U.S. patent application number 10/884062 was filed with the patent office on 2006-04-20 for compositions for manipulating the lifespan and stress response of cells and organisms.
Invention is credited to Konrad T. Howitz, Robert E. Zipkin.
Application Number | 20060084135 10/884062 |
Document ID | / |
Family ID | 33567706 |
Filed Date | 2006-04-20 |
United States Patent
Application |
20060084135 |
Kind Code |
A1 |
Howitz; Konrad T. ; et
al. |
April 20, 2006 |
Compositions for manipulating the lifespan and stress response of
cells and organisms
Abstract
Provided herein are methods and compositions for modulating the
activity of sirtuin deacetylase protein family members; p53
activity; apoptosis; lifespan and sensitivity to stress of cells
and organisms. Exemplary methods comprise contacting a cell with an
activating compound, such as a flavone, stilbene, flavanone,
isoflavone, catechin, chalcone, tannin or anthocyanidin; or an
inhibitory compound, such as a sphingolipid, e.g., sphingosine.
Inventors: |
Howitz; Konrad T.;
(Allentown, PA) ; Zipkin; Robert E.; (Wynnewood,
PA) |
Correspondence
Address: |
LICATLA & TYRRELL P.C.
66 E. MAIN STREET
MARLTON
NJ
08053
US
|
Family ID: |
33567706 |
Appl. No.: |
10/884062 |
Filed: |
July 1, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60532158 |
Dec 23, 2003 |
|
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|
60483949 |
Jul 1, 2003 |
|
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Current U.S.
Class: |
435/29 |
Current CPC
Class: |
A61P 17/08 20180101;
A61P 31/12 20180101; A61K 31/175 20130101; A61P 9/14 20180101; C12N
9/16 20130101; A61K 31/175 20130101; A61P 9/08 20180101; A61P 31/16
20180101; A61P 17/04 20180101; C12Q 1/34 20130101; A61P 27/12
20180101; A61P 27/14 20180101; A61P 1/16 20180101; G01N 2333/916
20130101; A61P 17/12 20180101; G01N 2500/02 20130101; A61P 9/12
20180101; A61P 25/16 20180101; A61P 25/02 20180101; A61P 35/00
20180101; A61P 29/00 20180101; A61P 43/00 20180101; A61P 25/28
20180101; A61P 13/08 20180101; A61P 9/10 20180101; A61P 17/06
20180101; A61P 27/02 20180101; A61P 35/02 20180101; A61K 45/06
20130101; A61P 37/08 20180101; A61K 31/00 20130101; A61P 3/06
20180101; A61P 17/14 20180101; A61P 9/00 20180101; A61P 17/16
20180101; A61P 31/10 20180101; A61P 31/22 20180101; A61K 2300/00
20130101; A61P 17/00 20180101; A61P 19/02 20180101; A61P 7/06
20180101; A61P 21/00 20180101; A61P 25/00 20180101; A61P 27/06
20180101; A61P 9/04 20180101; A61P 37/02 20180101; A61P 37/06
20180101; A61P 31/18 20180101 |
Class at
Publication: |
435/029 |
International
Class: |
C12Q 1/02 20060101
C12Q001/02 |
Claims
1. A method for activating a sirtuin deacetylase protein family
member, comprising contacting the sirtuin deacetylase protein
family member with an activating compound having a formula selected
from the group consisting of formulas 1-25 and 30.
2. The method of claim 1, wherein the compound is a polyphenol
compound or analog or derivative thereof.
3. The method of claim 1, wherein the plant compound is selected
from the group consisting of flavones, stilbenes, flavanones,
isoflavones, catechins, chalcones, tannins and anthocyanidins or
analog or derivative thereof.
4. The method of claim 1, wherein, if the compound is a naturally
occurring compound, it is not in a form in which it is naturally
occurring.
5. The method of claim 3, wherein the compound is selected from the
group consisting of resveratrol, butein, piceatannol,
isoliquiritgenin, fisetin, luteolin, 3,6,3',4'-tetrahydroxyflavone,
quercetin, and analogs and derivatives thereof.
6. The method of claim 1, wherein the sirtuin deacetylase protein
family member is SIRT1.
7. The method of claim 1, wherein the sirtuin deacetylase protein
family member is in a cell, and the method comprises contacting the
cell with the compound.
8. The method of claim 7, wherein the cell is in vitro.
9. The method of claim 7, wherein the cell is a cell of a
subject.
10. The method of claim 7, wherein the cell is in a subject and the
method comprises administering the compound to the subject.
11. The method of claim 1, further comprising determining the
activity of the sirtuin deacetylase protein family member.
12. The method of claim 10, further comprising determining the
activity of the sirtuin deacetylase protein family member.
13. The method of claim 7, wherein the cell is contacted with the
compound at a concentration of 0.1-100 .mu.M.
14. The method of claim 1, further contacting the cell with an
additional activating compound having a formula selected from the
group consisting of formulas 1-25 and 30.
15. The method of claim 14, comprising contacting the cell with a
least three different activating compounds having a formula
selected from the group consisting of formulas 1-25 and 30.
16. A method for inhibiting the activity of a sirtuin protein
family member, comprising contacting the sirtuin deacetylase
protein family member with an inhibiting compound having a formula
selected from the group consisting of formulas 26-29 and 31.
17. A method for shortening the lifespan of a cell or rendering it
resistant to stress, comprising contacting the cell with an
inhibiting compound having a formula selected from the group
consisting of formulas 26-29 and 31.
18. A composition comprising two compounds each having a formula
selected from the group of formulas 1-31.
19. A method for identifying a compound that modulates SIRT1 in
vivo, comprising (i) contacting a cell comprising a SIRT1 protein
with a peptide of p53 comprising an acetylated residue 382 in the
presence of an inhibitor of class I and class II HDAC under
conditions appropriate for SIRT1 to deacetylate the peptide and
(ii) determining the level of acetylation of the peptide, wherein a
different level of acetylation of the peptide in the presence of
the test compound relative to the absence of the test compound
indicates that the test compound modulates SIRT1 in vivo.
Description
[0001] This patent application claims the benefit of priority from
U.S. Provisional Patent Application No. 60/532,158, filed Dec. 23,
2003 and U.S. Provisional Patent Application No. 60/483,949, filed
Jul. 1, 2003, each of which is herein incorporated by reference in
its entirety.
BACKGROUND OF THE INVENTION
[0002] There is now good evidence from model organisms that the
pace of aging can be regulated (Kenyon, C. Cell 105, 165-168
(2001)). Longevity regulatory genes have been identified in many
eukaryotes, including rodents, flies, nematode worms and even
single-celled organisms such as baker's yeast (reviewed in
Sinclair, D. Mech Ageing Dev 123, 857-67 (2002); Hekimi, S. &
Guarente, L. Genetics and the specificity of the aging process.
Science 299, 1351-4 (2003)). These genes appear to be part of an
evolutionarily conserved longevity pathway that evolved to promote
survival in response to deteriorating environmental conditions
(Kenyon, C. Cell 105, 165-168 (2001); Guarente, L. and Kenyon, C.
Nature 408, 255-62. (2000). The yeast S. cerevisiae has proven a
particularly useful model in which to study cell autonomous
pathways of longevity (Sinclair, D. Mech Ageing Dev 123, 857-67
(2002)). In this organism, replicative lifespan is defined as the
number of daughter cells an individual mother cell produces before
dying. Yeast lifespan extension is governed by PNC1, a calorie
restriction (CR)--and stress-responsive gene that depletes
nicotinamide, a potent inhibitor of the longevity protein Sir2.
Both PNC1 and SIR2 are required for lifespan extension by CR or
mild stress (Lin et al. Science 289, 2126-8 (2000); Anderson et al.
Nature 423, 181-5 (2003)) and additional copies of these genes
extend lifespan 30-70% (Lin et al. Science 289, 2126-8 (2000);
Anderson et al. Nature 423, 181-5 (2003); Kaeberlein et al. Genes
Dev 13, 2570-80 (1999). Based on these results we proposed that CR
may confer health benefits in a variety of species because it is a
mild stress that induces a sirtuin-mediated organismal defense
response (Anderson et al. Nature 423, 181-5 (2003).
[0003] Sir2, a histone deacetylase (HDAC), is the founding member
of the sirtuin deacetylase family, which is characterized by a
requirement for NAD.sup.+ as a co-substrate (Landry et al. Proc
Natl Acad Sci USA 97, 5807-11 (2000); Imai et al. Nature 403,
795-800 (2000); Smith et al. Proc Natl Acad Sci USA 97, 6658-63
(2000); Tanner et al. Proc Natl Acad Sci USA 97, 14178-82 (2000);
Tanny et al. Cell 99, 735-45 (1999); Tanny, J. C. and Moazed, D.
Proc Natl Acad Sci USA 98, 415-20 (2001)). SIR2 was originally
identified as a gene required for the formation of
transcriptionally silent heterochromatin at yeast mating-type loci
(Laurenson, P. and Rine, J. Microbiol Rev 56, 543-60. (1992)).
Subsequent studies have shown that Sir2 suppresses recombination
between repetitive DNA sequences at ribosomal RNA genes
(rDNA)(Smith, J. S. and Boeke, J. D. Genes Dev 11, 241-54 (1997);
Bryk et al. Genes Dev 11, 255-69 (1997); Gottlieb, S. and Esposito,
R. E. Cell 56, 771-6 (1989)). Sir2 has also been implicated in the
partitioning of carbonylated proteins to yeast mother cells during
budding (Aguilaniu et al. Science (2003). Studies in C. elegans,
mammalian cells, and the single-celled parasite Leishmania,
indicate that the survival and longevity functions of sirtuins are
conserved (Tissenbaum, H. A. and Guarente, L. Nature 410, 227-30
(2001); Vaziri et al. Cell 107, 149-59 (2001); Luo et al. Cell 107,
137-48 (2001); Vergnes et al. Gene 296, 139-50 (2002)). In C.
elegans additional copies of sir-2.1 extend lifespan by 50% via the
insulin/IGF-1 signalling pathway, the same pathway recently shown
to regulate lifespan in rodents (Holzenberger et al. Nature 421,
182-7 (2003); Shimokawa et al. Faseb J 17, 1108-9 (2003); Tatar et
al. Science 299, 1346-51 (2003)).
SUMMARY OF THE INVENTION
[0004] Provided herein are methods for activating a sirtuin
deacetylase protein family member. The method may comprise
contacting a sirtuin deacetylase protein family member with a
compound having a structure selected from the group of formulas
1-25 and 31. Compounds falling within formulas 1-25 and 31 and
activating a sirtuin protein are referred to herein as "activating
compounds." The activating compound may be a polyphenol compound,
such as a plant polyphenol or an analog or derivative thereof.
Exemplary compounds are selected from the group consisting of
flavones, stilbenes, flavanones, isoflavones, catechins, chalcones,
tannins and anthocyanidins or analog or derivative thereof. In
illustrative embodiments, compounds are selected from the group of
resveratrol, butein, piceatannol, isoliquiritgenin, fisetin,
luteolin, 3,6,3',4'-tetrahydroxyflavone, quercetin, and analogs and
derivatives thereof. In certain embodiments, if the activating
compound is a naturally occurring compound, it may not in a form in
which it is naturally occurring.
[0005] The sirtuin deacetylase protein family member maybe the
human SIRT1 protein or the yeast Sir2 protein.
[0006] The sirtuin deacetylase protein family member may be in a
cell, in which case the method may comprise contacting the cell
with an activating compound or introducing a compound into the
cell. The cell may be in vitro. The cell may be a cell of a
subject. The cell may be in a subject and the method may comprise
administering the activating compound to the subject. Methods may
further comprise determining the activity of the sirtuin
deacetylase protein family member.
[0007] A cell may be contacted with an activating compound at a
concentration of 0.1-100 .mu.M. In certain embodiments, a cell is
further contacted with an additional activating compound. In other
embodiments, a cell is contacted with a least three different
activating compounds.
[0008] Other methods encompassed herein include methods for
inhibiting the activity of p53 in a cell and optionally protecting
the cell against apoptosis, e.g., comprising contacting the cell
with an activating compound at a concentration of less than about
0.5 .mu.M. Another method comprises stimulating the activity of p53
in a cell and optionally inducing apoptosis in the cell, comprising
contacting the cell with an activating compound at a concentration
of at least 50 .mu.M.
[0009] Also provided herein is a method for extending the lifespan
of a eukaryotic cell, such as by increasing its resistance to
stress, comprising contacting the cell with a compound selected
from the group consisting of stilbene, flavone and chalcone family
members. Such compounds are referred to as "lifespan extending
compounds." The compound may have the structure set forth in
formula 7. Other compounds may be activating compounds having a
structure set forth in any of formulas 1-25 and 30, provided they
extend lifespan or increase resistance to stress. The compound may
be selected from the group consisting of resveratrol, butein and
fisetin and analogs and derivatives thereof. In certain
embodiments, if the lifespan extending compound is a naturally
occurring compound, it is not in a form in which it is naturally
occurring. The method may further comprise determining the lifespan
of the cell. The method may also further comprise contacting the
cell with an additional compound or with at least three compounds
selected from the group consisting of stilbene, flavone and
chalcone family members or other lifespan extending compound. The
cell may be contacted with a compound at a concentration of less
than about 10 .mu.M or at a concentration of about 10-100 .mu.M.
The cell may be in vitro or in vivo, it may be a yeast cell or a
mammalian cell. If the cell is in a subject, the method may
comprise administering the compound to the subject.
[0010] Methods for inhibiting sirtuins; inhibiting deacetylation of
p53; stimulating apoptosis; shorting lifespan and rendering cells
and organisms sensitive to stress are also encompassed. One method
comprises contacting a sirtuin or cell or organism comprising such
with an inhibitory compound having a formula selected from the
group of formulas 26-29 and 31.
[0011] Also provided herein are compositions comprising, e.g., two
compounds each having a formula selected from the group of formulas
1-31. Further provided herein are screening methods for identifying
compounds, e.g., small molecules, that modulate sirtuins and/or
modulate the life span or resistance to stress of cells. Methods
may comprise (i) contacting a cell comprising a SIRT1 protein with
a peptide of p53 comprising an acetylated residue 382 in the
presence of an inhibitor of class I and class II HDAC under
conditions appropriate for SIRT1 to deacetylate the peptide and
(ii) determining the level of acetylation of the peptide, wherein a
different level of acetylation of the peptide in the presence of
the test compound relative to the absence of the test compound
indicates that the test compound modulates SIRT1 in vivo.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIGS. 1a through 1d show the effects of resveratrol on the
kinetics of recombinant human SIRT1. FIG. 1a shows resveratrol
dose-response of SIRT1 catalytic rate at 25 .mu.M NAD.sup.+, 25
.mu.M p53-382 acetylated peptide. Relative initial rates are the
mean of two determinations, each derived from the slopes of
fluorescence (arbitrary fluorescence units, AFU) vs. time plots
with data obtained at 0, 5, 10 and 20 min. of deacetylation. FIG.
1b shows the SIRT1 initial rate at 3 mM NAD.sup.+, as a function of
p53-382 acetylated peptide concentration in the presence (.DELTA.)
or absence (.box-solid.) of 100 .mu.M resveratrol. Lines represent
non-linear least-squares fits to the Michaelis-Menten equation.
Kinetic constants: K.sub.m(control, .box-solid.)=64 .mu.M,
K.sub.m(+resveratrol, .DELTA.)=1.8 .mu.M; V.sub.max(control,
.box-solid.)=1107 AFU/min., V.sub.max(+resveratrol, .DELTA.)=926
AFU/min. FIG. 1c shows the SIRT1 initial rate at 1 mM p53-382
acetylated peptide, as a function of NAD.sup.+ concentration, in
the presence (.DELTA.) or absence (.box-solid.) of 100 .mu.M
resveratrol. Lines represent non-linear least-squares fits to the
Michaelis-Menten equation. Kinetic constants: K.sub.m(control,
.box-solid.)=558 .mu.M, K.sub.m(+resveratrol, .DELTA.)=101 .mu.M;
V.sub.max(control, .box-solid.)=1863 AFU/min.,
V.sub.max(+resveratrol, .DELTA.)=1749 AFU/min. FIG. 1d shows
effects of resveratrol on nicotinamide inhibition of SIRT1. Kinetic
constants are shown relative to those of the control (no
nicotinamide, no resveratrol) and represent the mean of two
determinations. Error bars are standard errors of the mean. The
variable substrate in each experiment (N=NAD.sup.+, P=p53
acetylated peptide), the presence/absence of nicotinamide (.+-.)
and the resveratrol concentration (.mu.M) are indicated beneath
each pair of K.sub.m-V.sub.max bars.
[0013] FIG. 2a through 2d show the effects of polyphenols on Sir2
and S. cerevisiae lifespan. FIG. 2a shows the initial deacetylation
rate of recombinant GST-Sir2 as a function of resveratrol
concentration. Rates were determined at the indicated resveratrol
concentrations, either with 100 .mu.M `Fluor de Lys` acetylated
lysine substrate (FdL) plus 3 mM NAD.sup.+ (.DELTA.) or with 200
.mu.M p53-382 acetylated peptide substrate plus 200 .mu.M NAD.sup.+
(.box-solid.). FIG. 2b shows lifespan analyses determined by
micro-manipulating individual yeast cells as described Sinclair, D.
A. and Guarente (Cell 91, 1033-42 (1997)) in complete 2% glucose
medium with 10 .mu.M of each compound, unless otherwise stated.
Average lifespan was determined for wild type untreated
(.quadrature.), quercetin (.largecircle.) and piceatannol
(.circle-solid.). FIG. 2c shows the average lifespan for wild type
untreated (.quadrature.), fisetin (.largecircle.), butein (), or
resveratrol (.DELTA.). FIG. 2d shows average lifespan for wild type
untreated (.quadrature.), and growth with resveratrol at 10 .mu.M
(.DELTA.), 100 .mu.M (.circle-solid.), or 500 .mu.M
(.largecircle.).
[0014] FIGS. 3a through 3f show resveratrol extending lifespan by
mimicking CR and suppressing rDNA recombination. Yeast lifespans
were determined as in FIG. 2. FIG. 3a shows average lifespan for
wild type (wt) untreated (.quadrature.), wild type+resveratrol
(wt+R; .circle-solid.) and glucose-restricted+resveratrol (CR+R;
.largecircle.). FIG. 3b shows average lifespans for wild type
(.quadrature.), sir2(.DELTA.) sir2+resveratrol (sir2+R;
.tangle-solidup.), pnc1 (.largecircle.), and pnc1+resveratrol
(pnc1+R; .circle-solid.). FIG. 3c shows resveratrol suppressing the
frequency of ribosomal DNA recombination in the presence and
absence of nicotinamide (NAM). Frequencies were determined by loss
of the ADE2 marker gene from the rDNA locus (RDN1). FIG. 3d shows
that resveratrol does not suppress rDNA recombination in a sir2
strain. FIG. 3e show that resveratrol and other sirtuin activators
do not significantly increase rDNA silencing compared to a
2.times.SIR2 strain. Pre-treated cells (RDN1::URA3) were harvested
and spotted as 10-fold serial dilutions on either SC or SC with
5-fluororotic acid (5-FOA). In this assay, increased rDNA silencing
results in increased survival on 5-FOA medium. FIG. 3f show
quantitation of the effect of resveratrol on rDNA silencing by
counting numbers of surviving cells on FOA/total plated.
[0015] FIGS. 4a through 4e show resveratrol and other polyphenols
stimulating SIRT1 activity in human cells. FIG. 4a shows a method
for assaying intracellular deacetylase activity with a fluorogenic,
cell-permeable substrate, FdL (`Fluor de Lys`, BIOMOL). FdL (200
.mu.M) is added to growth media and cells are incubated for 1-3
hours to allow FdL to enter the cells and the lysine-deacetylated
product (deAc-FdL) to accumulate intracellularly. Cells are lysed
with detergent in the presence of 1 .mu.M TSA and 1 mM
nicotinamide. Addition of the non-cell-permeable Developer (BIOMOL)
releases a fluorophor, specifically from deAc-FdL. FIG. 4b shows
SIRT1 activating polyphenols stimulating TSA-insensitive, FdL
deacetylation by HeLa S3 cells. Cells were grown adherently in
DMEM/10% FCS and treated for 1 hour with 200 .mu.M FdL, 1 .mu.M TSA
and either vehicle (0.5% final DMSO, Control) or 500 .mu.M of the
indicated compound. Intracellular accumulation of deAc-FdL was then
determined as described briefly in FIG. 4a. The intracellular
deAc-FdL level for each compound (mean of six replicates) are
plotted against the ratios to the control rate obtained in the in
vitro SIRT1 polyphenol screen (see Table 1, Supplementary Tables 1
and 3). FIG. 4c shows U2OS osteosarcoma cells grown to .gtoreq.90%
confluence in DMEM/10% FCS exposed to 0 or 10 grays of gamma
irradiation (IR). Whole cell lysates were prepared 4 hours
post-irradiation and were probed by Western blotting with indicated
antibodies. FIG. 4d shows U2OS cells cultured as above and
pre-treated with the indicated amounts of resveratrol or a 0.5%
DMSO blank for 4 hours after which cells were exposed to 0 or 50
J/cm.sup.2 of UV radiation. Lysates were prepared and analyzed by
Western blot as in FIG. 4c. FIG. 4e shows human embryonic kidney
cells (HEK 293) expressing wild type SIRT1 or dominant negative
SIRT1-H363Y (SIRT1-HY) protein cultured as described above,
pre-treated with the indicated amounts of resveratrol or a 0.5%
DMSO blank for 4 hours, and exposed to 50 J/cm.sup.2 of UV
radiation as above. Lysates were prepared and analyzed as
above.
[0016] FIG. 5 shows the deacetylation site preferences of
recombinant SIRT1. Initial rates of deacetylation were determined
for a series of fluorogenic acetylated peptide substrates based on
short stretches of human histone H3, H4 and p53 sequence.
Substrates examined include: H3-4-9 with the sequence
K(Ac)QTARK(Ac) (SEQ ID NO:1); H3-9-14 with the sequence
K(Ac)STGGK(Ac) (SEQ ID NO:2); H3-9-14/pS with the sequence
K(Ac)--S(PO3)-TGGK(Ac) (SEQ ID NO:3); H3-14-18 with the sequence
K(Ac)APRK(Ac) (SEQ ID NO:4); H4-1-5 with the sequence SGRGK(Ac)(SEQ
ID NO:5); H4-12-16(Fluor de Lys-H4-AcK16) with the sequence
KGGAK(Ac) (SEQ ID NO:6); H4-12-16/diAc with the sequence
K(Ac)GGAK(Ac)(SEQ ID NO:7); p53-320 (Fluor de Lys-SIRT2) with the
sequence QPKK(Ac)(SEQ ID NO:8); p53-373 with the sequence
K(Ac)SKK(Ac)(SEQ ID NO:9); p53-382(Fluor de Lys-SIRT1 with the
sequence RHKK(Ac) (SEQ ID NO:10); p53-382/di-Ac (Fluor de
Lys-HDAC8) with the sequence RHK(Ac)K(Ac)(SEQ ID NO:11); and
.epsilon.-acetyl lysine (Fluor de Lys, Fdl) wit the sequence K(Ac).
All substrate were obtained from BIOMOL, Plymouth Meeting, Pa.).
Recombinant human SIRT1 (1 .mu.g, BIOMOL), was incubated for 10
minutes at 37.degree. C. with 25 .mu.M of the indicated fluorogenic
acetylated peptide substrate and 500 .mu.M NAD.sup.+. Reactions
were stopped by the addition of 1 mM nicotinamide and the
deacetylation-dependent fluorescent signal was determined.
[0017] FIG. 6a through 6c show intracellular deacetylation activity
measured with a cell-permeable, fluorogenic HDAC and sirtuin
substrate. HeLa S3 cells were grown to confluence in DMEM/10% FCS
and then incubated with fresh medium containing 200 .mu.M FdL for
the indicated times at 37.degree. C. Intracellular and medium
levels of deacetylated substrate (deAc-FdL) were determined
according to the manufacturer's instructions (HDAC assay kit,
BIOMOL). All data points represent the mean of two determinations.
FIG. 6a shows the concentration ratio of intracellular
([deAc-FdL].sub.i) to medium ([deAc-FdL].sub.o) concentrations in
the presence (.DELTA.) or absence (.box-solid.) of 1 .mu.M
trichostatin A (TSA). FIG. 6b shows total accumulation of
deacetylated substrate (deAc-FdL) in the presence (.DELTA.) or
absence (.box-solid.) of 1 .mu.M TSA. FIG. 6c shows intracellular
accumulation of deacetylated substrate (deAc-FdL) in the presence
(.DELTA.) or absence (.box-solid.) of 1 .mu.M TSA.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0018] As used herein, the following terms and phrases shall have
the meanings set forth below. Unless defined otherwise, all
technical and scientific terms used herein have the same meaning as
commonly understood to one of ordinary skill in the art.
[0019] The singular forms "a," "an," and "the" include plural
reference unless the context clearly dictates otherwise.
"Activating a sirtuin protein" refers to the action of producing an
activated sirtuin protein, i.e., a sirtuin protein that is capable
of performing at least one of its biological activities to at least
some extent, e.g., with an increase of activity of at least about
10%, 50%, 2 fold or more. Biological activities of sirtuin proteins
include deacetylation, e.g., of histones and p53; extending
lifespan; increasing genomic stability; silencing transcription;
and controlling the segregation of oxidized proteins between mother
and daughter cells.
[0020] An "activating compound" refers to a compound that activates
a sirtuin protein. Activating compounds may have a formula selected
from the group of formulas 1-25 and 30.
[0021] A "form that is naturally occurring" when referring to a
compound means a compound that is in a form, e.g., a composition,
in which it can be found naturally. For example, since resveratrol
can be found in red wine, it is present in red wine in a form that
is naturally occurring. A compound is not in a form that is
naturally occurring if, e.g., the compound has been purified and
separated from at least some of the other molecules that are found
with the compound in nature.
[0022] "Inhibiting a sirtuin protein" refers to the action of
reducing at least one of the biological activities of a sirtuin
protein to at least some extent, e.g., at least about 10%, 50%, 2
fold or more.
[0023] An "inhibitory compound" or "inhibiting compound" refers to
a compound that inhibits a sirtuin protein. Inhibitory compounds
may have a formula selected from the group of formulas 26-29 and
31.
[0024] A "naturally occurring compound" refers to a compound that
can be found in nature, i.e., a compound that has not been designed
by man. A naturally occurring compound may have been made by man or
by nature.
[0025] "Replicative lifespan" which is used interchangeably herein
with "lifespan" of a cell refers to the number of daughter cells
produced by an individual "mother cell." "Chronological aging," on
the other hand, refers to the length of time a population of
non-dividing cells remains viable when deprived of nutrients.
"Increasing the lifespan of a cell" or "extending the lifespan of a
cell," as applied to cells or organisms, refers to increasing the
number of daughter cells produced by one cell; increasing the
ability of cells or organisms to cope with stresses and combat
damage, e.g., to DNA, proteins; and/or increasing the ability of
cells or organisms to survive and exist in a living state for
longer under a particular condition, e.g., stress. Lifespan can be
increased by at least about 20%, 30%, 40%, 50%, 60% or between 20%
and 70%, 30% and 60%, 40% and 60% or more using methods described
herein.
[0026] "Sirtuin deacetylase protein family members;" "Sir2 family
members;" "Sir2 protein family members;" or "sirtuin proteins"
includes yeast Sir2, Sir-2.1, and human SIRT1 and SIRT2 proteins.
Other family members include the four additional yeast Sir2-like
genes termed "HST genes" (homologues of Sir two) HST1, HST2, HST3
and HST4, and the five other human homologues hSIRT3, hSIRT4,
hSIRT5, hSIRT6 and hSIRT7 (Brachmann et al. (1995) Genes Dev.
9:2888 and Frye et al. (1999) BBRC 260:273). Preferred sirtuins are
those that share more similarities with SIRT1, i.e., hSIRT1, and/or
Sir2 than with SIRT2, such as those members having at least part of
the N-terminal sequence present in SIRT1 and absent in SIRT2 such
as SIRT3 has.
[0027] The terms "comprise" and "comprising" are used in the
inclusive, open sense, meaning that additional elements may be
included.
[0028] The term "including"is used to mean "including but not
limited to". "Including" and "including but not limited to" are
used interchangeably.
[0029] The term "cis" is art-recognized and refers to the
arrangement of two atoms or groups around a double bond such that
the atoms or groups are on the same side of the double bond. Cis
configurations are often labeled as (Z) configurations.
[0030] The term "trans" is art-recognized and refers to the
arrangement of two atoms or groups around a double bond such that
the atoms or groups are on the opposite sides of a double bond.
Trans configurations are often labeled as (E) configurations.
[0031] The term "covalent bond" is art-recognized and refers to a
bond between two atoms where electrons are attracted
electrostatically to both nuclei of the two atoms, and the net
effect of increased electron density between the nuclei
counterbalances the internuclear repulsion. The term covalent bond
includes coordinate bonds when the bond is with a metal ion.
[0032] The term "therapeutic agent" is art-recognized and refers to
any chemical moiety that is a biologically, physiologically, or
pharmacologically active substance that acts locally or
systemically in a subject. Examples of therapeutic agents, also
referred to as "drugs", are described in well-known literature
references such as the Merck Index, the Physicians Desk Reference,
and The Pharmacological Basis of Therapeutics, and they include,
without limitation, medicaments; vitamins; mineral supplements;
substances used for the treatment, prevention, diagnosis, cure or
mitigation of a disease or illness; substances which affect the
structure or function of the body; or pro-drugs, which become
biologically active or more active after they have been placed in a
physiological environment.
[0033] The term "therapeutic effect" is art-recognized and refers
to a local or systemic effect in animals, particularly mammals, and
more particularly humans caused by a pharmacologically active
substance. The term thus means any substance intended for use in
the diagnosis, cure, mitigation, treatment or prevention of disease
or in the enhancement of desirable physical or mental development
and/or conditions in an animal or human. The phrase
"therapeutically-effective amount" means that amount of such a
substance that produces some desired local or systemic effect at a
reasonable benefit/risk ratio applicable to any treatment. The
therapeutically effective amount of such substance will vary
depending upon the subject and disease condition being treated, the
weight and age of the subject, the severity of the disease
condition, the manner of administration and the like, which can
readily be determined by one of ordinary skill in the art. For
example, certain compositions described herein may be administered
in a sufficient amount to produce a at a reasonable benefit/risk
ratio applicable to such treatment.
[0034] The term "synthetic" is art-recognized and refers to
production by in vitro chemical or enzymatic synthesis.
[0035] The term "meso compound" is art-recognized and refers to a
chemical compound which has at least two chiral centers but is
achiral due to a plane or point of symmetry.
[0036] The term "chiral" is art-recognized and refers to molecules
that have the property of non-superimposability of the mirror image
partner, while the term "achiral" refers to molecules that are
superimposable on their mirror image partner. A "prochiral
molecule" is a molecule that has the potential to be converted to a
chiral molecule in a particular process.
[0037] The term "stereoisomers" is art-recognized and refers to
compounds that have identical chemical constitution, but differ
with regard to the arrangement of the atoms or groups in space. In
particular, "enantiomers" refer to two stereoisomers of a compound
that are non-superimposable mirror images of one another.
"Diastereomers", on the other hand, refers to stereoisomers with
two or more centers of dissymmetry and whose molecules are not
mirror images of one another.
[0038] Furthermore, a "stereoselective process" is one that
produces a particular stereoisomer of a reaction product in
preference to other possible stereoisomers of that product. An
"enantioselective process" is one that favors production of one of
the two possible enantiomers of a reaction product.
[0039] The term "regioisomers" is art-recognized and refers to
compounds that have the same molecular formula but differ in the
connectivity of the atoms. Accordingly, a "regioselective process"
is one that favors the production of a particular regioisomer over
others, e.g., the reaction produces a statistically significant
increase in the yield of a certain regioisomer.
[0040] The term "epimers" is art-recognized and refers to molecules
with identical chemical constitution and containing more than one
stereocenter, but which differ in configuration at only one of
these stereocenters.
[0041] The term "ED.sub.50" is art-recognized. In certain
embodiments, ED.sub.50 means the dose of a drug that produces 50%
of its maximum response or effect, or alternatively, the dose that
produces a pre-determined response in 50% of test subjects or
preparations. The term "LD.sub.50" is art-recognized. In certain
embodiments, LD.sub.50 means the dose of a drug that is lethal in
50% of test subjects. The term "therapeutic index" is an
art-recognized term that refers to the therapeutic index of a drug,
defined as LD.sub.50/ED.sub.50.
[0042] The term "structure-activity relationship" or "(SAR)" is
art-recognized and refers to the way in which altering the
molecular structure of a drug or other compound alters its
biological activity, e.g., its interaction with a receptor, enzyme,
nucleic acid or other target and the like.
[0043] The term "aliphatic" is art-recognized and refers to a
linear, branched, cyclic alkane, alkene, or alkyne. In certain
embodiments, aliphatic groups in the present compounds are linear
or branched and have from 1 to about 20 carbon atoms.
[0044] The term "alkyl" is art-recognized, and includes saturated
aliphatic groups, including straight-chain alkyl groups,
branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl
substituted cycloalkyl groups, and cycloalkyl substituted alkyl
groups. In certain embodiments, a straight chain or branched chain
alkyl has about 30 or fewer carbon atoms in its backbone (e.g.,
C.sub.1-C.sub.30 for straight chain, C.sub.3-C.sub.30 for branched
chain), and alternatively, about 20 or fewer. Likewise, cycloalkyls
have from about 3 to about 10 carbon atoms in their ring structure,
and alternatively about 5, 6 or 7 carbons in the ring structure.
The term "alkyl" is also defined to include halosubstituted
alkyls.
[0045] Moreover, the term "alkyl" (or "lower alkyl") includes
"substituted alkyls", which refers to alkyl moieties having
substituents replacing a hydrogen on one or more carbons of the
hydrocarbon backbone. Such substituents may include, for example, a
hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a
formyl, or an acyl), a thiocarbonyl (such as a thioester, a
thioacetate, or a thioformate), an alkoxyl, a phosphoryl, a
phosphonate, a phosphinate, an amino, an amido, an amidine, an
imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a
sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a
heterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety.
It will be understood by those skilled in the art that the moieties
substituted on the hydrocarbon chain may themselves be substituted,
if appropriate. For instance, the substituents of a substituted
alkyl may include substituted and unsubstituted forms of amino,
azido, imino, amido, phosphoryl (including phosphonate and
phosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl
and sulfonate), and silyl groups, as well as ethers, alkylthios,
carbonyls (including ketones, aldehydes, carboxylates, and esters),
--CN and the like. Exemplary substituted alkyls are described
below. Cycloalkyls may be further substituted with alkyls,
alkenyls, alkoxys, alkylthios, aminoalkyls, carbonyl-substituted
alkyls, --CN, and the like.
[0046] The term "aralkyl" is art-recognized and refers to an alkyl
group substituted with an aryl group (e.g., an aromatic or
heteroaromatic group).
[0047] The terms "alkenyl" and "alkynyl" are art-recognized and
refer to unsaturated aliphatic groups analogous in length and
possible substitution to the alkyls described above, but that
contain at least one double or triple bond respectively.
[0048] Unless the number of carbons is otherwise specified, "lower
alkyl" refers to an alkyl group, as defined above, but having from
one to about ten carbons, alternatively from one to about six
carbon atoms in its backbone structure. Likewise, "lower alkenyl"
and "lower alkynyl" have similar chain lengths.
[0049] The term "heteroatom" is art-recognized and refers to an
atom of any element other than carbon or hydrogen. Illustrative
heteroatoms include boron, nitrogen, oxygen, phosphorus, sulfur and
selenium.
[0050] The term "aryl" is art-recognized and refers to 5-, 6- and
7-membered single-ring aromatic groups that may include from zero
to four heteroatoms, for example, benzene, pyrrole, furan,
thiophene, imidazole, oxazole, thiazole, triazole, pyrazole,
pyridine, pyrazine, pyridazine and pyrimidine, and the like. Those
aryl groups having heteroatoms in the ring structure may also be
referred to as "aryl heterocycles" or "heteroaromatics." The
aromatic ring may be substituted at one or more ring positions with
such substituents as described above, for example, halogen, azide,
alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl,
amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate,
carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido,
ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic
moieties, --CF.sub.3, --CN, or the like. The term "aryl" also
includes polycyclic ring systems having two or more cyclic rings in
which two or more carbons are common to two adjoining rings (the
rings are "fused rings") wherein at least one of the rings is
aromatic, e.g., the other cyclic rings may be cycloalkyls,
cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls.
[0051] The terms ortho, meta and para are art-recognized and refer
to 1,2-, 1,3- and 1,4-disubstituted benzenes, respectively. For
example, the names 1,2-dimethylbenzene and ortho-dimethylbenzene
are synonymous.
[0052] The terms "heterocyclyl" or "heterocyclic group" are
art-recognized and refer to 3- to about 10-membered ring
structures, alternatively 3- to about 7-membered rings, whose ring
structures include one to four heteroatoms. Heterocycles may also
be polycycles. Heterocyclyl groups include, for example, thiophene,
thianthrene, furan, pyran, isobenzofuran, chromene, xanthene,
phenoxanthene, pyrrole, imidazole, pyrazole, isothiazole,
isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine,
isoindole, indole, indazole, purine, quinolizine, isoquinoline,
quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline,
cinnoline, pteridine, carbazole, carboline, phenanthridine,
acridine, pyrimidine, phenanthroline, phenazine, phenarsazine,
phenothiazine, furazan, phenoxazine, pyrrolidine, oxolane,
thiolane, oxazole, piperidine, piperazine, morpholine, lactones,
lactams such as azetidinones and pyrrolidinones, sultams, sultones,
and the like. The heterocyclic ring may be substituted at one or
more positions with such substituents as described above, as for
example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl,
hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate,
phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,
ketone, aldehyde, ester, a heterocyclyl, an aromatic or
heteroaromatic moiety, --CF.sub.3, --CN, or the like.
[0053] The terms "polycyclyl" or "polycyclic group" are
art-recognized and refer to two or more rings (e.g., cycloalkyls,
cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls) in which
two or more carbons are common to two adjoining rings, e.g., the
rings are "fused rings". Rings that are joined through non-adjacent
atoms are termed "bridged" rings. Each of the rings of the
polycycle may be substituted with such substituents as described
above, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl,
cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido,
phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether,
alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an
aromatic or heteroaromatic moiety, --CF.sub.3, --CN, or the
like.
[0054] The term "carbocycle" is art-recognized and refers to an
aromatic or non-aromatic ring in which each atom of the ring is
carbon.
[0055] The term "nitro" is art-recognized and refers to --NO.sub.2;
the term "halogen" is art-recognized and refers to --F, --Cl, --Br
or --I; the term "sulfhydryl" is art-recognized and refers to --SH;
the term "hydroxyl" means --OH; and the term "sulfonyl" is
art-recognized and refers to --SO.sub.2.sup.-. "Halide" designates
the corresponding anion of the halogens, and "pseudohalide" has the
definition set forth on page 560 of "Advanced Inorganic Chemistry"
by Cotton and Wilkinson.
[0056] The terms "amine" and "amino" are art-recognized and refer
to both unsubstituted and substituted amines, e.g., a moiety that
may be represented by the general formulas: ##STR1## wherein R50,
R51 and R52 each independently represent a hydrogen, an alkyl, an
alkenyl, --(CH.sub.2).sub.m--R61, or R50 and R51, taken together
with the N atom to which they are attached complete a heterocycle
having from 4 to 8 atoms in the ring structure; R61 represents an
aryl, a cycloalkyl, a cycloalkenyl, a heterocycle or a polycycle;
and m is zero or an integer in the range of 1 to 8. In certain
embodiments, only one of R50 or R51 may be a carbonyl, e.g., R50,
R51 and the nitrogen together do not form an imide. In other
embodiments, R50 and R51 (and optionally R52) each independently
represent a hydrogen, an alkyl, an alkenyl, or
--(CH.sub.2).sub.m--R61. Thus, the term "alkylamine" includes an
amine group, as defined above, having a substituted or
unsubstituted alkyl attached thereto, i.e., at least one of R50 and
R51 is an alkyl group.
[0057] The term "acylamino" is art-recognized and refers to a
moiety that may be represented by the general formula: ##STR2##
wherein R50 is as defined above, and R54 represents a hydrogen, an
alkyl, an alkenyl or --(CH.sub.2).sub.m--R61, where m and R61 are
as defined above.
[0058] The term "amido" is art recognized as an amino-substituted
carbonyl and includes a moiety that may be represented by the
general formula: ##STR3## wherein R50 and R51 are as defined above.
Certain embodiments of amides may not include imides which may be
unstable.
[0059] The term "alkylthio" refers to an alkyl group, as defined
above, having a sulfur radical attached thereto. In certain
embodiments, the "alkylthio" moiety is represented by one of
--S-alkyl, --S-alkenyl, --S-alkynyl, and
--S--(CH.sub.2).sub.m--R61, wherein m and R61 are defined above.
Representative alkylthio groups include methylthio, ethyl thio, and
the like.
[0060] The term "carbonyl" is art recognized and includes such
moieties as may be represented by the general formulas: ##STR4##
wherein X50 is a bond or represents an oxygen or a sulfur, and R55
and R56 represents a hydrogen, an alkyl, an alkenyl,
--(CH.sub.2).sub.m--R61 or a pharmaceutically acceptable salt, R56
represents a hydrogen, an alkyl, an alkenyl or
--(CH.sub.2).sub.m--R61, where m and R61 are defined above. Where
X50 is an oxygen and R55 or R56 is not hydrogen, the formula
represents an "ester". Where X50 is an oxygen, and R55 is as
defined above, the moiety is referred to herein as a carboxyl
group, and particularly when R55 is a hydrogen, the formula
represents a "carboxylic acid". Where X50 is an oxygen, and R56 is
hydrogen, the formula represents a "formate". In general, where the
oxygen atom of the above formula is replaced by sulfur, the formula
represents a "thiolcarbonyl" group. Where X50 is a sulfur and R55
or R56 is not hydrogen, the formula represents a "thiolester."
Where X50 is a sulfur and R55 is hydrogen, the formula represents a
"thiolcarboxylic acid." Where X50 is a sulfur and R56 is hydrogen,
the formula represents a "thiolformate." On the other hand, where
X50 is a bond, and R55 is not hydrogen, the above formula
represents a "ketone" group. Where X50 is a bond, and R55 is
hydrogen, the above formula represents an "aldehyde" group.
[0061] The terms "alkoxyl" or "alkoxy" are art-recognized and refer
to an alkyl group, as defined above, having an oxygen radical
attached thereto. Representative alkoxyl groups include methoxy,
ethoxy, propyloxy, tert-butoxy and the like. An "ether" is two
hydrocarbons covalently linked by an oxygen. Accordingly, the
substituent of an alkyl that renders that alkyl an ether is or
resembles an alkoxyl, such as may be represented by one of
--O-alkyl, --O-alkenyl, --O-alkynyl, --O--(CH.sub.2).sub.m--R61,
where m and R61 are described above.
[0062] The term "sulfonate" is art recognized and refers to a
moiety that may be represented by the general formula: ##STR5## in
which R57 is an electron pair, hydrogen, alkyl, cycloalkyl, or
aryl.
[0063] The term "sulfate" is art recognized and includes a moiety
that may be represented by the general formula: ##STR6## in which
R57 is as defined above.
[0064] The term "sulfonamido" is art recognized and includes a
moiety that may be represented by the general formula: ##STR7## in
which R50 and R56 are as defined above.
[0065] The term "sulfamoyl" is art-recognized and refers to a
moiety that may be represented by the general formula: ##STR8## in
which R50 and R51 are as defined above.
[0066] The term "sulfonyl" is art-recognized and refers to a moiety
that may be represented by the general formula: ##STR9## in which
R58 is one of the following: hydrogen, alkyl, alkenyl, alkynyl,
cycloalkyl, heterocyclyl, aryl or heteroaryl.
[0067] The term "sulfoxido" is art-recognized and refers to a
moiety that may be represented by the general formula: ##STR10## in
which R58 is defined above.
[0068] The term "phosphoryl" is art-recognized and may in general
be represented by the formula: ##STR11## wherein Q50 represents S
or O, and R59 represents hydrogen, a lower alkyl or an aryl. When
used to substitute, e.g., an alkyl, the phosphoryl group of the
phosphorylalkyl may be represented by the general formulas:
##STR12## wherein Q50 and R59, each independently, are defined
above, and Q51 represents O, S or N. When Q50 is S, the phosphoryl
moiety is a "phosphorothioate".
[0069] The term "phosphoramidite" is art-recognized and may be
represented in the general formulas: ##STR13## wherein Q51, R50,
R51 and R59 are as defined above.
[0070] The term "phosphonamidite" is art-recognized and may be
represented in the general formulas: ##STR14## wherein Q51, R50,
R51 and R59 are as defined above, and R60 represents a lower alkyl
or an aryl.
[0071] Analogous substitutions may be made to alkenyl and alkynyl
groups to produce, for example, aminoalkenyls, aminoalkynyls,
amidoalkenyls, amidoalkynyls, iminoalkenyls, iminoalkynyls,
thioalkenyls, thioalkynyls, carbonyl-substituted alkenyls or
alkynyls.
[0072] The definition of each expression, e.g. alkyl, m, n, and the
like, when it occurs more than once in any structure, is intended
to be independent of its definition elsewhere in the same
structure.
[0073] The term "selenoalkyl" is art-recognized and refers to an
alkyl group having a substituted seleno group attached thereto.
Exemplary "selenoethers" which may be substituted on the alkyl are
selected from one of --Se-alkyl, --Se-alkenyl, --Se-alkynyl, and
--Se--(CH.sub.2).sub.m--R61, m and R61 being defined above.
[0074] The terms triflyl, tosyl, mesyl, and nonaflyl are
art-recognized and refer to trifluoromethanesulfonyl,
p-toluenesulfonyl, methanesulfonyl, and nonafluorobutanesulfonyl
groups, respectively. The terms triflate, tosylate, mesylate, and
nonaflate are art-recognized and refer to trifluoromethanesulfonate
ester, p-toluenesulfonate ester, methanesulfonate ester, and
nonafluorobutanesulfonate ester functional groups and molecules
that contain said groups, respectively.
[0075] The abbreviations Me, Et, Ph, Tf, Nf, Ts, and Ms represent
methyl, ethyl, phenyl, trifluoromethanesulfonyl,
nonafluorobutanesulfonyl, p-toluenesulfonyl and methanesulfonyl,
respectively. A more comprehensive list of the abbreviations
utilized by organic chemists of ordinary skill in the art appears
in the first issue of each volume of the Journal of Organic
Chemistry; this list is typically presented in a table entitled
Standard List of Abbreviations.
[0076] Certain compounds contained in compositions described herein
may exist in particular geometric or stereoisomeric forms. In
addition, compounds may also be optically active. Contemplated
herein are all such compounds, including cis- and trans-isomers, R-
and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the
racemic mixtures thereof, and other mixtures thereof. Additional
asymmetric carbon atoms may be present in a substituent such as an
alkyl group. All such isomers, as well as mixtures thereof, are
encompassed herein.
[0077] If, for instance, a particular enantiomer of a compound is
desired, it may be prepared by asymmetric synthesis, or by
derivation with a chiral auxiliary, where the resulting
diastereomeric mixture is separated and the auxiliary group cleaved
to provide the pure desired enantiomers. Alternatively, where the
molecule contains a basic functional group, such as amino, or an
acidic functional group, such as carboxyl, diastereomeric salts are
formed with an appropriate optically-active acid or base, followed
by resolution of the diastereomers thus formed by fractional
crystallization or chromatographic means well known in the art, and
subsequent recovery of the pure enantiomers.
[0078] It will be understood that "substitution" or "substituted
with" includes the implicit proviso that such substitution is in
accordance with permitted valence of the substituted atom and the
substituent, and that the substitution results in a stable
compound, e.g., which does not spontaneously undergo transformation
such as by rearrangement, cyclization, elimination, or other
reaction.
[0079] The term "substituted" is also contemplated to include all
permissible substituents of organic compounds. In a broad aspect,
the permissible substituents include acyclic and cyclic, branched
and unbranched, carbocyclic and heterocyclic, aromatic and
nonaromatic substituents of organic compounds. Illustrative
substituents include, for example, those described herein above.
The permissible substituents may be one or more and the same or
different for appropriate organic compounds. Heteroatoms such as
nitrogen may have hydrogen substituents and/or any permissible
substituents of organic compounds described herein which satisfy
the valences of the heteroatoms. Compounds are not intended to be
limited in any manner by the permissible substituents of organic
compounds.
[0080] The chemical elements are identified in accordance with the
Periodic Table of the Elements, CAS version, Handbook of Chemistry
and Physics, 67th Ed., 1986-87, inside cover. Also, the term
"hydrocarbon" is contemplated to include all permissible compounds
having at least one hydrogen and one carbon atom. In a broad
aspect, the permissible hydrocarbons include acyclic and cyclic,
branched and unbranched, carbocyclic and heterocyclic, aromatic and
nonaromatic organic compounds that may be substituted or
unsubstituted.
[0081] The term "protecting group" is art-recognized and refers to
temporary substituents that protect a potentially reactive
functional group from undesired chemical transformations. Examples
of such protecting groups include esters of carboxylic acids, silyl
ethers of alcohols, and acetals and ketals of aldehydes and
ketones, respectively. The field of protecting group chemistry has
been reviewed by Greene and Wuts in Protective Groups in Organic
Synthesis (2.sup.nd ed., Wiley: N.Y., 1991).
[0082] The term "hydroxyl-protecting group" is art-recognized and
refers to those groups intended to protect a hydrozyl group against
undesirable reactions during synthetic procedures and includes, for
example, benzyl or other suitable esters or ethers groups known in
the art.
[0083] The term "carboxyl-protecting group" is art-recognized and
refers to those groups intended to protect a carboxylic acid group,
such as the C-terminus of an amino acid or peptide or an acidic or
hydroxyl azepine ring substituent, against undesirable reactions
during synthetic procedures and includes. Examples for protecting
groups for carboxyl groups involve, for example, benzyl ester,
cyclohexyl ester, 4-nitrobenzyl ester, t-butyl ester,
4-pyridylmethyl ester, and the like.
[0084] The term "amino-blocking group" is art-recognized and refers
to a group which will prevent an amino group from participating in
a reaction carried out on some other functional group, but which
can be removed from the amine when desired. Such groups are
discussed by in Ch. 7 of Greene and Wuts, cited above, and by
Barton, Protective Groups in Organic Chemistry ch. 2 (McOmie, ed.,
Plenum Press, New York, 1973). Examples of suitable groups include
acyl protecting groups such as, to illustrate, formyl, dansyl,
acetyl, benzoyl, trifluoroacetyl, succinyl, methoxysuccinyl, benzyl
and substituted benzyl such as 3,4-dimethoxybenzyl, o-nitrobenzyl,
and triphenylmethyl; those of the formula --COOR where R includes
such groups as methyl, ethyl, propyl, isopropyl,
2,2,2-trichloroethyl, 1-methyl-1-phenylethyl, isobutyl, t-butyl,
t-amyl, vinyl, allyl, phenyl, benzyl, p-nitrobenzyl, o-nitrobenzyl,
and 2,4-dichlorobenzyl; acyl groups and substituted acyl such as
formyl, acetyl, chloroacetyl, dichloroacetyl, trichloroacetyl,
trifluoroacetyl, benzoyl, and p-methoxybenzoyl; and other groups
such as methanesulfonyl, p-toluenesulfonyl, p-bromobenzenesulfonyl,
p-nitrophenylethyl, and p-toluenesulfonyl-aminocarbonyl. Preferred
amino-blocking groups are benzyl (--CH.sub.2C.sub.6H.sub.5), acyl
[C(O)R1] or SiR1.sub.3 where R1 is C.sub.1-C.sub.4 alkyl,
halomethyl, or 2-halo-substituted-(C.sub.2-C.sub.4 alkoxy),
aromatic urethane protecting groups as, for example,
carbonylbenzyloxy (Cbz); and aliphatic urethane protecting groups
such as t-butyloxycarbonyl (Boc) or 9-fluorenylmethoxycarbonyl
(FMOC).
[0085] The definition of each expression, e.g. lower alkyl, m, n, p
and the like, when it occurs more than once in any structure, is
intended to be independent of its definition elsewhere in the same
structure.
[0086] The term "electron-withdrawing group" is art-recognized, and
refers to the tendency of a substituent to attract valence
electrons from neighboring atoms, i.e., the substituent is
electronegative with respect to neighboring atoms. A quantification
of the level of electron-withdrawing capability is given by the
Hammett sigma (.sigma.) constant. This well known constant is
described in many references, for instance, March, Advanced Organic
Chemistry 251-59 (McGraw Hill Book Company: New York, 1977). The
Hammett constant values are generally negative for electron
donating groups (.sigma.(P)=-0.66 for NH.sub.2) and positive for
electron withdrawing groups (.sigma.(P)=0.78 for a nitro group),
.sigma.(P) indicating para substitution. Exemplary
electron-withdrawing groups include nitro, acyl, formyl, sulfonyl,
trifluoromethyl, cyano, chloride, and the like. Exemplary
electron-donating groups include amino, methoxy, and the like.
[0087] The term "small molecule" is art-recognized and refers to a
composition which has a molecular weight of less than about 2000
amu, or less than about 1000 amu, and even less than about 500 amu.
Small molecules may be, for example, nucleic acids, peptides,
polypeptides, peptide nucleic acids, peptidomimetics,
carbohydrates, lipids or other organic (carbon containing) or
inorganic molecules. Many pharmaceutical companies have extensive
libraries of chemical and/or biological mixtures, often fungal,
bacterial, or algal extracts, which can be screened with any of the
assays described herein. The term "small organic molecule" refers
to a small molecule that is often identified as being an organic or
medicinal compound, and does not include molecules that are
exclusively nucleic acids, peptides or polypeptides.
[0088] The term "modulation" is art-recognized and refers to up
regulation (i.e., activation or stimulation), down regulation
(i.e., inhibition or suppression) of a response, or the two in
combination or apart.
[0089] The term "treating" is art-recognized and refers to curing
as well as ameliorating at least one symptom of any condition or
disease.
[0090] The term "prophylactic" or "therapeutic" treatment is
art-recognized and refers to administration of a drug to a host. If
it is administered prior to clinical manifestation of the unwanted
condition (e.g., disease or other unwanted state of the host
animal) then the treatment is prophylactic, i.e., it protects the
host against developing the unwanted condition, whereas if
administered after manifestation of the unwanted condition, the
treatment is therapeutic (i.e., it is intended to diminish,
ameliorate or maintain the existing unwanted condition or side
effects therefrom).
[0091] A "patient," "subject" or "host" to be treated by the
subject method may mean either a human or non-human animal.
[0092] The term "mammal" is known in the art, and exemplary mammals
include humans, primates, bovines, porcines, canines, felines, and
rodents (e.g., mice and rats).
[0093] The term "bioavailable" when referring to a compound is
art-recognized and refers to a form of a compound that allows for
it, or a portion of the amount of compound administered, to be
absorbed by, incorporated to, or otherwise physiologically
available to a subject or patient to whom it is administered.
[0094] The term "pharmaceutically-acceptable salts" is
art-recognized and refers to the relatively non-toxic, inorganic
and organic acid addition salts of compounds, including, for
example, those contained in compositions described herein.
[0095] The term "pharmaceutically acceptable carrier" is
art-recognized and refers to a pharmaceutically-acceptable
material, composition or vehicle, such as a liquid or solid filler,
diluent, excipient, solvent or encapsulating material, involved in
carrying or transporting any subject composition or component
thereof from one organ, or portion of the body, to another organ,
or portion of the body. Each carrier must be "acceptable" in the
sense of being compatible with the subject composition and its
components and not injurious to the patient. Some examples of
materials which may serve as pharmaceutically acceptable carriers
include: (1) sugars, such as lactose, glucose and sucrose; (2)
starches, such as corn starch and potato starch; (3) cellulose, and
its derivatives, such as sodium carboxymethyl cellulose, ethyl
cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt;
(6) gelatin; (7) talc; (8) excipients, such as cocoa butter and
suppository waxes; (9) oils, such as peanut oil, cottonseed oil,
safflower oil, sesame oil, olive oil, corn oil and soybean oil;
(10) glycols, such as propylene glycol; (11) polyols, such as
glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,
such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering
agents, such as magnesium hydroxide and aluminum hydroxide; (15)
alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18)
Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer
solutions; and (21) other non-toxic compatible substances employed
in pharmaceutical formulations.
[0096] The terms "systemic administration," "administered
systemically," "peripheral administration" and "administered
peripherally" are art-recognized and refer to the administration of
a subject composition, therapeutic or other material other than
directly into the central nervous system, such that it enters the
patient's system and, thus, is subject to metabolism and other like
processes.
[0097] The terms "parenteral administration" and "administered
parenterally" are art-recognized and refer to modes of
administration other than enteral and topical administration,
usually by injection, and includes, without limitation,
intravenous, intramuscular, intraarterial, intrathecal,
intracapsular, intraorbital, intracardiac, intradermal,
intraperitoneal, transtracheal, subcutaneous, subcuticular,
intra-articulare, subcapsular, subarachnoid, intraspinal, and
intrastemal injection and infusion.
Exemplary Methods and Compositions
[0098] Provided herein are methods for activating a sirtuin
deacetylase protein family member (referred to as "sirtuin
protein"). The methods may comprise contacting the sirtuin
deacetylase protein family member with a compound, such as a
polyphenol, e.g. a plant polyphenol, and referred to herein as
"activation compound" or "activating compound." Exemplary sirtuin
deacetylase proteins include the yeast silent information regulator
2 (Sir2) and human SIRT1. Other family members include proteins
having a significant amino acid sequence homology and biological
activity, e.g., the ability to deacetylate target proteins, such as
histones and p53, to those of Sir2 and SIRT1.
[0099] Exemplary activating compounds are those selected from the
group consisting of flavones, stilbenes, flavanones, isoflavanones,
catechins, chalcones, tannins and anthocyanidins. Exemplary
stilbenes include hydroxystilbenes, such as trihydroxystilbenes,
e.g., 3,5,4'-trihydroxystilbene ("resveratrol"). Resveratrol is
also known as 3,4'5-stilbenetriol. Tetrahydroxystilbenes, e.g.,
piceatannol, are also encompassed. Hydroxychalones including
trihydroxychalones, such as isoliquiritigenin, and
tetrahydroxychalones, such as butein, can also be used.
Hydroxyflavones including tetrahydroxyflavones, such as fisetin,
and pentahydroxyflavones, such as quercetin, can also be used.
[0100] In one embodiment, methods for activating a sirtuin protein
comprise an activating compound that is a stilbene or chalcone
compound of formula 1: ##STR15## wherein, independently for each
occurrence,
[0101] R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R'.sub.1,
R'.sub.2, R'.sub.3, R'.sub.4, and R'.sub.5 represent H, alkyl,
aryl, heteroaryl, alkaryl, heteroaralkyl, halide, NO.sub.2, SR, OR,
N(R).sub.2, or carboxyl;
[0102] R represents H, alkyl, or aryl;
[0103] M represents O, NR, or S;
[0104] A-B represents a bivalent alkyl, alkenyl, alkynyl, amido,
sulfonamido, diazo, ether, alkylamino, alkylsulfide, or hydrazine
group; and
[0105] n is 0 or 1.
[0106] In a further embodiment, the methods comprise a compound of
formula 1 and the attendant definitions, wherein n is 0. In a
further embodiment, the methods comprise a compound of formula 1
and the attendant definitions, wherein n is 1. In a further
embodiment, the methods comprise a compound of formula 1 and the
attendant definitions, wherein A-B is ethenyl. In a further
embodiment, the methods comprise a compound of formula 1 and the
attendant definitions, wherein A-B is
--CH.sub.2CH(Me)CH(Me)CH.sub.2--. In a further embodiment, the
methods comprise a compound of formula 1 and the attendant
definitions, wherein M is O. In a further embodiment, the methods
comprises a compound of formula 1 and the attendant definitions,
wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R'.sub.1,
R'.sub.2, R'.sub.3, R'.sub.4, and R'.sub.5 are H. In a further
embodiment, the method comprise a compound of formula 1 and the
attendant definitions, wherein R.sub.2, R.sub.4, and R'.sub.3 are
OH. In a further embodiment, the methods comprise a compound of
formula 1 and the attendant definitions, wherein R.sub.2, R.sub.4,
R'.sub.2 and R'.sub.3 are OH. In a further embodiment, the methods
comprise a compound of formula 1 and the attendant definitions,
wherein R.sub.3, R.sub.5, R'.sub.2 and R'.sub.3 are OH. In a
further embodiment, the methods comprise a compound of formula 1
and the attendant definitions, wherein R.sub.1, R.sub.3, R.sub.5,
R'.sub.2 and R'.sub.3 are OH. In a further embodiment, the methods
comprise a compound of formula 1 and the attendant definitions,
wherein R.sub.2 and R'.sub.2 are OH; R.sub.4 is
O-.beta.-D-glucoside; and R'.sub.3 is OCH.sub.3. In a further
embodiment, the methods comprise a compound of formula 1 and the
attendant definitions, wherein R.sub.2 is OH; R.sub.4 is
O-.beta.-D-glucoside; and R'.sub.3 is OCH.sub.3.
[0107] In a further embodiment, the methods comprise a compound of
formula 1 and the attendant definitions, wherein n is 0; A-B is
ethenyl; and R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R'.sub.1,
R'.sub.2, R'.sub.3, R'.sub.4, and R'.sub.5 are H (trans stilbene).
In a further embodiment, the methods comprise a compound of formula
1 and the attendant definitions, wherein n is 1; A-B is ethenyl; M
is O; and R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R'.sub.1,
R'.sub.2, R'.sub.3, R'.sub.4, and R'.sub.5 are H (chalcone). In a
further embodiment, the methods comprise a compound of formula 1
and the attendant definitions, wherein n is 0; A-B is ethenyl;
R.sub.2, R.sub.4, and R'.sub.3 are OH; and R.sub.1, R.sub.3,
R.sub.5, R'.sub.1, R'.sub.2, R'.sub.4, and R'.sub.5 are H
(resveratrol). In a further embodiment, the methods comprise a
compound of formula 1 and the attendant definitions, wherein n is
0; A-B is ethenyl; R.sub.2, R.sub.4, R'.sub.2 and R'.sub.3 are OH;
and R.sub.1, R.sub.3, R.sub.5, R'.sub.1, R'.sub.4 and R'.sub.5 are
H (piceatannol). In a further embodiment, the methods comprise a
compound of formula 1 and the attendant definitions, wherein n is
1; A-B is ethenyl; M is O; R.sub.3, R.sub.5, R'.sub.2 and R'.sub.3
are OH; and R.sub.1, R.sub.2, R.sub.4, R'.sub.1, R'.sub.4, and
R'.sub.5 are H (butein). In a further embodiment, the methods
comprise a compound of formula 1 and the attendant definitions,
wherein n is 1; A-B is ethenyl; M is O; R.sub.1, R.sub.3, R.sub.5,
R'.sub.2 and R'.sub.3 are OH; and R.sub.2, R.sub.4, R'.sub.1,
R'.sub.4, and R'.sub.5 are H (3,4,2',4',6'-pentahydroxychalcone).
In a further embodiment, the methods comprise a compound of formula
1 and the attendant definitions, wherein n is 0; A-B is ethenyl;
R.sub.2 and R'.sub.2 are OH, R.sub.4 is O-.beta.-D-glucoside,
R'.sub.3 is OCH.sub.3; and R.sub.1, R.sub.3, R.sub.5, R'.sub.1,
R'.sub.4, and R'.sub.5 are H (rhapontin). In a further embodiment,
the methods comprise a compound of formula 1 and the attendant
definitions, wherein n is 0; A-B is ethenyl; R.sub.2 is OH, R.sub.4
is O-.beta.-D-glucoside, R'.sub.3 is OCH.sub.3; and R.sub.1,
R.sub.3, R.sub.5, R'.sub.1, R'.sub.2, R'.sub.4, and R'.sub.5 are H
(deoxyrhapontin). In a further embodiment, the methods comprise a
compound of formula 1 and the attendant definitions, wherein n is
0; A-B is --CH.sub.2CH(Me)CH(Me)CH.sub.2--; R.sub.2, R.sub.3,
R'.sub.2, and R'.sub.3 are OH; and R.sub.1, R.sub.4, R.sub.5,
R'.sub.1, R'.sub.4, and R'.sub.5 are H (NDGA).
[0108] In another embodiment, methods for activating a sirtuin
protein comprise an activating compound that is a flavanone
compound of formula 2: ##STR16##
[0109] wherein, independently for each occurrence,
[0110] R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R'.sub.1,
R'.sub.2, R'.sub.3, R'.sub.4, R'.sub.5, and R'' represent H, alkyl,
aryl, heteroaryl, alkaryl, heteroaralkyl, halide, NO.sub.2, SR, OR,
N(R).sub.2, or carboxyl;
[0111] R represents H, alkyl, or aryl;
[0112] M represents H.sub.2, O, NR, or S;
[0113] Z represents CR, O, NR, or S; and
[0114] X represents CR or N; and
[0115] Y represents CR or N.
[0116] In a further embodiment, the methods comprise a compound of
formula 2 and the attendant definitions, wherein X and Y are both
CH. In a further embodiment, the methods comprise a compound of
formula 2 and the attendant definitions, wherein M is O. In a
further embodiment, the methods comprise a compound of formula 2
and the attendant definitions, wherein. M is H.sub.2. In a further
embodiment, the methods comprise a compound of formula 2 and the
attendant definitions, wherein Z is O. In a further embodiment, the
methods comprise a compound of formula 2 and the attendant
definitions, wherein R'' is H. In a further embodiment, the methods
comprise a compound of formula 2 and the attendant definitions,
wherein R'' is OH. In a further embodiment, the methods comprise a
compound of formula 2 and the attendant definitions, wherein R'' is
an ester. In a further embodiment, the methods comprise a compound
of formula 2 and the attendant definitions, wherein R.sub.1 is
##STR17## In a further embodiment, the methods comprise a compound
of formula 2 and the attendant definitions, wherein R.sub.1,
R.sub.2, R.sub.3, R.sub.4, R'.sub.1, R'.sub.2, R'.sub.3, R'.sub.4,
R'.sub.5 and R'' are H. In a further embodiment, the methods
comprise a compound of formula 2 and the attendant definitions,
wherein R.sub.2, R.sub.4, and R'.sub.3 are OH. In a further
embodiment, the methods comprise a compound of formula 2 and the
attendant definitions, wherein R.sub.4, R'.sub.2, R'.sub.3, and R''
are OH. In a further embodiment, the methods comprise a compound of
formula 2 and the attendant definitions, wherein R.sub.2, R.sub.4,
R'.sub.2, R'.sub.3, and R'' are OH. In a further embodiment, the
methods comprise a compound of formula 2 and the attendant
definitions, wherein R.sub.2, R.sub.4, R'.sub.2, R'.sub.3,
R'.sub.4, and R'' are OH.
[0117] In a further embodiment, the methods comprise a compound of
formula 2 and the attendant definitions, wherein X and Y are CH; M
is O; Z and O; R'' is H; and R.sub.1, R.sub.2, R.sub.3, R.sub.4,
R'.sub.1, R'.sub.2, R'.sub.3, R'.sub.4, R'.sub.5 and R'' are H
(flavanone). In a further embodiment, the methods comprise a
compound of formula 2 and the attendant definitions, wherein X and
Y are CH; M is O; Z and O; R'' is H; R.sub.2, R.sub.4, and R'.sub.3
are OH; and R.sub.1, R.sub.3, R'.sub.1, R'.sub.2, R'.sub.4, and
R'.sub.5 are H (naringenin). In a further embodiment, the methods
comprise a compound of formula 2 and the attendant definitions,
wherein X and Y are CH; M is O; Z and O; R'' is OH; R.sub.2,
R.sub.4, R'.sub.2, and R'.sub.3 are OH; and R.sub.1, R.sub.3,
R'.sub.1, R'.sub.4, and R'.sub.5 are H
(3,5,7,3',4'-pentahydroxyflavanone). In a further embodiment, the
methods comprise a compound of formula 2 and the attendant
definitions, wherein X and Y are CH; M is H.sub.2; Z and O; R'' is
OH; R.sub.2, R.sub.4, R'.sub.2, and R'.sub.3, are OH; and R.sub.1,
R.sub.3, R'.sub.1, R'.sub.4 and R'.sub.5 are H (epicatechin). In a
further embodiment, the methods comprise a compound of formula 2
and the attendant definitions, wherein X and Y are CH; M is
H.sub.2; Z and O; R'' is OH; R.sub.2, R.sub.4, R'.sub.2, R'.sub.3,
and R'.sub.4 are OH; and R.sub.1, R.sub.3, R'.sub.1, and R'.sub.5
are H (gallocatechin). In a further embodiment, the methods
comprise a compound of formula 2 and the attendant definitions,
wherein X and Y are CH; M is H.sub.2; Z and O; R'' is ##STR18##
R.sub.2, R.sub.4, R'.sub.2, R'.sub.3, R'.sub.4, and R'' are OH; and
R.sub.1, R.sub.3, R'.sub.1, and R'.sub.5 are H (epigallocatechin
gallate).
[0118] In another embodiment, methods for activating a sirtuin
protein comprise an activating compound that is an iso flavanone
compound of formula 3: ##STR19##
[0119] wherein, independently for each occurrence,
[0120] R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R'.sub.1,
R'.sub.2, R'.sub.3, R'.sub.4, R'.sub.5, and R''.sub.1, represent H,
alkyl, aryl, heteroaryl, alkaryl, heteroaralkyl, halide, NO.sub.2,
SR, OR, N(R).sub.2, or carboxyl;
[0121] R represents H, alkyl, or aryl;
[0122] M represents H.sub.2, O, NR, or S;
[0123] Z represents CR, O, NR, or S; and
[0124] X represents CR or N; and
[0125] Y represents CR or N.
[0126] In another embodiment, methods for activating a sirtuin
protein comprise an activating compound that is a flavone compound
of formula 4: ##STR20##
[0127] wherein, independently for each occurrence,
[0128] R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R'.sub.1,
R'.sub.2, R'.sub.3, R'.sub.4, and R'.sub.5, represent H, alkyl,
aryl, heteroaryl, alkaryl, heteroaralkyl, halide, NO.sub.2, SR, OR,
N(R).sub.2, or carboxyl;
[0129] R'' is absent or represents H, alkyl, aryl, heteroaryl,
alkaryl, heteroaralkyl, halide, NO.sub.2, SR, OR, N(R).sub.2, or
carboxyl;
[0130] R represents H, alkyl, or aryl;
[0131] M represents H.sub.2, O, NR, or S;
[0132] Z represents CR, O, NR, or S; and
[0133] X represents CR or N when R'' is absent or C when R'' is
present.
[0134] In a further embodiment, the methods comprise a compound of
formula 4 and the attendant definitions, wherein X is C. In a
further embodiment, the methods comprise a compound of formula 4
and the attendant definitions, wherein X is CR. In a further
embodiment, the methods comprise a compound of formula 4 and the
attendant definitions, wherein Z is O. In a further embodiment, the
methods comprise a compound of formula 4 and the attendant
definitions, wherein M is O. In a further embodiment, the methods
comprise a compound of formula 4 and the attendant definitions,
wherein R'' is H. In a further embodiment, the methods comprise a
compound of formula 4 and the attendant definitions, wherein R'' is
OH. In a further embodiment, the methods comprise a compound of
formula 4 and the attendant definitions, wherein R.sub.1, R.sub.2,
R.sub.3, R.sub.4, R.sub.5, R'.sub.1, R'.sub.2, R'.sub.3, R'.sub.4,
and R'.sub.5 are H. In a further embodiment, the methods comprise a
compound of formula 4 and the attendant definitions, wherein
R.sub.2, R'.sub.2, and R'.sub.3 are OH. In a further embodiment,
the methods comprise a compound of formula 4 and the attendant
definitions, wherein R.sub.2, R.sub.4, R'.sub.2, R'.sub.3, and
R'.sub.4 are OH. In a further embodiment, the methods comprise a
compound of formula 4 and the attendant definitions, wherein
R.sub.2, R.sub.4, R'.sub.2, and R'.sub.3 are OH. In a further
embodiment, the methods comprise a compound of formula 4 and the
attendant definitions, wherein R.sub.3, R'.sub.2, and R'.sub.3 are
OH. In a further embodiment, the methods comprise a compound of
formula 4 and the attendant definitions, wherein R.sub.2, R.sub.4,
R'.sub.2, and R'.sub.3 are OH. In a further embodiment, the methods
comprise a compound of formula 4 and the attendant definitions,
wherein R.sub.2, R'.sub.2, R'.sub.3, and R'.sub.4 are OH. In a
further embodiment, the methods comprise a compound of formula 4
and the attendant definitions, wherein R.sub.2, R.sub.4, and
R'.sub.3 are OH. In a further embodiment, the methods comprise a
compound of formula 4 and the attendant definitions, wherein
R.sub.2, R.sub.3, R.sub.4, and R'.sub.3 are OH. In a further
embodiment, the methods comprise a compound of formula 4 and the
attendant definitions, wherein R.sub.2, R.sub.4, and R'.sub.3 are
OH. In a further embodiment, the methods comprise a compound of
formula 4 and the attendant definitions, wherein R.sub.3, R'.sub.1,
and R'.sub.3 are OH. In a further embodiment, the methods comprise
a compound of formula 4 and the attendant definitions, wherein
R.sub.2 and R'.sub.3 are OH. In a further embodiment, the methods
comprise a compound of formula 4 and the attendant definitions,
wherein R.sub.1, R.sub.2, R'.sub.2, and R'.sub.3 are OH. In a
further embodiment, the methods comprise a compound of formula 4
and the attendant definitions, wherein R.sub.3, R'.sub.1, and
R'.sub.2 are OH. In a further embodiment, the methods comprise a
compound of formula 4 and the attendant definitions, wherein
R'.sub.3 is OH. In a further embodiment, the methods comprise a
compound of formula 4 and the attendant definitions, wherein
R.sub.4 and R'.sub.3 are OH. In a further embodiment, the methods
comprise a compound of formula 4 and the attendant definitions,
wherein R.sub.2 and R.sub.4 are OH. In a further embodiment, the
methods comprise a compound of formula 4 and the attendant
definitions, wherein R.sub.2, R.sub.4, R'.sub.1, and R'.sub.3 are
OH. In a further embodiment, the methods comprise a compound of
formula 4 and the attendant definitions, wherein R.sub.4 is OH. In
a further embodiment, the methods comprise a compound of formula 4
and the attendant definitions, wherein R.sub.2, R.sub.4, R'.sub.2,
R'.sub.3, and R'.sub.4 are OH. In a further embodiment, the methods
comprise a compound of formula 4 and the attendant definitions,
wherein R.sub.2, R'.sub.2, R'.sub.3, and R'.sub.4 are OH. In a
further embodiment, the methods comprise a compound of formula 4
and the attendant definitions, wherein R.sub.1, R.sub.2, R.sub.4,
R'.sub.2, and R'.sub.3 are OH.
[0135] In a further embodiment, the methods comprise a compound of
formula 4 and the attendant definitions, wherein X is CH; R'' is
absent; Z is O; M is O; and R.sub.1, R.sub.2, R.sub.3, R.sub.4,
R.sub.5, R'.sub.1, R'.sub.2, R'.sub.3, R'.sub.4, and R'.sub.5 are H
(flavone). In a further embodiment, the methods comprise a compound
of formula 4 and the attendant definitions, wherein X is C; R'' is
OH; Z is O; M is O; R.sub.2, R'.sub.2, and R'.sub.3 are OH; and
R.sub.1, R.sub.3, R.sub.4, R'.sub.1, R'.sub.4, and R'.sub.5 are H
(fisetin). In a further embodiment, the methods comprise a compound
of formula 4 and the attendant definitions, wherein X is CH; R'' is
absent; Z is O; M is O; R.sub.2, R.sub.4, R'.sub.2, R'.sub.3, and
R'.sub.4 are OH; and R.sub.1, R.sub.3, R'.sub.1, and R'.sub.5 are H
(5,7,3',4',5'-pentahydroxyflavone). In a further embodiment, the
methods comprise a compound of formula 4 and the attendant
definitions, wherein X is CH; R'' is absent; Z is O; M is O;
R.sub.2, R.sub.4, R'.sub.2, and R'.sub.3 are OH; and R.sub.1,
R.sub.3, R'.sub.1, R'.sub.4, and R'.sub.5 are H (luteolin). In a
further embodiment, the methods comprise a compound of formula 4
and the attendant definitions, wherein X is C, R'' is OH; Z is O; M
is O; R.sub.3, R'.sub.2, and R'.sub.3 are OH; and R.sub.1, R.sub.2,
R.sub.4, R'.sub.1, R'.sub.4, and R'.sub.5 are H
(3,6,3',4'-tetrahydroxyflavone). In a further embodiment, the
methods comprise a compound of formula 4 and the attendant
definitions, wherein X is C, R'' is OH;Zis O; M is O; R.sub.2,
R.sub.4, R'.sub.2, and R'.sub.3 are OH; and R.sub.1, R.sub.3,
R'.sub.1, R'.sub.4, and R'.sub.5 are H (quercetin). In a further
embodiment, the methods comprise a compound of formula 4 and the
attendant definitions, wherein X is CH; R'' is absent; Z is O; M is
O; R.sub.2, R'.sub.2, R'.sub.3, and R'.sub.4 are OH; and R.sub.1,
R.sub.3, R.sub.4, R'.sub.1, and R'.sub.5 are H. In a further
embodiment, the methods comprise a compound of formula 4 and the
attendant definitions, wherein X is C; R'' is OH; Z is O; M is O;
R.sub.2, R.sub.4, and R'.sub.3 are OH; and R.sub.1, R.sub.3,
R'.sub.1, R'.sub.2, R'.sub.4, and R'.sub.5 are H. In a further
embodiment, the methods comprise a compound of formula 4 and the
attendant definitions, wherein X is CH; R'' is absent; Z is O; M is
O; R.sub.2, R.sub.3, R.sub.4, and R'.sub.3 are OH; and R.sub.1,
R'.sub.1, R'.sub.2, R'.sub.4, and R'.sub.5 are H. In a further
embodiment, the methods comprise a compound of formula 4 and the
attendant definitions, wherein X is CH; R'' is absent; Z is O; M is
O; R.sub.2, R.sub.4, and R'.sub.3 are OH; and R.sub.1, R.sub.3,
R'.sub.1, R'.sub.2, R'.sub.4, and R'.sub.5 are H. In a embodiment,
the methods comprise a compound of formula 4 and the attendant
definitions, wherein X is C, R'' is OH; Z is O; M is O; R.sub.3,
R'.sub.1, and R'.sub.3 are OH; and R.sub.1, R.sub.2, R.sub.4,
R'.sub.2, R'.sub.4, and R'.sub.5 are H. In a further embodiment,
the methods comprise a compound of formula 4 and the attendant
definitions, wherein X is CH; R'' is absent; Z is O; M is O;
R.sub.2 and R'.sub.3 are OH; and R.sub.1, R.sub.3, R.sub.4,
R'.sub.1, R'.sub.2, R'.sub.4, and R'.sub.5 are H. In a further
embodiment, the methods comprise a compound of formula 4 and the
attendant definitions, wherein X is C, R'' is OH; Z is O; M is O;
R.sub.1, R.sub.2, R'.sub.2, and R'.sub.3 are OH; and R.sub.1,
R.sub.2, R.sub.4, R'.sub.3, R'.sub.4, and R'.sub.5 are H. In a
further embodiment, the methods comprise a compound of formula 4
and the attendant definitions, wherein X is C; R'' is OH; Z is O; M
is O; R.sub.3, R'.sub.1, and R'.sub.2 are OH; and R.sub.1, R.sub.2,
R.sub.4; R'.sub.3, R'.sub.4, and R'.sub.5 are H. In a further
embodiment, the methods comprise a compound of formula 4 and the
attendant definitions, wherein X is CH; R'' is absent; Z is O; M is
O; R'.sub.3 is OH; and R.sub.1, R.sub.2, R.sub.3, R.sub.4,
R'.sub.1, R'.sub.2, R'.sub.4, and R'.sub.5 are H. In a further
embodiment, the methods comprise a compound of formula 4 and the
attendant definitions, wherein X is CH; R'' is absent; Z is O; M is
O; R.sub.4 and R'.sub.3 are OH; and R.sub.1, R.sub.2, R.sub.3,
R'.sub.1, R'.sub.2, R'.sub.4, and R'.sub.5 are H. In a further
embodiment, the methods comprise a compound of formula 4 and the
attendant definitions, wherein X is CH; R'' is absent; Z is O; M is
O; R.sub.2 and R.sub.4 are OH; and R.sub.1, R.sub.3, R'.sub.1,
R'.sub.2, R'.sub.3, R'.sub.4, and R'.sub.5 are H. In a further
embodiment, the methods comprise a compound of formula 4 and the
attendant definitions, wherein X is C; R'' is OH; Z is O; M is O;
R.sub.2, R.sub.4, R'.sub.1, and R'.sub.3 are OH; and R.sub.1,
R.sub.3, R'.sub.2, R'.sub.4, and R'.sub.5 are H. In a further
embodiment, the methods comprise a compound of formula 4 and the
attendant definitions, wherein X is CH; R'' is absent; Z is O; M is
O; R.sub.4 is OH; and R.sub.1, R.sub.2, R.sub.3, R'.sub.1,
R'.sub.2, R'.sub.3, R'.sub.4, and R'.sub.5 are H. In a further
embodiment, the methods comprise a compound of formula 4 and the
attendant definitions, wherein X is C; R'' is OH; Z is O; M is O;
R.sub.2, R.sub.4, R'.sub.2, R'.sub.3, and R'.sub.4 are OH; and
R.sub.1, R.sub.3, R'.sub.1, and R'.sub.5 are H. In a further
embodiment, the methods comprise a compound of formula 4 and the
attendant definitions, wherein X is C; R'' is OH; Z is O; M is O;
R.sub.2, R'.sub.2, R'.sub.3, and R'.sub.4 are OH; and R.sub.1,
R.sub.3, R.sub.4, R'.sub.1, and R'.sub.5 are H. In a further
embodiment, the methods comprise a compound of formula 4 and the
attendant definitions, wherein X is C; R'' is OH; Z is O; M is O;
R.sub.1, R.sub.2, R.sub.4, R'.sub.2, and R'.sub.3 are OH; and
R.sub.3, R'.sub.1, R'.sub.4, and R'.sub.5 are H.
[0136] In another embodiment, methods for activating a sirtuin
protein comprise an activating compound that is an iso flavone
compound of formula 5: ##STR21##
[0137] wherein, independently for each occurrence,
[0138] R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R'.sub.1,
R'.sub.2, R'.sub.3, R'.sub.4, and R'.sub.5, represent H, alkyl,
aryl, heteroaryl, alkaryl, heteroaralkyl, halide, NO.sub.2, SR, OR,
N(R).sub.2, or carboxyl;
[0139] R'' is absent or represents H, alkyl, aryl, heteroaryl,
alkaryl, heteroaralkyl, halide, NO.sub.2, SR, OR, N(R).sub.2, or
carboxyl;
[0140] R represents H, alkyl, or aryl;
[0141] M represents H.sub.2, O, NR, or S;
[0142] Z represents CR, O, NR, or S; and
[0143] Y represents CR or N when R'' is absent or C when R'' is
present.
[0144] In a further embodiment, the methods comprise a compound of
formula 5 and the attendant definitions, wherein Y is CR. In a
further embodiment, the methods comprise a compound of formula 5
and the attendant definitions, wherein Y is CH. In a further
embodiment, the methods comprise a compound of formula 5 and the
attendant definitions, wherein Z is O. In a further embodiment, the
methods comprise a compound of formula 5 and the attendant
definitions, wherein M is O. In a further embodiment, the methods
comprise a compound of formula 5 and the attendant definitions,
wherein R.sub.2 and R'.sub.3 are OH. In a further embodiment, the
methods comprise a compound of formula 5 and the attendant
definitions, wherein R.sub.2, R.sub.4, and R'.sub.3 are OH.
[0145] In a further embodiment, the methods comprise a compound of
formula 5 and the attendant definitions, wherein Y is CH; R'' is
absent; Z is O; M is O; R.sub.2 and R'.sub.3 are OH; and R.sub.1,
R.sub.3, R.sub.4, R'.sub.1, R'.sub.2, R'.sub.4, and R'.sub.5 are H.
In a further embodiment, the methods comprise a compound of formula
5 and the attendant definitions, wherein Y is CH; R'' is absent; Z
is O; M is O; R.sub.2, R.sub.4, and R'.sub.3 are OH; and R.sub.1,
R.sub.3, R'.sub.1, R'.sub.2, R'.sub.4, and R'.sub.5 and H.
[0146] In another embodiment, methods for activating a sirtuin
protein comprise an activating compound that is an anthocyanidin
compound of formula 6: ##STR22##
[0147] wherein, independently for each occurrence,
[0148] R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8,
R'.sub.2, R'.sub.3, R'.sub.4, R'.sub.5, and R'.sub.6 represent H,
alkyl, aryl, heteroaryl, alkaryl, heteroaralkyl, halide, NO.sub.2,
SR, OR, N(R).sub.2, or carboxyl;
[0149] R represents H, alkyl, or aryl; and
[0150] A.sup.- represents an anion selected from the following:
Cl.sup.-, Br.sup.-, or I.sup.-.
[0151] In a further embodiment, the methods comprise a compound of
formula 6 and the attendant definitions, wherein A.sup.- is
Cl.sup.-. In a further embodiment, the methods comprise a compound
of formula 6 and the attendant definitions, wherein R.sub.3,
R.sub.5, R.sub.7, and R'.sub.4 are OH. In a further embodiment, the
methods comprise a compound of formula 6 and the attendant
definitions, wherein R.sub.3, R.sub.5, R.sub.7, R'.sub.3, and
R'.sub.4 are OH. In a further embodiment, the methods comprise a
compound of formula 6 and the attendant definitions, wherein
R.sub.3, R.sub.5, R.sub.7, R'.sub.3, R'.sub.4, and R'.sub.5 are
OH.
[0152] In a further embodiment, the methods comprise a compound of
formula 6 and the attendant definitions, wherein A.sup.- is
Cl.sup.-; R.sub.3, R.sub.5, R.sub.7, and R'.sub.4 are OH; and
R.sub.4, R.sub.6, R.sub.8, R'.sub.2, R'.sub.3, R'.sub.5, and
R'.sub.6 are H. In a further embodiment, the methods comprise a
compound of formula 6 and the attendant definitions, wherein
A.sup.-is Cl.sup.-; R.sub.3, R.sub.5, R.sub.7, R'.sub.3, and
R'.sub.4 are OH; and R.sub.4, R.sub.6, R.sub.8, R'.sub.2, R'.sub.5,
and R'.sub.6 are H. In a further embodiment, the methods comprise a
compound of formula 6 and the attendant definitions, wherein
A.sup.- is Cl.sup.-; R.sub.3, R.sub.5, R.sub.7, R'.sub.3, R'.sub.4,
and R'.sub.5 are OH; and R.sub.4, R.sub.6, R.sub.8, R'.sub.2, and
R'.sub.6 are H.
[0153] Methods for activating a sirtuin protein may also comprise a
stilbene, chalcone, or flavone compound represented by formula 7:
##STR23##
[0154] wherein, independently for each occurrence,
[0155] M is absent or O;
[0156] R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R'.sub.1,
R'.sub.2, R'.sub.3, R'.sub.4, and R'.sub.5 represent H, alkyl,
aryl, heteroary alkaryl, heteroaralkyl, halide, NO.sub.2, SR, OR,
N(R).sub.2, or carboxyl;
[0157] R.sub.a represents H or the two R.sub.a form a bond;
[0158] R represents H, alkyl, or aryl; and
[0159] n is 0 or 1.
[0160] In a further embodiment, the methods comprise an activating
compound represented by formula 7 and the attendant definitions,
wherein n is 0. In a further embodiment, the methods comprise an
activating compound represented by formula 7 and the attendant
definitions, wherein n is 1. In a further embodiment, the methods
comprise an activating compound represented by formula 7 and the
attendant definitions, wherein M is absent. In a further
embodiment, the methods comprise an activating compound represented
by formula 7 and the attendant definitions, wherein M is O. In a
further embodiment, the methods comprise an activating compound
represented by formula 7 and the attendant definitions, wherein
R.sub.a is H. In a further embodiment, the methods comprise an
activating compound represented by formula 7 and the attendant
definitions, wherein M is O and the two R.sub.a form a bond.
[0161] In a further embodiment, the methods comprise an activating
compound represented by formula 7 and the attendant definitions,
wherein R.sub.5 is H. In a further embodiment, the methods comprise
an activating compound represented by formula 7 and the attendant
definitions, wherein R.sub.5 is OH. In a further embodiment, the
methods comprise an activating compound represented by formula 7
and the attendant definitions, wherein R.sub.1, R.sub.3, and
R'.sub.3 are OH. In a further embodiment, the methods comprise an
activating compound represented by formula 7 and the attendant
definitions, wherein R.sub.2, R.sub.4, R'.sub.2, and R'.sub.3 are
OH. In a further embodiment, the methods comprise an activating
compound represented by formula 7 and the attendant definitions,
wherein R.sub.2, R'.sub.2, and R'.sub.3 are OH. In a further
embodiment, the methods comprise an activating compound represented
by formula 7 and the attendant definitions, wherein R.sub.2 and
R.sub.4 are OH.
[0162] In a further embodiment, the methods include contacting a
cell with an activating compound represented by formula 7 and the
attendant definitions, wherein n is 0; M is absent; R.sub.a is H;
R.sub.5 is H; R.sub.1, R.sub.3, and R'.sub.3 are OH; and R.sub.2,
R.sub.4, R'.sub.1, R'.sub.2, R'.sub.4, and R'.sub.5 are H. In a
further embodiment, the methods comprise an activating compound
represented by formula 7 and the attendant definitions, wherein n
is 1; M is absent; R.sub.a is H; R.sub.5 is H; R.sub.2, R.sub.4,
R'.sub.2, and R'.sub.3 are OH; and R.sub.1, R.sub.3, R'.sub.1,
R'.sub.4, and R'.sub.5 are H. In a further embodiment, the methods
comprise an activating compound represented by formula 7 and the
attendant definitions, wherein n is 1; M is O; the two R.sub.a form
a bond; R.sub.5 is OH; R.sub.2, R'.sub.2, and R'.sub.3 are OH; and
R.sub.1, R.sub.3, R.sub.4, R'.sub.1, R'.sub.4, and R'.sub.5 are
H.
[0163] Other compounds for activating sirtuin deacetylase protein
family members include compounds having a formula selected from the
group consisting of formulas 8-25 and 30 set forth below. ##STR24##
##STR25## ##STR26## ##STR27##
[0164] R.dbd.H, alkyl, aryl, heterocyclyl, or heteroaryl
[0165] R'.dbd.H, halogen, NO.sub.2, SR, OR, NR.sub.2, alkyl, aryl,
or carboxy ##STR28##
[0166] R.dbd.H, alkyl, aryl, heterocyclyl, or heteroaryl
##STR29##
[0167] wherein, independently for each occurrence,
[0168] R'.dbd.H, halogen, NO.sub.2, SR, OR, NR.sub.2, alkyl, aryl,
or carboxy
[0169] R.dbd.H, alkyl, aryl, heterocyclyl, or heteroaryl
##STR30##
[0170] wherein, independently for each occurrence,
[0171] L represents CR.sub.2, O, NR, or S;
[0172] R represents H, alkyl, aryl, aralkyl, or heteroaralkyl;
and
[0173] R' represents H, halogen, NO.sub.2, SR, OR, NR.sub.2, alkyl,
aryl, or carboxy. ##STR31##
[0174] wherein, independently for each occurrence,
[0175] L represents CR.sub.2, O, NR, or S;
[0176] W represents CR or N;
[0177] R represents H, alkyl, aryl, aralkyl, or heteroaralkyl;
[0178] Ar represents a fused aryl or heteroaryl ring; and
[0179] R' represents H, halogen, NO.sub.2, SR, OR, NR.sub.2, alkyl,
aryl, or carboxy. ##STR32##
[0180] wherein, independently for each occurrence,
[0181] L represents CR.sub.2, O, NR, or S;
[0182] R represents H, alkyl, aryl, aralkyl, or heteroaralkyl;
and
[0183] R' represents H, halogen, NO.sub.2, SR, OR, NR.sub.2, alkyl,
aryl, or carboxy. ##STR33##
[0184] wherein, independently for each occurrence,
[0185] L represents CR.sub.2, O, NR, or S;
[0186] R represents H, alkyl, aryl, aralkyl, or heteroaralkyl;
and
[0187] R' represents H, halogen, NO.sub.2, SR, OR, NR.sub.2, alkyl,
aryl, or carboxy.
[0188] Methods for activating a sirtuin protein may also comprise a
stilbene, chalcone, or flavone compound represented by formula 30:
##STR34##
[0189] wherein, independently for each occurrence,
[0190] D is a phenyl or cyclohexyl group;
[0191] R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R'.sub.1,
R'.sub.2, R'.sub.3, R'.sub.4, and R'.sub.5 represent H, alkyl,
aryl, heteroary, alkaryl, heteroaralkyl, halide, NO.sub.2, SR, OR,
N(R).sub.2, carboxyl, azide, ether; or any two adjacent R or R'
groups taken together form a fused benzene or cyclohexyl group;
[0192] R represents H, alkyl, or aryl; and
[0193] A-B represents an ethylene, ethenylene, or imine group;
[0194] provided that when A-B is ethenylene and R'.sub.3 is H:
R.sub.3 is not OH when R.sub.1, R.sub.2, R.sub.4, and R.sub.5 are
H; and R.sub.2 and R.sub.4 are not OMe when R.sub.1, R.sub.3, and
R.sub.5 are H; and R.sub.3 is not OMe when R.sub.1, R.sub.2,
R.sub.4, and R.sub.5 are H.
[0195] In a further embodiment, the methods include contacting a
cell with an activating compound represented by formula 30 and the
attendant definitions, wherein D is a phenyl group.
[0196] In a further embodiment, the methods include contacting a
cell with an activating compound represented by formula 30 and the
attendant definitions, wherein A-B is an ethenylene or imine
group.
[0197] In a further embodiment, the methods include contacting a
cell with an activating compound represented by formula 30 and the
attendant definitions, wherein A-B is an ethenylene group.
[0198] In a further embodiment, the methods include contacting a
cell with an activating compound represented by formula 30 and the
attendant definitions, wherein R.sub.2 is OH.
[0199] In a further embodiment, the methods include contacting a
cell with an activating compound represented by formula 30 and the
attendant definitions, wherein R.sub.4 is OH
[0200] In a further embodiment, the methods include contacting a
cell with an activating compound represented by formula 30 and the
attendant definitions, wherein R.sub.2 and R.sub.4 are OH.
[0201] In a further embodiment, the methods include contacting a
cell with an activating compound represented by formula 30 and the
attendant definitions, wherein D is a phenyl group; and A-B is an
ethenylene group.
[0202] In a further embodiment, the methods include contacting a
cell with an activating compound represented by formula 30 and the
attendant definitions, wherein D is a phenyl group; A-B is an
ethenylene group; and R.sub.2 and R.sub.4 are OH.
[0203] In a further embodiment, the methods include contacting a
cell with an activating compound represented by formula 30 and the
attendant definitions, wherein A-B is ethenylene; D is a phenyl
ring; R.sub.2 and R.sub.4 are OH; and R'.sub.3 is Cl.
[0204] In a further embodiment, the methods include contacting a
cell with an activating compound represented by formula 30 and the
attendant definitions, wherein A-B is ethenylene; D is a phenyl
ring; R.sub.2 and R.sub.4 are OH; and R'.sub.3 is OH.
[0205] In a further embodiment, the methods include contacting a
cell with an activating compound represented by formula 30 and the
attendant definitions, wherein A-B is ethenylene; D is a phenyl
ring; R.sub.2 and R.sub.4 are OH; and R'.sub.3 is H.
[0206] In a further embodiment, the methods include contacting a
cell with an activating compound represented by formula 30 and the
attendant definitions, wherein A-B is ethenylene; D is a phenyl
ring; R.sub.2 and R.sub.4 are OH; and R'.sub.3 is
CH.sub.2CH.sub.3.
[0207] In a further embodiment, the methods include contacting a
cell with an activating compound represented by formula 30 and the
attendant definitions, wherein A-B is ethenylene; D is a phenyl
ring; R.sub.2 and R.sub.4 are OH; and R'.sub.3 is F.
[0208] In a further embodiment, the methods include contacting a
cell with an activating compound represented by formula 30 and the
attendant definitions, wherein A-B is ethenylene; D is a phenyl
ring; R.sub.2 and R.sub.4 are OH; and R'.sub.3 is Me.
[0209] In a further embodiment, the methods include contacting a
cell with an activating compound represented by formula 30 and the
attendant definitions, wherein A-B is ethenylene; D is a phenyl
ring; R.sub.2 and R.sub.4 are OH; and R'.sub.3 is an azide.
[0210] In a further embodiment, the methods include contacting a
cell with an activating compound represented by formula 30 and the
attendant definitions, wherein A-B is ethenylene; D is a phenyl
ring; R.sub.2 and R.sub.4 are OH; and R'.sub.3 is SMe.
[0211] In a further embodiment, the methods include contacting a
cell with an activating compound represented by formula 30 and the
attendant definitions, wherein A-B is ethenylene; D is a phenyl
ring; R.sub.2 and R.sub.4 are OH; and R'.sub.3 is NO.sub.2.
[0212] In a further embodiment, the methods include contacting a
cell with an activating compound represented by formula 30 and the
attendant definitions, wherein A-B is ethenylene; D is a phenyl
ring; R.sub.2 and R.sub.4 are OH; and R'.sub.3 is
CH(CH.sub.3).sub.2.
[0213] In a further embodiment, the methods include contacting a
cell with an activating compound represented by formula 30 and the
attendant definitions, wherein A-B is ethenylene; D is a phenyl
ring; R.sub.2 and R.sub.4 are OH; and R'.sub.3 is OMe.
[0214] In a further embodiment, the methods include contacting a
cell with an activating compound represented by formula 30 and the
attendant definitions, wherein A-B is ethenylene; D is a phenyl
ring; R.sub.2 and R.sub.4 are OH; R'.sub.2 is OH; and R'.sub.3 is
OMe.
[0215] In a further embodiment, the methods include contacting a
cell with an activating compound represented by formula 30 and the
attendant definitions, wherein A-B is ethenylene; D is a phenyl
ring; R.sub.2 is OH; R.sub.4 is carboxyl; and R'.sub.3 is OH.
[0216] In a further embodiment, the methods include contacting a
cell with an activating compound represented by formula 30 and the
attendant definitions, wherein A-B is ethenylene; D is a phenyl
ring; R.sub.2 and R.sub.4 are OH; and R'.sub.3 is carboxyl.
[0217] In a further embodiment, the methods include contacting a
cell with an activating compound represented by formula 30 and the
attendant definitions, wherein A-B is ethenylene; D is a phenyl
ring; R.sub.2 and R.sub.4 are OH; and R'.sub.3 and R'.sub.4 taken
together form a fused benzene ring.
[0218] In a further embodiment, the methods include contacting a
cell with an activating compound represented by formula 30 and the
attendant definitions, wherein A-B is ethenylene; D is a phenyl
ring; and R.sub.4 is OH.
[0219] In a further embodiment, the methods include contacting a
cell with an activating compound represented by formula 30 and the
attendant definitions, wherein A-B is ethenylene; D is a phenyl
ring; R.sub.2 and R.sub.4 are OCH.sub.2OCH.sub.3; and R'.sub.3 is
SMe.
[0220] In a further embodiment, the methods include contacting a
cell with an activating compound represented by formula 30 and the
attendant definitions, wherein A-B is ethenylene; D is a phenyl
ring; R.sub.2 and R.sub.4 are OH; and R'.sub.3 is carboxyl.
[0221] In a further embodiment, the methods include contacting a
cell with an activating compound represented by formula 30 and the
attendant definitions, wherein A-B is ethenylene; D is a cyclohexyl
ring; and R.sub.2 and R.sub.4 are OH.
[0222] In a further embodiment, the methods include contacting a
cell with an activating compound represented by formula 30 and the
attendant definitions, wherein A-B is ethenylene; D is a phenyl
ring; and R.sub.3 and R.sub.4 are OMe.
[0223] In a further embodiment, the methods include contacting a
cell with an activating compound represented by formula 30 and the
attendant definitions, wherein A-B is ethenylene; D is a phenyl
ring; R.sub.2 and R.sub.4 are OH; and R'.sub.3 is OH.
[0224] Exemplary activating compounds are those listed in the
appended Tables having a ratio to control rate of more than one. A
preferred compound of formula 8 is Dipyridamole; a preferred
compound of formula 12 is Hinokitiol; a preferred compound of
formula 13 is L-(+)-Ergothioneine; a preferred compound of formula
19 is Caffeic Acid Phenol Ester; a preferred compound of formula 20
is MCI-186 and a preferred compound of formula 21 is HBED
(Supplementary Table 6).
[0225] Also included are pharmaceutically acceptable addition salts
and complexes of the compounds of formulas 1-25 and 30. In cases
wherein the compounds may have one or more chiral centers, unless
specified, the compounds contemplated herein may be a single
stereoisomer or racemic mixtures of stereoisomers.
[0226] In cases in which the compounds have unsaturated
carbon-carbon double bonds, both the cis (Z) and trans (E) isomers
are contemplated herein. In cases wherein the compounds may exist
in tautomeric forms, such as keto-enol tautomers, such as ##STR35##
and ##STR36## each tautomeric form is contemplated as being
included within the methods presented herein, whether existing in
equilibrium or locked in one form by appropriate substitution with
R'. The meaning of any substituent at any one occurrence is
independent of its meaning, or any other substituent's meaning, at
any other occurrence.
[0227] Also included in the methods presented herein are prodrugs
of the compounds of formulas 1-25 and 30. Prodrugs are considered
to be any covalently bonded carriers that release the active parent
drug in vivo.
[0228] Analogs and derivatives of the above-described compounds can
also be used for activating a member of the sirtuin protein family.
For example, derivatives or analogs may make the compounds more
stable or improve their ability to traverse cell membranes or being
phagocytosed or pinocytosed. Exemplary derivatives include
glycosylated derivatives, as described, e.g., in U.S. Pat. No.
6,361,815 for resveratrol. Other derivatives of resveratrol include
cis- and trans-resveratrol and conjugates thereof with a
saccharide, such as to form a glucoside (see, e.g., U.S. Pat. No.
6,414,037). Glucoside polydatin, referred to as piceid or
resveratrol 3-O-beta-D-glucopyranoside, can also be used.
Saccharides to which compounds may be conjugated include glucose,
galactose, maltose, lactose and sucrose. Glycosylated stilbenes are
further described in Regev-Shoshani et al. Biochemical J.
(published on Apr. 16, 2003 as BJ20030141). Other derivatives of
compounds described herein are esters, amides and prodrugs. Esters
of resveratrol are described, e.g., in U.S. Pat. No. 6,572,882.
Resveratrol and derivatives thereof can be prepared as described in
the art, e.g., in U.S. Pat. Nos. 6,414,037; 6,361,815; 6,270,780;
6,572,882; and Brandolini et al. (2002) J. Agric. Food.
Chem.50:7407. Derivatives of hydroxyflavones are described, e.g.,
in U.S. Pat. No. 4,591,600. Resveratrol and other activating
compounds can also be obtained commercially, e.g., from Sigma.
[0229] In certain embodiments, if an activating compound occurs
naturally, it may be at least partially isolated from its natural
environment prior to use. For example, a plant polyphenol may be
isolated from a plant and partially or significantly purified prior
to use in the methods described herein. An activating compound may
also be prepared synthetically, in which case it would be free of
other compounds with which it is naturally associated. In an
illustrative embodiment, an activating composition comprises, or an
activating compound is associated with, less than about 50%, 10%,
1%, 0.1%, 10.sup.-2% or 10.sup.-3% of a compound with which it is
naturally associated.
[0230] Sirtuin proteins may be activated in vitro, e.g., in a
solution or in a cell. In one embodiment, a sirtuin protein is
contacted with an activating compound in a solution. A sirtuin is
activated by a compound when at least one of its biological
activities, e.g., deacetylation activity, is higher in the presence
of the compound than in its absence. Activation may be by a factor
of at least about 10%, 30%, 50%, 100% (i.e., a factor of two), 3,
10, 30, or 100. The extent of activation can be determined, e.g.,
by contacting the activated sirtuin with a deacetylation substrate
and determining the extent of deacetylation of the substrate, as
further described herein. The observation of a lower level of
acetylation of the substrate in the presence of a test sirtuin
relative to the presence of a non activated control sirtuin
indicates that the test sirtuin is activated. The solution may be a
reaction mixture. The solution may be in a dish, e.g., a multiwell
dish. Sirtuin proteins may be prepared recombinantly or isolated
from cells according to methods known in the art.
[0231] In another embodiment, a cell comprising a sirtuin
deacetylase protein is contacted with an activating compound. The
cell may be a eukaryotic cell, e.g., a mammalian cell, such as a
human cell, a yeast cell, a non-human primate cell, a bovine cell,
an ovine cell, an equine cell, a porcine cell, a sheep cell, a bird
(e.g., chicken or fowl) cell, a canine cell, a feline cell or a
rodent (mouse or rat) cell. It can also be a non-mammalian cell,
e.g., a fish cell. Yeast cells include S. cerevesiae and C.
albicans. The cell may also be a prokaryotic cell, e.g., a
bacterial cell. The cell may also be a single-celled microorganism,
e.g., a protozoan. The cell may also be a metazoan cell, a plant
cell or an insect cell. The application of the methods decribed
herein to a large number of cell types is based at least on the
high convervation of sirtuins from humans to fungi, protozoans,
metazoans and plants.
[0232] In one embodiment, the cells are in vitro. A cell may be
contacted with a solution having a concentration of an activating
compound of less than about 0.1 .mu.M; 0.5 .mu.M; less than about 1
.mu.M; less than about 10 .mu.M or less than about 100 .mu.M. The
concentration of the activating compound may also be in the range
of about 0.1 to 1 .mu.M, about 1 to 10 .mu.M or about 10 to 100
.mu.M. The appropriate concentration may depend on the particular
compound and the particular cell used as well as the desired
effect. For example, a cell may be contacted with a "sirtuin
activating" concentration of an activating compound, e.g., a
concentration sufficient for activating the sirtuin by a factor of
at least 10%, 30%, 50%, 100%, 3, 10, 30, or 100.
[0233] In certain embodiments, a cell is contacted with an
activating compound in vivo, such as in a subject. The subject can
be a human, a non-human primate, a bovine, an ovine, an equine, a
porcine, a sheep, a canine, a feline or a rodent (mouse or rat).
For example, an activating compound may be administered to a
subject. Administration may be local, e.g., topical, parenteral,
oral, or other depending on the desired result of the
administration (as further described herein). Administration may be
followed by measuring a factor in the subject, such as measuring
the activity of the sirtuin. In an illustrative embodiment, a cell
is obtained from a subject following administration of an
activating compound to the subject, such as by obtaining a biopsy,
and the activity of the sirtuin is determined in the biopsy. The
cell may be any cell of the subject, but in cases in which an
activating compound is administered locally, the cell is preferably
a cell that is located in the vicinity of the site of
administration.
[0234] Also provided are methods for modulating the acetylation
level of p53 proteins. As shown herein (see, e.g., the Examples),
lysine 382 of p53 proteins in cells is deacetylated following
incubation of cells in the presence of low concentrations of
resveratrol. Accordingly, "p53 deacetylating concentrations" of
compounds include, e.g., concentrations of less than about 0.1
.mu.M, 0.5 .mu.M, 1 .mu.M, 3 .mu.M, 50 .mu.M, 100 .mu.M or 300
.mu.M. It has also been shown herein that p53 proteins in cells are
acetylated in the presence of higher concentrations of resveratrol.
Accordingly, "p53 acetylating concentrations" of compounds include,
e.g., concentrations of at least about 10 .mu.M, 30 .mu.M, 100
.mu.M or 300 .mu.M. The level of acetylation of p53 can be
determined by methods known in the art, e.g., as further described
in the Examples.
[0235] Other methods contemplated are methods for protecting a cell
against apoptosis. Without wanting to be limited to a particular
mechanism of action, but based at least in part on the fact that
acetylation of p53 proteins activates p53 proteins and that
activated p53 proteins induce apoptosis, incubating cells
comprising p53 proteins in the presence of a p53 deacetylating
concentration of an activating compound prevents the induction of
apoptosis of the cells. Accordingly, a cell can be protected from
apoptosis by activating sirtuins by contacting the cell with an
amount of an activating compound sufficient or adequate for
protecting against apoptosis, e.g., less than about 0.1 .mu.M, 0.5
.mu.M, 1 .mu.M, 3 .mu.M or 10 .mu.M. An amount sufficient or
adequate for protection against apoptosis can also be determined
experimentally, such as by incubating a cell with different amounts
of an activating compound, subjecting the cell to an agent or
condition that induces apoptosis, and comparing the extent of
apoptosis in the presence of different concentrations or the
absence of an enhancing compound and determining the concentration
that provides the desired protection. Determining the level of
apoptosis in a population of cells can be performed according to
methods known in the art.
[0236] Yet other methods contemplated herein are methods for
inducing apoptosis in a cell. Without wanting to be limited to a
particular mechanism of action, as shown in the Examples, at
certain concentrations of compounds, p53 proteins are acetylated
rather than deacetylated, thereby activating the p53 proteins, and
inducing apoptosis. Apoptosis inducing concentrations of compounds
may be, e.g., at least about 10 .mu.M, 30 .mu.M, 100 .mu.M or 300
.mu.M.
[0237] Appropriate concentrations for modulating p53 deacetylation
and apoptosis can be determined according to methods, e.g., those
described herein. Concentrations may vary slightly from one cell to
another, from one activating compound to another and whether the
cell is isolated or in an organism.
[0238] Cells in which p53 acetylation and apoptosis may be
modulated can be in vitro, e.g., in cell culture, or in vivo, e.g.,
in a subject. Administration of an activating compound to a subject
can be conducted as further described herein. The level of p53
acetylation and/or apoptosis in cells of the subject can be
determined, e.g., by obtaining a sample of cells from the subject
and conducting an in vitro analysis of the level of p53 acetylation
and/or apoptosis.
[0239] Also provided herein are methods for extending the lifespan
of a eukaryotic cells and/or increasing their resistance to stress
comprising, e.g., contacting the eukaryotic cell with a compound,
e.g., a polyphenol compound. Exemplary compounds include the
activating compounds described herein, such as compounds of the
stilbene, flavone and chalcone families. Although the Examples show
that quercetin and piceatannol, which activate sirtuins, were not
found to significantly affect the lifespan of eukaryotic cells, it
is believed that this may be the result of a lack of entry of the
compounds into the cell or potentially the existence of another
pathway overriding activation of sirtuins. Derivatives and analogs
of these compounds or administration of these compound to other
cells or by other methods are expected to activate sirtuins.
[0240] In one embodiment, methods for extending the lifespan of a
eukaryotic cell and/or increasing its resistance to stress comprise
contacting the cell with a stilbene, chalcone, or flavone compound
represented by formula 7: ##STR37##
[0241] wherein, independently for each occurrence,
[0242] M is absent or O;
[0243] R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R'.sub.1,
R'.sub.2, R'.sub.3, R'.sub.4, and R'.sub.5 represent H, alkyl,
aryl, heteroary alkaryl, heteroaralkyl, halide, NO.sub.2, SR, OR,
N(R).sub.2, or carboxyl;
[0244] R.sub.a represents H or the two R.sub.a form a bond;
[0245] R represents H, alkyl, or aryl; and
[0246] n is 0 or 1.
[0247] In a further embodiment, the methods comprise a compound
represented by formula 7 and the attendant definitions, wherein n
is 0. In a further embodiment, the methods comprise a compound
represented by formula 7 and the attendant definitions, wherein n
is 1. In a further embodiment, the methods comprise a compound
represented by formula 7 and the attendant definitions, wherein M
is absent. In a further embodiment, the methods comprise a compound
represented by formula 7 and the attendant definitions, wherein M
is O. In a further embodiment, the methods comprise a compound
represented by formula 7 and the attendant definitions, wherein
R.sub.a is H. In a further embodiment, the methods comprise a
compound represented by formula 7 and the attendant definitions,
wherein M is O and the two R.sub.a form a bond. In a further
embodiment, the methods comprise a compound represented by formula
7 and the attendant definitions, wherein R.sub.5 is H. In a further
embodiment, the methods comprise a compound represented by formula
7 and the attendant definitions, wherein R.sub.5 is OH. In a
further embodiment, the methods comprise a compound represented by
formula 7 and the attendant definitions, wherein R.sub.1, R.sub.3,
and R'.sub.3 are OH. In a further embodiment, the methods comprise
a compound represented by formula 7 and the attendant definitions,
wherein R.sub.2, R.sub.4, R'.sub.2, and R'.sub.3 are OH. In a
further embodiment, the methods comprise a compound represented by
formula 7 and the attendant definitions, wherein R.sub.2, R'.sub.2,
and R'.sub.3 are OH.
[0248] In a further embodiment, methods for extending the lifespan
of a eukaryotic cell comprise contacting the cell with a compound
represented by formula 7 and the attendant definitions, wherein n
is 0; M is absent; R.sub.a is H; R.sub.5 is H; R.sub.1, R.sub.3,
and R'.sub.3 are OH; and R.sub.2, R.sub.4, R'.sub.1, R'.sub.2,
R'.sub.4, and R'.sub.5 are H. In a further embodiment, the methods
comprise a compound represented by formula 7 and the attendant
definitions, wherein n is 1; M is absent; R.sub.a is H; R.sub.5 is
H; R.sub.2, R.sub.4, R'.sub.2, and R'.sub.3 are OH; and R.sub.1,
R.sub.3, R'.sub.1, R'.sub.4, and R'.sub.5 are H. In a further
embodiment, the methods comprise a compound represented by formula
7 and the attendant definitions, wherein n is 1; M is O; the two
R.sub.a form a bond; R.sub.5 is OH; R.sub.2, R'.sub.2, and R'.sub.3
are OH; and R.sub.1, R.sub.3, R.sub.4, R'.sub.1, R'.sub.4, and
R'.sub.5 are H.
[0249] The eukaryotic cell whose lifespan may be extended can be a
human, a non-human primate, a bovine, an ovine, an equine, a
porcine, a sheep, a canine, a feline, a rodent (mouse or rat) or a
yeast cell. A yeast cell may be Saccharomyces cerevisiae or Candida
albicans. Concentrations of compounds for this purpose may be about
0.1 .mu.M, 0.3 .mu.M, 0.5 .mu.M, 1 .mu.M, 3 .mu.M, 10 .mu.M, 30
.mu.M, 100 .mu.M or 300 .mu.M. Based at least on the high
conservation of Sir2 proteins in various organisms, lifespan can
also be prolonged in prokaryotes, protozoans, metazoans, insects
and plants.
[0250] The cell may be in vitro or in vivo. In some embodiments, a
life-extending compound is administered to an organism (e.g., a
subject) such as to induce hormesis, i.e., an increasing resistance
to mild stress that results in increasing the lifespan of the
organism. In fact, it has been shown that SIR2 is essential for the
increased longevity provided by calorie restriction, a mild stress,
that extends the lifespan of every organism it has been tested on
(Lin et al. (2000) Science 249:2126). For example, overexpression
of a Caenorhabditis. elegans SIR2 homologue, sir-2.1, increases
lifespan via a forkhead transcription factor, DAF-16, and a SIR2
gene has recently been implicated in lifespan regulation in
Drosophila melanogaster (Rogina et al. Science (2002) 298:1745).
Furthermore, the closest human Sir2 homologue, SIRT1, promotes
survival in human cells by down-regulating the activity of the
tumor suppressor p53 (Tissenbaum et al. Nature 410, 227-30 (2001);
Rogina et al. Science, in press (2002); and Vaziri, H. et al. Cell
107, 149-59. (2001)). The role of SIR2 in stress resistance and
cell longevity is further supported by the identification of PNC1
as a calorie restriction- and stress-responsive gene that increases
lifespan and stress resistance of cells by depleting intracellular
nicotinamide (Anderson et al. (2003) Nature 423:181 and Bitterman
et al. (2002) J. Biol. Chem. 277: 45099). Accordingly, compounds
may be administered to a subject for protecting the cells of the
subject from stresses and thereby extending the lifespan of the
cells of the subject.
[0251] Also encompassed are methods for inhibiting sirtuins;
inhibiting deacetylation of p53, e.g., for stimulating acetylation
of p53; stimulating apoptosis; reducing lifespan and/or rendering
cells or organisms more sensitive to stresses. Methods may include
contacting a cell or a molecule, such as a sirtuin or a p53
protein, with a compound that inhibits sirtuins, i.e., an
"inhibiting compound," such, a compound having a formula selected
from the group of formulas 26-29 and 31: ##STR38##
[0252] wherein, independently for each occurrence,
[0253] R' represents H, halogen, NO.sub.2, SR, OR, NR.sub.2, alkyl,
aryl, or carboxy;
[0254] R represents H, alkyl, aryl, aralkyl, or heteroaralkyl;
and
[0255] R'' represents alkyl, alkenyl, or alkynyl. ##STR39##
[0256] wherein, independently for each occurrence,
[0257] L represents O, NR, or S;
[0258] R represents H, alkyl, aryl, aralkyl, or heteroaralkyl;
[0259] R' represents H, halogen, NO.sub.2, SR, SO.sub.3, OR,
NR.sub.2, alkyl, aryl, or carboxy;
[0260] a represents an integer from 1 to 7 inclusively; and
[0261] b represents an integer from 1 to 4 inclusively.
##STR40##
[0262] wherein, independently for each occurrence,
[0263] L represents O, NR, or S;
[0264] R represents H, alkyl, aryl, aralkyl, or heteroaralkyl;
[0265] R' represents H, halogen, NO.sub.2, SR, SO.sub.3, OR,
NR.sub.2, alkyl, aryl, or carboxy;
[0266] a represents an integer from 1 to 7 inclusively; and
[0267] b represents an integer from 1 to 4 inclusively.
##STR41##
[0268] wherein, independently for each occurrence,
[0269] L represents O, NR, or S;
[0270] R represents H, alkyl, aryl, aralkyl, or heteroaralkyl;
[0271] R' represents H, halogen, NO.sub.2, SR, SO.sub.3, OR,
NR.sub.2, alkyl, aryl, or carboxy;
[0272] a represents an integer from 1 to 7 inclusively; and
[0273] b represents an integer from 1 to 4 inclusively.
##STR42##
[0274] wherein, independently for each occurrence,
[0275] R.sub.2, R.sub.3, and R.sub.4 are H, OH, or O-alkyl;
[0276] R'.sub.3 is H or NO.sub.2; and
[0277] A-B is an ethenylene or amido group.
[0278] In a further embodiment, the inhibiting compound is
represented by formula 31 and the attendant definitions, wherein
R.sub.3 is OH, A-B is ethenylene, and R'.sub.3 is H.
[0279] In a further embodiment, the inhibiting compound is
represented by formula 31 and the attendant definitions, wherein
R.sub.2 and R.sub.4 are OH, A-B is an amido group, and R'.sub.3 is
H.
[0280] In a further embodiment, the inhibiting compound is
represented by formula 31 and the attendant definitions, wherein
R.sub.2 and R.sub.4 are OMe, A-B is ethenylene, and R'.sub.3 is
NO.sub.2.
[0281] In a further embodiment, the inhibiting compound is
represented by formula 31 and the attendant definitions, wherein
R.sub.3 is OMe, A-B is ethenylene, and R'.sub.3 is H.
[0282] Also included are pharmaceutically acceptable addition salts
and complexes of the compounds of formulas 26-29 and 31. In cases
wherein the compounds may have one or more chiral centers, unless
specified, the compounds contemplated herein may be a single
stereoisomer or racemic mixtures of stereoisomers.
[0283] Exemplary inhibitory compounds are those set forth in the
appended Tables for which the "ratio to control rate" is lower than
one.
[0284] In cases in which the compounds have unsaturated
carbon-carbon double bonds, both the cis (Z) and trans (E) isomers
are contemplated herein. In cases wherein the compounds may exist
in tautomeric forms, such as keto-enol tautomers, such as ##STR43##
and ##STR44## each tautomeric form is contemplated as being
included within the methods presented herein, whether existing in
equilibrium or locked in one form by appropriate substitution with
R'. The meaning of any substituent at any one occurrence is
independent of its meaning, or any other substituent's meaning, at
any other occurrence.
[0285] Also included in the methods presented herein are prodrugs
of the compounds of formulas 26-29 and 31. Prodrugs are considered
to be any covalently bonded carriers that release the active parent
drug in vivo.
[0286] Inhibitory compounds may be contacted with a cell,
administered to a subject, or contacted with one or more molecules,
such as a sirtuin protein and a p53 protein. Doses of inhibitory
compounds may be similar to those of activating compounds.
[0287] Whether in vitro or in vivo, a cell may also be contacted
with more than one compound (whether an activating compound or an
inhibiting compound). A cell may be contacted with at least 2, 3,
5, or 10 different compounds. A cell may be contacted
simultaneously or sequentially with different compounds.
[0288] Also encompassed are compositions comprising one or more
activating or inhibiting compounds having a formula selected from
the group of formulas 1-31. Compounds may be in a pharmaceutical
composition, such as a pill or other formulation for oral
administration, further described herein. Compositions may also
comprise or consist of extracts of plants, red wine or other source
of the compounds.
[0289] Yet other methods contemplated herein include sceening
methods for identifying compounds that modulate sirtuins. Assays
may be conducted in a cell based or cell free format. For example,
an assay may comprise incubating (or contacting) a sirtuin with a
test compound under conditions in which a sirtuin can be activated
by an agent known to activate the sirtuin, and monitoring or
determining the level of activation of the sirtuin in the presence
of the test compound relative to the absence of the test compound.
The level of activation of a sirtuin can be determined by
determining its ability to deacetylate a substrate. Exemplary
substrates are acetylated peptides, e.g., those set forth in FIG.
5, which can be obtained from BIOMOL (Plymouth Meeting, Pa.).
Preferred substrates include peptides of p53, such as those
comprising an acetylated K382. A particularly preferred substrate
is the Fluor de Lys-SIRT1 (BIOMOL), i.e., the acetylated peptide
Arg-His-Lys-Lys. Other substrates are peptides from human histones
H3 and H4 or an acetylated amino acid (see FIG. 5). Substrates may
be fluorogenic. The sirtuin may be SIRT1 or Sir2 or a portion
thereof. For example, recombinant SIRT1 can be obtained from
BIOMOL. The reaction may be conducted for about 30 minutes and
stopped, e.g., with nicotinamide. The HDAC fluorescent activity
assay/drug discovery kit (AK-500, BIOMOL Research Laboratories) may
be used to determine the level of acetylation. Similar assays are
described in Bitterman et al. (2002) J. Biol. Chem. 277:45099. The
level of activation of the sirtuin in an assay may be compared to
the level of activation of the sirtuin in the presence of one or
more (separately or simultaneously) compounds described herein,
which may serve as positive or negative controls. Sirtuins for use
in the assays may be full length sirtuin proteins or portions
thereof. Since it has been shown herein that activating compounds
appear to interact with the N-terminus of SIRT1, proteins for use
in the assays include N-terminal portions of sirtuins, e.g., about
amino acids 1-176 or 1-255 of SIRT1; about amino acids 1-174 or
1-252 of Sir2.
[0290] In one embodiment, a screening assay comprises (i)
contacting a sirtuin with a test compound and an acetylated
substrate under conditions appropriate for the sirtuin to
deacetylate the substrate in the absence of the test compound; and
(ii) determining the level of acetylation of the substrate, wherein
a lower level of acetylation of the substrate in the presence of
the test compound relative to the absence of the test compound
indicates that the test compound stimulates deacetylation by the
sirtuin, whereas a higher level of acetylation of the substrate in
the presence of the test compound relative to the absence of the
test compound indicates that the test compound inhibits
deacetylation by the sirtuin.
[0291] Methods for identifying compounds that modulate, e.g.,
stimulate or inhibit, sirtuins in vivo may comprise (i) contacting
a cell with a test compound and a substrate that is capable of
entering a cell in the presence of an inhibitor of class I and
class II HDACs under conditions appropriate for the sirtuin to
deacetylate the substrate in the absence of the test compound; and
(ii) determining the level of acetylation of the substrate, wherein
a lower level of acetylation of the substrate in the presence of
the test compound relative to the absence of the test compound
indicates that the test compound stimulates deacetylation by the
sirtuin, whereas a higher level of acetylation of the substrate in
the presence of the test compound relative to the absence of the
test compound indicates that the test compound inhibits
deacetylation by the sirtuin. A preferred substrate is an
acetylated peptide, which is also prefeably fluorogenic, as further
described herein (Examples). The method may further comprise lysing
the cells to determine the level of acetylation of the substrate.
Substrates may be added to cells at a concentration ranging from
about 1 .mu.M to about 10 mM, preferably from about 10 .mu.M to 1
mM, even more preferably from about 100 .mu.M to 1 mM, such as
about 200 .mu.M. A preferred substrate is an acetylated lysine,
e.g., .epsilon.-acetyl lysine (Fluor de Lys, FdL) or Fluor de
Lys-SIRT1. A preferred inhibitor of class I and class II HDACs is
trichostatin A (TSA), which may be used at concentrations ranging
from about 0.01 to 100 .mu.M, preferably from about 0.1 to 10
.mu.M, such as 1 .mu.M. Incubation of cells with the test compound
and the substrate may be conducted for about 10 minutes to 5 hours,
preferably for about 1-3 hours. Since TSA inhibits all class I and
class II HDACs, and that certain substrates, e.g., Fluor de Lys, is
a poor substrate for SIRT2 and even less a substrate for SIRT3-7,
such an assay may be used to identify modulators of SIRT1 in vivo.
An exemplary assay is further described in the Examples and shown
in FIG. 4a.
[0292] Also provided herein are assays for identifying agents that
are capable of extending or reducing the lifespan of cells and/or
increasing or decreasing their resistance to stress. A method may
comprise incubating cells with a test compound and determining the
effect of the test compound on rDNA silencing and rDNA
recombination, wherein an increase in the frequency of rDNA
recombination and an absence of effect on rDNA silencing in the
presence of the test compound relative to the absence of the test
compound indicates that the test compound extends lifespan. This
assay is based at least on the observation that resveratrol reduced
the frequency of rDNA recombination by about 60% in a SIR2
dependent manner, but did not increasing rDNA silencing.
[0293] Also provided herein are methods for identifying the binding
site of activating or inhibitory compounds in sirtuin proteins. In
one embodiment, BML-232 (Table 10) is used. BML-232, has very
similar SIRT1 activating properties to resveratrol and contains a
phenylazide function. Phenylazide groups may be activated by the
absorption of ultraviolet light to form reactive nitrenes. When a
protein-bound phenylazide is light-activated it can react to form
covalent adducts with various protein functional groups in the site
to which it is bound. The photo cross-linked protein may then be
analyzed by proteolysis/mass spectrometry to identify amino acid
residues which may form part of the binding site for the compound.
This information, in combination with published three dimensional
structural information on SIRT1 homologs could be used to aid the
design of new, possibly higher affinity, SIRT1 activating
ligands.
Exemplary Uses
[0294] In one embodiment, cells are treated in vitro as described
herein to extend their lifespan, e.g., to keep them proliferating
longer and/or increasing its resistance to stress or prevent
apoptosis. That compounds described herein may increase resistance
to stress is based at least on the observation that Sir2 provides
stress resistance and that PNC1 modulates Sir2 activity in response
to cell stress (Anderson et al. (2003) Nature 423:181). This is
particularly useful for primary cell cultures (i.e., cells obtained
from an organism, e.g., a human), which are known to have only a
limited lifespan in culture. Treating such cells according to
methods described herein, e.g., by contacting them with an
activating or lifespan extending compound, will result in
increasing the amount of time that the cells are kept alive in
culture. Embryonic stem (ES) cells and pluripotent cells, and cells
differentiated therefrom, can also be treated according to the
methods described herein such as to keep the cells or progeny
thereof in culture for longer periods of time. Primary cultures of
cells, ES cells, pluripotent cells and progeny thereof can be used,
e.g., to identify compounds having particular biological effects on
the cells or for testing the toxicity of compounds on the cells
(i.e., cytotoxicity assays). Such cells can also be used for
transplantation into a subject, e.g., after ex vivo
modification.
[0295] In other embodiments, cells that are intended to be
preserved for long periods of time are treated as described herein.
The cells can be cells in suspension, e.g., blood cells, serum,
biological growth media, or tissues or organs. For example, blood
collected from an individual for administering to an individual can
be treated as described herein, such as to preserve the blood cells
for longer periods of time, such as for forensic purposes. Other
cells that one may treat for extending their lifespan or protect
against apoptosis include cells for consumption, e.g., cells from
non-human mammals (such as meat), or plant cells (such as
vegetables).
[0296] Compounds may also be applied during developmental and
growth phases in mammals, plants, insects or microorganisms, in
order to, e.g., alter, retard or accelerate the developmental
and/or growth process.
[0297] In another embodiment, cells obtained from a subject, e.g.,
a human or other mammal, are treated according to methods described
herein and then administered to the same or a different subject.
Accordingly, cells or tissues obtained from a donor for use as a
graft can be treated as described herein prior to administering to
the recipient of the graft. For example, bone marrow cells can be
obtained from a subject, treated ex vivo, e.g., to extend their
lifespan, and then administered to a recipient. The graft can be an
organ, a tissue or loose cells.
[0298] In yet other embodiments, cells are treated in vivo, e.g.,
to increase their lifespan or prevent apoptosis. For example, skin
can be protected from aging, e.g., developing wrinkles, by treating
skin, e.g., epithelial cells, as described herein. In an exemplary
embodiment, skin is contacted with a pharmaceutical or cosmetic
composition comprising a compound described herein. Exemplary skin
afflictions or skin conditions include disorders or diseases
associated with or caused by inflammation, sun damage or natural
aging. For example, the compositions find utility in the prevention
or treatment of contact dermatitis (including irritant contact
dermatitis and allergic contact dermatitis), atopic dermatitis
(also known as allergic eczema), actinic keratosis, keratinization
disorders (including eczema), epidermolysis bullosa diseases
(including penfigus), exfoliative dermatitis, seborrheic
dermatitis, erythemas (including erythema multiforme and erythema
nodosum), damage caused by the sun or other light sources, discoid
lupus erythematosus, dermatomyositis, skin cancer and the effects
of natural aging. The formulations may be administered topically,
to the skin or mucosal tissue, as an ointment, lotion, cream,
microemulsion, gel, solution or the like, as described in the
preceding section, within the context of a dosing regimen effective
to bring about the desired result. A dose of active agent may be in
the range of about 0.005 to about 1 micromoles per kg per day,
preferably about 0.05 to about 0.75 micromoles per kg per day, more
typically about 0.075 to about 0.5 micromoles per kg per day. It
will be recognized by those skilled in the art that the optimal
quantity and spacing of individual dosages will be determined by
the nature and extent of the condition being treated, the site of
administration, and the particular individual undergoing treatment,
and that such optimums can be determined by conventional
techniques. That is, an optimal dosing regimen for any particular
patient, i.e., the number and frequency of doses, can be
ascertained using conventional course of treatment determination
tests. Generally, a dosing regimen herein involves administration
of the topical formulation at least once daily, and preferably one
to four times daily, until symptoms have subsided.
[0299] Topical formulations may also be used as chemopreventive
compositions. When used in a chemopreventive method, susceptible
skin is treated prior to any visible condition in a particular
individual.
[0300] Compounds can also be delivered locally, e.g., to a tissue
or organ within a subject, such as by injection, e.g., to extend
the lifespan of the cells; protect against apoptosis or induce
apoptosis.
[0301] In yet another embodiment, a compound is administered to a
subject, such as to generally increase the lifespan of its cells
and to protect its cells against stress and/or against apoptosis.
It is believed that treating a subject with a compound described
herein is similar to subjecting the subject to hormesis, i.e., mild
stress that is beneficial to organisms and may extend their
lifespan. For example, a compound can be taken by subjects as a
food or dietary supplement. In one embodiment, such a compound is a
component of a multi-vitamin complex. Compounds can also be added
to existing formulations that are taken on a daily basis, e.g.,
statins and aspirin. Compounds may also be used as food
additives.
[0302] Compounds described herein could also be taken as one
component of a multi-drug complex or as a supplement in addition to
a multi-drug regimen. In one embodiment, this multi-drug complex or
regimen would include drugs or compounds for the treatment or
prevention of aging-related diseases, e.g., stroke, heart disease,
arthritis, high blood pressure, Alzheimer's. In another embodiment,
this multi-drug regimen would include chemotherapeutic drugs for
the treatment of cancer. In a specific embodiment, a polyphenol
compound could be used to protect non-cancerous cells from the
effects of chemotherapy.
[0303] Compounds may be administered to subject to prevent aging
and aging-related consequences or diseases, such as stroke, heart
disease, arthritis, high blood pressure, and Alzheimer's disease.
Compounds described herein can also be administered to subjects for
treatment of diseases, e.g., chronic diseases, associated with cell
death, such as to protect the cells from cell death. Exemplary
diseases include those associated with neural cell death or
muscular cell death, such as Parkinson's disease, Alzheimer's
disease, multiple sclerosis, amniotropic lateral sclerosis, and
muscular dystrophy; AIDS; fulminant hepatitis; diseases linked to
degeneration of the brain, such as Creutzfeld-Jakob disease,
retinitis pigmentosa and cerebellar degeneration; myelodysplasis
such as aplastic anemia; ischemic diseases such as myocardial
infarction and stroke; hepatic diseases such as alcoholic
hepatitis, hepatitis B and hepatitis C; joint-diseases such as
osteoarthritis; atherosclerosis; alopecia; damage to the skin due
to UV light; lichen planus; atrophy of the skin; cataract; graft
rejections; and etc.
[0304] Compounds described herein can also be administered to a
subject suffering from an acute disease, e.g., damage to an organ
or tissue, e.g., a subject suffering from stroke or myocardial
infarction or a subject suffering from a spinal cord injury.
Compounds can also be used to repair an alcoholic's liver.
[0305] Compounds can also be administered to subjects who have
recently received or are likely to receive a dose of radiation. In
one embodiment, the dose of radiation is received as part of a
work-related or medical procedure, e.g., working in a nuclear power
plant, flying an airplane, an X-ray, CAT scan, or the
administration of a radioactive dye for medical imaging; in such an
embodiment, the compound is administered as a prophylactic measure.
In another embodiment, the radiation exposure is received
unintentionally, e.g., as a result of an industrial accident,
terrorist act, or act of war involving radioactive material. In
such a case, the compound is preferably administered as soon as
possible after the exposure to inhibit apoptosis and the subsequent
development of acute radiation syndrome.
[0306] Based at least on the discovery that certain concentrations
of activating compounds prevent deacetylation of p53 in cells and
thereby may induce apoptosis in cells, the activating compounds can
also be administed to a subject in conditions in which apoptosis of
certain cells is desired. For example, cancer may be treated or
prevented. Exemplary cancers are those of the brain and kidney;
hormone-dependent cancers including breast, prostate, testicular,
and ovarian cancers; lymphomas, and leukemias. In cancers
associated with solid tumors, a activating compound may be
administered directly into the tumor. Cancer of blood cells, e.g.,
leukemia can be treated by administering a activating compound into
the blood stream or into the bone marrow. Benign cell growth can
also be treated, e.g., warts. Other diseases that can be treated
include autoimmune diseases, e.g., systemic lupus erythematosus,
scleroderma, and arthritis, in which autoimmune cells should be
removed. Viral infections such as herpes, HIV, adenovirus, and
HTLV-1 associated malignant and benign disorders can also be
treated by administration of compounds. Alternatively, cells can be
obtained from a subject, treated ex vivo to remove certain
undesirable cells, e.g., cancer cells, and administered back to the
same or a different subject.
[0307] In other embodiments, methods described herein are applied
to yeast cells. Situations in which it may be desirable to extend
the lifespan of yeast cells include any process in which yeast is
used, e.g., the making of beer, yogurt, and bakery items, e.g.,
bread. Use of yeast having an extended lifespan can result in using
less yeast or in having the yeast be active for longer periods of
time. Yeast or other mammalian cells used for recombinantly
producing proteins may also be treated as described herein.
[0308] Subjects that may be treated as described herein include
eukaryotes, such as mammals, e.g., humans, ovines, bovines,
equines, porcines, canines, felines, non-human primate, mice, and
rats. Cells that may be treated include eukaryotic cells, e.g.,
from a subject described above, or plant cells, yeast cells and
prokaryotic cells, e.g., bacterial cells. For example, activating
compounds may be administered to form animals to improve their
ability to withstand farming conditions longer.
[0309] Compounds may also be used to increase lifespan, stress
resistance, and resistance to apoptosis in plants. In one
embodiment, a compound is applied to plants, either on a periodic
basis or in fungi. In another embodiment, plants are genetically
modified to produce a compound. In another embodiment, plants and
fruits are treated with a compound prior to picking and shipping to
increase resistance to damage during shipping.
[0310] Compounds may also be used to increase lifespan, stress
resistance and resistance to apoptosis in insects. In this
embodiment, compounds would be applied to useful insects, e.g.,
bees and other insects that are involved in pollination of plants.
In a specific embodiment, a compound would be applied to bees
involved in the production of honey. Generally, the methods
described herein may be applied to any organism, e.g., eukaryote,
that may have commercial importance. For example, they can be
applied to fish (aquaculture) and birds (e.g., chicken and
fowl).
[0311] Higher doses of compounds may also be used as a pesticide by
interfering with the regulation of silenced genes and the
regulation of apoptosis during development. In this embodiment, a
compound may be applied to plants using a method known in the art
that ensures the compound is bio-available to insect larvae, and
not to plants.
[0312] Activated sirtuin proteins that are in vitro outside of a
cell may be used, e.g., for deacetylating target proteins, thereby,
e.g., activating the target proteins. Activated sirtuins may be
used, e.g., for the identification, in vitro, of previously unknown
targets of sirtuin deacetylation, for example using 2D
electrophoresis of acetyl labeled proteins.
[0313] At least in view of the link between reproduction and
longevity (Longo and Finch, Science, 2002), the compounds can be
applied to affect the reproduction of organisms such as insects,
animals and microorganisms.
[0314] Inhibitory compounds may be used for similar purposes as
high concentrations of activating compounds can be used for. For
example, inhibitory compounds may be used to stimulate acetylation
of substrates such as p53 and thereby increase apoptosis, as well
as to reduce the lifespan of cells and organisms and/or rendering
them more sensitive to stress.
Pharmaceutical Compositions and Methods
[0315] Pharmaceutical compositions for use in accordance with the
present methods may be formulated in conventional manner using one
or more physiologically acceptable carriers or excipients. Thus,
activating compounds and their physiologically acceptable salts and
solvates may be formulated for administration by, for example,
injection, inhalation or insufflation (either through the mouth or
the nose) or oral, buccal, parenteral or rectal administration. In
one embodiment, the compound is administered locally, at the site
where the target cells, e.g., diseased cells, are present, i.e., in
the blood or in a joint.
[0316] Compounds can be formulated for a variety of loads of
administration, including systemic and topical or localized
administration. Techniques and formulations generally may be found
in Remmington's Pharmaceutical Sciences, Meade Publishing Co.,
Easton, Pa. For systemic administration, injection is preferred,
including intramuscular, intravenous, intraperitoneal, and
subcutaneous. For injection, the compounds can be formulated in
liquid solutions, preferably in physiologically compatible buffers
such as Hank's solution or Ringer's solution. In addition, the
compounds may be formulated in solid form and redissolved or
suspended immediately prior to use. Lyophilized forms are also
included.
[0317] For oral administration, the pharmaceutical compositions may
take the form of, for example, tablets, lozanges, or capsules
prepared by conventional means with pharmaceutically acceptable
excipients such as binding agents (e.g., pregelatinised maize
starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose);
fillers (e.g., lactose, microcrystalline cellulose or calcium
hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or
silica); disintegrants (e.g., potato starch or sodium starch
glycolate); or wetting agents (e.g., sodium lauryl sulphate). The
tablets may be coated by methods well known in the art. Liquid
preparations for oral administration may take the form of, for
example, solutions, syrups or suspensions, or they may be presented
as a dry product for constitution with water or other suitable
vehicle before use. Such liquid preparations may be prepared by
conventional means with pharmaceutically acceptable additives such
as suspending agents (e.g., sorbitol syrup, cellulose derivatives
or hydrogenated edible fats); emulsifying agents (e.g., lecithin or
acacia); non-aqueous vehicles (e.g., ationd oil, oily esters, ethyl
alcohol or fractionated vegetable oils); and preservatives (e.g.,
methyl or propyl-p-hydroxybenzoates or sorbic acid). The
preparations may also contain buffer salts, flavoring, coloring and
sweetening agents as appropriate. Preparations for oral
administration may be suitably formulated to give controlled
release of the active compound.
[0318] For administration by inhalation, the compounds may be
conveniently delivered in the form of an aerosol spray presentation
from pressurized packs or a nebuliser, with the use of a suitable
propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In
the case of a pressurized aerosol the dosage unit may be determined
by providing a valve to deliver a metered amount. Capsules and
cartridges of e.g., gelatin, for use in an inhaler or insufflator
may be formulated containing a powder mix of the compound and a
suitable powder base such as lactose or starch.
[0319] The compounds may be formulated for parenteral
administration by injection, e.g., by bolus injection or continuous
infusion. Formulations for injection may be presented in unit
dosage form, e.g., in ampoules or in multi-dose containers, with an
added preservative. The compositions may take such forms as
suspensions, solutions or emulsions in oily or aqueous vehicles,
and may contain formulatory agents such as suspending, stabilizing
and/or dispersing agents. Alternatively, the active ingredient may
be in powder form for constitution with a suitable vehicle, e.g.,
sterile pyrogen-free water, before use.
[0320] The compounds may also be formulated in rectal compositions
such as suppositories or retention enemas, e.g., containing
conventional suppository bases such as cocoa butter or other
glycerides.
[0321] In addition to the formulations described previously, the
compounds may also be formulated as a depot preparation. Such long
acting formulations may be administered by implantation (for
example subcutaneously or intramuscularly) or by intramuscular
injection. Thus, for example, the compounds may be formulated with
suitable polymeric or hydrophobic materials (for example as an
emulsion in an acceptable oil) or ion exchange resins, or as
sparingly soluble derivatives, for example, as a sparingly soluble
salt.
[0322] Pharmaceutical compositions (including cosmetic
preparations) may comprise from about 0.00001 to 100% such as from
0.001 to 10% or from 0.1% to 5% by weight of one or more compounds
described herein.
[0323] In one embodiment, a compound described herein, is
incorporated into a topical formulation containing a topical
carrier that is generally suited to topical drug administration and
comprising any such material known in the art. The topical carrier
may be selected so as to provide the composition in the desired
form, e.g., as an ointment, lotion, cream, microemulsion, gel, oil,
solution, or the like, and may be comprised of a material of either
naturally occurring or synthetic origin. It is preferable that the
selected carrier not adversely affect the active agent or other
components of the topical formulation. Examples of suitable topical
carriers for use herein include water, alcohols and other nontoxic
organic solvents, glycerin, mineral oil, silicone, petroleum jelly,
lanolin, fatty acids, vegetable oils, parabens, waxes, and the
like.
[0324] Formulations may be colorless, odorless ointments, lotions,
creams, microemulsions and gels.
[0325] Compounds may be incorporated into ointments, which
generally are semisolid preparations which are typically based on
petrolatum or other petroleum derivatives. The specific ointment
base to be used, as will be appreciated by those skilled in the
art, is one that will provide for optimum drug delivery, and,
preferably, will provide for other desired characteristics as well,
e.g., emolliency or the like. As with other carriers or vehicles,
an ointment base should be inert, stable, nonirritating and
nonsensitizing. As explained in Remington's, cited in the preceding
section, ointment bases may be grouped in four classes: oleaginous
bases; emulsifiable bases; emulsion bases; and water-soluble bases.
Oleaginous ointment bases include, for example, vegetable oils,
fats obtained from animals, and semisolid hydrocarbons obtained
from petroleum. Emulsifiable ointment bases, also known as
absorbent ointment bases, contain little or no water and include,
for example, hydroxystearin sulfate, anhydrous lanolin and
hydrophilic petrolatum. Emulsion ointment bases are either
water-in-oil (W/O) emulsions or oil-in-water (O/W) emulsions, and
include, for example, cetyl alcohol, glyceryl monostearate, lanolin
and stearic acid. Exemplary water-soluble ointment bases are
prepared from polyethylene glycols (PEGs) of varying molecular
weight; again, reference may be had to Remington's, supra, for
further information.
[0326] Compounds may be incorporated into lotions, which generally
are preparations to be applied to the skin surface without
friction, and are typically liquid or semiliquid preparations in
which solid particles, including the active agent, are present in a
water or alcohol base. Lotions are usually suspensions of solids,
and may comprise a liquid oily emulsion of the oil-in-water type.
Lotions are preferred formulations for treating large body areas,
because of the ease of applying a more fluid composition. It is
generally necessary that the insoluble matter in a lotion be finely
divided. Lotions will typically contain suspending agents to
produce better dispersions as well as compounds useful for
localizing and holding the active agent in contact with the skin,
e.g., methylcellulose, sodium carboxymethylcellulose, or the like.
An exemplary lotion formulation for use in conjunction with the
present method contains propylene glycol mixed with a hydrophilic
petrolatum such as that which may be obtained under the trademark
Aquaphor.RTM. from Beiersdorf, Inc. (Norwalk, Conn.).
[0327] Compounds may be incorporated into creams, which generally
are viscous liquid or semisolid emulsions, either oil-in-water or
water-in-oil. Cream bases are water-washable, and contain an oil
phase, an emulsifier and an aqueous phase. The oil phase is
generally comprised of petrolatum and a fatty alcohol such as cetyl
or stearyl alcohol; the aqueous phase usually, although not
necessarily, exceeds the oil phase in volume, and generally
contains a humectant. The emulsifier in a cream formulation, as
explained in Remington's, supra, is generally a nonionic, anionic,
cationic or amphoteric surfactant.
[0328] Compounds may be incorporated into microemulsions, which
generally are thermodynamically stable, isotropically clear
dispersions of two immiscible liquids, such as oil and water,
stabilized by an interfacial film of surfactant molecules
(Encyclopedia of Pharmaceutical Technology (New York: Marcel
Dekker, 1992), volume 9). For the preparation of microemulsions,
surfactant (emulsifier), co-surfactant (co-emulsifier), an oil
phase and a water phase are necessary. Suitable surfactants include
any surfactants that are useful in the preparation of emulsions,
e.g., emulsifiers that are typically used in the preparation of
creams. The co-surfactant (or "co-emulsifer") is generally selected
from the group of polyglycerol derivatives, glycerol derivatives
and fatty alcohols. Preferred emulsifier/co-emulsifier combinations
are generally although not necessarily selected from the group
consisting of: glyceryl monostearate and polyoxyethylene stearate;
polyethylene glycol and ethylene glycol palmitostearate; and
caprilic and capric triglycerides and oleoyl macrogolglycerides.
The water phase includes not only water but also, typically,
buffers, glucose, propylene glycol, polyethylene glycols,
preferably lower molecular weight polyethylene glycols (e.g., PEG
300 and PEG 400), and/or glycerol, and the like, while the oil
phase will generally comprise, for example, fatty acid esters,
modified vegetable oils, silicone oils, mixtures of mono- di- and
triglycerides, mono- and di-esters of PEG (e.g., oleoyl macrogol
glycerides), etc.
[0329] Compounds may be incorporated into gel formulations, which
generally are semisolid systems consisting of either suspensions
made up of small inorganic particles (two-phase systems) or large
organic molecules distributed substantially uniformly throughout a
carrier liquid (single phase gels). Single phase gels can be made,
for example, by combining the active agent, a carrier liquid and a
suitable gelling agent such as tragacanth (at 2 to 5%), sodium
alginate (at 2-10%), gelatin (at 2-15%), methylcellulose (at 3-5%),
sodium carboxymethylcellulose (at 2-5%), carbomer (at 0.3-5%) or
polyvinyl alcohol (at 10-20%) together and mixing until a
characteristic semisolid product is produced. Other suitable
gelling agents include methylhydroxycellulose,
polyoxyethylene-polyoxypropylene, hydroxyethylcellulose and
gelatin. Although gels commonly employ aqueous carrier liquid,
alcohols and oils can be used as the carrier liquid as well.
[0330] Various additives, known to those skilled in the art, may be
included in formulations, e.g., topical formulations. Examples of
additives include, but are not limited to, solubilizers, skin
permeation enhancers, opacifiers, preservatives (e.g.,
anti-oxidants), gelling agents, buffering agents, surfactants
(particularly nonionic and amphoteric surfactants), emulsifiers,
emollients, thickening agents, stabilizers, humectants, colorants,
fragrance, and the like. Inclusion of solubilizers and/or skin
permeation enhancers is particularly preferred, along with
emulsifiers, emollients and preservatives. An optimum topical
formulation comprises approximately: 2 wt. % to 60 wt. %,
preferably 2 wt. % to 50 wt. %, solubilizer and/or skin permeation
enhancer; 2 wt. % to 50 wt. %, preferably 2 wt. % to 20 wt. %,
emulsifiers; 2 wt. % to 20 wt. % emollient; and 0.01 to 0.2 wt. %
preservative, with the active agent and carrier (e.g., water)
making of the remainder of the formulation.
[0331] A skin permeation enhancer serves to facilitate passage of
therapeutic levels of active agent to pass through a reasonably
sized area of unbroken skin. Suitable enhancers are well known in
the art and include, for example: lower alkanols such as methanol
ethanol and 2-propanol; alkyl methyl sulfoxides such as
dimethylsulfoxide (DMSO), decylmethylsulfoxide (C.sub.10 MSO) and
tetradecylmethyl sulfboxide; pyrrolidones such as 2-pyrrolidone,
N-methyl-2-pyrrolidone and N-(-hydroxyethyl)pyrrolidone; urea;
N,N-diethyl-m-toluamide; C.sub.2 -C.sub.6 alkanediols;
miscellaneous solvents such as dimethyl formamide (DMF),
N,N-dimethylacetamide (DMA) and tetrahydrofurfuryl alcohol; and the
1-substituted azacycloheptan-2-ones, particularly
1-n-dodecylcyclazacycloheptan-2-one (laurocapram; available under
the trademark Azone.RTM. from Whitby Research Incorporated,
Richmond, Va.).
[0332] Examples of solubilizers include, but are not limited to,
the following: hydrophilic ethers such as diethylene glycol
monoethyl ether (ethoxydiglycol, available commercially as
Transcutol.RTM.) and diethylene glycol monoethyl ether oleate
(available commercially as Softcutol.RTM.); polyethylene castor oil
derivatives such as polyoxy 35 castor oil, polyoxy 40 hydrogenated
castor oil, etc.; polyethylene glycol, particularly lower molecular
weight polyethylene glycols such as PEG 300 and PEG 400, and
polyethylene glycol derivatives such as PEG-8 caprylic/capric
glycerides (available commercially as Labrasol.RTM.); alkyl methyl
sulfoxides such as DMSO; pyrrolidones such as 2-pyrrolidone and
N-methyl-2-pyrrolidone; and DMA. Many solubilizers can also act as
absorption enhancers. A single solubilizer may be incorporated into
the formulation, or a mixture of solubilizers may be incorporated
therein.
[0333] Suitable emulsifiers and co-emulsifiers include, without
limitation, those emulsifiers and co-emulsifiers described with
respect to microemulsion formulations. Emollients include, for
example, propylene glycol, glycerol, isopropyl myristate,
polypropylene glycol-2 (PPG-2) myristyl ether propionate, and the
like.
[0334] Other active agents may also be included in formulations,
e.g., other anti-inflammatory agents, analgesics, antimicrobial
agents, antifungal agents, antibiotics, vitamins, antioxidants, and
sunblock agents commonly found in sunscreen formulations including,
but not limited to, anthranilates, benzophenones (particularly
benzophenone-3), camphor derivatives, cinnamates (e.g., octyl
methoxycinnamate), dibenzoyl methanes (e.g., butyl methoxydibenzoyl
methane), p-aminobenzoic acid (PABA) and derivatives thereof, and
salicylates (e.g., octyl salicylate).
[0335] In certain topical formulations, the active agent is present
in an amount in the range of approximately 0.25 wt. % to 75 wt. %
of the formulation, preferably in the range of approximately 0.25
wt. % to 30 wt. % of the formulation, more preferably in the range
of approximately 0.5 wt. % to 15 wt. % of the formulation, and most
preferably in the range of approximately 1.0 wt. % to 10 wt. % of
the formulation.
[0336] Topical skin treatment compositions can be packaged in a
suitable container to suit its viscosity and intended use by the
consumer. For example, a lotion or cream can be packaged in a
bottle or a roll-ball applicator, or a propellant-driven aerosol
device or a container fitted with a pump suitable for finger
operation. When the composition is a cream, it can simply be stored
in a non-deformable bottle or squeeze container, such as a tube or
a lidded jar. The composition may also be included in capsules such
as those described in U.S. Pat. No. 5,063,507. Accordingly, also
provided are closed containers containing a cosmetically acceptable
composition as herein defined.
[0337] In an alternative embodiment, a pharmaceutical formulation
is provided for oral or parenteral administration, in which case
the formulation may comprises an activating compound-containing
microemulsion as described above, but may contain alternative
pharmaceutically acceptable carriers, vehicles, additives, etc.
particularly suited to oral or parenteral drug administration.
Alternatively, an activating compound-containing microemulsion may
be administered orally or parenterally substantially as described
above, without modification.
[0338] Compounds described herein may be stored in oxygen free
environment according to methods in the art. For example,
resveratrol or analog thereof can be prepared in an airtight
capusule for oral administration, such as Capsugel from Pfizer,
Inc.
[0339] Cells, e.g., treated ex vivo with a compound described
herein, can be administered according to methods for administering
a graft to a subject, which may be accompanied, e.g., by
administration of an immunosuppressant drug, e.g., cyclosporin A.
For general principles in medicinal formulation, the reader is
referred to Cell Therapy: Stem Cell Transplantation, Gene Therapy,
and Cellular Immunotherapy, by G. Morstyn & W. Sheridan eds,
Cambridge University Press, 1996; and Hematopoietic Stem Cell
Therapy, E. D. Ball, J. Lister & P. Law, Churchill Livingstone,
2000.
Kits
[0340] Also provided herein are kits, e.g., kits for therapeutic
purposes or kits for modulating the lifespan of cells or modulating
apoptosis. A kit may comprise one or more activating or inhibitory
compounds described herein, and optionally devices for contacting
cells with the compounds. Devices include syringes, stents and
other devices for introducing a compound into a subject or applying
it to the skin of a subject.
[0341] The present description is further illustrated by the
following examples, which should not be construed as limiting in
any way. The contents of all cited references (including literature
references, issued patents, published patent applications as cited
throughout this application) are hereby expressly incorporated by
reference.
[0342] The practice of the present methods will employ, unless
otherwise indicated, conventional techniques of cell biology, cell
culture, molecular biology, transgenic biology, microbiology,
recombinant DNA, and immunology, which are within the skill of the
art. Such techniques are explained fully in the literature. See,
for example, Molecular Cloning A Laboratory Manual, 2.sup.nd Ed.,
ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor
Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N.
Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed.,
1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid
Hybridization (B. D. Hames & S. J. Higgins eds. 1984);
Transcription And Translation (B. D. Hames & S. J. Higgins eds.
1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc.,
1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal,
A Practical Guide To Molecular Cloning (1984); the treatise,
Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer
Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds.,
1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols.
154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And
Molecular Biology (Mayer and Walker, eds., Academic Press, London,
1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M.
Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse
Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y., 1986).
EXAMPLES
Example 1
Small Molecule Activators of SIRT1
[0343] To identify compounds that modulate SIRT1 activity, we
screened a number of small molecule libraries using a fluorescent
deacetylation assay in 96-well plates (Bitterman et al. J Biol Chem
277, 45099-107 (2002)). The substrate used in the assay was a
fluorogenic peptide based on the sequence encompassing the p53-K382
acetylation site, a known target of SIRT1 in vivo (Vaziri et al.
Cell 107, 149-59 (2001); Luo et al. Cell 107, 137-48 (2001);
Langley et al. EMBO J 21, 2383-2396 (2002)). This substrate was
preferred over a variety of other fluorogenic peptide substrates
that were based on other known HDAC targets (FIG. 5). The small
molecule libraries included analogues of nicotinamide,
.epsilon.-acetyl lysine, NAD.sup.+, nucleotides, nucleotide
analogues and purinergic ligands. From the initial screen, several
sirtuin inhibitors were found (Supplementary Table 7). However, the
most striking outcome was the identification of two compounds,
quercetin and piceatannol, that stimulated SIRT1 activity five and
eight-fold, respectively (Table 1). Both quercetin and piceatannol
have been previously identified as protein kinase inhibitors
(Glossmann et al. Naunyn Schmiedebergs Arch Pharmacol 317, 100-2
(1981); Oliver et al. J Biol Chem 269, 29697-703 (1994)).
[0344] Comparison of the structures of the two activating compounds
suggested a possible structure-activity relationship. Piceatannol
comprises two phenyl groups trans to one another across a linking
ethylene moiety. The trans-stilbene ring structures of piceatannol
are superimposable on the flavonoid A and B rings of quercetin,
with the ether oxygen and carbon-2 of the C ring aligning with the
ethylene carbons in piceatannol (see structures, Table 1). Further,
the 5,7,3' and 4' hydroxyl group positions in quercetin can be
aligned, respectively, with the 3,5,3' and 4' hydroxyls of
piceatannol.
[0345] Given the demonstrated longevity-enhancing effects of
sirtuin activity in S. cerevisiae( Kaeberlein et al. Genes Dev 13,
2570-80 (1999)) and C. elegans(Tissenbaum, H. A. and Guarente, L.
Nature 410, 227-30. (2001)), it was naturally of interest to
further explore the structure-activity relationship among compounds
that stimulate SIRT1. Both quercetin and piceatannol are
polyphenols, members of a large and diverse group of plant
secondary metabolites that includes flavones, stilbenes,
flavanones, isoflavones, catechins (flavan-3-ols), chalcones,
tannins and anthocyanidins (Ferguson, L. R. Mutat Res 475, 89-111
(2001); Middleton et al. Pharmacol Rev 52, 673-751 (2000)).
Polyphenols noteworthy with respect to potential
longevity-enhancing effects include resveratrol, a stilbene found
in red wine and epigallocatechin gallate (EGCG) from green tea.
Both have been suggested on the basis of epidemiological and
mechanistic investigations to exert cancer chemopreventive and
cardioprotective effects (Ferguson, L. R. Mutat Res 475, 89-111
(2001); Middleton et al. Pharmacol Rev 52, 673-751 (2000); and Jang
et al. Science 275, 218-20 (1997)). We therefore performed a
secondary screen encompassing resveratrol, EGCG and additional
representatives from a number of the polyphenol classes listed
above. The screen emphasized flavones due to the great number of
hydroxyl position variants available in this group (Middleton et
al. Pharmacol Rev 52, 673-751 (2000). The results of this screen
are summarized in Supplementary Tables 1-6. In the tables, a "ratio
to control rate" above 1 indicates that a compound with such a rate
is an activator of the sirtuin tested and a number under 1
indicates that a compound is an inhibitor.
[0346] Additional potent SIRT1 activators were found among the
stilbenes, chalcones and flavones (Table 1, Supplementary Tables 1
and 2). The six most active flavones had 3' and 4' hydroxyls
(Supplementary Table 2), although it should be noted that the most
active compound overall, resveratrol (3,5,4'-trihydroxystilbene),
was more active than piceatannol, which differs only by its
additional 3'-hydroxyl (Table 1). The importance of the 4'-hydroxyl
to activity is underscored by the fact that each of the 12 most
stimulatory flavones share this feature (Supplementary Tables 1 and
2).
[0347] Many, but not all of the most active compounds include
hydroxyls in the two meta positions (e.g. 5,7-dihydroxylated
flavones) of the ring (A ring), trans to that with the 4' or 3',4'
pattern (B ring, see Table 1, Supplementary Tables 1 and 2). A
potentially coplanar orientation of the trans phenyl rings may be
important for activity since catechins and flavanones, which lack
the 2,3 double-bond, have weak activity despite having equivalent
hydroxylation patterns to various stimulatory flavones (compare
Supplementary Tables 2 and 3 with 4 and 5). The absence of activity
in the isoflavone genistein, although hydroxylated in an equivalent
way to the stimulatory compounds apigenin and resveratrol (see
Supplementary Tables 1, 2 and 4), is consistent with the idea that
the trans positioning and spacing of the hydroxylated rings
contributes strongly to activity.
[0348] The biological effects of polyphenols are frequently
attributed to antioxidant, metal ion chelating and/or free-radical
scavenging activity (Ferguson, L. R. Mutat Res 475, 89-111 (2001);
Jang et al. Science 275, 218-20 (1997)). We considered the
possibility that the apparent polyphenol stimulation of SIRT1 might
simply represent the repair of oxidative and/or metal-ion induced
damage incurred during preparation of the recombinant protein. Two
features of our results argue against this being the case. First, a
variety of free-radical protective compounds, including
antioxidants, chelators and radical scavengers, failed to stimulate
SIRT1 (see Supplementary Table 6.). Second, among various
polyphenols of equivalent antioxidant capacity we observed diverse
SIRT1 stimulating activity (e.g. compare resveratrol, quercetin and
the epicatechins in Supplementary Tables 1, 2 and 5 and see
Stojanovic et al. Arch Biochem Biophys 391, 79-89 (2001)).
Example 2
Resveratrol's Effects on SIRT1 Kinetics
[0349] Detailed enzyme kinetic investigations were performed using
the most potent activator, resveratrol. Dose-response experiments
performed under the conditions of the polyphenol screening assays
(25 .mu.M NAD.sup.+, 25 .mu.M p53-382 acetylated peptide), showed
that the activating effect doubled the rate at .about.11 .mu.M and
was essentially saturated at 100 .mu.M resveratrol (FIG. 1a).
Initial enzyme rates, in the presence or absence of 100 .mu.M
resveratrol, were determined either as a function of acetyl-peptide
concentration with high NAD.sup.+ (3 mM NAD.sup.+, FIG. 1b) or as a
function of NAD.sup.+ concentration with high acetyl-peptide (1 mM
p53-382 acetylated peptide, FIG. 1c). Although resveratrol had no
significant effect on the two V.sub.max determinations (FIGS. 1b,
1c), it had pronounced effects on the two apparent K.sub.ms. Its
effect on the acetylated peptide K.sub.m was particularly striking,
amounting to a 35-fold decrease (FIG. 1b). Resveratrol also lowered
the K.sub.m for NAD.sup.+ over 5-fold (FIG. 1c). Since resveratrol
acts only on K.sub.m, it could be classified as an allosteric
effector of `K system` type (Monod et al. J. Mol. Biol. 12, 88-118
(1965)). This can imply that only the substrate binding affinity of
the enzyme has been altered, rather than a rate-limiting catalytic
step.
[0350] Our previous kinetic analysis of SIRT1 and Sir2 (Bitterman
et al. J Biol Chem 277, 45099-107 (2002)) and our genetic analysis
of Sir2's role in yeast lifespan extension (Anderson et al. Nature
423, 181-5 (2003); Anderson et al. J Biol Chem 277, 18881-90.
(2002)) have implicated nicotinamide (a product of the sirtuin
reaction) as a physiologically important inhibitor of sirtuin
activity. Therefore the effects of resveratrol on nicotinamide
inhibition were tested. In experiments similar to those of FIGS. 1b
and 1c, kinetic constants in the presence of 50 .mu.M nicotinamide
were determined either by varying the concentration of NAD.sup.+ or
that of the p53-382 acetylated peptide (FIG. 1d). Nicotinamide, in
contrast to resveratrol, affects the SIRT1 V.sub.max (note 30% and
36% V.sub.max decreases in absence of resveratrol, FIG. 1d and see
Bitterman et al. J Biol Chem 277, 45099-107 (2002)). In the
presence of 50 .mu.M nicotinamide, resveratrol appears to have
complex, concentration-dependent effects on the kinetics of SIRT1
(FIG. 1d). Apparent K.sub.m for NAD.sup.+ and acetylated substrate
appear to actually be raised by 5 .mu.M resveratrol when
nicotinamide is present. At 20 and 100 .mu.M, in the presence of 50
.mu.M nicotinamide, resveratrol lowers the K.sub.m for both
NAD.sup.+ and acetylated peptide, without reversing the
nicotinamide-induced V.sub.max decrease. It has been proposed that
sirtuins may bind nicotinamide at a second site, known as "the C
pocket", distinct from the "B" site that interacts with the
nicotinamide moiety of NAD.sup.+ (Bitterman et al. J Biol Chem 277,
45099-107 (2002)). In light of this potential complexity, further
kinetic studies, supplemented by structural/crystallographic
information, will likely be necessary to fully elucidate the
interplay between the effects of nicotinamide and polyphenols.
Example 3
Activating Compounds Extend Yeast Lifespan
[0351] To investigate whether these compounds could stimulate
sirtuins in vivo, we utilized S. cerevisiae, an organism in which
the upstream regulators and downstream targets of Sir2 are
relatively well understood. A resveratrol dose-response study of
Sir2 deacetylation rates (FIG. 2a) indeed reveals that resveratrol
stimulates Sir2 in vitro, with the optimum concentration of
activator being 2-5 .mu.M. Levels of activation were somewhat lower
than those for SIRT1, and unlike SIRT1, inhibition was seen at
concentrations greater than .about.100 .mu.M.
[0352] Resveratrol and four other potent sirtuin activators,
representatives of the stilbene, flavone, and chalcone families,
were tested for their effect on yeast lifespan. Due to the
potential impediment by the yeast cell wall or plasma membrane and
suspected slow oxidation of the compound in the medium, we chose to
use a concentration (10 .mu.M) slightly higher than the optimal
resveratrol concentration in vitro. As shown in FIG. 2b, quercetin
and piceatannol had no significant effect on lifespan. In contrast,
butein, fisetin and resveratrol increased average lifespan by 31,
55 and 70%, respectively, and all three significantly increased
maximum lifespan (FIG. 2c). Concentrations of resveratrol higher
than 10 .mu.M provided no added lifespan benefit and there was no
lasting effect of the compound on the lifespan of pre-treated young
cells (FIG. 2d).
[0353] For subsequent yeast genetic experiments we focused on
resveratrol because it was the most potent SIRT1 activator and
provided the greatest lifespan extension. Glucose restriction, a
form of CR in yeast, resulted in no significant extension of the
long-lived resveratrol-treated cells (FIG. 3a), indicating that
resveratrol likely acts via the same pathway as CR. Consistent with
this, resveratrol had no effect on the lifespan of a sir2 null
mutant (FIG. 3b). Given that resveratrol is reported to have
fungicidal properties at high concentrations (Pont, V. and Pezet,
R. J Phytopathol 130, 1-8 (1990)), and that mild stress can extend
yeast lifespan by activating PNC1 (Anderson et al. Nature 423,
181-5 (2003)), it was plausible that resveratrol was extending
lifespan by inducing PNC1, rather than acting on Sir2 directly.
However, resveratrol extended the lifespan of a pnc1 null mutant
nearly as well as it did wild type cells (FIG. 3b). Together these
data show that resveratrol acts downstream of PNC1 and requires
SIR2 for its effect. Thus, the simplest explanation for our
observations is that resveratrol increases lifespan by directly
stimulating Sir2 activity.
[0354] A major cause of yeast aging is thought to stem from the
inherent instability of the repetitive rDNA locus (Sinclair, D. A.
Mech Ageing Dev 123, 857-67 (2002); Lin et al. Science 289, 2126-8
(2000); Sinclair, D. A. and Guarente, L. Cell 91, 1033-42 (1997);
Defossez et al. Mol Cell 3, 447-55 (1999); Park et al. Mol Cell
Biol 19, 3848-56 (1999)). Homologous recombination between rDNA
repeats can generate an extrachromosomal circular form of rDNA
(ERC) that is replicated until it reaches toxic levels in old
cells. Sir2 is thought to extend lifespan by suppressing
recombination at the replication fork barrier of rDNA( Benguria et
al. Nucleic Acids Res 31, 893-8 (2003)). Consistent with the
lifespan extension we observed for resveratrol, this compound
reduced the frequency of rDNA recombination by .about.60% (FIG.
3c), in a SIR2-dependent manner (FIG. 3d). In the presence of the
Sir2 inhibitor nicotinamide, recombination was also decreased by
resveratrol (FIG. 3c), in agreement with the kinetic data (see FIG.
1d). Interestingly, we found that resveratrol and other sirtuin
activators had only minor effects on rDNA silencing (FIG. 3e and
f).
[0355] Another measure of lifespan in S. cerevisiae is the length
of time cells can survive in a metabolically active but nutrient
deprived state. Aging under these conditions (i.e. chronological
aging) is primarily due to oxidative damage (Longo, V. D. and
Finch, C. E. Science 299, 1342-6 (2003)). Resveratrol (10 .mu.M or
100 .mu.M) failed to extend chronological lifespan (not shown),
indicating that the sirtuin-stimulatory effect of resveratrol may
be more relevant in vivo than its antioxidant activity (Ferguson,
L. R. Mutat Res 475, 89-111 (2001); Middleton et al. Pharmacol Rev
52, 673-751 (2000)).
Example 4
Effects of Activators in Human Cells
[0356] To test whether these compounds could stimulate human SIRT1
in vivo, we first employed a cellular deacetylase assay that we had
developed. A schematic of the assay procedure is depicted in FIG.
4a. Cells are incubated with media containing the fluorogenic
.epsilon.-acetyl-lysine substrate, `Fluor de Lys` (FdL). This
substrate, neutral when acetylated, becomes positively charged upon
deacetylation and accumulates within cells (see FIG. 6a). Lysis of
the cells and addition of the non-cell-permeable `Developer`
reagent releases a fluorophor specifically from those substrate
molecules that have been deacetylated (FIG. 4a and see Methods).
With HeLa cells growing adherently, 5-10% of the signal produced in
this assay is insensitive to 1 .mu.M trichostatin A (TSA), a potent
inhibitor of class I and II HDACs but not sirtuins (class III)
(Denu, J. M. Trends Biochem Sci 28, 41-8 (2003)). (FIGS. 6b and
6c).
[0357] A selection of SIRT1-stimulatory and non-stimulatory
polyphenols were tested for their effects on this TSA-insensitive
signal (FIG. 4b). Cellular deacetylation signals in the presence of
each compound (y-axis, FIG. 4b) were plotted against their
fold-stimulations of SIRT1 in vitro (x-axis, FIG. 4b, data from
Supplementary Tables 1-3). For most of the compounds, the in vitro
activity roughly corresponded to the cellular signal. Compounds
with little or no in vitro activity clustered around the negative
control (Group A, FIG. 4b). Another grouping, of strong in vitro
activators is clearly distanced from the low activity cluster in
both dimensions (Group B, FIG. 4b). A notable outlier was butein, a
potent activator of SIRT1 in vitro which had no effect on the
cellular signal. With allowances for possible variation among these
compounds in properties unrelated to direct sirtuin stimulation,
such as cell-permeability and rates of metabolism, these data are
consistent with the idea that certain polyphenols can activate
native sirtuins in vivo.
[0358] One known target of SIRT1 in vivo is lysine 382 of p53.
Deacetylation of this residue by SIRT1 decreases the activity and
half-life of p53 (Vaziri et al. Cell 107, 149-59 (2001); Luo et al.
Cell 107, 137-48. (2001); Langley et al. EMBO J 21, 2383-2396
(2002)). To follow the acetylation status of K382 we generated a
rabbit polyclonal antibody that recognizes the acetylated form of
K382 (Ac-K382) on Western blots of whole cell lysates. As a control
we showed that the signal was specifically detected in extracts
from cells exposed to ionizing radiation (FIG. 4c), but not in
extracts from cells lacking p53 or where arginine had been
substituted for lysine 382 (data not shown). U2OS osteosarcoma
cells were pre-treated for 4 hours with resveratrol (0.5 and 50
.mu.M) and exposed to UV radiation. We consistently observed a
marked decrease in the level of Ac-K382 in the presence of 0.5
.mu.M resveratrol, compared to untreated cells (FIG. 4d). At higher
concentrations of resveratrol (>50 .mu.M) the effect was
reversed (FIG. 4d and data not shown), consistent with previous
reports of increased p53 activity at such concentrations (Dong, Z.
Mutat Res 523-524, 145-50 (2003)). The ability of low
concentrations of resveratrol to promote deacetylation of p53 was
diminished in cells expressing a dominant-negative SIRT1 allele
(H363Y) (FIG. 4e), demonstrating that SIRT1 is necessary for this
effect. This biphasic dose-response of resveratrol could explain
the dichotomy in the literature regarding the effects of
resveratrol on cell survival (Ferguson, L. R. Mutat Res 475, 89-111
(2001); Dong, Z. Mutat Res 523-524, 145-50 (2003); Nicolini et al.
Neurosci Lett 302, 41-4 (2001)).
[0359] Thus, we have discovered the first known class of small
molecule sirtuin activators, all of which are plant polyphenols.
These compounds can dramatically stimulate sirtuin activity in
vitro and promote effects consistent with increased sirtuin
activity in vivo. In human cells, resveratrol promotes
SIRT1-mediated p53 deacetylation of K382. In yeast, the effect of
resveratrol on lifespan is as great as any longevity-promoting
genetic manipulation (Anderson et al. Nature 423, 181-5 (2003)) and
has been linked convincingly to the direct activation of Sir2. The
correlation between lifespan and rDNA recombination, but not
silencing, adds to the body of evidence that yeast aging is due to
DNA instability (Sinclair, D. A. Mech Ageing Dev 123, 857-67
(2002); Lin et al. Science 289, 2126-8 (2000); Sinclair, D. A. and
Guarente, L. Cell 91, 1033-42. (1997); Defossez et al. Mol Cell 3,
447-55 (1999); Park et al. Mol Cell Biol 19, 3848-56 (1999)) not
gene dysregulation (Jazwinski, S. M. Ann N Y Acad Sci 908, 21-30
(2000)).
[0360] Sirtuins have been found in diverse eukaryotes, including
fungi, protozoans, metazoans and plants (Pandey et al. Nucleic
Acids Res 30, 5036-55 (2002); Frye, R. A. Biochem Biophys Res
Commun 273, 793-8 (2000)), and likely evolved early in life's
history (Kenyon, C. A conserved regulatory mechanism for aging.
Cell 105, 165-168 (2001)). Plants are known to produce a variety of
polyphenols, including resveratrol, in response to stresses such as
dehydration, nutrient deprivation, UV radiation and pathogens
(Soleas et al. Clin Biochem 30, 91-113 (1997); Coronado et al.
Plant Physiol 108, 533-542 (1995)). Therefore it is believed that
these compounds may be synthesized to regulate a sirtuin-mediated
plant stress response. This would be consistent with the recently
discovered relationship between environmental stress and Sir2
activity in yeast (Anderson et al. Nature 423, 181-5 (2003)).
Perhaps these compounds have stimulatory activity on sirtuins from
fungi and animals because they mimic an endogenous activator, as is
the case for the opiates/endorphins, cannabinols/endocannabinoids
and various polyphenols with estrogen-like activity (Ferguson, L.
R. Mutat Res 475, 89-111 (2001); Middleton et al. Pharmacol Rev 52,
673-751 (2000)). Alternatively, animal and fungal sirtuins may have
retained or developed an ability to respond to these plant
metabolites because they are a useful indicator of a deteriorating
environment and/or food supply.
Example 5
Materials and Methods for Examples 1-4
Compound Libraries and Deacetylation Assays
[0361] His.sub.6-tagged recombinant SIRT1 and GST-tagged
recombinant Sir2 were prepared as described by (Bitterman et al. J
Biol Chem 277, 45099-107. (2002). From 0.1 to 1 .mu.g of SIRT1 and
1.5 .mu.g of Sir2 were used per deacetylation assay (in 50 .mu.l
total reaction). SIRT1 assays and certain of those for Sir2
employed the p53-382 acetylated substrate (`Fluor de Lys-SIRT1',
BIOMOL) rather than FdL.
[0362] Themed compound libraries (BIOMOL) were used for primary and
secondary screening. Most polyphenol compounds were dissolved at 10
mM in dimethylsulfoxide (DMSO) on the day of the assay. For water
soluble compounds and negative controls, 1% v/v DMSO was added to
the assay. In vitro fluorescence assay results were read in white
1/2-volume 96-well microplates (Corning Costar 3693) with a
CytoFluor.TM.II fluorescence plate reader (PerSeptive Biosystems,
Ex. 360 nm, Em. 460 nm, gain=85). HeLa cells were grown and the
cellular deacetylation assays were performed and read, as above,
but in full-volume 96-well microplates (Corning Costar 3595).
Unless otherwise indicated all initial rate measurements were means
of three or more replicates, obtained with single incubation times,
at which point 5% or less of the substrate initially present had
been deacetylated. Calculation of net fluorescence increases
included subtraction of a blank value, which in the case of Sir2
was obtained by omitting the enzyme from the reaction and in the
case of SIRT1 by adding an inhibitor (200 .mu.M suramin or 1 mM
nicotinamide) to the reaction prior to the acetylated substrate. A
number of the polyphenols partially quenched the fluorescence
produced in the assay and correction factors were obtained by
determining the fluorescence increase due to a 3 .mu.M spike of an
FdL deacetylated standard (BIOMOL, catalog number KI-142). All
error bars represent the standard error of the mean.
Media and Strains
[0363] All yeast strains were grown at 30.degree. C. in complete
yeast extract/bactopeptone, 2.0% (w/v) glucose (YPD) medium except
where stated otherwise. Calorie restriction was induced in 0.5%
glucose. Synthetic complete (SC) medium consisted of 1.67% yeast
nitrogen base, 2% glucose, 40 mg/liter each of auxotrophic markers.
SIR2 was integrated in extra copy and disrupted as described by Lin
et al. (Science 289, 2126-8 (2000)). Other strains are described
elsewhere (Bitterman et al. J Biol Chem 277, 45099-107 (2002)). For
cellular deacetylation assays, HeLa S3 cells were used. U2OS
osteosarcoma and human embryonic kidney (HEK 293) cells were
cultured adherently in Dulbecco's Modified Eagle's Medium (DMEM)
containing 10% fetal calf serum (FCS) with 1.0% glutamine and 1.0%
penecillin/streptomycin. HEK 293 overexpressing dominant negative
SIRT1 H363Y was a gift of R. Frye (U. Pittsburgh).
Lifespan Determinations
[0364] Lifespan measurements were performed using PSY316AT
MAT.alpha. as previously described by Anderson et al. (J Biol Chem
277, 18881-90. (2002). All compounds for lifespan analyses were
dissolved in 95% ethanol and plates were dried and used within 24
hours. Prior to lifespan analysis, cells were pre-incubated on
their respective media for at least 15 hours. Following transfer to
a new plate, cells were equilibrated on the medium for a minimum of
4 hours prior to micro-manipulating them. At least 30 cells were
examined per experiment and each experiment was performed at least
twice. Statistical significance of lifespan differences was
determined using the Wilcoxon rank sum test. Differences are stated
to be significant when the confidence is higher than 95%.
Silencing and Recombination Assays
[0365] Ribosomal DNA silencing assays using the URA3 reporters were
performed as previously described by Bitterman et al. (J Biol Chem
277, 45099-107 (2002)). Ribosomal DNA recombination frequencies
were determined by plating W303AR cells (Sinclair, D. A. and
Guarente, L. Cell 91, 1033-42 (1997)) on YPD medium with low
adenine/histidine and counting the fraction of half-red sectored
colonies using Bio-Rad Quantity One software as described by
Anderson et al. (J Biol Chem 277, 18881-90. (2002)). At least 6000
cells were analyzed per experiment and all experiments were
performed in triplicate. All strains were pre-grown for 15 hours
with the relevant compound prior to plating.
Proteins and Western Analyses
[0366] Recombinant Sir2-GST was expressed and purified from E. coli
as previously described except that lysates were prepared using
sonication (Bitterman et al. J Biol Chem 277, 45099-107 (2002).
Recombinant SIRT1 from E. coli was prepared as previously described
(Bitterman et al. J Biol Chem 277, 45099-107 (2002). Polyclonal
antiserum against p53-AcK382 was generated using an acetylated
peptide antigen as previously described (Vaziri et al. Cell 107,
149-59 (2001) with the following modifications. Anti-Ac-K382
antibody was affinity purified using non-acetylated p53-K382
peptides and stored in PBS at -70.degree. C. and recognized an
acetylated but not a non-acetylated p53 peptide. Western
hybridizations using anti-acetylated K382 or anti-actin (Chemicon)
antibody were performed at 1:1000 dilution of antibody.
Hybridizations with polyclonal p53 antibody (Santa Cruz Biotech.)
used 1:500 dilution of antibody. Whole cell extracts were prepared
by lysing cells in buffer containing 150 mM NaCl, 1 mM MgCl.sub.2,
10% glycerol, 1% NP40, 1 mM DTT and anti-protease cocktail
(Roche).
Example 6
Localization of the Activation Domain of Sirtuins to their
N-Terminus
[0367] Yeast Sir2 and human SIRT1 are very homologous and differ
from human SIRT2 by the addition of an N-terminal domain that is
absent in SIRT2. The effect of resveratrol was assayed on human
recombinant SIRT2 as follows. Human recombinant SIRT2 was incubated
at a concentration of 1.25 .mu.g/well with 25 .mu.M of Fluor de
Lys-SIRT2 (BIOMOL cat. # KI-179) and 25 .mu.M NAD.sup.+ for 20
minutes at 37.degree. C., as described above. Results, indicate
that, in contrast to SIRT1, increasing concentrations of
resveratrol decrease SIRT2 activity. Thus, based on the difference
in structure of SIRT1 and SIRT2, i.e., the absence of an N-terminal
domain, it is believed that the N-terminal domain of SIRT1 and Sir2
is necessary for activation by the compounds described herein. In
particular, it is likely that the activator compounds described
herein interact with the N-terminal portion of sirtuins. The
N-terminal portion of SIRT1 that is necessary for the action of the
compounds is from about amino acid 1 to about amino acid 176, and
that of Sir2 is from about amino acid 1 to about amino acid
175.
Example 7
Resveratrol Extends the Lifespan of C. elegans
[0368] 50 C. elegans worms (strain N2) were grown in the presence
or absence of 100 .mu.M resveratrol for 17 days. On day 17, only 5
worms in the control group without resveratrol were alive, whereas
17 worms were alive in the group that was treated with resveratrol.
Thus, the presence of resveratrol in the growth media of C. elegans
extends their lifespan.
Example 8
Identification of Additional Activators of Sirtuins
[0369] Using the screening assay described in Example 1, five more
sirtuin activators have been identified. These are set forth in
supplementary Table 8.
Example 9
Identification of Inhibitors of Sirtuins
[0370] Using the screening assay described in Example 1, more
inhibitors were identified. These are set forth in the appended
supplementary Table 8, and correspond to the compounds having a
ratio to control rate of less than 1.
Example 10
Identification of Further Activators and Inhibitors of Sirtuins
[0371] Additional activators and inhibitors of sirtuins were
identified, and are listed in Tables 9-13. In these Tables, "SE"
stands for Standard error of the mean and N is the number of
replicates used to calculate mean ratio to the control rate and
standard error.
[0372] All SIRT1 rate measurements used in the calculation of
"Ratio to Control Rate" were obtained with 25 .mu.M NAD.sup.+ and
25 .mu.M p53-382 acetylated peptide substrate were performed as
described above and in K. T. Howitz et al. Nature (2003) 425: 191.
All ratio data were calculated from experiments in which the total
deacetylation in the control reaction was 0.25-1.25 .mu.M peptide
or 1-5% of the initial concentration of acetylated peptide.
[0373] Stability determinations (t.sub.1/2) derived from SIRT1 rate
measurements performed in a similar way to those described above,
except that 5 .mu.M p53-382 acetylated peptide substrate was used
rather than 25 .mu.M. The fold-stimulation (ratio to control)
obtained with a compound diluted from an aged stock solution was
compared to an identical dilution from a stock solution freshly
prepared from the solid compound. "t.sub.1/2" is defined as the
time required for the SIRT1 fold-stimulation of the compound from
the aged solution to decay to one-half of that obtained from a
freshly prepared solution. Ethanol stocks of resveratrol, BML-212
and BML-221 were prepared at 2.5 mM and the compounds were assayed
at a final concentration of 50 .mu.M. The water stock of
resveratrol was 100 .mu.M and the assay performed at 10 .mu.M.
Stocks were aged by storage at room 5 temperature, in glass vials,
under a nitrogen atmosphere.
[0374] The effect of some of these compounds on lifespan was
determined in yeast, C. elegans and D. melanogaster, as described
above. The results are set forth below in Table A: TABLE-US-00001 %
change in yeast % change in C. elegans % change in D. replicative
lifespan lifespan relative to melanogaster lifespan relative to
untreated untreated organisms relative to untreated Compound
organisms (10 .mu.M).sup.a (100/500 .mu.M).sup.b (100 .mu.M).sup.c
untreated 100% 100% 100% Resveratrol 170-180% 110% 130%
3,5,4'-Trihydroxy-trans-stilbene (from M. Tatar) Pinosylvin 114% ?
? 3,5-Dihydroxy-trans-stilbene BML-212 98% ? ?
3,5-Dihydroxy-4'-fluoro-trans-stilbene BML-217 90% ? ?
3,5-Dihydroxy-4'-chloro-trans-stilbene BML-221 165% >100%
(ongoing) ? 3,4'-Dihydroxy-5-acetoxy-trans-stilbene BML-233 ? 70%
(10) ? 3,5-Dihydroxy-4'-methoxy-trans-stilbene 50% (500)
.sup.aReplicative lifespans performed using 2% (w/v) glucose
standard yeast compete medium (YPD) under standard conditions.
.sup.bLifespan assays performed on N2 worms using E. coli as food
under standard conditions. .sup.cLifespan assays preformed using
1.5% yeast as food supply under otherwise standard conditions.
[0375] The results indicate that resveratrol significantly extends
lifespan in yeast, C. elegans and in D. melanogaster. Since BML-233
was shown to be a strong activator of 15 sirtuins (see above), the
results obtained in C. elegans may indicate that the compound is
toxic to the cells.
[0376] Without wanting to be limited to particular structures, it
appears that the following structure/activity relationships exist.
SIRT1 activation results from several of these new analogs
confirmed the importance of planarity, or at least the potential
for planarity, between and within the two rings of the active
compounds. Reduction of the double bond of the ethylene function,
between, the two rings essentially abolishes activity (compare
Resveratrol, Table A and Dihydroresveratrol, Table E). Replacement
of a phenyl moiety with a cyclohexyl group is nearly as detrimental
to SIRT1 stimulating activity (compare Pinosylvin, Table 9 and
BML-224, Table 12). Amide bonds are thought to have a partially
double bond character. However, replacement of the ethylene
function with a carboxamide abolished activity (compare Pinosylvin,
Table 9, with BML-219, Table 13). It is possible that this effect
could be due in part to the position that carbonyl oxygen must
assume in the conformation that places the two rings trans to one
another. If so, a compound in which the positions of the amide
nitrogen and carbonyl are reversed might be expected to have
greater activity.
[0377] In twelve of the analogs resveratrol's 4'-hydroxy was
replaced with various functionalities (see Tables 9 and 10, BML-221
in Table 11, BML-222 in Table 12). Although none of the
replacements tried led to substantial increases in SIRT1
stimulating activity, this parameter was, in general, remarkably
tolerant of substitutions at this position. Small groups (H-- in
Pinosylvin, Cl-- in BML-217, --CH.sub.3 in BML-228) did the least
to decrease activity. There is some evidence of a preference in the
enzyme's stilbene binding/activation site for unbranched (ethyl in
BML-225, azido in BML-232, --SCH.sub.3 in BML-230) and hydrophobic
functions (compare isopropyl in BML-231 to acetoxy in BML-221,
acetamide in BML-222). Solution stability relative to resveratrol
was strongly increased by one of the two 4'-substitutions (acetoxy,
BML-221) tested for this so far.
[0378] Resveratrol is currently the most potent known activator of
SIRT1. The collection of analogs described above, particularly the
group entailing substitutions at the 4' position, may be
instrumental in informing the design of new SIRT1 ligands with
improved pharmacological properties. One parameter that may be of
interest in this regard is stability. One 4'-substituted analog,
BML-221, displays a vast improvement in solution stability relative
to resveratrol and although diminished in vitro SIRT1 activating
ability, retains much of resveratrol's biological activity (see
lifespan data). The 4'-hydroxyl of resveratrol is thought to be of
primary importance to resveratrol's free-radical scavenging
reactivity (S. Stojanovic et al. Arch. Biochem. Biophys. 2001 391
79). Most of the 4'-substituted analogs have yet to be tested for
solution stability, but if resveratrol's instability in solution is
due to redox reactivity, many of the other analogs would be
expected to also exhibit improved stability.
[0379] The results obtained with 4'-substituted analogs may
indicate promising routes to explore while seeking to increase
SIRT1 binding affinity. For example, the efficacy of the 4'-ethyl
compound (BML-225) might indicate the presence of a narrow,
hydrophobic binding pocket at the SIRT1 site corresponding to the
4' end of resveratrol. Several new series of 4'-substituted analogs
are planned, the simplest comprising straight-chain aliphatic
groups of various lengths.
Example 11
Methods of Synthesis of the Compounds in Tables 9-13
[0380] Most of the resveratrol analogs were synthesized by the same
general procedure, from a pair of intermediates, a
benzylphosphonate and an aldehyde. The synthesis or sources of
these intermediates are described in section II. Section III.
describes the procedures for synthesizing the final compounds from
any of the benzylphosphonate/aldehyde pairs. The coupling reaction
(Section III. A.) is followed by one of two deprotection reactions
depending on whether the intermediates contained methoxymethyl
(Section III. B.) or methoxy (Section III. C.) protecting groups.
Section IV corresponds to Tables 14-18, which list the particular
benzylphosphonate and aldehyde used in the synthesis of particular
final compounds. Seven of the compounds--Resveratrol,
3,5-Dihydroxy-4'-methoxy-trans-stilbene, Rhapontin aglycone,
BML-227, BML-221, Dihydroresveratrol, BML-219--were not synthesized
by the general procedure and "N/A" appears next to their entries in
the table. Resveratrol was from BIOMOL and the syntheses of the
remaining compounds are described in Section V.
II. Synthetic Intermediates
A. Benzylphosphonates (Synthesized)
[0381] Synthesis of Diethyl 4-Acetamidobenzylphosphonate: To
diethyl 4-aminobenzylphosphonate in 1:1 methylene chloride/pyridine
was added catalytic DMAP and acetic anhydride (1.1 eq.). After 3
hours, the reaction was evaporated to dryness and purified via
flash chromatography (silica gel).
[0382] Synthesis of Diethyl 4-Methylthiobenzylphosphonate:
4-Methylthiobenzyl chloride was heated with triethylphosphite (as
solvent) at 120.degree. C. overnight. Excess triethyl phosphite was
distilled off under high vacuum and heat. Flash chromatography
(silica gel) yielded the desired product.
[0383] Synthesis of Diethyl 3,5-Dimethoxybenzylphosphonate: From
3-5-Dimethoxybenzyl bromide. See synthesis of Diethyl
4-Methylthiobenzylphosphonate.
[0384] Synthesis of Diethyl 4-Fluorobenzylphosphonate: From
4-Fluorobenzylphosphonate. See synthesis of Diethyl
4-Methylthiobenzylphosphonate.
[0385] Synthesis of Diethyl 4-azidobenzylphosphonate: To diethyl
4-aminobenzylphosphonate in acetonitrile (2.5 mL) at 0.degree. C.
was added 6M HCl (1 mL). Sodium nitrite (1.12 eq.) in water (1 mL)
was added drop wise and the resulting solution stirred at 0.degree.
C. for 30 mins. Sodium azide (8 eq.) in water (1 mL) added drop
wise (bubbling) and the solution stirred at 0.degree. C. for 30
mins., then at room temperature for 1 hour. The reaction was
diluted with ethyl acetate and washed with water and brine and
dried over sodium sulfate. Flash chromatography (silica gel)
yielded the desired product.
B. Aldehydes (Synthesized)
[0386] Synthesis of 3,5-Dimethoxymethoxybenzaldehyde: To
3,5-dihydroxybenzaldehyde in DMF at 0.degree. C. was added sodium
hydride (2.2 eq.). The reaction was stirred for 30 min. at
0.degree. C. Chloromethylmethyl ether (2.2 eq.) was added neat,
drop wise and the reaction allowed to warm to room temperature over
1.5 hrs. The reaction mixture was diluted with diethyl ether and
washed with water (2.times.) and brine (1.times.) and dried over
sodium sulfate. Flash chromatography (silica gel) yielded the
desired product.
C. Purchased Intermediates: Unless Listed Above, all Synthetic
Intermediates were Purchase from Sigma-Aldrich.
III. General Procedure for the Synthesis of Resveratrol
Analogues
A. Benzylphosphonate/Aldehyde Coupling Procedure
[0387] To the appropriate benzylphosphonate (1.2 eq.) in
dimethylformamide (DMF) at room temperature was added sodium
methoxide (1.2 eq.). This solution was allowed to stir at room
temperature for approximately 45 minutes. The appropriate aldehyde
(1 eq.) was then added (neat or in a solution of
dimethylformamide). The resulting solution was then allowed to stir
overnight at room temperature. Thin layer chromatography (TLC) was
used to determine completeness of the reaction. If the reaction was
not complete, the solution was heated at 45-50.degree. C. until
complete. The reaction mixture was poured into water and extracted
with ethyl acetate (2.times.). The combined organic layers were
washed with brine and dried over sodium sulfate. Flash
chromatography (silica gel) yielded the desired products.
B. General Procedure for the Deprotection of
Methoxymethylresveratrol Analogues
[0388] To the appropriate methoxymethylstilbene derivative in
methanol was added two drops of concentrated HCl. The resulting
solution was heated overnight at 50.degree. C. The solution was
evaporated to dryness upon completion of the reaction. Flash
chromatography (silica gel) yielded the desired product.
C. General Procedure for the Deprotection of Methoxyresveratrol
Analogues
[0389] To the appropriate methoxystilbene derivative in methylene
chloride was added tetrabutylammonium iodide (1.95 eq. per methoxy
group). The reaction was cooled to 0.degree. C. and boron
trichloride (1 M in methylene chloride; 2 eq. per methoxy group)
was added dropwise. Following the addition of boron trichloride,
the cooling bath was removed and the reaction allowed to stir at
room temperature until complete (as indicated by TLC). Saturated
sodium bicarbonate solution was added and the reaction vigorously
stirred for 1 hour. The reaction was poured into cold 1M HCl and
extracted with ethyl acetate (3.times.). The combined organic
layers were washed with water (1.times.) and brine (1.times.) and
dried over sodium sulfate. Flash chromatography (silica gel)
yielded the desired products.
V. Special Syntheses
[0390] Synthesis of BML-219 (N-(3,5-Dihydroxyphenyl)benzamide): To
benzoyl chloride (1 eq.) in dry methylene chloride at room
temperature was added triethylamine (1.5 eq.) and a catalytic
amount of DMAP followed by 3,5-dimethoxyaniline (1 eq.). The
reaction was allowed to stir overnight at room temperature. Upon
completion, the reaction was diluted with ethyl acetate and washed
with 1M HCl, water and brine and dried over sodium sulfate. Flash
chromatography (silica gel) yielded the methoxystilbene derivative.
To the methoxystilbene in dry methylene chloride at 0.degree. C.
was added tetrabutylammonium iodide (3.95 eq.) followed by boron
trichloride (4 eq.; 1M in methylene chloride). Upon completion of
the reaction (TLC), saturated sodium bicarbonate was added and the
mixture was vigorously stirred for 1 hour. The reaction was diluted
with ethyl acetate and washed with 1M HCl and brine and dried over
sodium sulfate. Flash chromatography (silica gel) yielded the
desired product.
[0391] Synthesis of BML-220 (3,3',5-trihydroxy-4'-methoxystilbene):
To Rhapontin in methanol was added catalytic p-toluenesulfonic
acid. The reaction was refluxed overnight. Upon completion of the
reaction (TLC), the reaction mixture was evaporated to dryness and
taken up in ethyl acetate. The organics were washed with water and
brine and dried over sodium sulfate. Flash chromatography (silica
gel) yielded the desired product.
[0392] Synthesis of BML-233 (3,5-Dihydroxy-4'-methoxystilbene): To
deoxyrhapontin in methanol was added catalytic p-toluenesulfonic
acid. The reaction was refluxed overnight. Upon completion of the
reaction (TLC), the reaction mixture was evaporated to dryness and
taken up in ethyl acetate. The organics were washed with water and
brine and dried over sodium sulfate. Flash chromatography (silica
gel) yielded the desired product.
[0393] Synthesis of BML-221 and 227 (4' and 3
monoacetylresveratrols): To resveratrol in tetrahydrofuran at room
temperature was added pyridine (1 eq.) followed by acetic anhydride
(1 eq.). After stirring for 48 hrs., another 0.25 eq. acetic
anhydride added followed by 24 hrs. of stirring. The reaction was
diluted with methylene chloride (reaction was not complete) and
washed with cold 0.5M HCl, water and brine. Organics were dried
over sodium sulfate. Flash chromatography yielded a mixture of 4'-
and 3-acetyl resveratrols. Preparative HPLC yielded both monoacetyl
resveratrols.
[0394] Synthesis of Dihydroresveratrol: To resveratrol in
argon-purged ethyl acetate in a Parr shaker was added 10% palladium
on carbon (10 wt %). The mixture was shaken under an atmosphere of
hydrogen (30 psi) for 5 hours. Filtration through a pad of celite
yielded the desired material.
Equivalents
[0395] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents of the specific embodiments described herein. Such
equivalents are intended to be encompassed by the following claims.
TABLE-US-00002 TABLE 1 APPENDIX OF TABLES: Stimulation of SIRT1
Catalytic Rate by Plant Polyphenols (100 .mu.M). Ratio to Control
Rate Compound Mean .+-. SE Structure Resveratrol
(3,5,4'-Trihydroxy-trans-stilbene) 13.4 .+-. 1.0 ##STR45## Butein
(3,4,2',4'-Tetrahydroxychalcone) 8.53 .+-. 0.89 ##STR46##
Piceatannol (3,5,3',4'-Tetrahydroxy-trans-stilbene) 7.90 .+-. 0.50
##STR47## Isoliquiritigenin (4,2',4'-Trihydroxychalcone) 7.57 .+-.
0.84 ##STR48## Fisetin (3,7,3',4'-Tetrahydroxyflavone) 6.58 .+-.
0.69 ##STR49## Quercetin (3,5,7,3',4'-Pentahydroxyflavone) 4.59
.+-. 0.47 ##STR50## Abbreviation: SE, Standard error of the mean.
Rate measurements with 25 .mu.M NAD.sup.+ and 25 .mu.M p53-382
acetylated peptide substrate were performed as described in
Methods. All ratio data were calculated from experiments in which
the total deacetylation in the control reaction was 0.25-1.25 .mu.M
peptide or 1-5% of the initial concentration of acetylated
peptide.
[0396] TABLE-US-00003 Supplementary Table 1. Effects of Stilbenes
and Chalcones (100 .mu.M) on SIRT1 Rate. Ratio to Control Rate
Compound Mean .+-. SE Replicates Structure Skeleton Resveratrol
(3,5,4'-Trihydroxy- trans-stilbene) Piceatannol # (3,5,3,'4'-
Tetrahydroxy-trans-stilbene) Deoxyrhapontin
(3,5-Dihydroxy-4'-methoxystilbene 3-O-.beta.-D-glucoside)
trans-Stilbene Rhapontin 3,3',5-Trihydroxy-4'-methoxystilbene
3-O-.beta.-D-glucoside cis-Stilbene 13.4 .+-. # 1.0 7.90 .+-. 0.50
1.94 .+-. 0.21 1.48 .+-. 0.15 1.40 .+-. 0.37- 1.14 .+-. 0.29 10 7 6
6 6 6 ##STR51## Butein (3,4,2',4'- Tetrahydroxychalcone) 4,2',4'-
Trihydroxychalcone 3,4,2',4',6'- Pentahydroxychalcone Chalcone 8.53
.+-. 0.89 7.57 .+-. 0.84 2.80 .+-. 0.32 1.34 .+-. 0.17 6 6 6 6
##STR52## Abbreviation: SE, Standard error of the mean. Rate
measurements with 25 .mu.M NAD.sup.+ and 25 .mu.M p53-382
acetylated peptide substrate were performed as described in
Methods. All radio data were calculated from experiments in which
the total deacetylation in the control reaction was 0.25-1.25 .mu.M
peptide or 1-5% of the initial concentration of acetylated
peptide.
[0397] TABLE-US-00004 Supplementary Table 2. Effects of Flavones
(100 .mu.M) on SIRT1 Rate (Part I). Ratio to Control Rate Compound
Mean .+-. SE Replicates Structure Skeleton Fisetin (3,7,3',4'-
Tetrahydroxyflavone) 5,7,3'4',5'- Pentahydroxyflavone Luteolin
(5,7,3',4'- Tetrahydroxyflavone) 3,6,3',4'- Tetrahydroxyflavone
Quercetin (3,5,7,3',4'- Pentahydroxyflavone) 7,3',4',5'-
Tetrahydroxyflavone 6.58 .+-. # 0.69 6.05 .+-. 0.98 5.66 .+-. 0.80
5.45 .+-. 0.57 4.59 .+-. 0.47 3.62 .+-. 0.56 9 6 6 12 16 6
##STR53## Kaempferol 3.55 .+-. 0.56 6 (3,5,7,4'-
Tetrahydroxyflavone) 6-Hydroxyapigenin 3.06 .+-. 0.29 6 (5,6,7,4'-
Tetrahydroxyflavone; Scutellarein) Apigenin 2.77 .+-. 0.40 6
(5,7,4'- Trihydroxyflavone) 3,6,2',4'- 2.10 .+-. 0.22 6
Tetrahydroxyflavone 7,4'-Dihydroxyflavone 1.91 .+-. 0.17 6
Abbreviation: SE, Standard error of the mean. Rate measurements
with 25 .mu.M NAD.sup.+ and 25 .mu.M p53-382 acetylated peptide
substrate were performed as described in Methods. All ratio data
were calculated from experiments in which the total deacetylation
in the control reaction was 0.25-1.25 .mu.M peptide or 1-5% of the
initial concentration of acetylated peptide.
[0398] TABLE-US-00005 Supplementary Table 3. Effects of Flavones
(100 .mu.M) on SIRT1 Rate (Part II). Ratio to Control Rate Compound
Mean .+-. SE Replicates Structure Skeleton 7,8,3,4'-
Tetrahydroxyflavone 3,6,2',3'- Tetrahydroxyflavone
4'-Hydroxyflavone 5,4'-Dihydroxyflavone 5,7-Dihydroxyflavone Morin
(3,5,7,2',4'- Pentahydroxyflavone) Flavone 1.91 .+-. # 0.39 1.74
.+-. 0.27 1.73 .+-. 0.12 1.56 .+-. 0.15 1.51 .+-. 0.18 1.461 .+-.
0.071 1.41 .+-. 0.23 6 6 6 6 6 6 6 ##STR54## 5-Hydroxyflavone 1.22
.+-. 0.19 6 Myricetin 0.898 .+-. 0.070 12 (Cannabiscetin;
3,5,7,3',4',5'- Hexahydroxyflavone) 3,7,3',4',5'- 0.826 .+-. 0.074
12 Pentahydroxyflavone Gossypetin 0.723 .+-. 0.062 6
(3,5,7,8,3',4'- Hexahydroxyflavone) Abbreviation: SE, Standard
error of the mean. Rate measurements with 25 .mu.M NAD.sup.+ and 25
.mu.M p53-382 acetylated peptide substrate were performed as
described in Methods. All ratio data were calculated from
experiments in which the total deacetylation in the control
reaction was 0.25-1.25 .mu.M peptide or 1-5% of the initial
concentration of acetylated peptide.
[0399] TABLE-US-00006 Supplementary Table 4. Effects of
isoflavones, Flavanones and Anthocyanidins (100 .mu.M) on SIRT1
Rate Ratio to Control Rate Compound Mean .+-. SE Replicates
Structure Skeleton Daidzein (7,4'-Dihydroxyisoflavone) (5,7,4'-
Trihydroxyisoflavone) 2.28 .+-. 0.74 1.109 .+-. 0.026 2 2 ##STR55##
Naringenin (5,7,4'- Trihydroxyflavanone) 3,5,7,3',4'-
Pentahydroxyflavanone Flavanone 2.10 .+-. 0.23 1.97 .+-. 0.22 1.92
.+-. 0.24 6 5 6 ##STR56## Pelargonidin chloride (3,5,7.4'-
Tetrahydroxyflavylium chloride) Cyanidin chloride (3,5,7,3',4'-
Pentahydroxyflavylium chloride) Delphinidin chloride
(3,5,7,3',4',5'- Hexahydroxyflavylium chloride) 1.586 .+-. # 0.037
0.451 .+-. 0.015 0.4473 .+-. 0.0071 2 2 2 ##STR57## Abbreviation:
SE, Standard error of the mean. Rate measurements with 25 .mu.M
NAD.sup.+ and 25 .mu.M p53-382 acetylated peptide substrate were
performed as described in Methods. All ratio data were calculated
from experiments in which the total deacetylation in the control
reaction was 0.25-1.25 .mu.M peptide or 1-5% of the initial
concentration of acetylated peptide.
[0400] TABLE-US-00007 Supplementary Table 5. Effects of Catechins
(Flavan-3-ols) (100 .mu.M) on SIRT1 Rate. Ratio to Control Rate
Compound Mean .+-. SE Replicates Structure Skeleton/Structure
(-)-Epicatechin (Hydroxy Sites: 3,5,7,3',4') (-)-Catechin (Hydroxy
Sites: 3,5,7,3',4') (-)-Gallocatechin (Hydroxy Sites:
3,5,7,3',4',5') (+)-Catechin (Hydroxy Sites: 3,5,7,3',4')
(+)-Epicatechin (Hydroxy Sites: 3,5,7,3',4') (-)-Epigallocatechin
(Hydroxy Sites: 3,5,7,3',4',5') 1.53 .+-. # 0.31 1.41 .+-. 0.21
1.35 .+-. 0.25 1.31 .+-. 0.19 1.26 .+-. 0.20 0.41 .+-. 0.11 4 4 4 4
4 4 ##STR58## (-)-Epigallocatechin Gallate (Hydroxy Sites:
3*,5,7,3',4',5'; *Position of gallate ester) 0.32 .+-. 0.12 4
##STR59## Abbreviation: SE, Standard error of the mean. Rate
measurements with 25 .mu.M NAD.sup.+ and 25 .mu.M p53-382
acetylated peptide substrate were performed as described in
Methods. All ratio data were calculated from experiments in which
the total deacetylation in the control reaction was 0.25-1.25 .mu.M
peptide or 1-5% of the initial concentration of acetylated
peptide.
[0401] TABLE-US-00008 Supplementary Table 6. Effects of Free
Radical Protective Compounds (100 .mu.M) on SIRT1 Rate. Ratio to
Control Rate Protective Compound Mean .+-. SE Replicates Mechanism
Hinokitiol 2.48 .+-. 0.15 2 Iron Chelatol (b-Thujaplicin; 2-
hydroxy-4-isopropyl-2,4,6- cycloheptatrien-1-one)
L-(+)-Ergothioneine 2.06 .+-. 0.48 2 Antioxidant,
((S)-a-Carboxy-2,3- Peroxynitrite dihydro-N,N,N-trimethyl-2-
Scavenger thioxo-1H-imidazole- 4-ethanaminium inner salt) Caffeic
Acid Phenyl Ester 1.80 .+-. 0.16 2 Iron Chelator MCI-186 1.2513
.+-. 0.0080 2 Radical (3-Methyl-1-phenyl-2- Scavenger
pyrazolin-5-one) and Antioxidant HBED 1.150 .+-. 0.090 2 Iron
Chelator (N,N'-Di-(2-hydroxy- benzyl)ethylenediamine- N,N'-diacetic
acid.HCl.H2O) Ambroxol 1.075 .+-. 0.0026 2 Radical
(trans-4-(2-Amino-3,5- Scavenger dibromobenzylamino)
cyclohexane.HCl) U-83836E 1.030 .+-. 0.055 2 "Lazaroid"
((-)-2-((4-(2,6-di-1- amino- Pyrrolidinyl-4-pyrimidinyl)- steroid,
1-piperazinyl)methyl)-3,4- Peroxidation dihydro-2,5,7,8- inhibitor
tetramethyl-2H-1- benzopyran-6-ol-2HCl) Trolox 0.995 .+-. 0.019 2
Antioxidant (6-Hydroxy-2,5,7,8- tetramethylchroman-2- carboxylic
acid) Abbreviation: SE, Standard error of the mean. Rate
measurements with 25 .mu.M NAD.sup.+ and 25 .mu.M p53-382
acetylated peptide substrate were performed as described in
Methods. All ratio data were calculated from experiments in which
the total deacetylation in the control reaction was 0.25-1.25 .mu.M
peptide or 1-5% of the initial concentration of acetylated
peptide.
[0402] TABLE-US-00009 Supplementary Table 7. Effects of
Miscellaneous Compounds (100 .mu.M) on SIRT1 Catalytic Rate. Ratio
to Control Rate Mean .+-. Repli- Compound SE cates Structure &
Activities Dipyridamole (2,6-bis- (Diethano- lamino)- 4,8-di-
piperidino- pyrimido[5,4- d]pyrimidine) 3.54 .+-.0.20 2 ##STR60##
Inhibitor of Adenosine Transport, Phosphodiesterase, 5-Lipoxygenase
Nicotinamide 0.428 .+-. 42 ##STR61## 0.019 Sirtuin Reaction
Product/Inhibitor NF279 0.0035 .+-. 3 ##STR62## 0.0011 Purinergic
Receptor Antagonist NF023 -0.0016 .+-. 3 ##STR63## 0.0015 G-protein
Antagonist Suramin -0.0002 .+-. 3 ##STR64## 0.0010 G-protein
Antagonist, Reverse Transcriptase Inhibitor Abbreviation: SE,
Standard error of the mean. Rate measurements with 25 .mu.M
NAD.sup.+ and 25 .mu.M p53-382 acetylated peptide substrate were
performed as described in Methods. All ratio data were calculated
from experiments in which the total deacetylation in the control
reaction was 0.25-1.25 .mu.M peptide or 1-5% of the initial
concentration of acetylated peptide.
[0403] TABLE-US-00010 Supplementary Table 8. Effects of Various
Modulators on SIRT1 Rate. Ratio to Compound, Control Rate
(Concentration) Mean .+-. SE Replicates Structure ZM 336372, (100
.mu.M) 3.5 .+-. 1.1 3 ##STR65## Camptothecin, (10 .mu.M) 2.92 .+-.
0.41 3 ##STR66## Coumestrol, (10 .mu.M) 2.30 .+-. 0.31 2 ##STR67##
NDGA, (100 .mu.M) 1.738 .+-. 0.088 3 ##STR68## Esculetin, (10
.mu.M) 1.737 .+-. 0.082 3 ##STR69## Sphingosine 0.069 .+-. 0.028 3
##STR70## Abbreviation: SE, Standard error of the mean. Rate
measurements with 25 .mu.M NAD.sup.+ and 25 .mu.M p53-382
acetylated peptide substrate were performed as described in
Methods. All ratio data were calculated from experiments in which
the total deacetylation in the control reaction was 0.25-1.25 .mu.M
peptide or 1-5% of the initial concentration of acetylated
peptide.
[0404] TABLE-US-00011 TABLE 9 SIRT1 Rate Effects of New Resveratrol
Analogs (100 .mu.M). Ratio to Stability in Control Rate Solution
Compound Mean .+-. SE N Structure t.sub.1/2, hrs. BML-217
(3,5-Dihydroxy- 4'-chloro-trans- stilbene) 10.6 .+-. 0.4 3
##STR71## Resveratrol (3,5,4'- Trihydroxy-trans- stilbene) 10.4
.+-. 0.5 43 ##STR72## 59 (ethanol), 20 (water) Pinosylvin
(3,5-Dihydroxy- trans-stilbene) 9.95 .+-. 0.45 3 ##STR73## BML-225
(3,5-Dihydroxy- 4'-ethyl-trans- stilbene) 9.373 .+-. 0.014 3
##STR74## BML-212 (3,5-Dihydroxy- 4'-fluoro-trans- stilbene) 8.20
.+-. 0.69 3 ##STR75## 66 (ethanol) BML-228 (3,5-Dihydroxy-
4'-methyl-trans- stilbene) 7.72 .+-. 0.12 3 ##STR76##
[0405] TABLE-US-00012 TABLE 10 SIRT1 Rate Effects of New
Resveratrol Analogs (100 .mu.M). Ratio to Stability in Control Rate
Solution Compound Mean .+-. SE N Structure t.sub.1/2, hrs. BML-232
(3,5-Dihydroxy- 4'-azido-trans- stilbene) 7.24 .+-. 0.12 3
##STR77## BML-230 (3,5-Dihydroxy- 4'-thiomethyl- trans-stilbene)
6.84 .+-. 1.26 6 ##STR78## BML-229 (3,5-Dihydroxy- 4'-nitro-trans-
stilbene) 6.78 .+-. 0.22 3 ##STR79## BML-231 (3,5-Dihydroxy-
4'-isopropyl- trans-stilbene) 6.01 .+-. 0.15 3 ##STR80## BML-233
3,5-Dihydroxy-4'- methoxy-trans- stilbene 5.48 .+-. 0.33 6
##STR81##
[0406] TABLE-US-00013 TABLE II SIRT1 Rate Effects of New
Resveratrol Analogs (100 .mu.M). Ratio to Stability in Control Rate
Solution Compound Mean .+-. SE N Structure t.sub.1/2, hrs.
Rhapontin aglycone (3,5,3'Trihydroxy- 4'-methoxy-trans- stilbene)
4.060 .+-. 0.069 3 ##STR82## BML-227 (3,4'-Dihydroxy-5-
acetoxy-trans- stilbene) 3.340 .+-. 0.093 3 ##STR83## BML-221
(3,5-Dihydroxy-4'- acetoxy-trans- stilbene) 3.05 .+-. 0.54 6
##STR84## 504 (ethanol) BML-218 (E)-1-(3,5- Dihydroxyphenyl)-
2-(2-napthyl) ethene 3.05 .+-. 0.37 6 ##STR85## 3-Hydroxystilbene
2.357 .+-. 0.074 3 ##STR86##
[0407] TABLE-US-00014 TABLE 12 SIRT1 Rate Effects of New
Resveratrol Analogs (100 .mu.M). Ratio to Stability in Control Rate
Solution Compound Mean .+-. SE N Structure t.sub.1/2, hrs. BML-226
(3,5-Dimethoxymethoxy- 4'-thiomethyl-trans- stilbene) 2.316 .+-.
0.087 3 ##STR87## BML-222 (3,5-Dihydroxy-4'- acetamide-trans-
stilbene) 1.88 .+-. 0.11 3 ##STR88## 3,4-Dihydroxy- trans-stilbene
1.64 .+-. 0.10 6 ##STR89## BML-224 (E)-1-(3,5- Dihydroxyphenyl)-
2-(cyclohexyl) ethene 1.297 .+-. 0.042 3 ##STR90## 3,4-Dimethoxy-
trans-stilbene 1.127 .+-. 0.019 3 ##STR91##
[0408] TABLE-US-00015 TABLE 13 SIRT1 Rate Effects of New
Resveratrol Analogs (100 .mu.M). Ratio to Stability in Control Rate
Solution Compound Mean .+-. SE N Structure t.sub.1/2, hrs.
Dihydroresveratrol (1-(3,5-Dihydroxyphenyl)- 2-(4-hydroxyphenyl)
ethane) 1.08 .+-. 0.14 4 ##STR92## 4-Hydroxy-trans- stilbene 0.943
.+-. 0.039 3 ##STR93## BML-219 N-phenyl-(3,5- dihydroxy)benzamide
0.902 .+-. 0.014 3 ##STR94## 3,5-Dihydroxy-4'- nitro-trans-stilbene
0.870 .+-. 0.019 3 ##STR95## 4-Methoxy-trans-stilbene 0.840 .+-.
0.089 3 ##STR96##
[0409] TABLE-US-00016 TABLE 14 Resveratrol Analog Synthetic
Intermediates Compound Benzylphosphonate Aldehyde Structure BML-217
(3,5-Dihydroxy- 4'-chloro-trans- stilbene) Diethyl 3-5-
dimethoxybenzyl phosphonate 4-Chlorobenzaldehyde ##STR97##
Resveratrol (3,5,4'- Trihydroxy-trans- stilbene) N/A N/A ##STR98##
Pinosylvin (3,5-Dihydroxy- trans-stilbene) Diethyl benzyl
phosphonate 3,5-Dimethoxy benzaldehyde ##STR99## BML-225
(3,5-Dihydroxy- 4'-ethyl-trans- stilbene) Diethyl 3-5-
dimethoxybenzyl phosphonate 4-Ethylbenzaldehyde ##STR100## BML-212
(3,5-Dihydroxy- 4'-fluoro-trans- stilbene) Diethyl 4-fluoro
benzylphosphonate 3,5-Dimethoxy benzaldehyde ##STR101## BML-228
(3,5-Dihydroxy- 4'-methyl-trans- stilbene) Diethyl 3-5-
dimethoxybenzyl phosphonate 4-Methylbenzaldehyde ##STR102##
[0410] TABLE-US-00017 TABLE 15 Resveratrol Analog Synthetic
Intermediates Compound Benzylphosphonate Aldehyde Structure BML-232
(3,5-Dihydroxy- 4'-azido-trans- stilbene) Diethyl 4-azido
benzylphosphonate 3,5-Dimethoxymethoxy benzaldehyde ##STR103##
BML-230 (3,5-Dihydroxy- 4'-thiomethyl- trans-stilbene) Diethyl
4-methylthio benzylphosphonate 3,5-Dimethoxymethoxy benzaldehyde
##STR104## BML-229 (3,5-Dihydroxy- 4-nitro-trans- stilbene) Diethyl
3-5- dimethoxybenzyl phosphonate 4-Nitrobenzaldehyde ##STR105##
BML-231 (3,5-Dihydroxy- 4'-isopropyl- trans-stilbene) Diethyl 3-5-
dimethoxybenzyl phosphonate 4-Isopropyl benzaldehyde ##STR106##
3,5-Dihydroxy- 4'-methoxy- trans-stilbene N/A N/A ##STR107##
[0411] TABLE-US-00018 TABLE 16 Resveratrol Analog Synthetic
Intermediates Compound Benzylphosphonate Aldehyde Structure
Rhapontin aglycone (3,5,3'Trihydroxy- 4'-methoxy-trans- stilbene)
N/A N/A ##STR108## BML-227 (3,4'-Dihydroxy-5- acetoxy-trans-
stilbene) N/A N/A ##STR109## BML-221 (3,5-Dihydroxy-4'-
acetoxy-trans- stilbene) N/A N/A ##STR110## BML-218 (E)-1-(3,5-
Dihydroxyphenyl)- 2-(2-napthyl) ethene Diethyl 3-5- dimethoxybenzyl
phosphonate 2-Naphthaldehyde ##STR111## BML-216 3-Hydroxystilbene
Benzylphosphonate 3-Methoxy benzaldehyde ##STR112##
[0412] TABLE-US-00019 TABLE 17 Resveratrol Analog Synthetic
Intermediates Compound Benzylphosphonate Aldehyde Structure BML-226
(3,5-Dimethoxymethoxy- 4'-thiomethyl- trans-stilbene) Diethyl
4-methylthio benzylphosphonate 3,5dimethoxymethoxy benzaldehyde
##STR113## BML-222 (3,5-Dihydroxy-4'- acetamide-trans- stilbene)
Diethyl 4-acetamido benzylphosphonate 3,5-dimethoxymethoxy
benzaldehyde ##STR114## BML-215 3,4-Dihydroxy- trans-stilbene
Benzylphosphonate 3,4-Dimethoxy benzaldehyde ##STR115## BML-224
(E)-1-(3,5- Dihydroxyphenyl)- 2-(cyclohexyl) ethene 3,5-Dimethoxy
benzylphosphonate Cyclohexane carboxaldehyde ##STR116##
3,4-Dimethoxy- trans-stilbene Benzylphosphonate 3,4-Dimethoxy
benzaldehyde ##STR117##
[0413] TABLE-US-00020 TABLE 18 Resveratrol Analog Synthetic
Intermediates Compound Benzylphosphonate Aldehyde Structure
Dihydroresveratrol (1-(3,5-Dihydroxyphenyl)- 2-(4-hydroxyphenyl)
ethane) N/A N/A ##STR118## BML-214 4-Hydroxy-trans- stilbene
Benzylphosphonate 4-Methoxy benzaldehyde ##STR119## BML-219
N-phenyl-(3,5- dihydroxy)benzamide N/A N/A ##STR120##
3,5-Dihydroxy-4-nitro- trans-stilbene 3,5-Dimethoxy
benzylphosphonate 4-Nitrobenzaldehdye ##STR121## 4-Methoxy-trans-
stilbene Benzylphosphonate 4-Methoxy benzaldehyde ##STR122##
[0414]
Sequence CWU 1
1
11 1 6 PRT Artificial sequence Synthetic 1 Lys Gln Thr Ala Arg Lys
1 5 2 6 PRT Artificial sequence Synthetic 2 Lys Ser Thr Gly Gly Lys
1 5 3 6 PRT Artificial sequence Synthetic 3 Lys Ser Thr Gly Gly Lys
1 5 4 5 PRT Artificial sequence Synthetic 4 Lys Ala Pro Arg Lys 1 5
5 5 PRT Artificial sequence Synthetic 5 Ser Gly Arg Gly Lys 1 5 6 5
PRT Artificial sequence Synthetic 6 Lys Gly Gly Ala Lys 1 5 7 5 PRT
Artificial sequence Synthetic 7 Lys Gly Gly Ala Lys 1 5 8 4 PRT
Artificial sequence Synthetic 8 Gln Pro Lys Lys 1 9 4 PRT
Artificial sequence Synthetic 9 Lys Ser Lys Lys 1 10 4 PRT
Artificial sequence Synthetic 10 Arg His Lys Lys 1 11 4 PRT
Artificial sequence Synthetic 11 Arg His Lys Lys 1
* * * * *