Sensitive kinetic analysis of small molecules binding to large drug targets

In this technical note, we show how the WAVEsystem, a Creoptix technology, can be used to accurately monitor binding kinetics of large target-to-analyte molecular weight (MW) ratios (>300:1), thanks to the expanded sensing field over which the Grating-Coupled Interferometry GCI) technology measures for high sensitivity.

Real-time, label-free analysis of interactions between small molecules and drug targets provides valuable information to guide the drug discovery process and identify promising drug candidates. However, weak binders and large target-to-analyte molecular weight (MW) ratios can make kinetic characterization extremely challenging, as they often require high sensitivity and the ability to confidently measure fast off-rates (koff). 

Using a proprietary Grating-Coupled Interferometry (GCI) technology, the WAVEsystem, a Creoptix technology, provides best-in-class sensitivity to allow highly accurate kinetic data to be generated at even low signal levels. Combined with robust microfluidic technology, GCI enables the characterization of molecular interactions in the presence of non-common solvents – such as acetonitrile or high concentrations of DMSO.

By measuring the binding kinetics of various small molecule inhibitors of Carbonic Anhydrase II (CAII), we show how the Creoptix WAVE achieves outstanding resolution for small, weakly binding drug molecules, for full kinetic characterization regardless of the target-to-analyte molecular weight ratio. 

1 Furosemide 2 4-Sulfamoylbenz. acid 3 Sulfanilamide 4 Methylsulfonamide 5a Acetazolamide 5b Acetazolamide
MW (Da) 330.74 201.20 172.20 95.12 222.25 222.25
Immob. level (pg/mm2) 6050 6050 6050 6050 6050 1450
Kinetic data
kon (M-1.s-1) 3.75x1044 2.98x104 2.67x104 1.15x104 1.31x106 1.22x106
koff (s-1) 0.051 0.040 0.180 3.37 0.038 0.031
km - - - - 1.17x107 -
Rmax (pg/mm2) 30.87 16.91 12.09 3.79 13.25 4.59
KD (μM) 1.4 1.3 293 293 0.029 0.026
Equilibrium data
Rmax (pg/mm2) 33.37 14.57 11.75 3.34 14.05 4.83
KD (μM) 2.4 3.8 6 270 0.048 0.028

Table 1: Kinetic and equilibrium data of various small molecule inhibitors of Carbonic Anhydrase II (CAII)

[Figure 1 TN201001-Creoptix-kinetic-analysis-small-molecule-binding.jpg] Figure 1 TN201001-Creoptix-kinetic-analysis-small-molecule-binding.jpg

Figure 1: Binding kinetics of various small molecule inhibitors of Carbonic Anhydrase II (CAII)


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