Why are recombinant systems helpful in GPCR screening?
Drug Discovery & Development magazine conducted a roundtable discussion with industry scientists to answer questions about GPCR. Below are the answers to a question related to recombinant cells. To review the full discussion, see all related articles.
James Netterwald: When you say recombinant cells, do you mean the GPCR is being overexpressed, under the control of some foreign promoter, or is it being tagged with something in order to facilitate further isolation later on?
Charles Lunn: Usually, recombinant cells means overexpressing the receptor to high above physiological levels within a host cell line, usually CHO HEK293, or U20S cells. The advantage of that approach is confidence that the observed signal derives from the overexpressed receptor. By analyzing the host cell lacking the recombinant receptor, you can confirm that your signal is derived from your recombinant protein. The problem with that approach is that the environment of the receptor is not as natural as in a natural cell type. Overexpressed receptors are can associate with each other and with other receptors. Your host cell may not contain relevant signaling factors, complicating your results. However, the advantage of knowing your signal derives from the target receptor is powerful.
Suresh Poda: Yes, I agree with Charles. A couple of years ago, we didn’t have too many tools to study GPCRs, so we have a lot of recombinant systems to do primary screens. I also agree that the advantages are many, but there are some disadvantages because sometimes when you overexpress GPCRs in cell lines, they tend to couple pathways that are different from normal signaling pathways. For example, some receptors that are naturally coupled to Gi pathway, but when we force-couple them and overexpress them, they tend to couple to Gq. So when you are screening, you are probably picking up more compounds towards the Gq pathway than the Gi pathway. So this is sometimes misleading, you are finding compounds that are not interesting to you.
Lunn: It speaks to the development strategy once a compound set is identified. Many investigators identify chemotypes that are interesting from a chemical point of view, and then characterize them in some biologic system that is considered to be disease-relevant. Sometimes you’ll find odd pharmacologies associated with these different chemotypes that may reflect exactly what Suresh was mentioning here—forced-coupling through a G-protein that does not reflect the disease-relevant phenotype. You can hopefully eliminate irrelevant compounds by this biological analysis.
Richard Eglen: Recombinant cell lines with GPCRs that are overexpressed have to be proved powerful workhorses. I think we’re starting to look at them with a hard eye now simply because the technology has proven to give options. But clearly having a signal unambiguously to the one receptor that’s against a very low background of other receptors is a significant advantage for high throughput screening.
Filed Under: Drug Discovery