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DESIGN, SYNTHESIS, AND EVALUATION OF ANTI-CANCER PRODRUGS TARGETING:

A. THE EGFRs(ErbB1, ErbB2, ErbB3, ErbB4)

B. THE ANNEXINS(Annexin VI)

ByZechariah MartingID No. 310056012

(PhD)

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BACKGROUND ON CANCER INFORMATION

Cancer was, is, and will remain a huge global health problem

Consider the following basic facts about cancer:

• About 30% of cancer deaths are due to the five leading behavioural and dietary risks:

high body mass index, low fruit and vegetable intake, lack of physical activity,tobacco use, alcohol use(WHO, February 2012)

• About 70% of all cancer deaths in 2008 occurred in low- and middle-incomecountries(WHO, February 2012)

• Cancer causing viral infections such as HBV/HCV and HPV are responsible for up to20% of cancer deaths in low- and middle-income countries(WHO, February 2012)

• Cancer is a leading cause of death worldwide, accounting for 7.6 million deaths(around 13% of all deaths) in 2008(Globocan, IARC, 2010)

• Deaths from cancer worldwide are projected to continue rising, with an estimated 13.1million deaths in 2030 (Globocan, IARC, 2010)

• The most frequent types of cancer differ between men and women(WHO, February

2012)• Tobacco use is the most important risk factor for cancer causing 22% of global cancer

deaths and 71% of global lung cancer deaths(WHO, February 2012)

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➔

Biological, e.g. HIV, HPV, etc.➔Chemical, e.g. benzene, nitrosamines,benzopyrenes, ROS, etc.

➔Endogenous, e.g. hormones. reactive oxygenspecies

➔Environmental, e.g. ecosystem imbalance,pollution, etc.

➔Lifestyle, e.g. poor diet, tobacco smoking, alcoholdrinking, drugs, age, etc.

➔Physical, e.g. radiation rays(high and/or low energy),age, air travel, etc.

Known Sources of Carcinogens and/or Mutagens

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Nitrosamines,

Reactive Oxygen species,

Benzopyrenes,

Gamma rays,

X-rays,

Amino acids,

Minerals,

Etc.

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Ligands:

EGF, AREG, EPGN, TGFα, BTC, EREG, HBEGF, NRG-1, NRG-2, NRG-3,

NRG-4

The Receptors:EGFR(EGFR1, HER1, ErbB1, ERBB1)

ErbB2(HER2, EGFR2, ERBB2)

ErbB3(ERBB3, HER3, EGFR3)

ErbB4(EGFR4, ERBB4, HER4)

THE CHEMICAL BASIS OF THE WORK

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Heterodimerization e.g.

EGFR1 + EGFR2

Homodimerization e.g.

EGFR1+ EGFR1

Autophosphorylation

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Activation of Downstream Signaling

Note:Internalization is the downstream and upstream biochemical cascades inclusion of other TKRs

Internalization

Cell Progression

Cell Adhesion

Protein Synthesis

Cell Survival

Increased Gene

Expression

Metabolism

AngiogenesisCellular Targets

Cell differentiation

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Lysosomal Degradation

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Activation of tyrosine kinase domains by mutations, e.g., EGFR

Dimerization of tyrosine kinase domains through fusion withdimerization motifs of other proteins, e.g., NGF and PDGF receptors

Binding to and Activation of pre-existing oligomers of its receptor byEGF

Being cell surface receptors with disulfide-linked (αβ)2, insulin receptor and IGF1 receptors are expressed as dimers, hence binding to either induces tyrosine kinase activity leading to signalling transduction

Mechanisms of Oncogenesis in the RTK Pathway

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Autocrine or paracrine stimulation by an excess of growth factors, e.g. EGF

Overexpression of a receptor, e.g. EGFR

Mutation in the transmembrane domain, e.g., neu in rat neuroblastoma

Covalent linkage of extracellular domain of receptor monomers. e.g., Ret

Mechanisms of Oncogenesis in the RTK PathwayCont'd

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CANCER THERAPY APPROACHES

➢ IMMUNOTHERAPY

➢ ADJUVANT THERAPY

➢ RADIATION

➢ HORMONAL

➢ SURGERY

➢ VACCINATION

➢ CHEMOTHERAPY

➢ PRODRUGS/TARGETED

1. APOPTOSIS INDUCING DRUGS

2. ANGIOGENESIS INHIBITING DRUGS

3. GROWTH SIGNAL INHIBITORS(e.g. EGFR)

4. ANNEXIN VI BLOCKERS

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DEVELOPING OUR OWN MODEL OF THEEGFR-LIGAND BIO-CASCADE REACTIONS

Source of crystal pictures: Wikipedia

EGFRs IN PICTURES

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EGFR + EGFR1 + EGF1 + EGF1

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General Comparison of the EGFRs

Receptor Gene Name and

Synonym

Similarities Differentiating

Features

Comments

EGFR1 ErbB1, HER1, ERBB1, Proto-

oncogene-like protein c-ErbB1

1.All have extracellular domain

with sub domains I-IV; one

transmembrane domain, and

Intracellular kinase domain 2. All

have splice variants whose

amplification and/or over-

expression are prominent in many

types of cancers 3. All are type I

transmembrane glycoproteins

Exhibits four major

downstream signaling:

RAS-RAF-MEK-ERK, PI3

kinase-AKT, PLCgamma-

PKC and STATs modules.

May also activate the NF-

kappa-B signaling cascade;

has about 1210 amino

acids

One known genetic isoform,

EGFRvIII(which is exclusively

expressed in human tumors);

the only receptor to bind EGF,

TGF-α, Amphiregulins, and

Epiregulin ligands

EGFR2 Neu, NGL, HER2, ErbB2,

ERBB2, Her-2/neu, Proto-

oncogene-like protein c-ErbB2

As above Cannot bind growth factors

due to the lack of its own

domain; It’s auto-inhibited

constitutively; has 862

amino acids

Needs a co-receptor for ligand

binding

EGFR3 HER3, c-erbB3, Proto-

oncogene-like protein c-

ErbB3, ERBB3

As above Has a neuregulin binding

domain and a defective

protein kinase domain;

has 1342 amino acids

No homodimerized moiety

actively known; it serves as a

low affinity receptor for the

heregulins(egfrs); E2+E3 forms

high affinity receptor complex

but low E4 and itself

EGFR4 ERBB4, ErbB4, Proto-

oncogene-like protein c-

ErbB4, HER4

As above Has a cysteine-rich domain;

phosphoinositol-3 kinase

binding site; has PDZ

domain binding motif; has

1308 amino acids

Highest overexpression in

brain, heart, and kidney; binds

itself with low affinity receptor

complex

A MODEL OF BIOCHEMICAL REACTIONS OF THE EGFRs WITH FOCUS ON EGFR1

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A MODEL OF BIOCHEMICAL REACTIONS OF THE EGFRs WITH FOCUS ON EGFR1

=

P+

n

G r B 2

G A B 2

P I 3 K

A k t

/

P K B

NCK

PAK

JNKK

JNK

ELK/JUN

S h c G r B 2

S o s

R a s

R a f

M E K

E R K

DNA

G r B 2

PKC

PI3

--\

--\

- - \

P+

Angiogenesis

B a d

Cell

survival

S T A T s

DNA

P+

P+

P+

P+

P+

P+

P+

P+

9

12 3 4

5678

+

H202

PTP

jun

fos

Key

Final Biochemical Pathway in the Cytoplasm

= Negative Feedback

= Aftermath of Oligomerization

= Internalization Outcome

= Impartation of Dimerization on a Non-receptor PRK

= Feedback Mechanism Directly on the Oligomer

+ = Positive Feedback Mechanism

----> = Inhibitory Path and Outcome

Note

a. Numbering is arbitrary

b. All biochemical reactions at the internalization stage Involve other PRKs

c. PTP = Protein Tyrosine Phosphatases

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Graphical Representation of all Dimerization Reactions of the EGFRswith Focus on the Most Informed(ErbB1+ErbB1 Homodimer)

EGFR1+EGFR1 9

EGFR1+EGFR2 8

EGFR1+EGFR3 0

EGFR1+EGFR4 0

EGFR2+EGFR2 2

EGFR2+EGFR3 3

EGFR2+EGFR4 4

EGFR3+EGFR3 0

EGFR3+EGFR4 0

EGFR4+EGFR4 3

PATHWAY E1+E2 E1+E3 E1+E4 E2+E2 E2+E3 E2+E4 E3+E3 E3+E4 E4+E4

1 X X X X X

2 X X X X X

3 X

4 X X* X*

5

6 X X*

7 X

8 X X

9 X

0

12

3

4

5

6

7

8

9

F r a c t i o n R e l a t i v e t o

H o m o d i m e r

E G F R 1

Hetero- and homo-dimers of the EGFRs

Series1

1. The most extensively studied E1+E1 has nine(9) pathways

2. No recorded pathways for E1+E3, E1+E4, E3+E3, and E3+E4

3. E1 through E4 = Represents Epidermal Growth Factor Receptors 1 to 4

4. X* = Provokes pathway 4 starting at PI3K only

MECHANISTIC MODELS OF DIMERIZATION IN TRK- LIGAND BINDING

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➔A monomeric ligand binds to a receptor monomer preparatory to complex state➔In the complex C, dimerization occurs due to covalency between the receptor dimer

➔A dimeric ligand binds to a receptor monomer leading to the complex C.➔In the complex C, a monomeric ligand mediates the autophosphrylation process through non-covalency


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➔A monomeric ligand mediates the receptor dimer through formation of a type of bond(e.g. H-bond)

➔In the complex C, there is no dimeric receptor

➔A dimeric ligand binds a monomeric ligand preparatory to complexation➔In complex C, the receptor dimer forms hence the dimerization

MECHANISTIC MODELS OF DIMERIZATION IN TRK LIGAND BINDINGCont'd

MECHANISTIC MODELS OF DIMERIZATION IN TRK LIGAND BINDING

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➔A dimeric ligand mediates the receptor dimer formation➔The complex C shows the receptor dimer-ligand dimer formation leading to the dimerization(Ullrich et al, 1990)

➔A monomeric ligand binds to a monomeric receptor preparatory to the complex formation➔In the complex C, the monomeric ligand mediates the monomeric receptors, held by a force(e.g. H-bond)

MECHANISTIC MODELS OF DIMERIZATION IN TRK- LIGAND BINDINGCont'd


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➔Monomeric ligand binds to a disulphide-stabilized receptor with subsequent intracomplex conformational change.➔The C complex need two ligands to accomplish dimerization(Ullrich et al , 1990)

Cont'd

➔Monomeric ligand binds to receptor extracellular ➔The complex C is a receptor dimer formation occupied by the two ligands(Ullrich et al, 1990)

SS S S

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Crystal Structure of the Complex of Human EpidermalGrowth Factor and Receptor Extracellular Domains

Domain 2

EGF

Domain 1

C Terminal

C Terminal

Domain 4

EGF

Domain 3

N Terminal

N Terminal

2 EGFRs + 2 EGFs

Crystal Structure of the Complex of Human Epidermal

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EGF

C Terminal

Domain 4

EGF

N Terminal

Domain 4

1 EGFR + 2 EGFs

Crystal Structure of the Complex of Human EpidermalGrowth Factor and Receptor Extracellular Domains

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METHODOLOGY

● Drug Modeling using Schrodinger's Suite 2012

● Chemical Databases in use:

High Throughout Virtual Screening of the Compounds

Extra Precision of the First Twenty Compounds

Identifying Predominant Amino Acid Residues Bonded to the

Compounds Common to the First 20

c. BUILT EGFR INHIBITORS

a. ZINC(CHEMBRIDGE) COMPOUNDS

b. ZINC(AMBINTER) COMPOUNDS

Extra Precision of the 30 Built and using the First 15

Identifying the Predominant Amino Acid Residues Bonded to by the Inhibitors

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METHODOLOGY Cont'd

d. RE-RUNNING THE EXTRA PRECISION USING THEPREDOMINANT AMINO ACID RESIDUES COMMON TO ALL WITHTHE 1IVO PREPARED GRID

THE CHEMICAL COMPOUND WITH THE HIGHEST GSCORE EMERGED

ZINC 04996316

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METHODOLOGY Cont'd

THE SAME APPROACH WAS FOLLOWED WITH 2J6M:

CRYSTAL STRUCTURE OF EGFR KINASE DOMAIN IN COMPLEX WITH AEE788

PURPOSE: AS A PROOF OF METHOD

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METHODOLOGY Cont'd

DRUG SYNTHESIS

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METHODOLOGY Cont'd

PRODRUG EVALUATION

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MY SUPERVISORS

PROF. DAVID E. HIBBS

PROF. PAUL W. GROUNDWATER

DR. THOMAS GREWAL

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SELECTED REFERENCES

Ferguson, K. M. Structure-Based View of Epidermal Growth Factor Regulation. Annu. Rev. Biophysics. 2008, 37:353-73

Boutin, J. A. Tyrosine Protein Kinase Inhibition and Cancer. Minireview. Int. J.Biochem. Vol. 26, No. 10/11, 1994, pp 1203-1226

Yang, R. Y. Targeting the Dimerization of ERBB Receptor Tyrosine Kinases,A Dissertation Presented to the Graduate School of Arts and Sciences of

Washington University. August 2009, St. Louis, Missouri

Choowongkomon, K., Sawatdichaikul, O., Songtawee, N., and Limtrakul, J. Receptor-Based Virtual Screening of EGFR Kinase Inhibitors from the NCI Diversity Database.

Molecule 2010, 15, 4041-4054

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THANK YOU

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COMMENTS, QUESTIONS, AND SUGGESTIONS

Email: [email protected]

THE CHALLENGE OF CANCER IS EVER PRESENT WITH US

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Name of Patient: Chen ZongtaoAge: 29 yr Weight of tumor: 154 pounds(70kg), right leg

Type of Tumor: NeurofibromaWhere: Lives in China in a remote villageWhy Late: Couldn't afford any medical attentionPresent Condition: On Therapy with Marked Progress

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ESTHESIONEUROBLASTOMA

Name: Chantal SebireStatus: Retired French Teacher Date of diagnosis: 2000Nature: Rare Form of Cancer Attitude: Refused all forms of TreatmentNext: Died, March 19, 2008 through euthanasia

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