Over production of certain proteins that control cellular responses in the body can lead to unregulated events, including various cancers. Proteins linked to tumor growth, known as oncoproteins, can be extremely difficult to inhibit by known small molecule drugs, owing to their relatively featureless binding surfaces, which lack well-defined small molecules. A sub-set of these proteins participate in protein-protein interactions.
Researchers in the Departments of Chemistry, and Biochemistry, and Molecular Biology at Colorado State University have used high-throughput screening to identify synthetic proteins that bind to gankyrin. Gankyrin is an oncoprotein that, when overexpressed leads to increased cellular levels of gankyrin-dependent protein-protein interactions. Cell signaling as a result of these interactions leads to genome instability and various cancers, which include breast, liver, oral, pancreatic, and colorectal cancers, as well as esophageal squamous cell carcinoma. Here at Malvern, we were excited to see two of our MicroCal instruments being used in the characterization of binding interactions between new gankyrin binding proteins, which represent protein therapeutic leads.
Isothermal titration calorimetry (ITC), was used to prove that 1st generation protein drug leads specifically bound to Gankyrin with 6 µM affinity. This interaction was measured with the native proteins without the need of any other probes or modifications, a hallmark of the technology. We can be assured that the proteins, therefore, have a direct binding event.
Differential scanning calorimetry (DSC), was used to investigate the stability of the syntheticproteins. Indeed, the proteins were as stable as their parent protein which unfolds at a high temperature of 91.1 ºC; synthetic proteins unfold at a temperature only 4 ºC lower. Precise measurement of individual domain unfolding patterns is a specialty of the DSC. Other experiments supported that the proteins interact inside living cells.
A paper outlining the research* said: “These new gankyrin-binding proteins are thermostable, express well in E. coli as soluble proteins, and represent the first synthetic proteins that recognize gankyrin in vitro and in complex cellular environments. These proteins likely represent valuable starting points for further optimizing affinity to gankyrin and modulating gankyrin-dependent oncogenic cell function and fate.”
These new synthetic gankyrin binding protein are an exciting invention that could lead to future advances in the arena of cancer pathway regulation. Being part of such important research is what drives Malvern to keep innovating and improving the capabilities and quality of our instruments.
*Resurfaced Shape Complementary Proteins That Selectively Bind the Oncoprotein Gankyrin. Alex M. Chapman† and Brian R. McNaughton*,†,‡†Department of Chemistry and ‡Department of Biochemistry & Molecular Biology, Colorado State University, Fort Collins, Colorado 80523, United States. ACS Chemical Biology In press.
From hit to lead – accelerate early drug discovery with ITC (Recorded Webinar)
Solve stability problems in preformulation and process development using DSC (Recorded Webinar)