Sensitive kinetic analysis of small molecules binding to large drug targets

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 (k off ).

Using a proprietary Grating-Coupled Interferometry (GCI) technology, the Creoptix ® WAVE 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/mm 2 ) 6050 6050 6050 6050 6050 1450 Kinetic data k on (M -1 .s -1 ) 3.75x1044 2.98x104 2.67x104 1.15x104 1.31x106 1.22x106 k off (s -1 ) 0.051 0.040 0.180 3.37 0.038 0.031 k m - - - - 1.17x107 - R max (pg/mm 2 ) 30.87 16.91 12.09 3.79 13.25 4.59 K D (μM) 1.4 1.3 293 293 0.029 0.026 Equilibrium data R max (pg/mm 2 ) 33.37 14.57 11.75 3.34 14.05 4.83 K D (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: Binding kinetics of various small molecule inhibitors of Carbonic Anhydrase II (CAII)

Einloggen