Microscale Thermophoresis: Drug-screening Assay Based on GPCRs
Receptor proteins located within the cell membrane serve to convey molecules or information into the interior. Because numerous drugs function by binding to such receptors, their interactions play important roles in the fight against various diseases. For example, the coupling can activate a signal chain which in the end specifically changes the metabolism of malignant cells. A collaboration between biophysicists from the LMU and researchers at the Massachusetts Institute of Technology (MIT) in Boston has found a very elegant and potentially revolutionary new way to screen for drug binding to membrane receptors. The Munich part of the project was led by scientists of the Nanosystems Initiative Munich (NIM) and of NanoTemper, a commercial spin-off of the LMU. The new approach will allow screening of the most common class of receptors (G protein-coupled receptors, GPCRs) for the binding of new drugs directly in solution. The study appears online in the recent Proceedings of the National Academy of Sciences of America (PNAS).
Membrane receptors coordinate communications between cells - whether between single-celled organisms or the cells of a complex organism such as the human body. The huge receptor proteins traverse the cell membrane from the exterior surface to the inside. When a messenger molecule - or a pharmaceutical - successfully binds at the external surface, the protein conformation changes. This, in turn, activates a signal chain inside the cell. As potential drug targets, receptor proteins are highly in demand for testing the efficacy of new pharmaceuticals. Of special interest are the G protein-coupled receptors (GPCRs) because a wide range of diseases can be successfully treated with GPCR-targeting drugs.
But there are two major challenges: First, receptor proteins are very fragile and normally need to be embedded in a membrane. Second, drug candidates are typically much smaller than their potential receptors. Therefore it is nearly impossible to test the binding directly by conventional methods which depend on measuring changes in mass or size. Alternatives, such as indirect cell culture experiments, take much more time and consume more material.
The researchers from Boston and Munich combined two innovative techniques to overcome these barriers.
The MIT group specializes in putting G protein-coupled receptors (GPCRs) into membrane-like structures made from artificial peptides. In this way the artificially produced receptors remain correctly folded and soluble.
The biophysicists of the LMU used microscale temperature gradients to detect drug binding. This method is based on the fact that molecules - as for example the receptors - in solution move along a temperature gradient in a characteristic way. The scientists established a microscopic temperature gradient by localized laser heating of a test tube containing about 1 microliter sample solution. They compared the movement of the pure receptor with the molecule's behavior after addition of a test drug. If the drug binds to the receptor molecule, the characteristics of its movement change. Not only is the setup of the so-called microscale thermophoresis (MST) binding assay very robust. The method is so sensitive that small, binding-induced changes in the conformation or shape of a GPCR can be detected using very small amounts of protein and test sample. By varying the amount of drug, the efficacy of the binding can be determined quantitatively.
These results document the successful combination of soluble GPC receptors with the microscale thermophoresis (MST) recently developed by the NanoTemper startup company. The new approach has great potential to become a simple and rapid standard assay for pharmaceutical and basic research (NIM).
Xiaoqiang Wang, Karolina Corin, Philipp Baaske, Christoph J. Wienken, Moran Jerabek-Willemsen, Stefan Duhr, Dieter Braun, Shuguang Zhang: Peptide surfactants for cell-free production of functional G protein-coupled receptors. Proceedings of the National Academy of Sciences of America (PNAS), Published online May 9, 2011.