Mechanistic evaluation of a TRPV6 Ca2+channel inhibitor

Calcium is a crucial divalent cation, relevant in physiological processes as haemostaseology or immune defence. It enters the cell via specific calcium selective ion channels, among which the transient receptor potential vanilloid 6 channel (TRPV6) represents a highly calcium selective member of the TRP family. It is located in the plasma membrane and for ms a tetrameric channel complex. Although the channel is expressed in various tissues, as the intestine or placenta, it is also proposed to play a role in several malignancies, e.g. breast and prostate cancer. While it is not expressed in normal prostate tissue, it is up-regulated in prostate cancer cells. This and the matter of fact, that to date only one TRPV6-targeted in- human phase-I study has been published, render the TRPV6 channel an attractive target for the development of a selective inhibitory substance to therapeutically interfere with cancer. Recently, a specific small molecule inhibitor of TRPV6, called cis-22a, has been developed. This project aims at characterising the effect of this blocker and the comparison with other potential TRPV6 inhibitors on TRPV6 over expressing HEK293 cells, employing the patch clamp as well as calcium imaging techniques. Moreover, the effect of cis-22a will be tested on breast and prostate cancer cell lines expressing endogenous TRPV6 channels. The overall impact of potential TRPV6 inhibition on the invasive capacity of cancer cells will then be evaluated by measuring cell migration and proliferation. Another part of the project aims at utilising opto-pharmacology to target TRPV6 channels via a photo-switchable compound. Here, a photo-switchable derivate of cis-22a, interfering with TRPV6 activity upon application of light pulses, will be established and analysed. In silico evaluation of the binding site of cis- 22a on the TRPV6 channel by MD simulations will be confirmed by functional analysis of mutated TRPV6 channels (structure-guided mutagenesis). In summary, this project aims at characterising the properties and the binding site of the cis-22a inhibitor and at establishing a specific, photo-switchable TRPV6 inhibitor, which might path the way for novel anti-cancer therapies.

Calcium is a divalent cation and is involved in many physiological processes, such as immune defense and hemostaseology. The TRPV6 channel (transient receptor potential vanilloid 6), a member of the TRP superfamily, is highly calcium-selective and localized in the form of a tetramer in the plasma membrane. Expression in intestinal or placental cells as well as overexpression in malignant tumors of the breast and prostate show the involvement of TRPV6 in important cell signaling pathways. Although TRPV6 is not expressed in normal prostate tissue, it is upregulated in prostate cancer cells. TRPV6 ion channels are therefore an interesting target for the development of new inhibitors. In addition, there is only one in-human phase I study with a TRPV6 blocker to date. Only recently a new inhibitor, cis-22a, has been developed. The aim of this project was to investigate this blocker and compare it with other potential inhibitors in TRPV6 expressing HEK293 cells using calcium fluorescence imaging and the patch clamp technique. In silico studies of the cis-22a binding site using molecular dynamics simulations were subsequently confirmed by functional analysis of these mutant TRPV6 channels and complemented by analysis of the crystal structure of a TRPV6 channel in complex with cis-22a. This revealed a new, additional cis-22a binding site at the pore exit, which is physiologically utilized by the calcium-binding protein calmodulin to inactivate TRPV. This was followed by a detailed analysis of the regulation of the TRPV6 channel via lipids, in particular phosphatidylinostitol-4,5-bisphosphate (PIP2). Methods for interference of the channel-PIP2 interaction were used for this purpose. In combination with cis-22a as an instrument to analyze the lipid binding site and the pore, experimental evidence for an allosteric coupling of the lipid binding site and the pore was obtained. In addition, it was shown that there is also feedback from the pore to the lipid binding site, with the exception of the pore outlet. In summary, the results of the project aimed to characterize the properties and binding site of the cis-22a blocker as well as the overlapping regulation of the TRPV6 channel via lipids, thereby identifying potential targets for further inhibitors.