current programs in medicinal/biological chemistry 1.nitric oxide mimetic drug discovery...
TRANSCRIPT
Current Programs in Medicinal/Biological Chemistry
1. Nitric oxide mimetic drug discovery• Alzheimer’s and neurodegenerative disorders
• Colon cancer chemoprevention
2. Selective estrogen receptor modulators• chemical toxicology & cancer promotion/prevention
• postmenopausal antidepressants (inc. botanical)
NIH/NCI CA 102590NIH/NCCAM AT002299
NIH/NIA AG027425Institute for Study of Aging-Elan Pharmaceuticals
A Brief Presentation of Research from the Thatcher Group
Better Living Through ChemistryBetter Living Through Chemistry
1.Nitric oxide mimetic drug discovery
►Clinical drugs: nitroglycerin (GTN); isosorbide dinitrate (ISDN); organic nitrates-angina (1874); CHF; cardioprotection (2005)
Biological activity mimics NO: bioactivation to NO in vivo?
►NO biology: Nobel Prize 1998 Medicine: “NO sex; NO wonder; NO way”NO signaling ubiquitous (diffusible free radical gas binds to Fe-heme)Endogenous NO: from Arg +NO synthase (NOS)
eNOS: smooth muscle relaxation, anti-atherogeniciNOS: immune response; antibacterial
nNOS: neurotransmission, learning & memoryNO is essential for normal physiological function in the CNS, including learning & memory, & is compromised in disease states including neurodegenerative disorders
RONO2 + 3e- + 3H+ NO
Better Living Through ChemistryBetter Living Through Chemistry
1.Nitric oxide mimetic drug discovery►Clinical drugs: nitroglycerin (GTN); isosorbide dinitrate (ISDN); organic nitrates
-angina (1874); CHF; cardioprotection (2005)
GT 1061 in Phase 1 clinical trials for Alzheimer’s (FDA approved IND)GT 1061 in Phase 1 clinical trials for Alzheimer’s (FDA approved IND)
NO is essential for normal physiological function in the CNS, including learning & memory, & is compromised in disease states including neurodegenerative disorders
Better Living Through ChemistryBetter Living Through Chemistry
2. Selective estrogen receptor modulators-tissue specific estrogen agonist effect: bone and lipids
-tissue specific antiestrogen effect: mammary and uterine -Anti-osteoporosis; hormone replacement therapy (HRT); chemopreventive; -anticancer; antiinflammatory
-endometrial cancer; breast cancer; thrombosis + strokeRisk: long-term use in healthy (menopausal) women
► SERMs are polyaromatic phenols: oxidative metabolism by oxidase/peroxidase
Are oxidative metabolites responsible for toxicity or therapeutic activity?
Can new SERMs be designed to improve efficacy and safety profile?
Common Themes & VisionCommon Themes & Vision1. Nitric oxide mimetic drug discovery
2. Selective estrogen receptor modulators
• both families are reactive molecules; metabolism is important for activity and potentially toxicity; these are good drugs clinically proven over decades or a century
• NO signaling and biological redox systems are intrinsic to the actions of both families
Aims► Molecules hitting multiple targets; diseases are multifactorial► Moderate potency, multiple mechanisms, high safety► Translational research: bench-to-bedside
Tools of the Trade Tools of the Trade
1. Synthetic organic chemistry: drug candidates; model compounds; reactive intermediates; novel biological probes Examples
2. Physical - Mechanistic organic chemistry: reaction mechanisms; kinetics; identification of reaction intermediates; computational methodsExamples
3. Cell biology and proteomics: Perturbation of cell growth and protein/gene biomarkers; ROS and NOx production; LC-MS-MS Examples
4. Animal models- cancer*, cardio** and cerebrovascular**: murine tissue pathology; immunohistochemistry; ex vivo function Examples
5. Animal models- behavior: antidepressant; cognition enhancement; anxiolytic; spatial working and reference memory; transgenics** Examples
* extradepartmental collaborations at UIC** extramural collaborations
Br
OH
OH
i. HNO3, H2SO4, DCM
ii. Na2S2O3, CH3OH
iii. H2O2, H2SO4ONO2
ONO2
S S
ONO2
ONO2
GT 015
i, ii, iii
SH
CO2H
1. EtOH, PTSA,
2. Allyl bromide,3. AgNO3, I2, CH3CN
ONO2
ONO2
EtO2C
S
ONO2
EtO2C
S
O2NO
SH
CO2Et
Toluene, reflux
K2CO3, Acetone
S
CO2Et
+
GT 947
GT 794
1 2 3
SH
CO2H
1. LiAlH4, THF 2. HNO3, (CH3CO)2OS S
ONO2
ONO2
GT 4294
SH
OH
H3CO SH+
Br
BrO
KOH
EtOH-AcOEtH3CO S
O
Br
PPA
SH3COBr
SH3COBr
Brbromoacetamide
DCM-EtOH
H2O2
DCM-TFA SH3COBr
BrOH O
N
NaH, DMF
SH3COBr
O
O
N
O
O
SH3COBr
O
O
N
PPh3, TMSCl1) HCl
2) BBr3
SHOBr
O
O
N
5M NaOCH3, AcOEtCuI, DMF-MeOH, 120oC
SHOOMe
O
O
N
SHOSO2Me
O
O
N
CuI, proline, NaOHCH3SO2Na, DMSO
120oC
1Return
2Return
HPLC Chromatogram of raloxifene (0.05 mM), rat liver microsomes (1.0 nmol P450/mL), GSH (0.5 mM), and a NADPH generating system in 50 mM phosphate buffer ( 37 °C , 30 min). For control incubations, either GSH or NADP+ was omitted.
SOH
HO
O(H2C)5N(H2C)2OPh
SOH
O
O(H2C)5N(H2C)2OPh
O
SO
O
O(H2C)5N(H2C)2OPh
SOH
HO
O(H2C)5N(H2C)2OPh
Raloxifene
HO
OH
TyrP450 P450
TyrP450
MAJORMINOR
GSHnucleoside
conjugatesadducts
GSHnucleoside
conjugatesadducts
R1R
2
R3
TS1 TS2TS3
R
a
P
a
RbPb/R2
TSaTSb
R1 TS1 R2 TS2 TS3 R310
15
20
25
30
G
(kcal/
mo
l)
Ra TSa Pa R2 TSb Rb0
5
10
15
20
25
G
(kcal/
mo
l)
3Return
1.25 1.35 1.45 1.55 1.65 1.75 1.85 1.95 2.050
25
50
75
100
log ([GT094], uM)
rel.
in
cres
e in
cel
ln
um
ber
as
%
Fig 9. Concentration response of cell number with GT094 in Caco-2 cell (48hr) assayed by sulforhodamine B dye staining: EC50 = 40 μM.
Time, h 0 6 12 18 24% G1 36 ± 0.7 17.0 ± 6.4 28.5 ± 6.939.1 ± 2.326.4 ± 7.0% S 48.8 ± 2.555.0 ± 4.6 38.1 ± 2.536.5 ± 17.3 61.0 ± 5.6% G2-M 14.9 ± 3.128.1 ± 1.8 33.5 ± 9.424.6 ± 15.1 12.7 ± 1.5
0 6 12 18 24-5
0
5
10
15
20
25
t, hours
rel
% o
f ce
lls
in G
2/M
Figure 8. Cell cycle FACS analysis of propidium bromide treated Caco-2 cells incubated with GT 094 (100 uM, 48 h).
75
250
50
25
band 3
band 1
band 2
20
15
10
PBS wash
coomassieblue staining
Western blotAnalysis by HRP-Streptavidin
Biotineluate
PBS wash
Biotineluate
MW
78 kDa glucose-regulated protein (GRP 78; Bip)72 kDa, protein disulfide isomerase A4 precursor
57 kDa, protein disulfide isomerase, precursor57 kDa, protein disulfide isomerase, A3 precursor
17 kDa, mGST 1
MALDI-TOF + anti-GRP78 + rec. hGRP78 & PDIA4
LC-MS-MS
anti-mGST
75
250
50
25
band 3
band 1
band 2
20
15
10
PBS wash
coomassieblue staining
Western blotAnalysis by HRP-Streptavidin
Biotineluate
PBS wash
Biotineluate
MW
75
250
50
25
band 3
band 1
band 2
20
15
10
75
250
50
25
band 3
band 1
band 2
20
15
10
PBS wash
coomassieblue staining
Western blotAnalysis by HRP-Streptavidin
Biotineluate
PBS wash
Biotineluate
MW
78 kDa glucose-regulated protein (GRP 78; Bip)72 kDa, protein disulfide isomerase A4 precursor
57 kDa, protein disulfide isomerase, precursor57 kDa, protein disulfide isomerase, A3 precursor
17 kDa, mGST 1
MALDI-TOF + anti-GRP78 + rec. hGRP78 & PDIA4
LC-MS-MS
anti-mGST
Cell cycle andproliferation
Proteomic identification ofmodified proteins in rat liver
Colon Cancer ChemopreventionColon Cancer Chemoprevention
0
10
20
30
40
50
60
8 week drug treatment
no
. o
f A
CF
s p
er c
olo
n
Aberrant Crypt Foci (ACF) correlate with progression to tumors in animal models
1. GT094 significantly reduces ACFs compared to no drug and ASA treatment.
2. GT 094 significantly reduces colon cell proliferation (p27 elevated) and tumor weight and multiplicity in a 30 week AOM study
100
AOM Treated Colon GT-094
0
AOM Exposure Time (30 weeks)
75
50
25
GT-094
iNOS
130 kDa
*
iNO
SQ
uant
ity A
.U
P=0.013
100
AOM Treated Colon GT-094
0
AOM Exposure Time (30 weeks)
75
50
25
GT-094
iNOS
130 kDa
*
iNO
SQ
uant
ity A
.U
P=0.013
4Return
5Return
-P
erce
nt
corr
ect
Per
cen
t co
rrec
t
DonepezilDonepezil (mg/kg)(mg/kg)
GT1061GT1061 (mg/kg)(mg/kg)
* * P< 0.01 vs PostsurgeryP< 0.01 vs Postsurgery
50
60
70
80
***
* **