“Ion channels are terrific molecular targets, and many drugs have been targeted to them,” stated David Clapham, M.D., Ph.D., Aldo R. Castenada professor of cardiovascular research at Children’s Hospital Boston. Yet, one of the biggest challenges is the gold standard assay—the patch clamp.
This is a time-consuming technique—single cell membranes must be broken open and the current must be recorded while controlling voltage in the cell. Although HT assays exist, not all ion channels are suited to them. “The most promising are the very fast, voltage-dependent channels with large, rapid changes and ones less amenable are ones that are similar to each other in their properties, like TRP channels—these are more difficult.”
Dr. Clapham also presented information on what he thought were good, fairly recent, ion-channel targets and included some recent data on some of his work with these targets.
Many TRP channels are involved in sensory functions, like smell, taste, and hearing. TRPV3 is an ion channel that is well expressed in skin. Dr. Clapham demonstrated that both skin barrier formation and some aspects of hair formation are altered by this ion-channel’s activation or block.
It is activated by subtle temperature changes—temperatures about 32ºC—indicating TRPV3 is sensing heat at the skin surface and relating that to the nerves. This indicates it may help regulate body temperature. Growth factors such as EPGR potentiates TRPV3 to bring calcium into karatinocytes, and, in turn, TRPV3 potentiates EPGR, so there’s a positive feedback loop.
“This is important for the proper formation of skin barriers, so that there is normally a cycle of karatinocytes maturing from deeper in the skin to the surface of the skin.” Dr. Clapham added that TRP channels are difficult to work with because they are fairly slow and their properties are often difficult to distinguish. In addition, they are often small in size, and there is a lack of known ways to activate them.
Additional ion channels that Dr. Clapham thought were worth pursuing were the NAV1.7 to NAV1.9 pain targets, which are voltage-gated sodium channels. A new chloride channel, TMAM16-A, and the ORAI channel, which is important in the immune system, were also on the list. An interesting new target for contraception, called CATSPER, is an ion channel only present in mature sperm and required for male fertility. “This may be a good method of contraception without hormones,” said Dr. Clapham.
“Our job is to find new targets and new molecules, and then other people can work with those molecules to target diseases.”
There are many challenges for the generation of new GPCRs, said Michel Bouvier, Ph.D., professor and chairman in the department of biochemistry at the University of Montreal. These include selectivity and ligand-biased signaling, where one receptor can couple to different signaling pathways in a cell.
“The problem with this is that you are trying to monitor the efficacy of a compound toward one signaling pathway, but since there are multiple ones, we don’t necessarily know which one to follow that will correlate with a disease or particular activity.” His approach is to develop one assay that could encapsulate in one reading all the signaling pathways and by dissecting the signatures, provide information about the pathways being engaged by a receptor.
Utilizing Roche’s label-free xCELLigence platform, his group is able to measure cell impedence. Each well of the plate has electrodes. As the cells grow, the impedance increases, and when the cells are treated with compounds that bind to receptors, many different pathways are triggered.
The readout reflects changes in impedance from the compound over time—providing a global assessment of the various pathways. Different compounds generate different curve shapes. “We can use this technology to differentiate classes of compounds that have different relative selectivity toward different pathways. It’s generating a simpler way to classify compounds in different efficacy profiles toward different signaling pathways.”
Dr. Bouvier added that they can now, using selective inhibitors of different pathways such as the generation of cyclic AMP, show how the inhibition influences the shape of the impedance curve. “Not only can we start classifying the ligands in different categories or compounds, but we can start making predictions on which pathways these compounds will be actively inhibiting. His group is planning to develop algorithms to apply to the curve and thus, provide a response as to which pathway is being affected. “We first need to confirm which portion of the curve informs us about each pathway.”
This approach can be used for almost any receptors, reported Dr. Bouvier. It provides a big time savings—one assay instead of four or five. However, he added, “we don’t know yet if all signaling pathways will respond to changes in impedance—from our data so far, we haven’t encountered such a pathway.”