What have we learned from the enthalpy database of 200 ... · What have we learned from the enthalpy database of 200 compound binding to 8 carbonic anhydrase isoforms as determined

What have we learned from the enthalpy database of200 compound binding to 8 carbonic anhydrase isoforms

as determined by ITC

Daumantas Matulis

Department of Biothermodynamics and Drug DesignInstitute of Biotechnology, Vilnius University

September 26, 2016

Daumantas Matulis (Vilnius University) 2nd Annual European MicroCal Meeting September 26, 2016 1 / 42

Outline

Protein - ligand interactions: Thermodynamics and Kinetics(Fluorescent thermal shift assay, Isothermal titration calorimetry,Enzymatic inhibition assays, Surface plasmon resonance)

Intrinsic - observed binding parameters

Structure - binding thermodynamics correlations (compound chemicalstructure - binding affinity (close to SAR), compound chemicalstructure - binding enthalpy, X-ray crystal structure ofprotein-compound complex - binding ∆G , ∆H, T∆S , ∆Cp, ∆V , andother


Protein - compound binding


Protein – Ligand binding

Every component in water!Daumantas Matulis (Vilnius University) 2nd Annual European MicroCal Meeting September 26, 2016 4 / 42

Protein – Ligand binding


What types of energy can we measure for binding?

VT

A

PT

G

TP

G

PT

S

PP

S

PT

V

T

CP

TP P

S

T

V

A

G

S

V

Y

U

2/T

T

TG

P

2TT

Y

P

P

T

Y

/1 PT

H

P

P

T

C

P

P

T

C

H PC

2/T

T

TA

P

VT

U

VCP

V

T

C

P

V

T

C

Free energy 1st derivative 2nd derivative 3rd derivative

TP

V


Protein - compound binding assays


Fluorescent thermal shift assay (ThermoFluor®, FTSA)


Fluorescent thermal shift assay (ThermoFluor®, FTSA)


Strong vs Weak Ligand Dosing Curves


Wide range of affinities by FTSA


Advantage of TSA over ITC: no limit in affinity

ITC experiment: FTSA experiment:


Inhibition of CA XII enzymatic activity


Comparison of IC50 with Kd


CA XIV char. by ITC using strongly-binding dorzolamide)


CA XIV characterization by TF at various pH


CA XIV inhibitor dosing curves by SFA


Determination of CA XIV active protein fraction 96 ± 5 %of 500 nM


Intrinsic vs observed binding thermodynamics


Intrinsic binding enthalpy of VD12-05 to hCA II


General scheme of Intrinsic parameter determination bysubtracting protonation-linked effects from observedparameters


Observed ∆H dependence on buffer and pH

Ethoxzolamide binding to hCAXII

Jogaite et al., Bioorg. & Med. Chem. 21 (2013) 1431–6


Observed ∆Hobs as a function of pH and buffer

Ethoxzolamide binding to hCAXIII

Baranauskiene and Matulis BMC Biophysics 5:12 (2012)Daumantas Matulis (Vilnius University) 2nd Annual European MicroCal Meeting September 26, 2016 23 / 42

Deprotonation enthalpy of inhibitor

RSO2NH– + H+ ↔ RSO2NH2

∆H = −6.2 kcal/mol


Inhibitor pKa by spectrophotometry

pKa = 9.4 ± 0.2

O

SNH2

S

O

O N

N

6 7 8 9 10 11 12pH

A(267nm)/A(247nm) 1.0

0.0

0.5

pKa = 8.9 ± 0.2

O

SNH2

S

ClO

O N

N4b 3b

240 250 260 270 280 2900.0

0.2

0.4

0.6

0.8

1.0

Abs

orpt

ion

Wavelength,,nm

,pH,7.0,,pH,7.5,pH,8.0,pH,8.5,pH,9.0,pH,9.5,pH,10.0,pH,10.5,pH,11.0,pH,11.5,pH,12.0

247 267

Kisonaite et al. Plos One 9(12): e114106 (2014)


Intrinsic ∆G of EZA binding to CA XIV


Observed vs intrinsic affinity to CA I

4e 3e1

10

100

1000

10000

1.5 k

SO2NH2

F

F

S

F

F

SO2NH2

SKd, p

M stebimoji K

d (pH 7.0)

tikroji Kd

150 k

Grey - observed, red - intrinsic affinity. Fluorine does not affect the (intrinsic)

affinityDaumantas Matulis (Vilnius University) 2nd Annual European MicroCal Meeting September 26, 2016 27 / 42

ITC of all 12 human CA isoforms

δH (k

J/mol

)-80

-60

-40

-20

0

Pow

er (

μJ/

s)

14.4

14.6

14.8

15.0

15.2

Time (min)0 20 40 60 80 100

Pow

er (

μJ/s

)

14.2

14.4

14.6

14.8

15.0

15.2

Time (min)0 20 40 60 80

Pow

er (

μJ/s

)

14.8

15.0

15.2

15.4

15.6

15.8

Time (min)0 20 40 60 80

Pow

er (

μJ/

s)

14.5

15.0

15.5

Time (min)0 20 40 60 80 100

Pow

er (

μJ/s

)4.5

5.0

5.5

Time (min)0 20 40 60 80

Pow

er (

μJ/s

)

13.2

13.4

13.6

13.8

14.0

Time (min)0 20 40 60 80 100

Molar ratio

0 0.5 1 1.5 2 2.5

Pow

er (

μJ/s

)

5.5

6.0

6.5

7.0

Time (min)0 20 40 60 80 100

Pow

er (

μJ/

s)

20.8

21.0

21.2

21.4

21.6

Time (min)0 20 40 60

δH (k

J/mol

)

-80

-60

-40

-20

0

δH (k

J/mol

)

-80

-60

-40

-20

0

δH (k

J/mol

)

-80

-60

-40

-20

0

Molar ratio

0 0.5 1 1.5 2 2.5

Molar ratio

0 0.5 1 1.5 2 2.5

Pow

er(μ

J/s)

6.0

6.5

7.0

7.5

Time (min)0 20 40 60 80 100

CA I, pH 8.0 CA II, pH 7.9 CA III, pH 8.0

CA IV, pH 8.0 CA VA, pH 8.0 CA VB, pH 8.0

CA VI, pH 8.0 CA VII, pH 8.0 CA IX, pH 8.0

CA XII, pH 8.0

CA XIII, pH 8.0

CA XIV, pH 8.0


ThermoFluor of all 12 human CA isoforms

CA III

T m(o C)

50

55

60

65

70

75

80

CA II, pH 6.0

Lt (M)1×10−31×10−6 1×10−5 1×10−40

CA XIV, pH 6.2

Fluo

resc

ence

(a. u

.)

6

8

10

12

t (oC)40 50 60 70

EZA(μM)07.8026.3200

0Lt (M)

1×10−31×10−6 1×10−5 1×10−4

CA XIII, pH 6.0

Fluo

resc

ence

(a. u

.)

0

20

40

60

80

80

[EZA] (μM)011.717.6200

t (oC)40 50 60 70

0

Lt (M)1×10−31×10−6 1×10−5 1×10−4

CA XII, pH 6.0

Fluo

resc

ence

(a. u

.)

2

4

6

8

10 [EZA](μM)011.726.3200

t (oC)40 50 60 70 80

CA IX, pH 6.2

Fluo

resc

ence

(a. u

.)

0

10

20

30

t (oC)40 50 60 70 80

[EZA] (μM)017.626.3133

CA VII, pH 6.0

Fluo

resc

ence

(a. u

.)

5

10

15

t (oC)40 50 60 70 80

[EZA](μM)012.525.0200

CA VI, pH 5.7

Fluo

resc

ence

(a. u

.)

5

10

15

20

t (oC)40 50 60 70 80

[EZA] (μM)0

5025

100

CA VA, pH 6.0

Fluo

resc

ence

(a. u

.)

10

15

20

25

t (oC)40 50 60 70 80

[EZA](μM)06.2525.0100

CA IV, pH 6.1

Fluo

resc

ence

(a. u

.)

5

10

15

t (oC)40 50 60 70 80

[EZA](μM)011.717.6200

Fluo

resc

ence

(a.

u.)

10

20

30

40

t (oC)40 50 60 70 80

[EZA](μM)013.229.6100

CA I, pH 6.0

Fluo

resc

ence

(a. u

.)

0

10

20

30

40

t (oC)40 50 60 70 80

[EZA](μM)20026.3411.710

T m(o C)

50

55

60

65

70

75

80

T m(o C)

50

55

60

65

70

75

80

T m(o C)

50

55

60

65

70

75

80

Fluo

resc

ence

(a. u

.)

5

10

15

20

25

( C)40 50 60 70 80

[EZA](μM)012.550.0100

CA VB, pH 6.0

o


U-shapes of all 12 CA isoform for ∆G of EZA+CA

CA VA

ΔbGintr

Δ bG

(kJ/m

ol)

−70

−60

−50

−40

−30

−20CA III

ΔbGintr

CA I

ΔbGintr

CA II

ΔbGintr

Δ bG

(kJ/m

ol)

−70

−60

−50

−40

−30

−20CA IV

ΔbGintr

CA VB

ΔbGintr

CA VII

ΔbGintr

CA IX

ΔbGintr

pH5.0 6.0 7.0 8.0 9.0 10.0

ΔbG

(kJ/

mol

)

−70

−60

−50

−40

−30

−20CA XII

ΔbGintr

CA XIII

ΔbGintr

CA XIV

ΔbGintr

CA VI

ΔbGintrΔ bG

(kJ/m

ol)

−70

−60

−50

−40

−30

−20

pH5.0 6.0 7.0 8.0 9.0 10.0

pH5.0 6.0 7.0 8.0 9.0 10.0

pKa =7.1

pKa=6.5 pKa=7.2

pKa=6.2 pKa=7.0pKa=6.8

pKa=7.0 pKa=8.3pKa=6.8


X-shapes of all 12 CA isoforms for ∆H of EZA+CA

CA VA

CAIII

pH5.0 6.0 7.0 8.0 9.0 10

CA XIV

ΔbH

(kJ/

mol

)

−100

−80

−60

−40

−20

0

Pi

TRIS

ΔbHintr

CA I

Pi

TRIS

ΔbHintr

CA II

ΔbH

(kJ/

mol

)

−100

−80

−60

−40

−20

0

Pi

TRIS

ΔbHintr

CA IV

Pi

TRIS

ΔbHintr

CA VB

ΔbH

(kJ/

mol

)

−100

−80

−60

−40

−20

0

Pi

TRIS

ΔbHintr

CA VI

Pi

TRIS

ΔbHintr

CA VII

Pi

TRIS

ΔbHintr

CA IX

pH5.0 6.0 7.0 8.0 9.0 10

ΔbH

(kJ/

mol

)

−100

−80

−60

−40

−20

0

Pi

TRIS

ΔbHintr

CA XII

pH5.0 6.0 7.0 8.0 9.0 10

Pi

TRIS

ΔbHintr

CA XIII


Structure - thermodynamics correlations


Compound structure – thermodynamics correlation map


Enthalpy - entropy compensation


Compound structure – kinetics correlation map

100 nM

1000 nM

5

5.5

6

6.5

7

-3 -2.5 -2 -1.5 -1 -0.5 0

2-1-b

2-1-b

2-1-b

2-1-b

2-1-b2-1-b

2-1-a 2-1-a

2-1-a

2-1-a

2-1-a

2-1-c2-1-c2-1-c2-1-c

2-1-c

2-1-c

log(kd)

log(ka)

10 nM1 nM

5

5.5

6

6.5

7

-3 -2.5 -2 -1.5 -1 -0.5

1-1-b

1-1-b

1-1-b

1-1-b1-1-b

1-1-b

1-1-a

1-1-a1-1-a

1-1-a

1-1-a

1 nM 10 nM

log(ka)

log(kd)

100 nM

1000 nM

A

C

4.5

5

5.5

6

6.5

-2 -1.5 -1 -0.5 0 0.5

2-2-a

2-2-b

2-2-a

2-2-a

2-2-a

2-2-a

2-2-b

2-2-b

2-2-b

2-2-b 2-2-b

log(kd)

log(ka)

1 nM 10 nM 100 nM

1000 nM

D

CA I

CA II

CA VII

CA IXcd

CA XIIcd

CA XIII

log(kd)

5

5.5

6

6.5

7

-2.2 -1.8 -1.4 -1 -0.6

log(ka)

3-3-a

3-3-a

3-3-a

3-3-a

3-3-a

3-1-a

3-1-a

3-1-a

3-2-c

3-2-c

3-2-c

3-2-c3-2-c 3-4-a

3-4-a3-4-a3-2-a

3-2-a

3-2-b

3-2-b

3-2-d

3-2-d

E1 nM 10 nM

100 nM

1000 nM

4

4.5

5

5.5

6

6.5

-1.5 -1 -0.5 0

1-2-a

1-2-a

1-2-a

1-2-a

1-2-a

1-2-a

1-2-b1-2-b

1-2-b

1-2-b

1-2-c

1-2-c

1-2-c

1-2-c

1000 nM

100 nM

10 nM1 nM

log(kd)lo

g(ka)

B

Talibov et al. J. Med. Chem. 2016


X-ray structure – thermodynamics correlation map

Kisonaite et al. Plos One 9(12): e114106 (2014)Daumantas Matulis (Vilnius University) 2nd Annual European MicroCal Meeting September 26, 2016 36 / 42

Crystal structures of 1 and 3 bound to chCA IX

Dudutiene et al., J. Med. Chem. 57 (2014) 2435–46


Selective CA IX inhibitors

0 10-9

10-7

10-5

0

25

50

75

100

Act

ivity

(%

)

Lt (M)

0 40 80

0.2

0.4

0.6

Ab

sorb

an

ce(a

.u.)

Time (s)

0 0.5 1.0 1.5 2.0Molar ratio

0 0.5 1.0 1.5 2.0

-30

-20

-10

0

Molar ratio

40 80-0.08

-0.04

0.00

Po

we

r(µ

cal/s)

Time (min)40 80

-0.08

-0.04

0.00

Po

we

r(µ

cal/s)

Time (min)

0 10-9

10-7

10-5

Lt (M)

0 40 80

0.2

0.4

0.6

Abs

orba

nce

(a.u

.)

Time (s)

0 10-6

10-5

10-4

Lt (M)

0 10-6

10-5

10-4

0

5

10

15

20

∆Tm

(o C)

Lt (M)

0.0

0.5

1.060 80T (oC)

No

rma

lize

dflu

ore

sce

nce

(a.

u.)

0.0

0.5

1.060 80T (oC)

No

rma

lize

dflu

ore

sce

nce

(a.

u.)

F

F

O

S

F

N

SO2NH2F

F

O

S

F

N

SO2NH2

VD12-09 VD11-4-2

Res

pons

e (R

. U.)

-1

0

1

2

3

Time (s)-50 0 50 100 150 200

Resp

onse

(R. U

.)

0

4

8

12

Time (s)-50 0 50 100 150

Resp

onse

(R.U

.)

0

2

4

6

8

10

Time (s)-40 0 40 80 120

VD11-4-2

VD12-09

Dudutiene et al., J. Med. Chem. 57 (2014) Talibov et al., J. Med. Chem. 59(5) (2016)


Building CA IX inhibitors – SAR


Conclusions

All binding and inhibition assays will provide only the observedthermodynamic parameters. It is important to distinguish observedfrom intrinsic binding parameters

Only intrinsic parameters may be correlated with structure

TSA is a very good assay to determine tight binding

A series of CA IX inhibitors were designed using biothermodynamicand structural methods with the potential to be developed asanticancer drugs


Acknowledgment

Collaborations:Peteris TrapencierisWen-Yih ChenNadine MartinetJohn E. LadburySeppo ParkkilaJoachim W. DeitmerHelena DanielsonMatthew J. ToddDaiva TauraiteAlgirdas BrukstusRolandas Meskys

Funding:FP6 Marie CurieFP7-Regpot “MoBiLi”EEA-Norway GrantsCOSTResearch Council of LithuaniaDotacijos GrantLithuania-Latvia-Taiwan GrantHealthy Aging Grant


Thank you for your attenton!


Documents

What have we learned from the enthalpy database of 200 ... · What have we learned from the enthalpy database of 200 compound binding to 8 carbonic anhydrase isoforms as determined