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

***

* **