Structure Activity

Relationship of Carbonic
Anhydrase Inhibitors
using Surface Plasmon
Resonance
By Nat Dempkowski
 Materials
• Biacore T100
• Biacore CM5 Sensor Chip
• Human Carbonic Anhydrase I
• Bovine Carbonic Anhydrase II
• Binding and Buffer Solutions
o EA, EDC, NHS, Acetate, PBS, HEPES, NaOH,
Sulfamate
• Carbonic Anhydrase Inhibitors Human Carbonic
(Sulfonamides) Anhydrase II
 Inhibitors

Sulfanilamide Acetazolamide Saccharin

Ammonium Sulfamate Topiramate Sulpiride

Hydrochlorothiazide Methazolamide
Celecoxib
 Protein Immobilization
Procedure
• Prime CM5 sensor chip by
running NaOH and CM5 Sensor Chip
Sulfamate into the flow cells
• Using EA, EDC, NHS
solutions, immobilize
Carbonic Anhydrase on the
Biacore T100
chip
• Measure Response Units of
Carbonic Anhydrase
immobilized on chip
 Inhibitor Response
Measurement
Procedure
• Dilute inhibitors in buffer solution
• Dilute different concentrations of inhibitors from
~500µM to ~1µM
• Inject solutions into flow cells with immobilized protein
from lowest concentration to highest
• Run buffer injections after high concentrations to clear
accumulation of inhibitors
• Measure response units using Surface Plasmon
Resonance and model kinetics of binding
 15
10
10
5
5

Response from Inhibitors

Response
0
0

-5 -5
-100 -50 0 50 100 150 200 250 300 350 400 -100 -50 0 50 100 150 200 250 300 350 400
RU Tim e s Tim e s
RU
16
14
Acetazolamide 25
Celecoxib
20
12
10 15
8
10
6
4 5
2

Response
0
0
-2 -5
-100 -50 0 50 100 150 200 250 300 350 400 RU -100 -50 0 50 100 150 200 250 300 350 400
RU Tim e s
14 Tim e s
12
12
10

8
Hydrochlorothiazide 10 Methazolamide
8
6
6
4 4
2 2
Response

0 0
-2 -2
-100 -50 0 50 100 150 200 250 300 350 400 -100 -50 0 50 100 150 200 250 300 350 400
Tim e s Tim e s

Sulfanilamide Topiramate
 Kinetics Data Kd
Mw Ka (dissociatio KD
(g/mol) (association) n) (affinity) Rmax (RU)
Acetazolamide 222.3 5.42E+06 0.05723 1.06E-08 16.82
Celecoxib 381.4 8.13E+04 0.1267 1.56E-06 26.31
Hydrochlorothiazide 297.7 1.16E+04 0.03752 3.25E-06 22.46
Methazolamide 236.3 5.79E+06 0.07977 1.38E-08 19.51
Sulfanilamide 172.2 2.40E+04 0.05141 2.14E-06 14.99
Topiramate 339.4 8.75E+04 0.02324 2.66E-07 13.32
Ammonium
Sulfamate 114.1 No Binding Observed
Sodium saccharin 205.2 No Binding Observed
Sulpiride 341.4 No Binding Observed
 Association Rates
Kinetic Association Constant
1.00E+07

1.00E+06
ka (M-1,sec-1)

1.00E+05

1.00E+04

1.00E+03
Acetazolamide Celecoxib Hydrochlorothiazide Methazolamide Sulfanilamide Topiramate
 Binding Affinity
Binding Affinity Constant
1.00E-05

1.00E-06

1.00E-07
KD

1.00E-08

1.00E-09
Acetazolamide Celecoxib Hydrochlorothiazide Methazolamide Sulfanilamide Topiramate
 Conclusion
• Binding Affinity is determined largely by the
Stiffness/Flatness of Molecule
• Double bonded nitrogen group increases association
rates greatly.

Acetazolamide
Methazolamide