Transcript Kristoffer Brännström

Enthalpic Forces Correlate with
the Selectivity of TransthyretinStabilizing Ligands in Human
Plasma.
Kristoffer Brännström
Umeå University
Umeå
Structure and function of
human transthyretin
• is mainly produced in liver and choroid plexus
• transport of T4
Thyroxine Binding Site (TBS) together with T4
Structure and function of
human transthyretin
• transport of T4 and holo-retinol binding protein
Transthyretin = a transporter of thyroxine
and retinol binding protein
Pathological side of TTR
Pathological side of TTR
ATTRwt
FAP
ATTR –TTR-related amyloidosis:
ATTRwt – wild-type TTR amyloidosis
FAP - Familial amyloid polyneuropathy
Therapeutic strategies for TTR
amyloidosis
Kinetic stabilisation
Aim of study
• to select and evaluate new
TTR tetramer stabilizers
• to elucidate the properties
that influence efficacy of
ligands to stabilize TTR
tetramer in human plasma
Selection of new TTR kinetic stabilizers
• Drug development is a highly expensive and timeconsuming process
• A shortcut is to repurpose already FDA-approved
drugs
1200 FDA-approved drugs
Prestwick Chemicals
Primary screen with turbidimetry
new
new
new
The drug must be selective…
TTR
Assay in plasma - specificity
test for TTR-binders
1. Incubate drug
in plasma (~5 µM
TTR) 1 h.
2. Add 4 M
urea,
incubate ON.
Binding:
stable
tetramer.
No binding:
dissociation of
tetramer and
unfolding.
Native like gel
Separates monomer
from tetramers
Monomer
13
Determination of Binding
affinity (KD) with ITC
Luteolin
Luteolin
KD = 70nM
ΔH = -11.2 kcal/mol
ΔS = -1.4 cal/mol/deg
binding
affinity
to TTR
(KD), nM
IC50
plasma,
μM
enthalpy
(ΔH),
kcal
entropy
(ΔS),
kcal 25
°C
free
energy
(ΔG),
kcal/mol
(25 °C)
LUT
API
DIF
TOA
MEA
NIF
DIC
ACL
GEM
70
250
580
150
480
260
1000
1200
100
7±3
18 ±3
25 ± 12
40 ± 11
50 ± 4
130 ± 40
140 ± 38
170 ± 39
180 ± 27
–11.2
–9.6
–7.8
–9.4
–6.8
–7.0
–3.8
–6.6
–1.0
–1.4
–0.6
0.7
–0.1
1.8
2
4.3
1.4
8.6
–9.8
–9.0
–8.5
–9.3
–8.6
–9.0
–8.1
–8.0
–9.6
Binding affinity (KD) vs Selectivity
Binding Affinity (KD) is a poor predictor
of Selectivity
Enthalpy (△H) correlates better with
selectivity
Conclusions
• Plasma components strongly affect the level of
nonspecific binding and efficacy of substances
• Binding affinity (KD) cannot exclusively be used as
parameter to predict specificity
• Enthalpy (△H) correlates better with selectivity
Acknowledgements
•
•
•
•
•
•
Anders Olofsson
Irina Iakovleva
Lina Nilsson
Malin Walfridsson
Afshan Begum
Elisabeth Sauer-Eriksson
Therapeutic strategies for TTR
amyloidosis