2 - delsuc.net

Utilisation de la RMN du fluor
pour la mesure d'interaction
ligand-protéine
Marc-André Delsuc
• Strasbourg - juin-2013 •
Fluor et protéine
• étude d’interaction
• marquage de la protéine
50
40
Dalvit, C., et al
Fluorine-NMR experiments for highthroughput screening: theoretical
aspects, practical considerations, and
range of applicability.
J Am Chem Soc 125, 7696–7703 (2003).
30
20
10
0
2000 2002 2004 2006 2008 2010 2012
NMR & Protein & Fluorine
• Strasbourg - juin-2013 •
In-house and
commercial
collections
Selected scaffolds
with/without
fluorine for parallel
chemistry
Filters
1. Virtual enumeration
2. Selection of linkers
and R, RF groups
Data set of
fluorinated
fragments
Design of small
fragment library
per each scaffold
1. Clustering
2. Selection
Synthesis
Known ligands
Peptide chemistry
Defragmentation
Reviews ! POST SCREEN
Synthesis
!
s
i
t
r
a
v
o
N
Chez
LEF-DOS library
L-R
LEF library
Fn
Fn
Chemical synthesis
and / or analogs
selection
Fn
Fn
L’-R’
CF or CF3 tagged
potential ligands
Fn
Fn
Fn
L’-R’
Fn
L-R
19F-NMR
Fluorinated
peptides or
peptide mimetics
library
n
N
H
L-R
L’-R’
RF
O
Fn
O
H
N
O
R
N
H
direct binding assay ± protein
Binders identification & druggability assessment
19F-NMR
competition binding
assay + known ligand
SPR or ITC or fluorescence
Spy molecules selection
19F-NMR
competition binding assay
Fragment optimization
Hit identification
SAR-by-Archive
Focused-, targetbased libraries
HTS hits
Natural products
Binding site
Potency, Solubility
Fluorine scan
Validation &
characterization
Drug Discovery Today
FIGURE 1
Workflow for the screening with the 19F NMR-based binding assay. Abbreviations: HTS: high-throughput screening; ITC: isothermal titration calorimetry; LEF: local
environment of fluorine; LEF-DOS: local environment of fluorine-diversity oriented synthesis; SAR: structure–activity relationship; SPR: surface plasmon resonance.
Thelocal
molecules
of the from
fluorine
libraries
in Drug
large Discovery
mixand739–746
in the presence
absence
1.! Vulpetti, A. & Dalvit, C. Fluorine
environment:
screening
to are
drugtested
design.
Today 17,
(2012). of the protein. The binding molecules
• Strasbourg - juin-2013 •
RMN du Fluor
• Quelques Faits
• 19F : abondance
• rapport gyromagnétique
• sensibilité théorique
• largeur spectrale
• background
• sensibilité pratique
100%
0.941 p/rp à 1H
85% p/rp à 1H
200 - 400 ppm
absent
x2-x10 p/rp à 1H
• Relaxation
• R2 dominé par le CSA
• très sensible à τc
‣ «tumbling time» : temps de retournement de la molécule ~ MW
‣ interaction => élargissement
• Strasbourg - juin-2013 •
déplacements chimiques
hexa-F benzène : -158 ppm
mono-F benzène : -71ppm
• Strasbourg - juin-2013 •
ed colloidal systems in biomedical applications. credible range of biological effects, from complete
N metabolic F
N to very highly enhanced
inertness
credible
range
of
biological
effects,
from
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metabolic
inertness
to very
highly
enhanced
ls with impressive biological activity include I (diflucan,
flucospecificity
for binding at a particular receptor site. Thus, it is not surprising
that Nthe
preparation
and
specificity
for
binding
at
a
particular
receptor
site.
Thus,
it
is
not
surprising
that
the
preparation
and
popular magazines as a one-dose treatment for study
vaginal
yeast
of fluorinated
inhibitors, substrate analogs, anti-metabolites, "transition state analogs", suicide
CH3 suicide
study
of
fluorinated
inhibitors,
substrate
anti-metabolites,
state
analogs",
ich are antibacterials that have been developed in
responseand
to inhibitors, and
substrates
other analogs,
organofluorine
compounds"transition
is a highly
active
research area
substrates
and
other
organofluorine
compounds
is a Seebach,
highly active
research areaH2N
F 1996;
l infections to currently used drugs; Lefenuron
(IV),
a and
growth
(Resnati
andinhibitors,
Soloshonok,
1996;
Ojima,
McCarthy, and
Welch,
1990; Ramachandran,
NH
2
and
Soloshonok,
1999;
Banks,
ousehold pets; and pyrrole V, which finds (Resnati
application
as an2000). 1996; Ojima, McCarthy, and Welch,I 1996; Seebach, 1990; Ramachandran,
II
1999;
Banks,an2000).
o a potential drug or agricultural chemical can
produce
infrom complete metabolic inertness to very highly enhanced
O
O
OH
O
receptor site. Thus, it is not surprising that the preparationNand
N
F
COOH
O
COOH
OH
F
F
Cl
N
CCH
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2
2
ate analogs, anti-metabolites, "transition state analogs",N suicide
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F
COOH
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COOH
F
O
O
N N CH2CCH2 N F
N
organofluorine compounds is a highly active research area
N
N
N
N
N
F
N
C
NH
C
NH
OCF
2CHCF3
ma, McCarthy, and Welch, 1996; Seebach, 1990; Ramachandran,
N
CF
N
N
quelques molécules
N
CH3 F
F
O
F
F
O
F
COOH
COOH
I
N
N
I
N
CH3
O
H2N
NH2
II
III
O
Cl
C O
NH C NH
IV
F
IV
control : CF3
TFA
TFE
3-fluoro-phenol
...
CN
CN
Br
CHCF2001.
3
Copyright OCF
by the2author,
OCF2CHCF3
Cl
Cl
CF3
CF3
N
NH
H
Cl
V
Cl
V
CN
Br
OCF2CHCF3
III F
III F
Br
C NH C NH
F
F
IV
H2N
Cl
O
F
Cl
H2N
II
F
F
II
NH2
N
N
Cl
NH2
F
F
N
CH3
N
Copyright by the author, 2001.
H
Copyright
Cl by the author, 2001.
V
• Strasbourg - juin-2013 •
(a)
(b)
O
His57
F
F
HN
F
Compound (1)
0% Inhibition
at 1mM
Trp215
Ser214
Ser195
- protein
+ protein
Cys191
difference
–64.5
–65.0
–65.5
–66.0
–66.5
ppm
δ 19F
19F
NMR-based binding assay performed with the LEF library against bovine trypsin. Only the signal of the molecule interacting
withDiscovery
the protein
Drug
Today
is visible in the difference spectrum (obtained by subtracting the spectra in the absence and presence of the protein) and its chemical shift
enables on-the-fly identification of fragment (1) as binder. (b) High resolution (1.15 A ̊) X-ray structure (PDB code: 3NK8) of the NMR identified
2
ligand in complex with bovine trypsin [42]. The short interactions of the CF3 group with the protein are displayed with black dashed lines.
LEF:
local environment
of LEF
fluorine;
PDB:
Proteinbovine
Database
Bank Only the signal of the molecule interacting with the protein is visible
MR-basedAbbreviations:
binding assay
performed
with the
library
against
trypsin.
e spectrum (obtained by subtracting the spectra in the absence and presence of the protein) and its chemical shift enables on-the-fly identificat
(1) as binder. (b) High
resolution
(1.15
A˚) X-ray
(PDB
3NK8)
the NMR
identified
ligand
in complex
1.! Vulpetti,
A. & Dalvit,
C. Fluorine
localstructure
environment:
from code:
screening
to drugof
design.
Drug Discovery
Today
17, 739–746
(2012). with bovine trypsin [42]. The
ns of the CF3 group with the protein are displayed with black dashed lines. Abbreviations: LEF: local environment of fluorine; PDB: Protein Database
• Strasbourg - juin-2013 •
Proteins. Several fluorinated analogs of the aromatic amino acids can be obtained commercially,
including 4-fluorophenylalanine (VI), 6- and 5-fluorotryptophan (VII, VIII), and 3-fluorotyrosine (IX).
(See http://synthetech.clipper.net/specialty.html, http://www.fluorochemusa.com and http://
www.sigma-aldrich.com.) Synthetic methods are available for preparation of fluorinated derivatives
of most of the common amino acids, as well as a variety of fluorinated nucleotides and sugars.
Placement of these fluorinated materials into biopolymers has been accomplished by a number of
different strategies, including chemical synthesis and biosynthetic incorporation by organisms.
marquage de peptides
• acides aminés fluorés
ARTICLES
H
H2N
H
C COOH
H
Papeo et al.
H
of the
Intermediate
Amines 4-6a
H2NPrimary
C COOH
C Scheme
COOH 1. Synthesis
H2N C
COOH
H2N
CH2
CH2
CH2
CH2
F
N
H
F
F
C. Dalvit / Progress in Nuclear Magnetic Resonance Spectroscopy 51 (2007) 243–271
VI
VII
VIII
OH
IX
Conditions and reagents: (a) [bis(trifluoroacetoxy)iodo]benzene, pyridine, DMF, H2O, room temp, 70%; (b) DCM, TFA, room temp, quant
(5), 98% (6).
Copyright by the author, 2001.
Ser/Thr
Kinase
substrates containing
theseAkt-1
amino acids
were successfully
performed. Although we have shown their application only to
the 3-FABS experiments, these novel fluorinated amino acids
can now find useful application also in the FAXS (fluorine
chemical shift anisotropy and exchange for screening) experiment,29 a 19F-based competition binding assay, for the detection
in particular of molecules that inhibit the interaction between
two proteins.
2003 American Chemical Society.
of the originally formulated technique proved to be the low
sensitivity compared to that of other fluorescence-based21 and
radioactive-based22 procedures. In order to overcome this
problem, two possible strategies could be envisaged: (1) use
of cryogenic probe technology23 optimized to 19F detection24
0.120 0.105and
ppm (2) use of substrates containing magnetically equivalent
multiple CF3- moieties.24,25 As more magnetically equivalent
30
fluorine atoms are present in the substrate the throughput
Br
25
increases, less amount of enzyme is required, and weaker
20
IC = This
0.72 ±0.05µM
inhibitors can be identified.
last point is particularly relevant
15
in a fragment-based screening approach performed with 3-FABS.
HN
H89
10
Fragments are low molecular weight Nmolecules (typically <250
5
NH
Da) that display a high binding efficiency index
(BEI ) (-log
SO
0
KD)/MW
or BEI1 ) (-log 10
IC50)/MW with MW expressed in
0.1
26-28
[H89]
µM to their small size have a weak affinity for
kDa),
but indue
theand
receptor.
The detection
of these
affinity
molecules
eening and deconvolution (top)
IC measurement
(bottom) for compound
H89 weak
performed
with 3-FABS
at 564 MHz
F
a
Figure 1. Chemical structures of polyfluorinated glycine (PFG) and
polyfluorinated amino acids (PFAs).
[Sw ] in µM
N
H
Results and Discussion
50
1.! Dalvit, C. Ligand- and substrate-based 19F NMR screening: Principles and
Synthesis toofdrug
Polyfluorinated
Amino
Acids.
NR-Fmoc-PFAs
applications
discovery. Progress
in Nuclear
Magnetic
Resonance …
51, 243–271
were
secured(2007).
by performing a mono- or double-reductive
amination reaction on the corresponding primary amines. Thus,
the suitable intermediates 4-6 were prepared either by Hofmann
amide degradation (4) from NR-Fmoc-glutamine30 or by Boc
F NMR
• Strasbourg
protective group removal (5, 6)31 from NR-Fmoc-N
δ-Boc- - juin-2013 •
The enzyme is the Ser/Thr kinase Akt-1. The five compounds (identified with ‘‘cpds’’) present in the positive mixture are (2-amino-6
50
!
2
19
BL21.
g L!1
cillin)
PO4,
dium
ptical
37 1C
meyer
on of
supuoro
e and
g L!1
L of
of hrs
244.1
oro.
and
cetic
and
and
ning
while
e rise
f the
ously
han6
marquage de la protéine
• Biblio récente
• Crowley, P. B., Kyne, C. & Monteith, W. B. Simple and inexpensive
incorporation of 19F-Tryptophan for protein NMR spectroscopy.
Chem. Commun. 48, 10681 (2012).
• Salwiczek, M., Nyakatura, E., Gerling, U., Ye, S. & Koksch, B.
Fluorinated amino acids: compatibility with native protein structures
and effects on protein–protein interactions. Chem Soc Rev 41,
2135–2171 (2012).
• Vulpetti, A. & Dalvit, C. Fluorine local environment: from screening to
drug design. Drug Discovery Today 17, 739–746 (2012).
19F
NMR spectra of ... (C) Cell lysate with
over-expressed GB1 from 50 mL of E. coli
culture grown on minimal medium containing
5-fluoroindole. The cell lysate sample (C)
contained ~0.1 mM GB1 and the spectrum was
obtained with 2048 transients. Spectra were
referenced to a 10% trifluoroacetic acid sample.
Fig. 2
• Strasbourg - juin-2013 •
Communications
même avec l’ARN
DOI: 10.1002/anie.201207128
1.!Fauster, K., Kreutz, C. & Micura, R. 2′SCF 3Uridine-A Powerful Label for
Probing Structure and Function of
RNA by 19F NMR Spectroscopy.
Angew. Chem. Int. Ed. 51, 13080–
13084 (2012).
bel for Probing Structure and
pectroscopy**
Micura*
or
sd
ly.
e
or
at
r,
e
e
n
al
e-
Figur
stem
(blue
(Prot
mixe
(red)
tein]
Figure to
3. Structure probing of a bistable RNA. A) Unmodified RNA;[17]
Figure 1. New concept for fluorine labeling of RNA with respect
19
secondary structure model of full-length (5) and reference (5 a) RNA
F NMR spectroscopic applications.
adde
(left); imino proton NMR spectra (right). B) Same as (A), but for 2’repr
SCF3 labeled analogues. C) Assignment of folds 6’ and 6’’ of RNA 6 by
defin
19
NMR spectroscopy. D) Same as C, but in E. coli cell lysate.
molar range; less material is needed and potentialF aggregashift
Conditions for A–C: [RNA] = 0.3 mm, [Na2HAsO4] = 25 mm, pH 7.0,
tion problems are minimized. Moreover, the 2’-SCF
beha
3 group
H2O/D
O
=
9:1,
298
K;
conditions
for
D:
[RNA]
=
10
mm,
E.
coli
lysate/
2
resp
represents an isolated spin system, therefore proton
decouD2O = 9:1, 298 K (for lysate preparation, see the Supporting Informathat
pling (as, for example, required for 2’-F labels)
tion). is not
• Strasbourg - juin-2013inter
•
A
6
m.
h
h
r
Figure 1.
F spin-echo spectra recorded as a function of the HSA
m
19F spin-echo
each spectrum. The
data were
with an exponential
function ofas1 a func
Figure
2. multiplied
spectra recorded
fo
concentration. The CF3 resonance of the control molecule (2) is at +15.46
Hz before Fourier
transformation. The
The concentration
of of
thethe
twospy
molecules
concentration.
CF resonance
molecule (3)2
ppm, and the CF3 resonance of the spy molecule (1) is at +14.62was
ppm.
25 µM, whereas
the spectra),
concentration
HSA
from top to bottom,
(lower
andforthe
CFwas,
3 resonance of the control bm
The spectra were acquired with a total spin-echo period of 320 ms
with500, 700, and 900 nM. The signal intensity ratio I(1)/I(2) is, from
0, 300,
+15.46 ppm (upper spectra). The spectra were acquiredµ
topwith
to bottom, 0.86, 0.66, 0.38, 0.21, and 0.07.
an interval between the 180° pulses (2τ) of 40 ms. A total of 96 scans
echo period of 80 ms with an interval between the 180°0
a repetition time of 3.5 s and a spectral width of 25 ppm were acquired for
p
ms.
ATtotal
of
96
scans
were
recorded
for the lower spe
A
R
I
C
L
E
S
thiadiazol-2-yl]piperazine
(2)
recorded
with
proton
decoupling
each spectrum. The data were multiplied with an exponential function of 1
for the upper spectra with a repetition time of 3.5 s and a
during the acquisition period in the presence of different
Hz before Fourier transformation. The concentration of the two molecules
25 ppm. The data were multiplied with an exponentialF
was 25 µM, whereas the concentration for HSA was, from top to bottom,
concentrationsbefore
of HSA
are shown
in FigureThe
1. ITC
measure- of 3 and
c
Fourier
transformation.
concentration
nM
HSA
0, 300, 500, 700, and 900 nM. The signal intensity ratio I(1)/I(2) is,ments
from performed with 2 did not find any evidence of binding
µM, respectively, whereas the concentration for HSA wasth
top to bottom, 0.86, 0.66, 0.38, 0.21, and 0.07.
c
0, 150, 300, 450, and 600 nM. The signal intensity rat
im
plotted scale intensity is, from left to right, 0.94, 0.69, 0.5
thiadiazol-2-yl]piperazine (2) recorded with proton decoupling
d
e
during the acquisition period in the presence of different
For a weak affinity ligand the exchange term
concentrations of HSA are shown in Figure 1. ITC measurecontribute significantly to the line width of the ro
s
un example d’interaction
ments performed with 2 did not find any evidence of binding
the presence of the protein. The fluorine signal i
25 µM
coupled with several protons
and therefore
improvement it is necessary to record the spec
to HSA (only heat of dilution was detected with 8 µL injections
duringin acquisition.
2 shows
of 800 µM of decoupling
2 into 30 µM HSA)
agreement withFigure
the NMR
echo fluorine
forboth
themolecules
spy molecule
2-h
results. A concentration
of onlyspectra
25 µM for
was
used in the NMR
experiments.
Theand
lowcontrol
concentration
of the
robenzoic
acid (3)
molecule
(2) record
spy molecule avoids problems arising from nonspecific binding
and aggregation. Disadvantages with these molecules are
d
represented by the rapid rotation of the fluorine atoms about
w
the C3 axis of the group observed even in the bound state. This
th
results in a limited difference in line width for the CF3 signal
to HSA (only heat of dilution was detected with 8 µL injections
c
of the spy molecule between the free and bound state. However
of 800 µM of 2 into 30 µM HSA) in agreement with the NMR
the exchange contribution to the line width can be large.
results. A concentration of only 25 µM for both molecules was
o
Molecules with a CF Group. Molecules with a CF group are
fu
used in the NMR
experiments. The low concentration of
the
particularly
suited for the competition ligand based screening
19
F problems
spin-echo
spectra
recorded binding
asexperiments.
a function
of the HSA
F
spyFigure
molecule2.avoids
arising
from nonspecific
The 19F CSA can be very large therefore
increasing
50 µM
is
concentration.
The
CF
resonance
of
the
spy
molecule
(3)
is
at
-64.06
ppm
the
difference
in
line
width
between
the
free
and
bound state
and aggregation. Disadvantages with these molecules are
m
decoupling
as
a
function
of
HSA
concentratio
of
the
spy
molecule
according
to
eq
1.
For
example
the
CSA
(lower
spectra),
and
the
CF
resonance
of
the
control
molecule
(2)
is
at
3
represented by the rapid rotation of the fluorine atoms about
b
for
an
aromatic
CF
ranges
from
71
ppm
for
monofluoro-benzene
with
these
molecules
is
the
required
higher
co
ppm
spectra).even
Theinspectra
were
acquired
with a total spin- 34
the+15.46
C3 axis of
the (upper
group observed
the bound
state.
This
c
19F CSA
to 158 ppm forthe
hexafluoro-benzene.
Inspectra
addition,ofthe
experiments.
The
Figure
4
were
r
echoinperiod
of difference
80 ms with
an interval
the
180°
pulses
(2τ)
of
40
sp
results
a limited
in line
width forbetween
the CF3 signal
• Strasbourg
•
of the spy molecule in the bound state can increase
from- juin-2013
an
(
)
[EL]signal of a spy molecule
[EL] are similar in
s
of a fluorine or proton
emerging from Table 1 is the large number
Dfree
(3)
Dbound + 1 Dobs )
m
magnitude. The transverse
R2] of the fluorine
[LTOT] relaxation rate
[LTOT
containing molecules present in the MDDR librar
.
signal has an additional contribution originating from the large
logical search within this library demonstrates tha
19
29 of the spy
e
where
Dbound andofDthe
are
the
diffusion
coefficients
F
atom
that
is
given
by
CSA
interaction
free
20 years the percentage of compounds in developm
ARTI CLES
mxperimentsmolecule in the bound and free states, respectively. [EL]/[LTOT]
ing
at
least
one
fluorine
atom
has
doubled. A ste
2 fraction of bound and free ligand,
and (1 - [EL]/[LTOT]) are
the
ηCSA
a
1 with Measured
2 Binding
from
10.9% in the
1981-1985
period to 19.4% i
2
CSA
2 42and2Its Comparison
e-Point
NMR-Derived
Constant
Fluorescence
Value
respectively.
n
R2
) ∆σ 1 +
Bfor
γ
τ
+
0 F c
2 2
2000 period
is observed.
The fluorine atom
has been
15
3
3
2(1
+
ω
R
,
the
transverse
relaxation
rate
for
the
weak-affinity
[LTOT]
[ETOT]
[EL]/[LTOT]
[EL]
KDapp
KINMR
KIfluo
n KD
F τc ) spy
2,obs[I]
introduced in the process of lead optimization f
(1)
is
given
by
the
equation
s 44.3 molecule,
25
50
0.6
0.00159
0.080
326.8
3.9 ( 0.9
3.3 ( 0.3
potency,
physical-chemical
properties,
and metab
25
50
0.6
0.00171
0.086
300.4
3.6
(
0.8
s 37.7
where ∆σ is
the CSA of the 19F atom
against enzyme attack.
[EL]
[EL]and is given by ∆σ ) σzz
n
) σand
+ 1R2,free
+ K is theofbinding
RThe
fluo is the binding
2,obs
2,bound
-R(σ
different
σ’s
are
the
components
the constantInofthe
or
the
concentration
binding
constants
are
expressed
in
µM.
3
derived
from
ITC,
and
K
xx + [L
yy)/2.
D
I
selection
of
the
two
molecules
particula
]
[L
]
serived from fluorescence.
TOT
TOT
NMR
[LTOT],The
and [Easymmetry
of
4, 3, )
and HSA, respectively. The KI
is the binding constant for
TOT] are the concentration
chemical shift [I],
tensor.
parameter
η
CSA
be
given
to
their
solubility
since
theintensity
presence
of a
2
2
NMR is due to an estimated (5% error in the
24a,b
rhe NMR measurements as previously
24π
described.
The(δ
error
of
the
K
signal
ratio
δ
)
I
[EL]
[EL]
free
bound
(3/2)(σxx - σyy)/∆σ, and for1 an
increasesPhénomène
the lipophilicity
of a compound. Molec
- axially symmetric chemical shift
(4)
t
d’amplification
K-1 field, γF is
] strength
[LTOTof] the magnetic
the
tensor ηCSA ) 0. B[L
0 is
TOT
not very soluble in aqueous solution are not
h
the fluorine gyromagnetic ratio, ωF is the fluorine Larmor
screening experiments since they might bind in a
e
R2,free
are the time.
transverse relaxation rate
where R2,bound
is the
correlation
frequency,
and τcand
manner to the receptor. Therefore proton and flu
s
constants
for theperformed
ligand in the
bound and
free states,
respectively.
A simulation
assuming
an axially
symmetric
CSA
and proton WaterLOGSY spectra for the poten
The last term is the exchange term, where δbound and δfree are
,
tensor
and assuming an equal CSA for the free and bound state
control molecules are recorded in the absence of
;
the
isotropic
chemical
shifts
of
the
fluorine
resonance
of
the
.
of a ligand indicates that the difference in line width of the 19F
concentration typically 2-4 times higher (i.e., 10
spy molecule in the bound and free states, respectively and 1/K-1
signal of the spy molecule between the free and bound state
than the concentration used in the screening p
,
is
the
residence
time
of
the
ligand
bound
to
the
protein.
Equation
.
from just the CSA contribution alone can be very large.27 This
molecules that according to the NMR spectra are
4 is valid only when the experiments are performed with a long
,
difference
the ).size
of the receptor and with the
do not aggregate at these concentrations are c
2τ period increases
(where τ with
. 1/K
-1 Experiments recorded with τ <
square
the magnetic
fieldcontribution
strength. High
magnetic
fields
potential candidates for the spy and control molec
5/K-1 of
result
in a reduced
of the
exchange
termcan
to
,
lead
to extremely
broad relaxation
line widths
(>200
Hz) for fluorine
33
;
the screening.
the observed
transverse
rate.
;
signals
of either
macromolecules
(e.g.,
protein
selectively
A library of 19F-containing molecules that fulfi
Therefore
screening
is performed
by ausing
a long
2τ per;
28 Such line
h
F) or strongly
protein-bound
ligands.
labeled
withis19possible
iod. This
because
the evolution
under the
heterodescribed above has been generated in our labor
1H-the
19F direct
widths
fluorine resonances
nuclearmake
scalar detection
couplings of
is refocused
at the end of
of the
the
molecules are tested in mixtures against the r
.
macromolecule
or high-affinity
scheme. However,
the 2τ periodligands
should impractical
not be very for
longthe
in
WaterLOGSY for the identification of a potential s
.
purposes
of screening.
contrast, the
strong
magnetic
fields
order
minimize
signalInattenuation
originating
from the spatial
.ction limits
Other direct methods with 1H or 19F detection can
of to
FAXS.
Experiments
performed with
the
• Strasbourg - juin-2013 •
are particularly well-suited for competition binding experiments
(
relaxation
)
{
(
(
)
)
}
Principe de la méthode FAXS (Fluorine chemical
shift Anisotropy and eXchange for Screening)
Screening par compétition entre une molécule dite “espion” et un mélange de molécules
candidates.
Utilisation d’une molécule de contrôle
Observation des variations d’intensités des raies de la molécule espion et de la molécule de
contrôle.
1.! Dalvit, C., Fagerness, P., Hadden, D., Sarver, R. & Stockman, B. Fluorine-NMR experiments for high-throughput screening:
theoretical aspects, practical considerations, and range of applicability. J Am Chem Soc 125, 7696–7703 (2003).
• Strasbourg - juin-2013 •
Principe de la méthode FAXS
Fluorine chemical shift Anisotropy and eXchange for Screening
19F
τ
[
τ]
n
1H
τres = 1/koff
R
α
= [EL]
/[ LSpectroscopy
T]
ogress in Nuclear Magnetic
Resonance
51 (2007) 243–2712,app
2τ≫τres
1.0
T2 = 1.2s
Spy Molecule Signal Intensity
ine atom or a COOH
to the fluorine atom
the component of the
to the aromatic ring
This ortho effect (see
arge contribution of
e simulations previor the observed high
e to perform FAXS
ng a protein concensize of the target pro! 25 kDa) (data not
the biomolecular tarenically cooled probe
tion 6.2, and/or with
to the square depen-
= αR2,lié + (1-α)R
2,libre +Réch
251
0.8
Difference
0.6
0.4
0.2
T2 = 0.3s
0.0
0.0
0.5
1.0
CPMG Filter (s)
1.5
2.0
• Strasbourg - juin-2013 •
Avantages de la méthode FAXS
• Seule la molécule espion est observée
• Les molécules criblées ne portent pas nécessairement
d’atome de fluor
• Absence de superposition de raies : possibilité de cribler un
grand nombre de molécules à la fois
• Le fluor a un CSA très important : influence très significative
sur la largeur des raies
• Strasbourg - juin-2013 •
Autres techniques...
SAR
STD
Spin Labelling
Diffusion
Editing
Inverse NOE
Pumping
Water-LOGSY
FAXS
Large Potein
(> 30 kDa)
limited
yes
yes
no
yes
yes
yes
Small Protein
(<10 kDa)
yes
no
yes
yes
no
no
yes
labeled
protein
yes
no
no
no
no
no
no
protein (nmol)
25
0.1
1
100
25
25
1-10
KD tight
no limit
100pM
100pM
100nM
1nM
100pM
100pM
KD weak
1mM
10mM
10mM
1mM
1mM
10mM
10mM
deconvolution
no
yes
yes
yes
yes
yes
yes
• Strasbourg - juin-2013 •
premier example
• Hexokinase HIV
• enzyme impliquée dans l’homeostasie du glucose
• forte coopérativité
• molécules «activateurs»
Servier
J. Boutin
G. Ferry
M. Antoine
NMRTEC
O. Assemat
J-P Starck
• Enzyme
• système dynamique
• buffer complexe
• Strasbourg - juin-2013 •
super-open inactive form
slow exchange
closed active form
glucose
binding pocket
• Strasbourg - juin-2013 •
GK
E* + glucose
k-1
glucose < 5mM
k1
E*.glucose
k-2
k2
E.glucose
k3
glucose > 8mM
+ATP
k-3
E + glucose
E.glucose.ATP
G6P+ATP
E.G6P.ADP
Étude par fast-flow fluorescence
5470
Biochemistry, Vol. 48, No. 23, 2009
Biochemistry, Vol. 48, No. 23, 2009 5469
KSO and GKO are the low- and high-affinity conformers described
GKA dependence of kobs4 GKA and GKA glucokinase activator:
del
ound A or LY2121260.
to the cyclic four-step preexisting equilibrium model
ibed by Scheme 4 and eq 8.
kobs ¼
k3 Glc þ ½Glc$ KDk2GlcGlcSO
1 þ KD½Glc$
Glc SO
bed
tor:
þ
k -3 Glc þ ½Glc$ KkD-2GlcGlcO
1 þ K½Glc$
D Glc O
kobs ¼ kon ½GKA$ þ koff
! Antoine, M., Boutin, J. A. & Ferry, G.
Binding Kinetics of Glucose and Allosteric Activators to Human
Glucokinase Reveal Multiple Conformational States.
Biochemistry 48, 5466–5482 (2009).
ð6Þ
ð7Þ
del
¼
k3 GKA þ ½GKA$ KkD12 GKA
GKA
ð6Þ
ð7Þ
1
þ K½GKA$
D1 GKA
þ
GKA
k-3 GKA þ ½GKA$ Kk-2
D2 GKA
1 þ K½GKA$
D2 GKA
ð8Þ
netic Simulations. Demo version of Berkeley Madonna
KinTek Explorer were used for modeling and testing of
ent kinetic mechanisms for ligand binding to GK.
ð8Þ
ULTS
eady-State Kinetics and Equilibrium Glucose Binding.
nna
recombinant
human GK used in this study is the panof
c isoform, deleted from its 11 first residues, His-tagged
s N-terminus, and purified to homogeneity. Steady-state
ic behavior of the His-tagged truncated GK was deterdng.for glucose
ATP. GK glucose-induced
showed positive
cooperativity
FIGUREand
1: Time-resolved
conformational
change
by GK
intrinsic=
fluorescence
(λexcwhile
= 295hyperbolic
nm). RepresenanK0.5
8.5 mM)
glucose monitored
(nH = 1.8,
Glc
FIGURE 2: Dependence of kobs1 Glc, kobs2 Glc, kobs3 Glc, and kobs4 Glc on glucose conc
parameters k3 Glc = 0.13 s-1, k-3 Glc = 0.63 s-1, KD Glc SO = 30 mM, k2 Glc = 0.8
semilogarithmic scale for kobs3 Glc.
and the four exponential fit were 560 and 94, respectively, clearly
conforme
indicating
that
the
four
exponential
fit
model
is
largely
preferred.
(GKSO) a
FIGURE 1: Time-resolved glucose-induced conformational change
et al. (12)
Using
high nm).
glucose
concentration, the third phase accounted
monitored by GK intrinsic fluorescence (λexc
= 295
Represensilent, the
for two-thirds of the total amplitude, while the first, second, and
tative fluorescence transient recorded after
rapid mixing of apo-GK
isomeriza
fourth phase represented each 5-10% of the total amplitude.
(1 μM) and glucose (50 mM) in buffer A. The
upper frame shows the
binding o
Thus, only the third phase was observable for the lowest glucose
recorded transient; lower frames show theconcentrations
residuals forused.
the best
fit
to
(k3 Glc + k
When plotted against glucose concentraone, two, three, and four exponentials. tion, total fluorescence intensity yielded an hyperbolic trend,
route A. T
glucose to
readily fitted to eq 2 and led to an apparent KDapp Glc value of
4.3 mM,
in agreement
with the microplate equilibrium
data- juin-2013
unimolecu
• Strasbourg
•
Pre-Steady-State Glucose Binding
Kinetics.
Pre-steady-
Premier «espion»
MWI119
• Strasbourg - juin-2013 •
Deuxième «espion»
O
O
O
O
OH
N
H
H
N
O
F
Human pancreatic glucokinase was diluted to a concentration of 3.85 µM in a 40 mM Hepes buffer containing 35 mM KCl, 1.8 mM
MgCl2, 72 mM glucose, 1.4 mM TCEP, 4.5 % glycerol.
Spy and screened molecules were dissolved in DMSO, and added to the protein solution so that final experimental conditions were
protein 3.7 µM, spy molecule 8 µM, 3-fluorophenol (used as en internal calibration) 8 µM, screened molecule 30 µM, 10% DMSO
• Strasbourg - juin-2013 •
Optimisation
Simulation
[L] : 250µM
Kd : 50µM
T2(cpx) : 30msec
First a series of T2 measurements were
carried out to validate the approach, and
to find the best conditions. Experimental
T2 are shown on the side, along with
simulated T2 curves, showing a good
agreement between results and
simulation
The best conditions were then chosen.
Simulation
[GK] : 6µM
Kd : 50µM
T2(cpx) : 30msec
Simulation
[GK] : 20µM
Kd : 50µM
T2(cpx) : 30msec
• Strasbourg - juin-2013 •
Molécules testées
O
Chiral
CH3
Chiral
O
O
H3C
H3C
O
N
N
N
O
H
O
H3C
H
O
H3C
O
H3C
H
N
H
N
H
N
N
H
H3 C
CH3
O
H
O
44276
O
CH3
N
O
CH3
S
O
O
H3C
Cl
46183
45077
Chiral
O
O
CH3
Chiral
O H3C
O
AKY5
H
O
H
N
H3C
N
H
N
N
H
H3C
CH3
N
H
S
H
O
H 3C
diastereoisomere 2 racemique
OH
N
H
46267
H
N
H
N
H
N
O
Chiral
44520
45132
H
O
O
H3C
O
O
N
H
N
H
N
N
S
CH3
N
H3C
Cl
H
O
CH3
S
O
O
O
N
O
SGN74
OH
O
H
N
H3C
45077
O
CH3
S
CH3
O
CH3
S
AKY5
H3C
H 3C
O
O
Cl
O
N
CH3
O
H
S
O
N
H
N
O
O
O
O
H3C
diastereoisomere 1 racemique
H3C
O
CH3
O
OS
O
O
N
O
N
OH
H 3C
OH
O
CH3
O
diastereoisomere
1 racemique
O
O
O
46267
AKY5
45077
O
S
CH3
O
Chiral
SGN
• Strasbourg - juin-2013 •
Mesure
30 = Io /ICPMG30ms
ß30 value
• à 600 MHz sur une
sonde cryo-19F
Test enzymatique
• Strasbourg - juin-2013 •
• Strasbourg - juin-2013 •
Deuxième example
• Sa Majesté le Ribosome 70S
Thermus thermophilus
IGBMC
M. Yusupov
N. Garreau de Loubresse
B. Kieffer
R. Recht
• Strasbourg - juin-2013 •
Site adapteur du tRNA
• Strasbourg - juin-2013 •
Ribosome 5mg/ml
• Strasbourg - juin-2013 •
Résultats préliminaires
cinetic
wait 120h!
lb4 ESBS4 21 (19F)
lb4 ESBS4 21 (19F)
lb4 ESBS4 41 (19F)
lb4 ESBS4 41 (19F)
lb4 ESBS4 61 (19F)
lb4 ESBS4 61 (19F)
lb4 ESBS4 21 (19F)
35
lb4 ESBS4 41 (19F)
lb4 ESBS4 61 (19F)
60
lb4 ESBS4 101 (19F)
lb4 ESBS4 126 (19F)
lb4 ESBS4 101 (19F)
lb4 ESBS4 101 (19F)
lb4 ESBS4 126 (19F)
lb4 ESBS4 126 (19F)
lb4 ESBS4 130 (19F)
lb4 ESBS4 130 (19F)
30
lb4 ESBS4 130 (19F)
25
70
50
80
90
100
Shigemi'230'µl'
298K'
512'scans'(~20min)'
'
600'MHz'+'
Cryoprobe'
70
40
No
Ribosome!
+20µl
Lincomycin! cine%c&wait&
17h&
[1 mM]!
+20 µl
Ribosome!
[1,4 µM]!
+20 µl
Ribosome!
[0,7 µM]!
0
0
0
10
10
5
20
10
20
30
40
15
30
50
20
40
60
'
ppm
-74,8
-75
-75,2
-75,4
-75,6
-75,8
-76
-76,2
-76,4
-76,6
-76,8
ppm -77
-110,2
-77,2
-110,4
-77,4
-110,6
-77,6-110,8
-77,8 -111
-111,2
24)µM)
.112.4)ppm)
-111,4
-111,6
-111,8
Linezolid)
%
24)µM)
).76.6)ppm))
3FP)
%
%
TFE)
ppm
-112
-112,2
-112,4
-112,6
-119,2
-112,8
-113
-119,4
-113,2
-119,6
-119,8
12)µM)
.120.8)ppm)
-120
-120,2
-120,4
-120,6
-120,8
-121
-121,2
-121,4
-121,6
-121,8
-122
-122,2
• Strasbourg - juin-2013 •
Remerciements
• GK
• Servier
‣ Jean Boutin
‣ Gilles Ferry
‣ Matthias Antoine
• NMRTEC
‣ Olivier Assemat
‣ Jean-Philippe Starck
• Ribosome
• équipe Ribosome
‣ Marat Yusupov
‣ Nicolas Garreau de Loubresse
• équipe RMN
‣ Bruno Kieffer
‣ Raphael Recht
• Conectus
• Strasbourg - juin-2013 •
Remerciements
• GK
• Servier
‣ Jean Boutin
‣ Gilles Ferry
‣ Matthias Antoine
• NMRTEC
‣ Olivier Assemat
‣ Jean-Philippe Starck
• Ribosome
• équipe Ribosome
‣ Marat Yusupov
‣ Nicolas Garreau de Loubresse
• équipe RMN
‣ Bruno Kieffer
‣ Raphael Recht
• Conectus
• Strasbourg - juin-2013 •