1. Kristoffer Brännström
Enthalpic Forces Correlate with the Selectivity of Transthyretin-Stabilizing Ligands in Human Plasma.
Kristoffer Brännström

2. Umeå University
Umeå

3. Structure and function of human transthyretin
is mainly produced in liver and choroid plexus
transport of T4
Thyroxine Binding Site (TBS) together with T4

4. Structure and function of human transthyretin
transport of T4 and holo-retinol binding protein
Transthyretin = a transporter of thyroxine
and retinol binding protein

5. Pathological side of TTR
TTR is also belong to famaly of proteins denoted amyloid
We dont know what exactly triggers them to aggregates There are several factors that was suggest to underlay the aggregation

6. Pathological side of TTR
ATTRwt
FAP
TTR is also belong to famaly of proteins denoted amyloid
We dont know what exactly triggers them to aggregates There are several factors that was suggest to underlay the aggregation
ATTR –TTR-related amyloidosis:
ATTRwt – wild-type TTR amyloidosis
FAP - Familial amyloid polyneuropathy

7. Therapeutic strategies for TTR amyloidosis
Kinetic stabilisation

8. 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

9. Selection of new TTR kinetic stabilizers
Drug development is a highly expensive and time-consuming process
A shortcut is to repurpose already FDA-approved drugs
Prestwick Chemicals
1200 FDA-approved drugs

10. Primary screen with turbidimetry
new
new
new

11. The drug must be selective…
TTR

12. Assay in plasma - specificity test for TTR-binders
Binding: stable tetramer.
2. Add 4 M urea, incubate ON.
1. Incubate drug in plasma (~5 µM TTR) 1 h.
No binding: dissociation of tetramer and unfolding.

13. Native like gel
Separates monomer from tetramers
Monomer

14. Determination of Binding
affinity (KD) with ITC

15. Luteolin
Luteolin
KD = 70nM
ΔH = kcal/mol
ΔS = -1.4 cal/mol/deg

16. Binding affinity (KD) vs Selectivity
LUT
API
DIF
TOA
MEA
NIF
DIC
ACL
GEM
binding affinity to TTR (KD), nM 70
250
580
150
480
260
1000
1200
100
IC50 plasma, μM
7 ± 3
18 ±3
25 ± 12
40 ± 11
50 ± 4
130 ± 40
140 ± 38
170 ± 39
180 ± 27
enthalpy (ΔH), kcal
–11.2
–9.6
–7.8
–9.4
–6.8
–7.0
–3.8
–6.6
–1.0
entropy (ΔS), kcal 25 °C
–1.4
–0.6
0.7
–0.1
1.8 2
4.3
1.4
8.6
free energy (ΔG), kcal/mol (25 °C)
–9.8
–9.0
–8.5
–9.3
–8.6
–8.1
–8.0
And in order to estimate the efficasy of the drugs in plasma we develop in our group assay there we could to estimate the Ic50 for each ligand
Binding affinity (KD) vs Selectivity

17. Binding Affinity (KD) is a poor predictor of Selectivity

18. Enthalpy (△H) correlates better with selectivity

19. 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

20. Acknowledgements Anders Olofsson Irina Iakovleva Lina Nilsson
Malin Walfridsson
Afshan Begum
Elisabeth Sauer-Eriksson

22. Therapeutic strategies for TTR amyloidosis