Transcript Ghavamix

‫به نام‬
‫مهربانتر‬
In the name of the‫ین‬most
compassionate
1
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
2
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.
3
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 cells4
Angiogenesis and Metastasis
• Tumor growth requires nutrients
– Active nutrient search
• Phase 4:
– 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
5
Angiogenesis
Angiogenesis :
Sprouting of new tubes
off of pre-existing tubes
6
The Angiogenesis Signaling Cascade
Cancer cell
VEGF (or bFGF)
Receptor protein
Relay
proteins
Endothelial
cell surface
Genes are
activated in
cell nucleus
Proteins stimulate
new endothelial cell
growth
7
Activators of Angiogenesis
Tumor Angiogenic Factors (TAF)
VEGF : vascular endothelial growth factor
bFGF: basic fibroblast growth factor
8
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
9
Multiple Isoforms of VEGF-A are Generated
from Exon Splicing
Heparin-binding
domain
VEGFR-binding
domain
206
1 VEGF-A206
Highest molecular weight isoform bound to extracellular matrix
189
1 VEGF-A189
Sequestered in the extracellular matrix
165
1 VEGF-A165
Most abundant isoform expressed in humans
121
1 VEGF-A121
Highly diffusible isoform
VEGF8-109 is a segment from VEGF-A
10
VEGF8-109
•
•
•
•
Homodimeric glycoproteinis
Held together by two intermolecular disulfide bonds
MW 24 KD
Have receptor binding domains in each monomer
11
VEGF and VEGF Receptors
12
VEGF Receptors
 VEGFR I, Flt-1
Higher affinity to VEGF
Act primarily as a decoy receptor
Modulating the availability of VEGF
to VEGFRII
 VEGFR II, KDR
Lower affinity to VEGF
The 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
13
KDR Mechanism of Action
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
video.mp4
14
Angiogenesis inhibitors
Inhibitors of
o Growth factors
o Endothelial cell signal
transduction
o Endothelial cell proliferation
o Endothelial cell migration
o Matrix metalloproteinase
o Endothelial cell survival
o Bone marrow precursor cells
Given its central role in promoting many cancers, VEGF provides
an attractive target for therapeutic intervention
15
Strategies for VEGF Inhibition
• Targeting the ligand
(e.g. VEGF antibodies
such as Avastin)
• Targeting the
receptor (e.g. VEGF
antagonists)
Video
16
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
165
121
VEGFl3 /VEGFl2
VEGFl1/VEGFl2
Hv1
scVEGF
(William Leenders, et al.)
(Germaine Fuh, et al.)
(Niv Papo, et al.)
(Gerhard Siemeister, et al.)
Problem of previous studies:
Purification
Highe IC50
17
In this study
-
Mutation Design
-
Modeling and MD Simulation
-
Molecular Docking
-
Gene Synthesis
-
Expression, Refolding & Purification of heterodimeric VEGF
-
CD & Fluorescence Analysis
-
HuVEC Proliferation & Capillary Tube Formation Assay
18
Identification of KDR Binding Site by Using
ASA Analysis
Variation of the mean accessible surface areas,
∆ASA (Å2), with the amino acid residues number
for VEGF RBD (PDB ID: 3V2A)
(Brozzo, M.S., et al., Blood, 2012. 119(7): p. 1781-1788).
140
∆ ASA (Å)
120
100
80
60
40
20
0
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
Residues Number
75
80
85
90
95
100
105
110
115
19
Identification of Amino Acids Involved in
dimerization by Using ASA Analysis
Variation of the mean accessible surface
areas, ∆ASA (Å2), with the amino acid
residues number for VEGF RBD (PDB
ID: 2VPF)
140
∆ ASA (Å)
120
100
80
60
40
20
0
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
Residues Number
75
80
85
90
95
100
105
110
115
20
140
2VPF
120
3V2A
∆ASA Å2
100
80
60
40
20
0
0
20
40
60
80
100
120
Residues nmmber
21
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
22
Mutation Design


Segment 41-46
Segment 81-87
Wild type VEGF
GQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPD
EIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESN I
TMQIMRIKPHQGQHIGEMSFLQHNKCECRPKKD
Mutant VEGF
GQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPGE
GDEILTFKPSCVPLMRCGGCCNDEGLECVPTEESNI
T MQIHGLRPSVAGQ HIG EMSFLQH NKCECRPKKD
23
Selected Segments for Mutation
12Å
•
13Å
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
24
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
Zhang, Y. BMC bioinformatics, 2008. 9(1): p. 40
Roy, 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.
25
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
26
Stability Investigations for MD Simulation
0.1
 RMSD was stabilized nearly around
100 ps (MD production)
RMSD(nm)
0.08
0.06
0.04
0.02
0
0
100
200
 Total energy was stabilized nearly
around 10 ps (Equilibration phase)
-243000
600
700
800
900
1000
Potential Energy
60000
-290000
58000
-292000
56000
-294000
54000
-296000
52000
-298000
50000
-245000
1
11
21
31
41
51
Time (ps)
61
71
81
91
-300000
0
20
40
60
Time (ps)
80
100
27
P-energy (Kcal/mol)
-241000
500
Kinetic Energy
K-energy (Kcal/mol)
Total energy (Kcal/mol)
-239000
400
Time(ps)
-235000
-237000
300
Stability Investigations for MD Simulation
 Temperature & Density was stabilized at 300 oK & 1.02 gr/cm3 (Equilibration phase)
400
1040
Density (kg/m3)
Temperature (K)
1030
350
300
1020
1010
1000
990
250
0
200
400
600
800
0
1000
200
400
600
800
1000
Time (ps)
Time (ps)
 Radius of gyration was stabilized
Radius of Gyration (nm)
1.8
1.797
1.794
1.791
1.788
1.785
1.782
0
200
400
600
Time (ps)
800
1000
28
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
29
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.
30
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
31
Sequencing
 Plasmid extraction and sequencing was
used to confirm the fidelity of the amplified
fragment
32
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
33
Refolding
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
34
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
35
Purification By Two Step Affinity
Chromatography System
His tagged & Strep tagged heterodimeric variant
N-VEGF
HD-VEGF
H
M-VEGF
S
H
S
H
S
36
Purification By Two Step Affinity
Chromatography System
Purification of His-tagged and Strep-tagged heterodimeric protein was carried out
using Ni-NTA Agarose & Strep-Tactin column
300
250
0.8
A280
200
0.6
150
0.4
100
0.2
Imidazole (mM)
1
50
0
0
0
5
10
15
20
Volume (ml)
3
2.5
0.8
2
A280
0.6
1.5
0.4
1
0.2
0.5
0
0
0
2
4
Volume (ml)
6
8
10
37
Destiobiotin (mM)
1
Strep- Tactin
38
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.
39
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.
40
Far-UV CD
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
20000
Native homodimer
[Ɵ](deg.cm2.dmol-1)
Mutannt homodimer
Heterodimer
0
-20000
190
200
210
220
230
240
wavelength (nm)
N-VEGF
HD-VEGF
M-VEGF
Helix
12%
12%
12%
Sheet
55%
53.5%
52%
Random coil
33%
34.5%
36%
41
Fluorescence spectroscopy
 Intrinsic fluorescence
Mutant homodimer
Native homodimer
Heterodimer
Native homodimer
Mutant heterodimer
Mutant homodimer
8
120
250
200
150
100
50
0
7
100
6
80
f0/f
Flourescnce Intensity
300
60
315
335
355
Wavelength
375
395
5
4
3
40
2
20
1
0
0
295
Native homodimer
Heterodimer
Heterodimer
350
Fluorescence Intensity
 Quenching fluorescence
 Extrinsic fluorescence
400
450
500
Wavelength (nm)
550
600
0
0.05
0.1
0.15
0.2
[Acrylamid] (M)
No significant structural changes in the HD-VEGF in compare with
native and mutant homodimer.
Correct structure of HD-VEGF
42
0.25
HuVEC Proliferation Assay
HD-VEGF
IC50 (ng/ml)
M-VEGF
HD-VEGF
M-VEGF
33±2
373±3
43
HuVEC Capillary Tube Formation Assay
HD-VEGF
Control
60 ng/ml HD-VEGF
M-VEGF
80 ng/ml HD-VEGF
IC50 (ng/ml)
480 ng/ml M-VEGF
HD-VEGF
M-VEGF
24±1
329±3
44
Previous studies
IC50 ng/ml
Proliferation assay
Tub formation assay
Refrences
H-VEGF 8-109
33
24
VEGFl3165/VEGFl2121
150
-
Gerhard Siemeister, et al. 1998
VEGFl1/VEGFl2
200
-
William Leenders, et al. 2002
Hv1
550
-
Germaine Fuh, et al.1998.
scVEGF
-
227
Niv Papo, et al. 2011.
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
45
Conclusion III

CD analysis showed secondary structural similarity between HD-VEGF and NVEGF 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.
46
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 antitumor effect of HD-VEGF
47
Thank you
48