به نام مهربانتر ین In the name of the most compassionate 1

به نام مهربانت In the nameرین

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Design and Construction of Antagonistic VEGF Variant for Inhibition of Angiogenesis

By:Fahimeh Ghavamipour

Supervisors:Dr. R. H. Sajedi

Dr. M. Aghamaali

Advisors: Dr. S. S. Arab

Dr. K. Mansouri

Dept. of Biology, Faculty of Sciences, University of Guilan

Cancer

• The division of normal cells is precisely controlled. New cells are only formed for growth or to replace dead ones.

• Cancerous cells divide repeatedly out of control even though they are not needed, they crowd out other normal cells and function abnormally. They can also destroy the correct functioning of major organs.

Tumor Growth

• Phase 1: Genetic mutations– Cellular cycle and apoptosis disruption– Uncontrolled reproduction, no cell death

• Phase 2: Interaction with immune system – Cancer cells inhibit immune system

• Phase 3: Solid tumor– Cancer cell diffusion– Necrotic zones– Solid tumor diameter 1-2 mm

Necrotic zone

Uncontrolled Reproduction

Healthy cells

Angiogenesis and Metastasis

• Tumor growth requires nutrients– Active nutrient search

• Phase 4: Angiogenesis– Segregation of proteins which promote

blood vessel growth– Aberrant vascular network

• Phase 5: Metastasis– Cancer cells enter in blood network– New colonies in healthy regions

Angiogenesis

Angiogenesis :

Sprouting of new tubes

off of pre-existing tubes

The Angiogenesis Signaling Cascade

Genes are activated in cell nucleus

Cancer cell

VEGF (or bFGF)

Endothelial cell surfaceRelay

proteins

Receptor protein

Proteins stimulate new endothelial cell growth

Activators of Angiogenesis

Tumor Angiogenic Factors (TAF)

VEGF : vascular endothelial growth factor

bFGF: basic fibroblast growth factor

VEGF & Angiogenesis

•VEGF or VEGFA is the most potent & dominant stimulator of angiogenesis

• A key factor in tumor growth, progression and metastasis

•Highly expressed in several tumor types

•4 isoforms:VEGF121, 165, 189, 206

Multiple Isoforms of VEGF-A are Generated from Exon Splicing

Highly diffusible isoform

1 121VEGF-A121

Highest molecular weight isoform bound to extracellular matrix

VEGFR-bindingdomain

Heparin-bindingdomain

VEGF-A206

Sequestered in the extracellular matrix

1 189VEGF-A189

Most abundant isoform expressed in humans

VEGF-A165

VEGF8-109 is a segment from VEGF-A

• Homodimeric glycoproteinis• Held together by two intermolecular disulfide bonds• MW 24 KD• Have receptor binding domains in each monomer

VEGF8-109

VEGF and VEGF Receptors

VEGF Receptors

VEGFR I, Flt-1Higher affinity to VEGFAct primarily as a decoy receptorModulating the availability of VEGF to VEGFRII

VEGFR II, KDRLower affinity to VEGFThe principal receptor for VEGF signaling

This mutational analysis implicates KDR, but not Flt-1, has a critical role in VEGF

induction of endothelial cell proliferation

KDR Mechanism of Action

video.mp4

oDimeric structure of VEGF is a prerequisite of receptor activation

oOne VEGF molecule bridges two receptors via two similar recognition sites

oVEGFR-2 induces angiogenesis through activation of the classical extracellular regulated kinase pathway, leading to signal transduction

Inhibitors of

o Growth factorso Endothelial cell signal transductiono Endothelial cell proliferationo Endothelial cell migrationo Matrix metalloproteinaseo Endothelial cell survivalo Bone marrow precursor cells

Given its central role in promoting many cancers, VEGF provides an attractive target for therapeutic intervention

Angiogenesis inhibitors

Strategies for VEGF Inhibition

• Targeting the ligand (e.g. VEGF antibodies such as Avastin)

• Targeting the receptor (e.g. VEGF antagonists)

The Aim of Study

Construction of a heterodimeric antagonistic VEGF variant (HD-VEGF)

• Binding domains for the VEGF-receptor KDR/Flk-1 is present at one pole of the dimer, whereas the other pole carries domain swap mutations

• HD-VEGF blocks KDR dimerization and KDR-mediated VEGF activities that are crucial in the angiogenic process

VEGFl3165/VEGFl2

121 (William Leenders, et al.)

VEGFl1/VEGFl2 (Germaine Fuh, et al.)

Hv1 (Niv Papo, et al.)

scVEGF (Gerhard Siemeister, et al.)

Problem of previous studies:PurificationHighe IC50

In this study

Theoretical study

- Mutation Design

- Modeling and MD Simulation

- Molecular Docking

HD-VEGF construction

- Gene Synthesis

- Expression, Refolding & Purification of heterodimeric VEGF

Structural & Functional Studies

- CD & Fluorescence Analysis

- HuVEC Proliferation & Capillary Tube Formation Assay

Variation of the mean accessible surface areas, ∆ASA (Å2), with the amino acid residues number for VEGF RBD (PDB ID: 3V2A)

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 1150

Residues Number

)Identification of KDR Binding Site by Using

ASA Analysis

Theoretical study

(Brozzo, M.S., et al., Blood, 2012. 119(7): p. 1781-1788).

Variation of the mean accessible surface areas, ∆ASA (Å2), with the amino acid residues number for VEGF RBD (PDB ID: 2VPF)

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 1150

Residues Number

Theoretical study

Identification of Amino Acids Involved in dimerization by Using ASA Analysis

0 20 40 60 80 100 1200

Residues nmmber

Theoretical study

Identification of KDR Binding Domains

By using crystal structure of :

VEGF RBD (PDB ID: 2VPF)

VEGF-KDR complex (PDB ID: 3V2A)

Alanine scanning studies

The amino acids involved in KDR binding and protein dimerization were analyzed

Theoretical study

Segment 41-46 Segment 81-87

Mutant VEGFG Q N H H E V V K F M D V Y Q R S Y C H P I E T L V D I F Q E Y P G E G D E I L T F K P S C V P L M R C G G C C N D E G L E C V P T E E S N I T M Q I H G L R P S V A G Q H I G E M S F L Q H N K C E C R P K K D

Wild type VEGF G Q N H H E V V K F M D V Y Q R S Y C H P I E T L V D I F Q E Y P D

E I E Y I F K P S C V P L M R C G G C C N D E G L E C V P T E E S N I T M Q I M R I K P H Q G Q H I G E M S F L Q H N K C E C R P K K D

Mutation Design

Theoretical studyTheoretical study

12Å 13Å

Selected Segments for Mutation

• These suitable sequences were searched in

Protein Data Bank with some criteria such as:

•Having similar dihedral angles•The same distance between amino and carboxy terminals in segments•Their conformations

Modeling

• A 3D structure model of mutant VEGF was constructed based on wild type VEGF (PDB ID:2VPF) structure by using the I-TASSER program

• I-TASSER is best program in casp7 & casp8

• The model of designed mutant VEGF was compared to native one after energy minimization process

Theoretical study

Zhang, Y. BMC bioinformatics, 2008. 9(1): p. 40Roy, A., A. Nature protocols, 2010. 5(4): p. 725-738.Roy, A., J. Yang, and Y. Zhang, Nucleic Acids Research, 2012. 40(W1): p. W471-W477.

Molecular Dynamic Simulation

The selected model of mutant VEGF was used as initial structures for MD.

MD was done by GROMACS software Minimization was done during 5000 steps The simulation was performed in 300 °K GROMOS43a1 force field was used The simulation was run with 2 fs Time steps The explicit model H2O with 8 Å thickness was used for the

simulation The simulation was run for 1 ns

Theoretical study

Stability Investigations for MD Simulation

RMSD was stabilized nearly around 100 ps (MD production)

Total energy was stabilized nearly around 10 ps (Equilibration phase)

0 100 200 300 400 500 600 700 800 900 1000

-0.0200000000000001

-4.16333634234434E-17

Time(ps)

1 11 21 31 41 51 61 71 81 91

-245000

-243000

-241000

-239000

-237000

-235000

Time (ps)

0 10 20 30 40 50 60 70 80 90 10050000

-300000

-298000

-296000

-294000

-292000

-290000

Kinetic EnergyLinear (Kinetic Energy)

Time (ps)

Theoretical study

Temperature & Density was stabilized at 300 oK & 1.02 gr/cm3 (Equilibration phase)

Radius of gyration was stabilized

Theoretical study

Stability Investigations for MD Simulation

0 200 400 600 800 1000250

Time (ps)

0 200 400 600 800 1000990

Time (ps)

0 100 200 300 400 500 600 700 800 900 10001.78200000000001

1.78500000000001

1.78800000000001

1.79100000000001

1.79400000000001

1.79700000000001

1.80000000000001

Time (ps)

Molecular Docking

• The proposed structural model of HD-VEGF and KDR crystal structure

were used for docking

• Docked molecular complexes were constructed using 3D-Dock program

• 10000 complex were suggested by 3D-Dock as the best complexes

Theoretical study

Conclusion I

Identification of KDR binding domain was performed and replace by

suitable segments

A 3D structure model of mutant VEGF was constructed successfully

The model was refined through MD simulation

MD simulation showed good stability in Stability Investigations

Study on the docked complexes in compare with native complex shows

having incorrect contact or wrong orientation between complex of

VEGF mutant model and KDR.

Theoretical study

Gene Synthesis & Transformation

• The receptor binding domain of mutant VEGF gene was designed, synthesized and inserted in pET-21a+ expression vector

• Via NheI and BamHI sites with additional N-Terminal Strep tag

• Plasmid was transformed into the competent cells of E. coli (BL21) expression system. using Sambrook chemical method

Sequencing

Plasmid extraction and sequencing was

used to confirm the fidelity of the amplified

fragment

Expression Of Wild type & Mutant VEGF

• Inducing of positive colony by 1mM IPTG & 2mM lactose in 27 oC

• Monitoring of wild type & mutant VEGF expression using reducing

SDS-PAGE analysis

• The receptor binding domain of Native & mutant VEGF was expressed

as inclusion body in E. coli

To production of heterodimeric VEGF

The mutant and Native VEGF were mixed in an approximately equimolar ratio, and the refolding was performed on the mixture through multi-step refolding procedure

After the refolding the solution was expected to contain [1/4 NativeVEGF homodimer (N-VEGF) , 1/2 heterodimeric VEGF (HD-VEGF), 1/4 mutant VEGF homodimer (M-VEGF) ]

N-VEGF HD-VEGF M-VEGF

RefoldingHD-VEGF construction

Reducing & Non-reducing SDS-PAGE

Considering to the fact that VEGF dimerization is essential for its

receptor binding and biological activity, we followed dimerization of

the protein in the refolding process using reducing and non-reducing

SDS-PAGE.

Refolded protein was present primarily in the dimeric form with little

amount of monomer

Reducing Non reducing

N-VEGF HD-VEGF M-VEGF

Purification By Two Step Affinity Chromatography System

His tagged & Strep tagged heterodimeric variant

Purification By Two Step Affinity Chromatography System

0 2 4 6 8 10 12 14 16 18 200

Volume (ml)A

0 1 2 3 4 5 6 7 8 9 100

Volume (ml)

Purification of His-tagged and Strep-tagged heterodimeric protein was carried out using Ni-NTA Agarose & Strep-Tactin column

Strep- TactinHD-VEGF construction

Purified HD-VEGF

Non-reducing SDS-PAGE showed a single 28 kDa band indicating that the protein was purified to homogeneity

Gerhard Siemeister, et al. 1998.

Conclusion II

Sequencing confirmed the construction of mutants VEGF

The mutant VEGF was expressed as inclusion bodies successfully.

The mutation had no effect on protein dimerization.

Native and mutant VEGF were successfully refolded and dimerized. Also the heterodimeric variant was obtained in dimeric form.

Purified HD-VEGF variant was obtain whit very good quality by using two step affinity chromatography system.

As we aspect, the Far-UV CD spectra of M-VEGF, HD-VEGF & N-VEGF Shows that HD-VEGF forms are nearly similar to that of N-VEGF

M-VEGF HD-VEGF N-VEGF

12% 12% 12% Helix52% 53.5% 55% Sheet36% 34.5% 33% Random coil

Structural Analysis

Far-UV CD

190 200 210 220 230 240

-20000

Native homodimer

Mutannt homodimer

Heterodimer

wavelength (nm)

Fluorescence spectroscopy

Intrinsic fluorescence

400 420 440 460 480 500 520 540 560 580 6000

Native homodimer Mutant homodimer

Heterodimer

Wavelength (nm)

0 0.05 0.1 0.15 0.2 0.250

Mutant heterodimerLinear (Mutant heterodimer)Native homodimerLinear (Native homodimer)

[Acrylamid] (M)

Extrinsic fluorescence Quenching fluorescence

No significant structural changes in the HD-VEGF in compare with native and mutant homodimer.Correct structure of HD-VEGF

Structural Analysis

295 315 335 355 375 3950

Mutant homodimer Heterodimer

Native homodimer

Wavelength

HuVEC Proliferation Assay

M-VEGF HD-VEGF

373±3 33 2± IC50 (ng/ml)

Functional study

HD-VEGF M-VEGF

HuVEC Capillary Tube Formation Assay

M-VEGF HD-VEGF

3±329 1±24 IC50 (ng/ml)

Functional study

Control 60 ng/ml HD-VEGF

HD-VEGF

M-VEGF

80 ng/ml HD-VEGF 480 ng/ml M-VEGF

Previous studies

The privilege of our HD-VEGF

• Using of two step affinity chromatography system• Precise determination of amino acids involved in receptor binding• Deliberate design considering to the less effect on protein structure

and dimerization

Functional study

IC50 ng/mlRefrences Tub formation assay Proliferation assay

24 33 H-VEGF 8-109

Gerhard Siemeister, et al. 1998 - 150 VEGFl3165/VEGFl2

William Leenders, et al. 2002 - 200 VEGFl1/VEGFl2

Germaine Fuh, et al.1998 . - 550 Hv1

Niv Papo, et al. 2011. 227 - scVEGF

Conclusion III

CD analysis showed secondary structural similarity between HD-VEGF and N-

VEGF protein and it was in agreement with the theoretical study.

Fluorescence spectroscopy showed No significant structural changes in the

HD-VEGF variant in compare with native and mutant homodimer, and it was

in agreement with the results of CD analysis

Investigation of anti-angiogenic properties of heterodimeric variant revealed

that this variant can significantly inhibit the proliferation and capillary tube

formation of endothelial cells in vitro

Moreover this variant possesses much higher inhibitory effect than other

VEGF antagonists which have been so far constructed

Purified HD-VEGF variant was obtain whit very good quality by using two

step affinity chromatography system.

Structural & Functional study

Future works

• The mutation of Flt-1 receptor binding sites in order to decrease the IC50 value

• the mutation in KDR binding site in another monomer.• The precise design to modify the receptor binding domain of HD-VEGF in

order to induce higher binding affinity and subsequently lower IC50• Investigation of heterodimeric variant interaction with KDR receptor using

some techniques such as ELISA, SPR and spectroscopic methods• Supplementary anti-angiogenic and anti-tumor tests• Animal trials and in-vivo studies to investigate the anti-angiogenic and anti-

tumor effect of HD-VEGF

48Thank you

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