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Spezielle Methoden der pharmazeutischen Chemie 3Spezielle Methoden der pharmazeutischen Chemie 3
Asymmetrische Naturstoff und Arzneistoffsynthesey y(für Diplomanden und Dissertanten im Fach Pharmazeutischen Chemie/Synthese)
Inhibition of the Protein Synthesis by Antisense Oligonucleotides
DNA
T i ti Inhibition of the transcription
PrimaryRNA Transcript
Transcription Inhibition of the transcriptionby triple helix formation
RNA-Transcript
Processing Blockade of the processing factors
mRNA
TransportInhibition of the transport by double strand formation
mRNA
p by double strand formation
Enzymatic degradationof double strands
Nucleus
Cytoplasma
Translation Inhibition of the translation by double strand formation
Protein
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Possible Binding Positions of Antisense Oligonucleotides
Nucleusgenomic DNA
TranscriptionantisenseOligonucleotide
genomic DNA
primary Transcriptcappingregion
polyadenylationsite
exon-introntransition
RNA ProcessingantisenseOligonucleotide
t T i tribosomal
binding siteAUG
TranslationantisenseOli l tid
mature Transcriptbinding site
Oligonucleotide
CytoplasmaProtein
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y
Catalytic Effect of Antisense Oligonucleotides
Nucleusgenomic DNA
Transcription
ge o c
primary Transcriptcappingregion
polyadenylationsite
exon-introntransition
RNA Processing
t T i tribosomal
binding siteAUG
mature Transcriptbinding site
antisenseOligonucleotide Degradation by RNAse H
Cytoplasma
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Specific Action of Antisense Oligonucleotides
- sequence dependent inhibition of the gene sequence of the target proteinof the gene sequence of the target protein
- inhibition of genes of other proteins should be avoided g p(BLAST data bank)
~ 4.000.000.000 base pairs consist in the human genoman 18mer statistically occurs onlyone time in 68.000.000.000 base pairs
but sequence of bases is not statistical in the genomand pairing can occur even if there are 3 mismatches
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Unspecific Action of Antisense Oligonucleotides
- typical action of the polyanionic backbonehaemostasis activation of the complement systemhaemostasis, activation of the complement systemoccuring on high dosage beyond the therapeutic range
- caused by special structure features:immune stimulation by the CpG motiveimmune stimulation by the CpG motiveimmune stimulation by the phosphorthioate backbone
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Choice of the Target Sequence
- exclusion of sequences pairing with other mRNA (gene data bank)- not more that 2 CpG motivesp- no hairpin structures- no repetitive guanosines as targets
- no sequence as target, which is hindered by the secundary or tertiary structure of mRNAy y y
due to the matter of fact, that predictions of th d t ti t t f RNA till li blthe secundary or tertiary structure of mRNA are still unreliable the empirical method („gene walk“) is the method of choice
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Synthesis of Oligonucleotides - the Phosphoroamidite Process
Synthesis of Phosphorothioates - Sulfurization Reagents
Sulfurization TimeDescriptionReagent
N SS
S
N
15 minTetraethylthiuramdisulfide (= TETD Reagent)
S 0.5 M in anhydrous acetonitril
1 minBenzoyltetrasulfide
SS
O
SS
O0.4 M in THF or dichloromethane
y
S S 15 secTetrakis-(2-methylethyl)-thioperoxy-POO
PO
OS S
0.5 M in acetonitril
diphosphate
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Phosphorodithioates
O Base-1O
O Base-1O
O
O
O
OBase-2
PO
S
HSO
Base-2P
OO
HO
OO
HO P
OBase-3O
HS
S
P
OBase-3O
HO
O
P
OO
PhosphorodithioatesDNA
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Methylphosphonates
OO Base-1
OO Base-1
O
O
O
O
OBase-2
PO
HO
OBase-2
PO
O
HO
OO
HO P
OBase-3O
P
OBase-3O
HO
O
PHO
OO
MethylphosphonatesDNA
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Phosphoric Acid Triesters
O Base-1O
O Base-1O
O
O
O
OBase-2
PO
O
ROO
Base-2P
OO
HO
OO
HO P
OBase-3O
RO
O
P
OBase-3O
HO
O
P
OO
Phosphoric Acid TriestersDNA
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Phosphoramidates
OO Base-1
OO Base-1
O
O
O
O
OBase-2
PO
O
NO
Base-2P
OO
HO RH
OO
HO P
H
R
OBase-3O
N
O
P
OBase-3O
HO
O
PH
OO
PhosphoramidatesDNA
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Biophysical Characterisation of OligonucleotidesKonformation of the DNA-Backbone
S l Ch i i f A liStructural Characteristics of DNA-Helices
Circular Dichroism Spectra of DNA-Modificationsp
Temperature Dependence of Circular Dichroism Spectra
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Konformation of the DNA-Backbone
O O
α
--
P
OO O
PO O
O
γ
β
2´ 4´
5´
O
BH
O
O
O
B
ε
δ
1´
2
3´
4- OH
PO
O
HO
OH
H
ξ-
O
P
OOOH
H
A C3´ endo B: C2´-endo
O
A: C3 -endo B: C2 -endo
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Structural Characteristics of DNA-Helices
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Structural Characteristics of DNA-Helices
A DNA B DNA Z DNAA-DNA B-DNA Z-DNAsugar conformation C3'-endo C2'-endo C2'-endo (only dC)
i t ti i ht i ht l ftorientation right right leftdiameter (Å) 26 20 18base pairs per winding 11 10 3 – 10 6 12 (6 dimers)base pairs per winding 11 10,3 – 10,6 12 (6 dimers)rotation per nucleotide (Å) 32,7° 36° -60° (per dimer)distance per turn (Å) 28,2 33,7 45p ( )distance per base pair (Å) 2,56 3,37 3,62 – 3,72inclination of the base 20° -5,9° -7°major groove (Å; width; depth) 2,7; 13,5 11,7; 8,5 -minor groove (Å; width; depth) 11,0; 2,8 5,7; 7,5 2,7; 9,9
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Circular dichroism (CD)
Right-hand and left-hand polarised light runs through chiral media with differentRight hand and left hand polarised light runs through chiral media with different speed and is absorbed in a different degreeand is absorbed in a different degree.The absorption coefficients of right-hand polarised light (εR) and left-hand polarised light (ε ) are different! This effect is named circular dichroismus (CD)light (εL) are different! This effect is named circular dichroismus (CD).
Δε = εL - εRL R
positive CD bands: Δε > 0negati e CD bands: Δε < 0negative CD bands: Δε < 0
The difference of the absorption coefficients is determined by measuring a CD spectrum.
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a) UV spectrum measured with linear polarised light b) UV spectrum measured with left-hand polarised lightb) UV spectrum measured with left hand polarised light c) UV spectrum measured with right-hand polarised light
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Comparison of UV, ORD and CD Absorption Curves:
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Application of CD Spectroscopy:• for the deteremination of configuration of chiral natural products• for the conformation analysis of chiral compounds• for the anaysis of high molecular chiral natural products (proteins, nucleotides, carbohydrates) (p y )
• for the anaysis of interactions between drugs and receptor proteins and nucleotides
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CD Spectra of DNA-Modifications
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CD Spectra and Duplex Formation
3,0
2,0
0 0
1,0
mde
g)
-1,0
0,0
CD
(m
-2,0
-3,0
00 30 60 90 202 2 2 2 3
Wavelength (nm)
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Temperature Dependence of CD Spectra
3,0
2,0
0 0
1,0
mde
g)
-1,0
0,0
CD
(m
-2,0
-3,0
200
240
280
320
Wavelength
10° 20° 30° 40° 50° 60° 70° 80°
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Circular Dichroism Spectra and Transition Temperatures (Tm)
Vitravene TM - solution for injection (6.6 mg Fomivirsen sodium / ml)j ( g )
Developer: Isis and CIBA-VisionProducer: Novartis Ophthalmics EuropeProducer: Novartis Ophthalmics EuropeApplication: CMV - Retinitis (Cytomegalovirus)Target: Major Immediate Early Region 2 (IE2) of the human CMVTarget: Major Immediate Early Region 2 (IE2) of the human CMVEC 50: 0,34 ±0,25 μM
for virus antigen production in human fibrinoblastsfor virus antigen production in human fibrinoblasts0,03 ±0,02 μM for virus antigen production in human retina pigment epithelg p p g p
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Dosage Dependence of Side Effects of Phosphorothioates
Dosage Side effects
100 mg / kg80 mg / kg
immune stimulation and hepatotoxicity in miceclotting and complement effects in monkeysg g g p yproximal tubular degradation
20 mg / kg10 mg / kg
lymphoid hyperplasia in miceinhibition of clotting timescomplement activationatrophic and regenerative changes in proximal tubules
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Purification of the Artificial Oligonucleotides by HPLC
0,30
0,20
0,25
Vol
ts 0,15
0,05
0,10
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
0,00
11,7
83
HPLC-Conditions: gradient elution, linear gradient 10-40% B in 0-30 min
Minutes
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
A 0,1M triethylammonium acetate (TEAA) in WaterB 0,1M TEAA in Water / acetonitril (20:80)
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CD Spectra of the modified Oblimersen Derivatives
3,0
4,0
1,0
2,0
deg)
0,0
1,0
CD
(md
-2,0
-1,0
-3,0
200
230
260
290
320
Wavelength
Oblimersen free carboxy group aminofluorescein
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CD Spectra of the modified Oblimersen Derivatives
3,0
4,0
1 0
2,0
eg)
0,0
1,0
CD
(mde
-2,0
-1,0
-3,0
200
230
260
290
320
2 2 2 2 3
Wavelength
Oblimersen 1 lysine 2 lysine 3 lysine
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Transition Temperatures (Tmm)
78,780,0
69,568,4
67 4 67 6 68 270,0
75,0
pera
ture
65,5
,67,4 67,6 68,2
65,0
Tem
p
60,0
n ster
n oate
roup
cein
ine
ine
ine
Obl
imer
sen
osph
odie
s
Obl
imer
sen
osph
orth
io
carb
oxy
gr
noflu
ores
c
1 ly
s
2 ly
s
3 ly
s
OPh
o OPh
o
free
c
amin
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Cell Culture Test for Antisense OligonucleotidesCell Culture Test for Antisense Oligonucleotides
- Transfection reagent necessary for membrane penetration
- Measurement of the expression inhibition by determination of the target mRNA concentration (Northern Blot)the target mRNA concentration (Northern Blot)
- Measurement of the expression inhibition by determination of the target protein concentration (Western Blot)g p ( )
- Controll experiments with not pairing oligonucleotides(sense oligonucleotide, scrambled oligonucleotide, reverse
oligonucleotide)
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Cell Culture Test Cell Culture Test of the modified Oblimersen Derivatives
- human melanoma cell line 607B
t f ti ith Li f ti ( i t f k ti i li id )- transfection with Lipofectin (a mixture of kationic lipids)
- incubation for 48 hours
- protein extraction by lysis of the cells
- quantitative determination of BCL-2 by Western Blotq y
- standardisation with Actin
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Western Blot
80
607080
)
304050
CL-
2 (%
)
102030B
C
0
ence
scei
n
rsen
ysin
e
ysin
e
ysin
e
grou
p
rsed
seq
ue
min
oflu
ores
Obl
ime
1 l y
2 ly
3 ly
carb
oxy
g
reve
r
amfree
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The Research Team
P f D Ch i ti N
I tit t f Ph Ch i t I tit t f Ph Ch i t D t t f D t l
Prof. Dr. Christian Noe
Institute of Pharm. ChemistryUniversity of Frankfurt / Main
Atmaca-Abdel Aziz Serap
Institute of Pharm. ChemistryUniversity of Vienna
Bauchinger Arnulf
Department of Dermatology University of Vienna
Pehamberger HubertAtmaca-Abdel Aziz Serap Gilbert MatthiasKaufhold Lucius
Kock Michael
Bauchinger ArnulfSaadat KarminNovak Clemens
Winkler Johannes
Pehamberger Hubert
Department of Clinical PharmacologyUniversity of ViennaKock Michael