Protein Small Molecule interactions

Protein-Small Molecule Interactions

Protein small molecule interactions are a challenge for any label free binding technique, as small molecules create only small changes upon binding to a protein. Imagine a 30 kDa molecular weight protein kinase immobilized on the surface. The small molecule staurosproine (MW 467 Da) would be expected to bind. At KD concentration, at equilibrium, one expects only a 0.8% change in the binding signal. As may be seen below, SKi Pro offers the superior sensitivity necessary to be able to characterize these interactions. Additionally, SKi Pro offers the MIK-MS technique in which small molecule binding is detected by mass spectrometry with very high fidelity.

CAII Furosemide

Figure 1 Sensorgram of furosemide interacting with carbonic anhydrase II.

Carbonic Anhydrase II

There is a history in the field of label free binding to study the performance of the system by immobilizing the enzyme carbonic anhydrase II (usually from bovine erythrocytes) on the surface of the chip and then interacting different concentrations of sulfonamides. This is easily done with SKi Pro. As shown in figure 1 a high quality fit (orange lines) is obtained for the gray data during a titration of 0.1–30 µM furosemide (MW 331 Da). One clearly sees the change in shape of the curves expected during the association phase, and the common shape of the dissociation as expected for two state binding.

Customer Data

As with the CAII-furosemide test system, SKi Pro may also be used to characterize customer interactions. Typically to obtain data of this quality, a moderate to high amount of protein must be immobilized on the surface of the SKi sensors in such a way that the binding properties of the protein are unchanged.

Figure 2 shows examples of two small molecule inhibitors targeted against a customer kinase. The inhibitors have a molecular weight of between 400–600 Da and the kinase weighs approximately 35 kDa between the concentrations of 25–200 µM.

numKD (µM)kon (M-1sec-1)koff (sec-1)
A1904.5x102 8.4x10-2
B60 1.3x103 7.8x10-2

High quality fits are obtained even in the case of weak interactions such as these. These interactions are hundreds of times weaker than the interaction with furosemide shown in figure 1, and up to 100,000 times weaker than the strongest antibody-antigen interaction shown in the antibody interactions page.

Weak small molecule interactions from customer data

Figure 2 Two examples of weak interactions from customer data.