The design synthesis and biological application of tools to enable the wavelength-dependent control of receptor activation and inhibition.

We are interested in making chemicals that will help us to understand how certain biological processes work. We are particularly interested in the proteins that the active component of cannabis interacts with the so called 'cannabinoid receptors'. We are also interested in the proteins that the 'hot' component of chilli peppers acts on, which is called TRPV1. These proteins have many interesting biological effects that we would like to understand in more detail. One of the problems with studying these proteins is that they are located in the cell membrane and so it is difficult to investigate them with out disturbing their environment. Another problem is controlling when and where we turn the proteins 'on'. If we simply apply chemicals to activate these proteins then it will not always be possible to control where the chemicals go and hence we may activate some proteins that we don't want to interfere with. One way of solving these problems is by making chemicals that are not biologically active until they are illuminated. This is good as light does not disrupt the cell's environment too much. By using a very small and accurate source of light (a laser) we can control when and where the chemical is released in the cell very accurately. The technology to do this already exists, but so far no one has been able to release two different chemicals (say one that turns the protein 'on' and another one that turns it 'off') in the cell at different times. We aim to do this by altering the wavelength of light that is required to activate each chemical. Light of different colours has different wavelengths, so you can think of us using one colour of light to turn the protein 'on' and another to turn it 'off'. This technology will be very powerful, as it will give us a level of control over activation of the protein that is not currently possible.

In previous work we have demonstrated that it is possible to removed one photolabile protecting (caging) group in the presence of another in a wavelength dependent manner. Our initial studies focussed on the caging of a TRPV1 agonist and wavelength orthogonal photolysis studies were analysed using 1H NMR. We now aim to extend this work to the orthogonal caging of a TRPV1 agonist and a TRPV1 antagonist. The photolysis of these compounds will initially be studied using 1H NMR and then we will conduct photolysis in vitro. It is our aim to be able to use one wavelength of light to selectively photolyse the TRPV1 agonist (hence activating TRPV1) and a second wavelength of light to selectively photolyse the TRPV1 antagonist (hence inhibiting the actions of the TRPV1 agonist). This will be the first demonstration of wavelength orthogonal photolysis in vitro. The use of lasers as the source of radiation potentially offers unprecedented temporal and spatial control over the release of agonists and antagonists in vitro. Once the concept of in vitro wavelength orthogonal photolysis is proven we will seek to apply this technology to the study of the cellular messenger anandamide. Anandamide acts as an agonist at both the cannabinoid (CB) receptors and TRPV1 and its actions seem to depend to some extent on its location. Therefore, the ability to temporally and spatially control where anandamide is released, through the use of a caged anandamide derivative, will allow study of this compound in an unprecedented manner. Caging or a TRPV1 antagonist in a wavelength orthogonal manner to the anandamide would allow dissection of the activities of anandamide on the TRPV1 and CB receptors. This project aims to prove concept on the powerful technology of wavelength orthogonal in vitro photolysis and will allow preliminary data for more substantial projects focussing on the study of anandamide and also the design of biologically compatible caging groups.