Introduction
Differentiating lead molecules by affinity for target has been a cornerstone in the development of pharmaceutical compounds. Increasingly it is recognized that differentiating by kinetics (on-rates and off-rates) may lead to longer residence time in the patient and be a point of differentiation for a pharmaceutical product (1, 2, 3). Therefore the ability to measure kinetics of small molecule binding to proteins is becoming integrated earlier into the drug discovery process. Surface plasmon resonance allows the elucidation of kinetic constants in a label-free manner. The BiOptix 404Pi™ instrument can measure small molecules down to ~100 daltons (Da).
The carbonic anhydrases are a family of enzymes whose primary function in animals is to interconvert carbon dioxide and bicarbonate to maintain acid-base balance in blood and other tissues (4). 4-aminomethyl benzenesulfonamide (ABS or Mafenide) is a carbonic anhydrase II inhibitor used to treat severe burns (5). It has been shown to inhibit bacterial reproduction which aids the wound healing process. In this application note, the ability of the BiOptix 404Pi SPR instrument to measure affinity and kinetic rate constants for the interaction of the small molecule ABS (223 Da) with carbonic anhydrase II is demonstrated. Additionally the reproducibility of the measurement is reported.
Materials and Methods
Immobilization of Carbonic Anhydrase
Carbonic anhydrase II (Sigma-Aldrich) was immobilized onto channels 3 and 4 of a BiOptix CMD200m sensor chip using standard NHS/EDC amine coupling with blocking of the remaining active esters with ethanolamine. Approximately 8500 RU was loaded onto both flow cells. The reference channels 1 and 2 were activated with EDC/NHS and blocked with ethanolamine.
ABS Kinetic Measurement Protocol
4-aminomethyl benzenesulfonamide (ABS) (Fluka) was 3-fold serially diluted from 100 μM to 1.23 μM into running buffer, phosphate buffered saline with 0.05% TWEEN 20, pH 7.4 (PBST) (TEKnova). ABS injections were performed in 2×2 mode and blanks were included every third injection to enable double referencing. The flow rate was 100 μL/min, association time was 60 seconds and dissociation time was 140 seconds and flow cell temperature was 20°C. All concentrations were run in duplicate.
Results
Kinetic Analysis of ABS binding to Carbonic Anhydrase II
Data from duplicate injections of ABS over immobilized carbonic anhydrase II were double referenced and analyzed using Scrubber 2.0 software (6) and were highly reproducible. All the sensorgrams were described well by a 1:1 kinetic binding model (Figure 1). An association rate constant (ka) of 10,000 M⁻¹s⁻¹ and a dissociation rate constant (kd) of 0.0984s⁻¹ were measured on channel 3 which yielded a KD value of 9.8 μM. Nearly identical results were obtained on channel 4 (ka = 9,676 M⁻¹s⁻¹, kd = 0.0950 s⁻¹, KD = 9.8 μM).