Download NMR to characterise protein-ligand interaction

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NMR to characterise protein-ligand
interaction
Applications in drug discovery
Rational drug design – the concept
• Traditional drug design …
Observation (1928)
•
Drug (1942)
Target (1965)
Rational drug design ….
N
N
N
N
N
O
N
N
Identify potential attack point
target
drug
What makes a good drug?
siRNA strategies
(protein)
target
antibodies
Small molecule inhibition
Ideally a good (oral) small molecule drug is “rule of 5”
compliant ….
… MW < 500
… ClogP < 5
larger molecules diffuse more slowly
water/octanol partition coefficient
small logP means: soluble in octanol AND water
… < 5 H bond donors
… < 10 H bond acceptors
… < 5 rotatable bonds
restricts degrees of conformational freedom
Drug discovery
• Bringing a new medicine to market is a long, complex,
expensive, risky and difficult process
• It takes on average over 10 years and costs around $1-5bn to
bring a new medicine to market
NMR
> 10 years
• Most projects fail: Of every 100 drug research projects
started, only 1 will make it as a drug
What options do we have?
Protein observe methods
define binding sites
Ligand observe methods
find ligands in mixtures
not-binder
binder
116
117
118
STD
119
120
121
1D
122
123
124
10.4
10.0
9.6
9.2
• identifies binding sites
• needs larger amounts of protein
• protein size limitation
• requires isotopic labelling (15N or 13C)
9.0
8.8
8.6
8.4
8.2
8.0
• no labelling
• minimal amounts of protein
• no protein size limitation
7.8
7.6
ppm
Ligand binding equilibrium
+
N
N
E+L
Association constant
Dissociation constant
NB
KA=
KD=
EL
[EL]
[E] [L]
[E] [L]
[EL]
(hardly ever used)
pKD=-log KD
KD
mM
µM
nM
pM
pKD
3
6
9
12
Enzymatic tests often use IC50 (50% inhibition)
Conversion through Cheng-Prusoff eqn: K i =
IC50
1+ K[ S ]
M
Affinity ranges
KD
mM
pKD 3
µM
6
nM
9
pM
12
Typical biochemical interactions
Marketed drugs
Fragments
Practical detection limit
Detection range ligand-observe NMR
Detection range protein-observe NMR
How can we describe the KD?
… thermodynamically
.. by the mass law
[E] [L]
KD=
[EL]
∆GD=∆HD+∆SD=-RTlnKD
KD
… kinetically
Off rate
KD= On rate
The thermodynamic description
∆GD=∆HD+T∆SD=-RTlnKD
dissociation
1 mM
1 µM
18 kJ/mol @ 37C
36 kJ/mol
1 nM
53 kJ/mol
binding
40 kJ/mol
Loss of translational entropy
40 kJ/mol
Loss of rotational
entropy
6 kJ/mol per
uncharged H bond
19 kJ/mol per
charged H bond
110 J/mol Å2
Hydrophobic
surface
Concept of fragments ….
A weakly binding fragment is
actually doing a pretty good
job …
nM
µM
mM
… a few additional interactions
might turn it into a proper drug!
nM
µM
mM
Pocket properties must match …
… not too big, not too small
… shape complementary to small molecules (not too shallow - “convexity”)
… availability of H bond donors/acceptors
Concept of druggability
1. binding of a small molecule requires
certain properties of the binding
pocket
2. if such a pocket is available, we have
a high chance to find fragments
indeed many fragments are found
to bind to “natural” binding sites
3. if we find no fragments, there is
probably no such pocket
4. Without a suitable “small molecule”
pocket this target might not bind to
small molecules
From: Hajduk et al., J.
Med. Chem. (2005) 48,
2518
NMR fragment screening hitrate is predictive for
success in small molecule approaches!
The mass law description
[E] [L]
KD=
[EL]
[ EL]
fb =
[ E ]tot
Relevant for protein
observe NMR
Fraction bound
[ EL]
fb =
[ L]tot
Relevant for ligand
observe NMR
([ E ]tot + [ L]tot + K D ) 2 − 4[ E ]tot [ L]tot
[ E ]tot + [ L]tot + K D
−
[ EL] =
2
2
• fraction bound, KD and concentrations are linked
• optimal experimental concentration ranges depend on
• which KD range?
• ligand observe or protein observe?
• achievable concentrations for
ligand (solubility? DMSO conc?)
Protein (supply? Stability? Solubility?)
Fraction bound (protein) depending on [L] and KD
100.00%
80.00%
100 µM protein (typical concentrations)
problematic ….
… with 100 mM stocks it means 5%
DMSO in solution
… many ligands are not that soluble
60.00%
40.00%
20.00%
KD=0.05 mM
KD=0.5 mM
0.00%
0.01 0.05
mM mM
0.1
mM
0.5
mM
[L]
Practical detection limit for a
binder ~20%
KD=5 mM
1 mM 5 mM
More realistic maximal ligand
concentration
Millimolar binders will be difficult to detect and characterise
Difficult to achieve full titration curve
(and get KD)
100.00%
90.00%
80.00%
70.00%
60.00%
50.00%
40.00%
30.00%
20.00%
10.00%
0.00%
0.01 0.05
mM mM
[L]
KD=0.05 mM
KD=0.5 mM
0.1
mM
0.5
mM
KD=5 mM
1 mM 5 mM
A KD=5 mM ligand will only just
be visible @ 1 mM ligand/0.1
mM protein
Tight binders will quickly be saturated
Quickly saturated ….
100.00%
80.00%
60.00%
40.00%
20.00%
KD=0.05 mM
0.00%
0.01 0.05
mM mM
[L]
KD=0.5 mM
0.1
mM
0.5
mM
KD=5 mM
1 mM 5 mM
Very tight binders follow a step function
Increasing saturation only monitors [L]
100.00%
90.00%
80.00%
70.00%
60.00%
50.00%
40.00%
30.00%
20.00%
10.00%
0.00%
0.01 0.05
mM mM
[L]
KD=5 nM
KD=0.5 mM
0.1
mM
0.5
mM
KD=5 mM
1 mM 5 mM
Typical concentrations in practice
Protein observe - screening
•
[E] > 50 µM
Protein observe - titration
•
• Practical detection limit for
reasonable throughput
• Solubility? Availability?
•
[L] = 1 mM
• Titration might have to be
repeated with a more suitable [E]
•
Lowest [L] should be ½ [E]
•
Concentrations should give good
coverage at minimum no. of points –
‘rule of 3’
• Safely detects mM binders
Ligand observe
•
[L] > 100 µM
• Practical detection limit for
reasonable throughput
•
[E] ~ 1 …. 5 µM
• Typically [L]: [E]=20 ..50:1
• Depends on size and method
used
[E] ~ KD
• e.g. 50 – 150 – 450 – 1350 µM
•
Protein should reach >80%
occupancy at highest [L]
•
CHECK THE CONCENTRATIONS
(i.e. run a 1D NMR – many
compounds are not very soluble!)
The kinetic description
Off rate
KD=
On rate
In many practical
cases (at least for
fragments …) the on
rate will be diffusion
controlled
For molecules of
similar structure and
binding mode:
KD ∝ off rate
Always the slower step (otherwise
there would not be binding)
Slow exchange
(often observed
for tight binders)
Fast exchange
(often observed for
weaker binders)
Kinetics of ligand binding: protein observe NMR
Fast exchange
In true fast
exchange ∆δ is
a measure of
fraction bound
200 µM protein
+100 µM ligand (1:2)
+300 µM ligand (3:2)
+900 µM ligand (9:2)
+2700 µM ligand (27:2)
Fit uses parameters
KD and δ(complex)
Kinetics of ligand binding: protein observe NMR
Slow exchange
Slow exchange
∆δ>koff
Rel. occupancy
determined by fB
Classification as “fast”,
“intermediate” or “slow”
is ‘per resonance’, not
‘per system’!
Intermediate exchange
∆δ~koff
200 µM protein
+100 µM ligand (1:2)
+300 µM ligand (3:2)
+900 µM ligand (9:2)
Complex lineshape
Kinetics of ligand binding: ligand-observe NMR
In fast exchange all observed
parameters are weighted
averages:
δav = fB δbound + (1-fB)δfree
T1 av= fB Τ1 bound + (1-fB)Τ1 free
T2 av= fB Τ2 bound + (1-fB)Τ2 free
Much shorter than T2 free
Influences lineshape even
for small fB’s (<5%)
Effect can be experimentally
increased through T1ρ filter
[Protein]
Ligand fB
Kinetics of ligand binding: ligand-observe NMR
In fast exchange all observed
parameters are weighted
averages:
Transferred NOE
δav = fB δbound + (1-fB)δfree
T1 av= fB Τ1 bound + (1-fB)Τ1 free
T2 av= fB Τ2 bound + (1-fB)Τ2 free
NOE av= fB ΝΟΕ bound + (1-fB)NOE free
large and -ive
-prot
+prot
WaterLOGSY
+prot
small and +ive
-prot
Logistics
4
1 Compound library on
384-well plates
• stock solutions in
DMSO-d6
• the more concentrated
the better – but there is a
limit!
• QC control of screening
libraries?
2 protein
• need to plan protein
supply before screening
• how much do I need?
(100s of mgs for protein
observe screening)
• stability? Can it be
recycled?
• basic project
management skills are
useful
3
Robotic
sample
preparation
5
Automatic acquisition
Summary
• Rational drug design usually means optimising an interaction between a
small molecule and a protein target
• NMR can support rational design by characterising this interaction
• In order to do so we need to understand the mass law and its
implications
• Just like in any other biophysical technique!
• Choose the right concentrations depending on KD
• Choose the right method depending on the exchange regime
• Don’t underestimate the logistical aspect!
• While NMR is capable of delivering structures, crystallography is
usually the preferred method
• Localising binding sites is easy though!
• We might get binding modes from more sophisticated experiments
Structural information from NMR
• protein-ligand structures yield binding modes - very valuable to
understand ligand SAR (structure-activity relationship)
• crystallography is the preferred option
• a soakable system can yield structures in high throughput (and
even be used for screening)
Develop
crystal
system
Apo
structure
develop
soakable
system
complex
structure
• NMR structures take long time ….. (apo and even more so as a
complex)
Structural information from NMR
CSPs can map binding sites onto a crystal structure ….
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N
S
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N
ME
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assigned
unassigned
CSP
This information comes “for free” with all protein observe methods
… but - how does the ligand bind?
Kinetics of ligand binding: ligand-observe NMR
• No need to label protein
• Can use protein from natural source or insect cell expression
• Minimal protein requirements
• At least one order of magnitude less than protein observe
• ligand observe methods rely on fast exchange
• Cannot find tight or covalent binders!!
What if I want to characterise these – but cannot label the target?
Ligand observe
reporter screen
Reporter ligand (known affinity)
Test ligand (affinity to be determined)
Ligand observe reporter screen
Dalvit et al
JACS 2002, 124, 7702-7709
Reporter ligand (known affinity)
Test ligand (affinity to be determined)
[Protein]
Ligand fB
“calibration”
Competition measurement
KI=47 µM
(KD=41 µM)