Medicinal Chemistry

February - June, 2018

Prof. Elena Goun

Copyright: www.nature.com web.rollins.edu/~jsiry/memb; blogs.nature.com

Imaging beyond the Proteome

Elena Dubikovskaya

12/15/10

Laboratory of Bioorganic Chemistry and Molecular Imaging (LCBIM)

http://lcbim.epfl.ch

Short Biography

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Please introduce yourself

What is your name?

Where are you from?

What is your field of study?

What are your career plans?

Course Objective

Objective:

To provide students with relevant background inthe field of medicinal chemistry, drug development,and diagnostics.

The Main Book*

* The course will also includeadditional material not includedin the book

Copyright: wiley.com

The second book*

* The course will also includeadditional material not includedin the book

Copyright: amazon.com

Grading system

Exam:• Written exam• 70% of the grade

Homework:• Presentation in the class – 30% of the grade

• 20-30 min, 3 people per group• any medicinal chemistry topic of your choice• Will take place 8-9 week of the semester• Need to select the title before week 7

Imaging beyond the Proteome

Elena Dubikovskaya

12/15/10

Novel Chemical Tools and Their Application in Medicine

Laboratory of Bioorganic Chemistry and Molecular Imaging

Molecular Imaging enables the interrogation of molecules in living systems

Copyright: www.nature.com web.rollins.edu/~jsiry/memb; blogs.nature.com

Molecular Imaging enables the interrogation of molecules in living systems

Copyright: http://www.scienceblogs.de/lindaunobel/GFP.jpg

2008 Nobel Prize in Chemistry

Molecular Imaging enables the interrogation of molecules in living systems

Copyright: cas.uoregon.edu/photos/Sciencephoto, www.eurekalert.org www.hoxfulmonsters.com bsc.ua.edu/site/images www.newscientist.com

Development of Probes

Copyright: millville.sps.edu

Cell Surface Glycosylation

Fatty Acid Transport

Protein functions

H2O2 signaling

Development of probes

Activity of GlucoseTransporters

Image guided surgery

Copyright: micro.magnet.fsu.edu; wikimedia.org/wikipedia; nature medicine

Mitochondria membranepotential

Small ligand-receptorinteractions

15

• Near-Infrared imaging uses light in the range of 650-900 nm:- minimum absorption by tissues - low interference from tissue autofluorescence

• It allows increasing tissue depth penetration and minimizing background signal

The NIR window in biological tissues

Frangioni J.V., Curr. Opin. Chem. Biol., 2003, 7, 626-634. Weissleder R. Nature Biotech., 2001, 19, 316-317.

Near-infrared probes for Imaging of fatty acid uptake

Developing of Probes for Image-Guided-

Surgery and Diagnostic in Oncology

http://www.licor.com/translational/

What is Probe?

- Antibody- Peptide- Small molecule

Conjugates of Interest

• Optical probes

• Optoacoustic probes

• PET probes• Dual modality probes

(PET/Optical)

• Drug conjugates

Development of tumor-specific fluorescent probes

Cancer-

targeting

molecule

“clickable” NIR fluorescent dyeSmall molecules, peptides, antibodies...

Conjugation via “click chemistry”

Cancer-

targeting

molecule

PROBE for intra-operative imaging

Tumor Detection in Ovarian Cancer

van Dam GM et al. Nature Medicine 2011

Folate receptor-α (FA-α) is overexpressed in

90-95 % of epithelial ovarian cancers

FA-α binds folate with high affinity

which is then endocytosed and shuttled into

the cytoplasm

Folate + Fluoresceine = folate-FITC

Tumor Detection in Ovarian Cancer

van Dam GM et al. Nature Medicine 2011

Tumor Detection in Ovarian Cancer

van Dam GM et al. Nature Medicine 2011

Tumor Detection in Ovarian Cancer

van Dam GM et al. Nature Medicine 2011

What is Probe?

- Antibody- Peptide- Small molecule

Integrin Receptors

• Transmembrane receptors to recognize the

components of extracellular matrix

• Critical for cell adhesion, migration, proliferation and

angiogenesis

• Upon activation triggers a number of signaling

cascades (FAK, Src, ERK and AKT pathways) leading

to new blood vessel formation

• Highly expressed in aggressive solid tumors and their

function is important for the invasion and metasthesis

of cancer cells

• RGD is a tripeptide composed of L-arginine, glycine, and L-aspartic acid

• αvβ3 integrins are counter parts of RGD-Peptides

E. Ruoslahti, M.D. Pierschbacher, Cell 1986, 44 (4), 517–18

RGD peptide: introduction

RGD (Arg-Gly-Asp)

Extracellular segment of integrin αVβ3 in complex with an

Arg-Gly-Asp ligand

Possible other markers for image-guided surgery of GBM: RGD peptides

“Integrins αvβ3 and αvβ5 were expressed in many glioma vessels;the intensity of vascular expression of >vA3 increased with gradeof malignancy…”

Possible other markers for image-guided surgery of GBM: Cilengitide

• Cilengitide was in clinical trials the treatment of GBM

• Small molecule and potent binder (high intratumoralconcentrations were detected in humans)

• Conjugates of Cilengitide with ICG might comprise a valuable tool for image-guided surgery of GBM

• Biological evaluation in animal models of GBM

Diagnostic

Copyright: www.medscape.com

• RGD-Ga68 imaging reagent had been previously developed and validated in clinic by Dr. John Prior (CHUV)

Multimeric cRGD

1-cRGD-Cy7

2-cRGD-Cy7

4-cRGD-Cy7

Cy7

Copyright: themyersonthemove.blogspot.com

Copyright: www.pinterest.com

Multimeric cRGD

1-cRGD-Cy7

2-cRGD-Cy7

4-cRGD-Cy7

Cy7

Advantages of Multimeric cRGD

Copyright: themyersonthemove.blogspot.comIn collaboration with Dr. Anita Wolfer

RGD peptide: a multimeric approach

RGD-ICG

2RGD-IR820

Fatty acid uptake plays a key role in biology of many diseases

• Extensive uptake is linked:

• Insulin resistance in skeletal muscle – leads to type 2 diabetes• Cardiomyopathy (heart muscle disease – leads to heart failure)• Fatty liver disease (large vacuoles of triglyceride fat

accumulate in liver cells –liver failure)• Lipotoxicity in heart, liver, and pancreatic beta cells

(major cause of heart failure, obesity, and diabetes)

Other pathologies:

• cardiac hypoxia• intestinal melabsorption

… and many others

39

Using ICG-FA probe for imaging of GBM?

Brains withTumor

ICG-FA

Healthy brain

ICG-FA

y665

y667

• ICG-FA injected IV (200 uL, 100 uM)• Mice were sacrificed 2 h postinjection

Accumulation of ICG-FA in tumor vs healthy brains

High grade astrocytoma tumor models (pathology is GBM-like)

Mouse model: GFAPHaRas;GFAP-CRE;GFAP-LUC;p53WT/FLOX line that crosse-bred and then bred 10N on FVBNJ background

- In collaboration with Doug Hanahan

U87 brain tumor WT nude mice

U87 brain tumor WT nude mice

BLI

BLI

ICG

In collaboration with PerkinElmer

Accumulation of ICG-FA in U87 tumor vs healthy brains

Targeting of brain tumors with ICG-FA probe

In collaboration with PerkinElmer

ICG-FA DLIT Reconstruction

Copyright: millville.sps.edu

Cell Surface Glycosylation

Fatty Acid Transport

Protein functions

H2O2 signaling

Development of Probes

Urgent need to develop new assaysfor drug discovery

- No methods that would allow studies of fatty acid fluxes in living organisms exist

Problem:

A novel approach for imagingmetabolic fluxes in living animals

with Prof. Andreas Stahl GroupNutritional Science and Toxicology Department,

UC Berkeley

Obesity is a real problem worldwide

• Obesity is associated with about 30 diseases

– heart disease

– high blood pressure

– arthritis

– sleep apnea

– stroke

– type 2 diabetes

• It is the second leading cause of death in the US

• Economic costs are estimated of 123 billion per year

Health effects of obesity

Body Mass Index Categories:

Copyright: freebmicalculator.net

Underweight = <18.5Normal weight = 18.5–24.9 Overweight = 25–29.9 Obesity = BMI of 30 or greater

Trends of obesity among different countries

Copyright: http://tomatotalk.earthfare.com

Trend of obesity among US adults

45%

33%

67%

Copyright: http://tomatotalk.earthfare.com

What are the molecular signatures of obesity related disorders that can be targeted to create new therapeutics?

Fatty acids are a very important source of energy

Copyright: http://en.wikipedia.org/wiki/Fatty_acid

= ENERGY FAT

Uptake of fatty acids into cells

OH

O

Cytosol

Fatty Acid Transporter Proteins (FATP)

Passive diffusion

Pflugers Arch. (2004) 447, 722

Blocking FATP in the intestine reduces caloric uptake

OH

O

Cytosol

X = Decreased Fatty Acid uptake

Pflugers Arch. (2004) 447, 722

High Fat Diet

6 weeks

Knock out FATP High Fat Diet

6 weeks

Mice with no muscle/fat FATP are protected from diet induced obesity

These mice are also protected from insulin desensitization

J. Lipid Res. (2005), 46, 597

Development of assays that can be used for drug discovery

- No methods that would allow studies of fatty acid fluxes in vivo exist

Problem:

Bioluminescent imaging (BLI)

• 10-17 mol/L

• 100,000 times more sensitive than fluorescence

HO

N

S

N

S

CO2H Fireflyluciferase

OxyluciferinFirefly LuciferinATP, O2,

Mg2+

AMP, PPi,

CO2

S

N

S

N O

HO +

60

Bioluminescence in Nature

___________________________________________Chemical & Engineering News, 2006, 84, 36-38; Science, 2005, 309, 263

• Find Food

• Attract Mates

• Defend against predators

• Camouflage

Molecular Imaging enables the interrogation of molecules in living systems

Luciferin

Molecular Imaging enables the interrogation of molecules in living systems

Luciferin

Applications of BLI to probe molecular signatures of target tissues

(1) Nat Methods, 2006, 3, 295. (2) Mol Ther, 2005, 11, 926. (3) Proc. Natl. Acad. Sci. 2010, (4) Angew. Chem. Int. Ed. 2007, 46, 7031.

X

N

S

N

S

CO2H

X = O, NH

CAGEUNCAGING

HX

N

S

N

S

CO2H Fireflyluciferase

OxyluciferinFree Luciferin

+

ATP, O2,

Mg2+

AMP, PPi,

CO2

S

N

S

N O

HO

CAGE

SAR of FATP in respect to their substrates

J. Lipid Res. (2005), 46, 597

n > 10

O

O

R

HO

O

R

not a substrate for FATP

NB

N

FFHO

O

BODIPY - Lauric acid

FATP

Cytosol

S

N

S

N CO2H

OS O

O

SHO

O

Glutathione (GSH)SH

HO

O

S

N

S

N CO2H

OHS O

O

S O

OS

N

S

N CO2H

HO

Luciferese

Mg 2+, ATP

+ oxyluciferin

FATP

CCD Camera

S

N

S

N CO2H

OS O

O

S

FFA (LIPID) Releasable Linker

Luciferin (IMAGING PROBE)

HO

O

S

N

S

N CO2H

OS O

O

FFA (LIPID) Linker

Luciferin (IMAGING PROBE)

HO

O

FATP

Cytosol

S

N

S

N CO2H

HO

Luciferese

Mg 2+, ATPoxyluciferin

FATP

CCD Camera

S

N

S

N CO2H

OS O

O

HO

O

Hydrolysis S OHHO

O

+ CO2

+

Imaging Intestinal absorption

Probes behave just like a natural fatty acid

Small Intestine

Colon

Copyright: https://www.aalaslearninglibrary.org

Intestinal fatty acid uptake in real time in live animals

Where does the light come from?

The duodenum is the first section of the small intestine

Stomach

Colon

Duodenum

MicroCT and BLI overlay of uptake

BaSO4

radiocontrast agent

Comparison to the control compound

60 min 60 min

S

N

S

N CO2H

OS O

O

HO

O

Copyright: www.classymommy.com

Polyethylene Glycol

Brown adipose tissue generates body heat

Copyright: www.nature.com http://www.adiposetissue.org http://4.bp.blogspot.com www.ehow.com/facts

White adipocyte Brown adipocyte

The only known mammals lacking BAT (Boars)

Recent discovery of BAT in adult humans

Copyright: http://en.wikipedia.org N Engl J Med, (2009) 360, 1509

If activated, brown adipose tissue…

Copyright: sce.uhcl.edu/puzdrowski/vitruvian

Drug

~ 25% of daily calories

next promising target for the treatment of obesity

N Engl J Med, (2009) 360, 1509

Current technologies to image BAT

• Positron Emission (PET) and Single Photon emission Computed Tomography (SPEC)

• Both require radioisotope labeled tracers

(18F and 123I)

• Very expensive and not suitable for high throughput screening of drugs

Imaging of Brown adipose tissue in living mice

control FFA-luc

Copyright: https://www.aalaslearninglibrary.org www.typefreediabetes.com

- first example of imaging BAT without using radioactivity

FFA-luc + CL 316243

- first detection of FFA flux in live mice

Increases BAT thermogenesis

and metabolic rate

CL 316243Cl

HN

HO

O

O CO2H

CO2H

78

• Near-Infrared imaging uses light in the range of 650-900 nm:- minimum absorption by tissues - low interference from tissue autofluorescence

• It allows increasing tissue depth penetration and minimizing background signal

The NIR window in biological tissues

Frangioni J.V., Curr. Opin. Chem. Biol., 2003, 7, 626-634. Weissleder R. Nature Biotech., 2001, 19, 316-317.

Near-infrared probes for Imaging of fatty acid uptake

79

Optoacoustic imaging

Copyright: http://www.ithera-medical.com/technology/msot-principle.html

• Based on laser energyabsorption by tissues andultrasound waves

• ICG containing moleculesrepresent an ideal class ofcompounds for optoacousticimaging

• Conjugation of fatty acidwith ICG allows creation ofnovel NIR probe for imagingof fatty acid uptake

Indocyanine Green (ICG)(FDA approved in 1959)

Current status:

40 microns and 2 cm penetration depth

Imaging BAT Activation with ICG-FA Probe

• CL31624 was injected i.p.

• After 20 minutes mice were injected with ICG-FA and 1 hour later biodistribution was determined

With and without β-3 adrenergic stimulation (CL316243)

81

Interscapular BATNo Stimulation

Interscapular BATCL316243 Stimulation

In collaboration with Vasilis Ntziachristos (TUM)

Imaging BAT Activation with ICG-FA probeWith and without β-3 adrenergic stimulation (CL316243)

82

Interscapular BATNo Stimulation

Interscapular BATCL316243 Stimulation

Conclusion: the probe mimics natural fatty acids uptake

In collaboration with Vasilis Ntziachristos (TUM)

Developing of Probes for Image-Guided-

Surgery and Diagnostic in Oncology

Conclusions

Copyright: millville.sps.edu

Cell Surface Glycosylation

Fatty Acid Transport

Protein functions

H2O2 signaling