In this technical note we show how the WAVEsystem, a Creoptix technology, can be used to accurately measure fast off-rate kinetics of weakly binding molecules. Thanks to a cartridge design that enables ultra-fast transition times of 150 ms, low potency hits can now be easily spotted for more successful drug discovery.
Weak binders, such as those found in fragment-based screening libraries, are typically ranked by affinity rather than kinetics. This is because standard instrumentation lacks the capacity to measure fast off-rates. However, measuring affinity rather than off-rates can generate large numbers of false positive results, extending workflows and incurring unnecessary costs.
Employing a proprietary Grating-Coupled Interferometry (GCI) technology, the Creoptix WAVEsystem provides resolution of fast off-rates up to 10s-1. This enables accurate, early stage selection of true hits to greatly increase efficiencies.
By measuring the binding kinetics of methyl sulfonamide to Carbonic Anhydrase II, we show that the WAVEsystem achieves outstanding resolution at extremely fast off-rates, even for large target-to-analyte molecular weight ratios.
| Kinetic Data | |||
|---|---|---|---|
| Creoptix WAVE | Biacore™ S51 | ||
| kon (M-1 s-1) | 9.36 x 103 | kon (M-1 s-1)
|
8.05 x 103 |
| koff (s-1) | 2.86 | koff (s-1)
|
2.21 |
| KD (uM) | 306 | KD (uM) | 274 |
Table 1: Kinetic data compared to published data in Myszka, David G. “Analysis of small-molecule interactions using Biacore S51 technology.” Analytical biochemistry 329.2 (2004): 316-323.
Legend: (A) Sensorgram of methyl sulfonamide (95.1 Da) binding to Carbonic Anhydrase II (29 kDa) immobilized by amine-coupling on a WAVEchip PCH at 8500 pg/mm2 on flow cell 1. Data was acquired at 40Hz with a flow rate of 200 μl/min. (B) Zoom into the association phase (left) and dissociation phase (right) with the respective residual distribution plots below showing the high and clear resolution of the kinetics.
![[Figure 1 TN201101-Creoptix-fast-off-rates-to-reduce-false-positives.jpg] Figure 1 TN201101-Creoptix-fast-off-rates-to-reduce-false-positives.jpg](https://p3.aprimocdn.net/malvernpanalytical/8ddb9b09-3dfd-400a-91e2-ae360106df9b/Figure%201%20TN201101-Creoptix-fast-off-rates-to-reduce-false-positives_Original%20file.jpg)
Figure 1: Methylsulfonamide (95.1 Da) binding to Carbonic Anhydrase II (CAII). 1 pg/mm2 = 1 RU
