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1
High-Throughput Screening (HTS) by Immunoassay Tests
Service de Marquage Moléculaire et de Chimie Bioorganique
CEA Saclay
Service de Pharmacologieet d ’Immunologie
CEA Saclay (J. Grassi)
Laboratoire de Synthèse BioorganiqueUMR 7514
ULP / CNRS Illkirch
High-Throughput Synthesis and Screening (HTS)
HT Synthesis of molecules, catalysts, materials ...
HT Screening for: molecules, macromoleculesproperties (biological, physical)(pure compounds or mixtures)
2
H T S for antioxidantsprotective agents against oxidative stressselection of Norbadione A
H T S for enantioselective catalysts : yields & ee’s
HTSby immunoassay tests
Total synthesis of Norbadione A
Structure of an antibody
3
Polyclonal responseSelection of the mouse with the best response
Generation of poly- and monoclonal antibodies (Ab)
Chemistry Biology
109 Ac.
Injection of the immunogen
109 Ac. 109 Ac.
Cell fusion~ 103 hybridomes
selection of anti- AbH
Multiplication by cloning~ 10 Monoclonal Ab
anti- H
Synthesis of a fonctionalderivative of
N
O
O
O
O
H
O
NH
BSA
Synthesis of the immunogen
NH2 BSA
H
Hapten H
H
Competitive EIA (Enzyme ImmunoAnalysis)
Capacity > 1000 analysis/ day
YY
Y E
E
E
H
H
H
Capture
S
Color (DO)
YY
Y E
WashH
H
H
H
= Acethylcholinesterase (AChE)E
Color (DO): Ellman reagent
4
Competitive EIA (Enzyme ImmunoAnalysis)
YE
H
0
25
50
75
100
0,001 0,01 0,1 1
Log [H]
B/B
o (
%)
H
= Acethylcholinesterase (AChE)E
Color (DO): Ellman reagent
+
Y
H
Y
EH
+
K*
K
B0DO max
B/B0
Color
(µm)
YELLOW COLOR
Principle of AChE detection with Ellman reagent
Reading of the DO
2 HO
O
S NS
NO
SS
O2N
O2CO2C
O2N
SO2C
O2N
O2N
O2C SS
N
+
+
+Acethylcholine
esterase
5
New chemoluminescent probes
Design and synthesis of chemoluminescentprobes for the detection of cholinesterase
activityJ. Am. Chem. Soc. 2002, 124, 4874-4880
2 HO
O
S NS N
O
R S S N
NO2
O OO
SZ
O OO
SZ
S
EtO
O
Z
O
++
*
O OO
SS
O2N
R
Z
(R = H, NO2)hν
Acethylcholine
esterase
P.Y. Renard
Combinatorial approach
Conception of a library of catalysts
ParallelSynthesis
High-throughputscreening
Screening for new catalysts
A BCatalyst
Classical approach
Conception of a catalyst
SynthesisEvaluation
6
R
O COOH
R
OH COOH
R
COOHOH+
Asymmetriccatalyst
eeyieldee
Combinatorialchemistry
7
5 AB recognize bothenantiomers of MA
5 AB are selective for MA-S
1 AB is selective for MA-R
B/B
o(%
)
mAb-15
0
25
50
75
100
0,1 1 10 100
Log [Competitor] [mM]
(S)-MA
(R)-MA
BF
OH C OOH
OH COOH
O COO H
mAb-8
0
25
50
75
100
0,001 0,01 0,1 1
Log [Competitor] [mM]
B/B
o(%
)
BF
(S)-MA
(R)-MA
O COO H
OH COOH
OH C OOH
11 Monoclonalantibodies (Ab)
COOHOH
NR
+
*
R
O COOH
R
OH COOH
R
COOHOH+
Asymmetriccatalyst
ee
yield yield
mAb-15
mAb-8
mAb-15Combinatorial
chemistry
8
2112 experiments
2 Sourcesof hydrogen =
HCOOH/TEAKOH/iPrOH
6 Solvants = DMF, DMF/H2O, EtOH, DMSODMF/EtOH, DMF/DMSO
[RuCl2 (p-cym.)]2[RuCl2 (p-benz.)]2[RhCl2 (Cp)]2[IrCl2 (Cp)]2
4 M =
(2 solvants)
22 Ligands =
NHR1
NHR2
NHR2
NHR1
NHR1
NHR2
NHR2
NHR1
Conception catalysts’ library
CO2HO
NN R2R1
M
CO2HHHO
*BF MA
H2 source
No LL1 L2 L3 L4 L5 L6 L7 L8 L9 L10 L11
No H2
BA
C
D
L12L13L14L15L16L17L18 L19L20L21L22
M2
M1
M3
M4
M2
M1
M3
M4
Yields
0-10%10-30%
50-70%
30-50% 90-100%70-90%
High-Throughput Screeningof Enantioselective Catalysts by Immunoassay .Angew.Chem. Int. Ed.2002, 114, 132-135.
Screening resultsM/L* (1:1.2) 1.6%
DMF/H2O; 25°C
BF MA (S)
CO2HO CO2HHHO
No H2
No L
0-10%10-30%
45-60%
30-45%60-75%
ee(S)
75-90%
L1 L2 L3 L4 L5 L6 L7 L8 L9 L10 L11
L12L13L14L15L16L17L18L19L20L21L22
M : RuCl2(p-c y m)]2
L* :NH
NH2
SO2CF3
9
HTS tests for the ee determination
- IR thermography (Reetz and coll. 1999)
- Capillary aray electrophoresis (Reetz and coll. 2000)
- CD-HPLC (Mikami and coll.2001)
- Electrospray ionization with isotopically labeledsubstrates (Reetz and coll. 1999)
- Immunoassay
H T S for antioxidantsprotective agents against oxidative stressselection of Norbadione A
H T S for enantioselective catalysts : yields & ee’s
HTS
Total synthesis of Norbadione A
10
Antioxidants
RadioprotectorsDecorporation
UV-protectors
Protections against the oxidative stress
Fe ; Cu
H2O2
H2O
Chelates
Free radicals.OH ; .OOH ; O2
.
H2O2
γradiations
UVradiations
Oxidative Stress
.OH
.OOH
O2.
Active OxygenSpecies
HN
N
NH2
O
O
O
HN
N
O
O
O
OP
OO
O OH
HN
N
NH2
O
O
OP
O
O OH
HN
N
O
H2N
O
OP
O
O OH
N
N
NC T A G
DNA strand break
Base degradation
Abasic siteformation
Addition of lipidperoxidation
products
CO2H
OHC CHO
DNA-proteinlink formations
PROTEIN
H2N
Lys
11
3 AnticorpsMonoclonaux
Antibodies’ generation
mAb-62
0
25
50
75
100
0,01 0,1 1 10
Log [Thymidine] [µM]
B/B
o(%
)3 monoclonalantibodies
HN
N
O
O
O
OH
HO
HN
N
O
O
O
O
HO
RThymidine
DO
- Competitivebinding
- wash
EE
- Competitivebinding
- wash
High-throuput sceening of new protective agentsagainst oxidative stress
H2O2
UVFe/EDTA
H2O
137Cs
HO. ; HOO.
protectiveagent
=
=
=
protectiveagent
= Degradated thymidine
HN
N
O
O
O
OH
HO
12
Strategy for the discovery of new protective agentsagainst oxidative stress
Libraries ofMolecules
in vitro screeningantioxidant activity
Lead identification
different sources:Plants, mushrooms, algaes...
SynthesisExtraction
Lead optimisationfor the
synthesis of analogs
Toxicological evaluations Physical determinationsMechanism of action studies
Biological activities:- anti-inflamatory-anti-proliferative-anti-cancer-anti-aging- radioprotective
Tests:CellularTissularIn vivo
Mushroom extracts – UV degradation tests
% o
f thy
mid
ine
prot
ectio
n
20
40
60
80
100
Sarcodon Repandum
Cantharellus Cibarius
Pleurotus Ostreatus
Amethystus PisolithusTinctorus
Extractions :- MeOH / Acetone / HCl- CH2Cl2 then MeOH
Experimental conditions:Thymidine (70µM) ; H2O2 (5mM) ; irradiation at 254nm 1.75 J/cm2
Tp Phosphate 25mM pH 7.4
Norbadione A
13
Flavones AnthocyanesFlavanones
OH
HO
OH
R
ResveratrolPiceatannol
R3
OH
R1
O
OH
HO
OR
OH
Catechine
O
OH
HO
R1
R3
OHOH
O
R2
O
OH
HO
OH
R
OHOH
O
O
OHOH
R1
HO R3
OH
R2
+
O OHO
R2
R1
R3 R4R5
OO
OH
O
HO
O
R
Curcumin O
O
OMe
OMe
HOO
HC O2H
Célastrol
R2
OOHOH
HO
R1R3
Purpurogallin
N
N
N
N
OH
OH
Lumazine
Coumarins
R3
OHR1R5
R2R4
OHR1HO
R2
R3
R4
R2
OHOHHO
R1R3HN
HO NH2
Sérotonine
Stilbene derivatives
Quinones
Hindered phenolsPhenols Aza-phenols
R2
R3
O
HO
R1
Theaflavins Chromenes
Library of antioxidants
β -Diketones
O
OH
HO
OR
R
Cinnamic acid derivatives
R
N
OHN
N
OH
R
N
OH
NHO
R
O
HOCOOH
TroloxR2R1
HOR1
Flavanols
OHO
R1R2
R3
(Melatonine)
O
OHCO2H
OHOH
O OH
OHOH
O OH
OH
NH
OH NH2
OHOH
O O
OH
OHOH
OHO
OH
OHOH
O
OH
Trolox 3,4 -Dihydroxybenzoic acid Gallic acidSérotonine
Propyl GallateDL-3,4 -Dihydroxy -mandelic acid
3,4 -dihydroxy -phenylacetic acid
O
OO
OHOH
O
OHOH
Ellagic acid
OH
NH2
4-Aminophenol
OH
OH
O
NH2
4-Aminosalicylic acid
O
OH
OH
OH
O
Naringenin (trihydroxyflavanone)
O
OH
OH
OOH
OHOH
OOH
OOH
OHOH
O
OH
OH
OOH
OHOH
OH
O
OH
OH
OO
OHOH
O O O
OHOH
OH
OHOH
OH
Quercetin Fisetin
Myricetin
(+)-Rutin
O
OH
OH
OHOH
OH
O
OH
OH
OHOH
OH
Catechin (-)-Epicatechin
OO
OH
O
OH
O
Curcumin
O
OH
OH
OH
O
O
OHOH
OH
OH
OH
O
OH
OH
OH
OH
OH
OH
O
OH
(-)-Gallocatechin gallate (-)-Gallocatechin
Flavanol
O
OH
OH
OH
OH
O O
OH
OH
O
Apigenin 7-glucoside
O OOH
OO
OH
OH
OH
OH Esculin
OHNH2
NH2
OH
OH
OH
2,-Diaminophenol
Resveratrol
O
O
OMe
OMe
2,3 -Dimethoxy -5-methyl-1,4 -benzoquinone
N+
ODMPO
O
OH
H
CO2H
Celastrol
O OHOHOH
OH
Purpurogallin
OOH OH
OH
OH
O
OH
OH
O
OH
OH
OH
O
Gossypin
O
OH O
OH
OH
OH
OH
O
OH O
OH
OH
O
OH O
OH
OH
OH
Taxifolin Apigenin
Kaempferol
OOH
OH
O
Esculetin
O
OH
OH
OH
OH
OHCyanidin chloride
+ O
OH
OH
OH
OH
Pelargonidine chloride
+
N
N
N
N
OH
OHLumazine
O
OH
OH
OOH
BaicaleinO
OH
OH
OH
OHO
OH
O
OH
OH
OH
O
OHMorin Luteolin
OH O
OH
OH
OO
O
OH
OH
OH
OH
OH
Quercitrine
O
OH
OMe
OH OH
Xanthohumol
OO
OH
O
OOOH
HO2C
OH
O
OH
OH
CO2H
Norbadione A
OO
OHCO2H
OO
OHCO2H
MeO
OMeO
O
OHOH
BnO
ONO2
OH O
7-Hydroxy -4-methyl-8-nitrocoumarine
O
OH
OH
O
Chrysin
O
OH O
OH
OHOMeHesperetin
O
OH O
O
OH
rut inose
OMeHesperitin
Pulvinic acidderivatives
14
Protection(%)
0-10%
10-30%
50-80%
30-50%
80-100%
A
BC
DE
FGH
7 864 531 2A
BC
DE
FGH
7 864 531 2
UV/H2O2137Cs
O
OO OH
HO
O
CO2 KKO2C
HO
OH
HOO
O
Norbadione A
E
protecting agents
N
NH
O
OH
O
O
OH
Oxidative stress70 commercial molecules10 synthetic molecules10 natural extracts
tested in 4h1 highly efficient molecule found
γ Radiations
UV, Fenton
1- Norbadione
2- Quercitine
3- Fisetin
4- Myricetin
5- Catechin
6- 4-hydroxy-4-methyl-8-nitrocoumarine
7- Trolox
Thymidine 70µ molUV 254nm 1.75 J/cm2
H2O2 5mM in H2Oantiox. 100µ m
Fe2SO4 0.35 µ mol +H2O2 35 µ mol in H 2O
γ Ray 3h
10
20
30
40
50
60
70
80
90
UV
10
20
30
40
50
60
70
80
90
% p
rote
ctio
n T
hym
idin
e
FENTON
10
20
30
40
50
60
70
80
90
γ Ray
Antioxidative properties
1 4 5 62 3 7
1 4 5 62 3 7
1 4 5 62 3 7
15
DNA protection against γ radiations
Plasmide pUC18, phosphate buffer pH 7,4Irradiation 137Cs, 30 min. (60Gy)
Norbadione Analogue
Norbadione AnaloguepUC18
pUC18 + γ
O
OO OH
HO
O
CO2KKO2C
HO
OH
HOO
O
O
HO
O
KO2C
MeO
OMe
0
500
1000
1500
2000
2500
3000
SR
B a
rbitr
ary
units
of f
luor
esce
nce)
0 25 50 75 100 125
Cisplatine (µM)
NOR-03Cells : K1 wt
Test : SRB / 48 h
Nor : 20 µg/ml
SRB/NOR-B +
SRB/NOR-B -
SRB/NOR-A +
SRB/NOR-A -
CisplatineCisplatine
Cytotoxicity of cisplatinelowered by Norbadione A
Norbadione A +human thyroid carcinoma
Cisplatine(increasing conc.)
2 days
sulfo-rhodamine testliving cells count
RadioRadio-- & & chemoprotecting effects of Norbadionechemoprotecting effects of Norbadione A A
P. Bischoff (Strasbourg)X RaysX Rays
6 days
X Ray15 mV, 8Gy
RDM4 cells +increasing conc .Norbadione A
« uptiblue» testLiving cells count
Survival rate of cells increased with Norbadione A
0
250
500
750
1000
1250
1500
1750
Arb
itrai
ry u
nits
of f
luor
esce
nce
0 0,150,3 0,6 1,2 2,5 5 10 20
µg/ml
NOR-B/8Gy
NOR-A/8Gy
16
Mushrooms and radioactivity
Pisolithe (Pisolithus tinctorius )
Bolet bai (Xerocomus badius )
In 1986, radioactive particles from the electrical plan of Tchernobyl contaminated several european countries including several french regions. Bolet bai , a commestiblemushroom, contains high concentrations in cesium 137.In 1989, Steglich found that cesium 137 is selectively localised in the pigments present on the top of the mushroom in association with norbadione A.Norbadione A can be extracted in higher quantities from a other mushroom Pisolithe (M. Gill).
(Steglich, Gill)
NORBADIONE A
O
OO OH
HO
O
CO2KKO2C
HO
OH
HOO
O
Cesium complexationCesium complexation byby NorbadioneNorbadione A:A:mass spectrum studiesmass spectrum studies
O
HOOCO
OHHO
COOH OH
H
O
O
HOO
O
(M =H, M =K)
COOKKOOCC s+
COOCsKOOCC s+
COOKKOOC
COOKKOOC
+ C s+
+ 2 C s+
β1
β2
complex 1:1
complex 1:2
β1 = 4.9 l.mol-1
β2 = 9.5 l.mol-1+ K+
Speciation of norbadione in presence of Cs +
-1,00E-05
0,00E+00
1,00E-05
2,00E-05
3,00E-05
4,00E-05
5,00E-05
6,00E-05
7,00E-05
8,00E-05
9,00E-05
1,00E-04
0 0,5 1 1,5 2 2,5
nb eq Cs+
ccn
(M)
M. DesageB. AmekrazC. Moulin
free nba
1:1 complex
1:2 complex
17
Absortion of Norbadione A
Elimination of the Norbadione
complexed cesium in the urine and faeces
DecontaminationDecontamination byby decorporationdecorporationwithwith a a chelatingchelating agentagent
Individual contaminated by the radioelement
Norbadione A, a radioprotective agent with a double mechanim of action:
1- detoxification byspecific chelation and elimination of 137Cs (decorporation)
2- antioxidant properties to capture the reactive oxygen species generated by γ Ray emitted from 137Cs
18
H T S for antioxidantsprotective agents against oxidative stressselection of Norbadione A
H T S for enantioselective catalysts : yields & ee’s
HTS
Total synthesis of Norbadione A
Langercyclisation
ClCl
O
O
OPOMe
R3SiO
OSiMe3MeO+
O
O
POPO
CO2P
OP
+
Diels-Alder
Norbadione A: retrosynthetic scheme
HO
O
OHO
O OHR1O
O
CO2HHO2C
OO
HO
O
R1O
O
HO2C OP
TfOO
O
B(OMe)2
B(OMe)2
+
Suzuki-Miyamacoupling
Suzuki-Miyamacoupling
19
MeOOMe
OO
OBn
Me3SiR1O
OSiMe3
OMeO
OBn
TMSOTf 54%
Cl ClO
O
OO
HO OMeCO2 Me
OBn
OO
TfO OMeCO2Me
OBn
1- Et3 N, TMSCl
2- LDA, T MSCl 95%
Tf2O, Py
100%Z 68%
CO2 Et
O O
O
BnO
O
CO2Et
OH
CO2Me
iPr3 SiO
OSi(iPr3)
OHOH
OBnO
O
OTfOTf
OBnO
O
Cl ClO
O
OH
BnO
OH
CO2Et
OH
O
Cl
O
CO2Et
OH
B(OR)2
B(OR)2
OBnO
O
TiPSOTf, 2eq. Et3 N, 4eq.
CH2Cl2 , RT 70%
BnOLi, 7eq.
THF, -40?C to RT
77%
60%
Na2S2O4 sat.
Acetone, RT, 1h
TsOH, cat.
Tol. reflx, 5h 45%
X Ray structure
(CF3SO2)2O Py, CH2Cl2
0?C to RT quant.
20
OTfOTf
OBnO
OMe3SnSnMe3
PdCl2 (PPh3)
SnMe3
SnMe3
OBnO
OO
O
OMeCO2Me
OBn
O
OO OMe
M eO
O
CO2M e
MeO2 C
B nO
OBn
OBnO
O
O
OO OH
HO
O
CO2 H
HO2C
HO
OH
OHO
O
1- TMSI / CH2Cl2
PdCl2 (PPh3), cat.Na2CO3 aq.THF, reflx, 3h
55%
2- LiOH / THF3- H+
LiCl, dioxane60°C / 50%
2eq.
Norbadione Aacid form
TfO
Conclusions
Two high-throughput screenings (HTS) by immunoassay testsfor the selection of :
- catalytic systems- selection of highly active antioxidant agents
Total synthesis of Norbadione A a potent radioprotective agentselected by HTS immunoassay tests
21
Acknowledgments
Frédéric Taran Thierry Le Gall Alain ValleixStéphane Meunier Marine Lesage Céline Caussignac Jean-Michel Siaugue Stephanie Nowaczyk Sophie DézardJean-Marie GomisPierre-Yves RenardStéphane Sabelle
Laure BuscarletJacques GrassiPhilippe PradellesChristophe CréminonHervé Volland
Alain WagnerBarbara Mohar
SMM
SPI
CNRS
22
Validation of the Enzyme Immuno Assays (EIA)
Validation on n = 42 samples
0
20
40
60
80
100
0 20 40 60 80 100
M1/L2
HPLC / %
EIA / %
y = 1,0312x - 0,464R = 0,9324
ee’s
0
20
40
60
80
100
0 20 40 60 80 100
HPLC / %
M1/L2
EIA / %
y = 0,9836x - 0,5699R = 0,9711
Yields
Antioxidant Tests
Radical sources
Target molecule
idem but signal inhibition
+ Antioxidant
UV Abs. LuminescenceElectrophoresis RPEHPLC
concentration determination ofone of the oxidized product
degradationproducts
conc. determinationof the unafected target
idem but higher conc.of unaffected target
+ Antioxidant
Immunoanalysis
23
Tests for the evaluation of antioxidant properties of single compounds or mixtures
. TBA method (thiobarbituric acid) : inhibition of the oxidation of deoxyribose (1959)
. HPLC : inhibition of the hydroxylation aromatic compouds (1984)
. TRAP method (total peroxyl radical trapping parameter) (1987)
. RPE : inhibition of the DMPO-OH radical generation (1990)
. Randox-TEAC method (Trolox equivalent antioxydant capacity) : decoloration of the ABTS radical (1993)
. Electrophoresis : inhibition of the split of DNA strand (1993)
1 J / cm2 - 5 min
0,4
0,3
0,2
0,1
EIA
Time (minutes)
[Thy
mid
ine]
eq. (
µM)
0 20 40 60 80 100
0,01
0,03
0,05
0,07
0 20 40 60 80 100
N
NH
O
OH
O
O
OH
HPLC
AU
(267
nm)
Time (minutes)
UV Degradation
UV (254nm)H2O2 5mM
HN
N
O
O
O
OH
HO
Phosphate Buffer
HN
N
O
O
O
OH
HO
HN
N
O
O
O
OH
HO
+ OH +
24
Degradation by Radiolysis
[Thy
mid
ine]
eq. (
µM)
0,4
0,2
0,6
0,8EIA
time (minutes)
0 20 40 60 80 100
Irradiation 1h – 150 Gy.
AU
(267
nm)
0,01
0,02
0,03
0 20 40 60 80 100
N
NH
O
OH
O
O
OH
HPLC
time (minutes)
γradiations
137CsHN
N
O
O
O
OH
HO
Phosphate Buffer
HN
N
O
O
O
OH
HO
HN
N
O
O
O
OH
HO
+ OH +
0,4
0,2
Time (minutes)
0,6
EIA
0 20 40 60 80 100
[Thy
mid
ine]
eq. (
µM)
AU
0,05
0,1
0,15
0,2
0 20 40 60 80 100
N
NH
O
OH
O
O
OH
HPLC
Time (minutes)
Fenton type degradation
Fe/EDTA/H2O2(1:1:100) 5min.
Fenton HN
N
O
O
O
OH
HO
Phosphate Buffer
HN
N
O
O
O
OH
HO
HN
N
O
O
O
OH
HO
+ OH +
25
OTf
OTf
BO
OB
O
O
1- TMSI / CH2Cl2O
OO OH
HO
O
CO2HHO2C
HO
OH
BB
O OO
O OO
TfO OMeCO2 Me
OBn
O
OO OM e
M eO
O
CO2M e
M eO2 C
BnO
OBn
+ 2
PdCl2 (PPh3 ), cat . Na2CO 3 aq.
THF, reflx, 3h 63%
PdCl2 (dppf) KOAc
2- LiOH / THF
3- H+