates that in some embodiments of the invention, any feature or
combination of features set forth herein can be excluded or omitted. To illustrate, if the
specification states that a complex comprises components A, B and C, it is specifically intended
that any of A, B or C, or a ation thereof, can be omitted and disclaimed.
As used herein, the transitional phrase “consisting essentially of” (and grammatical
variants) is to be interpreted as encompassing the recited als or steps “and those that do not
materially affect the basic and novel characteristic(s)” of the claimed invention. Thus, the term
“consisting essentially of” as used herein should not be interpreted as equivalent to “comprising.”
The term “about,” as used herein when referring to a measurable value, such as,
for example, an amount or concentration and the like, is meant to ass variations of ± 20%,
± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of the specified amount. A range provided herein for
a measureable value may include any other range and/or individual value therein.
Having thus described certain embodiments of the present ion, it is to be
understood that the invention defined by the appended claims is not to be limited by particular
details set forth in the above description as many apparent ions thereof are possible without
departing from the spirit or scope thereof as hereinafter claimed.
Claims (40)
1. A method of identifying a mitoketoscin using virtual high-throughput screening and phenotypic drug screening, the method comprising; identifying a compound that targets a mitochondrial ketone re-utilization enzyme using virtual high-throughput ing; and testing the compound for mitochondrial inhibition activity.
2. The method of claim 1, n the mitoketoscin binds to OXCT1.
3. The method of claim 1, wherein the mitoketoscin binds ACAT1.
4. The method of any one of claims 1 to 3, wherein the phenotypic drug screening is in vitro drug screening.
5. The method of any one of claims 1 to 3, wherein the ypic drug ing comprises an ATP-depletion assay.
6. The method of any one of claims 1 to 3, wherein the phenotypic drug screening comprises an extracellular acidification rate assay.
7. The method of any one of claims 1 to 3, n the phenotypic drug screening comprises an oxygen consumption rate assay.
8. The method of any one of claims 1 to 7, further comprising testing the mitoketoscin for anti-cancer activity.
9. The method of claim 8, wherein the anti-cancer activity tested is mammosphere
10. The method of claim 8, wherein the anti-cancer activity tested is cell migration.
11. The method of any one of claims 1 to 10 further comprising g the mitoketoscin for anti-microbial activity.
12. A mitoketoscin comprising the general formula: , wherein R may be selected from the group consisting of chlorine, bromine, iodine, yl, alkanes, cyclic alkanes, alkenes, cyclic alkenes, s, ketones, aldehydes, ylic acids, , esters, amines, amides, monocyclic or clic arene, heteroarenes, phenols and benzoic acid.
13. The mitoketoscin of claim 12, wherein the mitoketoscin possesses anti-aging activity.
14. The mitoketoscin of claim 12, wherein the mitoketoscin possesses at least one of radiosensitizing activity and photosensitizing activity.
15. The mitoketoscin of claim 12, wherein the mitoketoscin possesses anti-microbial activity.
16. The mitoketoscin of claim 12, wherein the mitoketoscin sensitizes cancer stem cells to chemotherapeutic agents.
17. The mitoketoscin of claim 12, wherein the toscin sensitizes cancer stem cells to natural substances.
18. The mitoketoscin of claim 12, wherein the mitoketoscin sensitizes cancer stem cells to caloric restriction.
19. The mitoketoscin of any one of claims 12 to 18, wherein the mitoketoscin binds to at least one of OXCT1, OXCT2, ACAT1, and ACAT2.
20. Use of a mitoketoscin in the manufacture of a medicament for treating cancer, wherein the mitoketoscin comprises the general formula: , wherein R may be ed from the group consisting of hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkanes, cyclic alkanes, alkenes, cyclic s, alkynes, ketones, aldehydes, carboxylic acids, ethers, esters, amines, amides, monocyclic or polycyclic arene, heteroarenes, phenols and benzoic acid.
21. The use of claim 20, wherein R is fluorine.
22. Use of a mitoketoscin in the manufacture of a medicament for treating a microbial infection, wherein the mitoketoscin comprises the general a: , wherein R may be selected from the group ting of hydrogen, fluorine, chlorine, e, iodine, carboxyl, alkanes, cyclic alkanes, alkenes, cyclic s, alkynes, ketones, aldehydes, carboxylic acids, ethers, esters, amines, amides, clic or polycyclic arene, heteroarenes, phenols and benzoic acid.
23. The use of claim 22, wherein R is ne.
24. Use of a mitoketoscin in the manufacture of a ment for treating an age-related condition, wherein the mitoketoscin comprises the general formula: , wherein R may be selected from the group consisting of hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkanes, cyclic alkanes, alkenes, cyclic alkenes, alkynes, ketones, aldehydes, carboxylic acids, ethers, , amines, amides, monocyclic or polycyclic arene, heteroarenes, s and benzoic acid.
25. The use of claim 24, wherein R is fluorine.
26. A pharmaceutical composition comprising, as an active ingredient, a toscin comprising the general formula: , wherein R may be selected from the group consisting of hydrogen, fluorine, chlorine, bromine, iodine, yl, alkanes, cyclic alkanes, alkenes, cyclic alkenes, alkynes, ketones, aldehydes, carboxylic acids, ethers, esters, amines, amides, monocyclic or polycyclic arene, heteroarenes, s, c acid; and a pharmaceutically able carrier.
27. The pharmaceutical composition of claim 26, wherein R is fluorine.
28. A mitoketoscin comprising the general formula: , wherein R may be selected from the group consisting of hydrogen, , chlorine, bromine, iodine, carboxyl, alkanes, cyclic alkanes, alkenes, cyclic alkenes, alkynes, ketones, aldehydes, carboxylic acids, ethers, esters, amines, amides, monocyclic or polycyclic arene, heteroarenes, phenols and benzoic acid.
29. The mitoketoscin of claim 28, wherein the mitoketoscin possesses anti-aging activity.
30. The mitoketoscin of claim 28, wherein the mitoketoscin possesses radiosensitizing activity.
31. The mitoketoscin of claim 28, wherein the mitoketoscin possesses photosensitizing activity.
32. The mitoketoscin of claim 28, wherein the mitoketoscin sensitizes cancer stem cells to chemotherapeutic agents.
33. The mitoketoscin of claim 28, wherein the mitoketoscin sensitizes cancer stem cells to natural substances.
34. The mitoketoscin of claim 28, wherein the toscin sensitizes cancer stem cells to c restriction.
35. The mitoketoscin of any one of claims 28 to 34, wherein the mitoketoscin binds to at least one of OXCT1, OXCT2, ACAT1, and ACAT2.
36. Use of a mitoketoscin in the manufacture of a ment for treating cancer, wherein the mitoketoscin comprises the general formula: , n R may be selected from the group consisting of hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkanes, cyclic alkanes, alkenes, cyclic alkenes, s, ketones, aldehydes, ylic acids, ethers, esters, amines, , monocyclic or polycyclic arene, heteroarenes, phenols and benzoic acid.
37. The use of claim 36, wherein R is fluorine.
38. The use of claim 36 or claim 37, n the medicament further comprises a mitochondrial support substrate comprising at least one of glucose, pyruvate, lactate, fatty acids and acetyl-carnitine.
39. A pharmaceutical composition comprising, as an active ingredient, a mitoketoscin comprising the general formula: , n R may be selected from the group consisting of hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, s, cyclic s, alkenes, cyclic alkenes, alkynes, ketones, des, carboxylic acids, ethers, esters, amines, amides, monocyclic or polycyclic arene, heteroarenes, phenols, benzoic acid; and a pharmaceutically acceptable carrier.
40. The pharmaceutical composition of claim 39, wherein R is fluorine. Drug Screening & Validation Virtual high-throughput screening (vHTS) Step 1. Selection of two molecular libraries of 1000 compounds each (both for OXCT1 and ACAT1) from a small molecular screening collection of = : 30,000 compounds Step 2. Further analysis of predicted binding affinity and visual inspection Compounds performing well in all is steps were selected for assay Phenotypic drug screening pletion assays on human breast cancer cells (MCF7) 84 Compounds (OXCTl); 143 nds (ACATl) Observed 8 hits at 20 uM Functional validation Mammosphere assays Metabolic Flux is OCR/ECAR Mitoketoscins Mitoketoscins Compound 1 Compound 5 F? CI H N <0) 2 N ZLOVQ/FHo N Compound 2 Compound 6 ng 9 H \v" \ N N F30 H OE Compound 3 Compound 7 Q N / o N 0 m \ (1flag NJLS / N N\__jN N 3D-Spheroid CSC Assay spheres) ctrl) versus counts 80 Mammosphere 60 Control 10 [1M 25 [1M 50 [1M ctrl) versus 00O counts 0'1O Mammosphere J:o Control 10 [1M 25 [1M 50 [1M WO 05698 control 100 counts 80 Mammosphere ** Control 25 [1M 50 [1M 100 uM ctrl) versus 80 counts 60 Mammosphere 40 Control 25 [1M 50 [1M 100 uM control 100 counts 00O Mammosphere 0'1O Control 25 [1M 50 [1M 100 uM ctrl) versus 100 counts 00O Mammosphere 0'1O Control 25 [1M 50 [1M 100 uM WO 05698 WControl azflsésee Compound 2 20 40 60 8O Time (min) -o— Control AEQEEBEE «\w 2.5 pM ““5 HM Com D.ou nd 8 \\§. 20 40 60 80 Time (min) *Control ~\\\\\\\\\\‘25 ulVl xxxwm uM n: ““5 uM -— 2O o 20 40 60 80 Time (min) 30 +Contro| \\10 [1M \\\\\\\\\\'5 “M m \\\\\\\\\“2.5 “M -— 20 O 20 4O 6O 80 Time (min) 30 Cm0.wm 2 Im.mm 25 N 1pM axfigéaee Nmu.M 50 1.0 MN\\\\ m 5 ______________________________________________________ mmum m N\\\\\\\\\mm m §§m.w kmmk.m VWN\\\\\\\\\\\\\\\\\\\\\\\\\\\\\Nmmmam mm capacity 30 Compound 8 I Control Ammm\:_E\_oE& 2 5 2.5 pM 20 NSpM Basal Proton Leak ATP-link Maximal Spare respiration ation respiration respiratory capacfly FIG. SB C0m D.0U nd 5 I Control 25 ®5pM axfigésee 10 pM 25 pM Basal Proton Leak ATP-link Maximal Spare respiration respiration respiration respiratory capacity 30 Compound 3 I COntrd 25 § 2.5 pM Ammm\:_E\_oE& 20 * §@ 51 u.0Mu.M 0 \\\\\\\\\m \m Spare respiration respiration atory capacfly FIG. GB
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