We are synthesizing novel ruthenium-modified adenosine triphosphate (ATP) analogues. These molecules are being designed so that they can be used by kinases to phosphorylate protein-substrates with a ruthenium-modified phosphate group. Additionally, the ruthenated-phosphate group must be inaccessible to phosphatases such that once the protein-substrate is irreversibly phosphorylated. We plan to use these molecules to study and disrupt intracellular communication with a specific focus on cancerous cells.(The image above shows that one of the ruthenium-modified ATP molecules will look like (left) as well as a rendering of a protein structure with a tyrosine that has been phosphorylated with our modified ATP (right))
(You can see what we’ve been doing by checking out our on-line notebooks.)
Post-translational modifications (PTMs) are some of the most important means for activating protein functionality in vivo. Of all of the different types of PTMs, protein phosphorylation is one of the most abundant processes and plays a major role in communication within and between cells. Phosphorylation occurs when a kinase transfers the terminal phosphate from ATP onto a protein substrate. This process is reversible in cells with protein phosphatases dephosphorylating the protein-substrate. Phosphorylation is generally used to activate or deactivate protein function and can even be utilized as a way to target proteins for degradation. Numerous diseases, including several types of cancer, have been noted for the observation that the regulatory machinery controlling phosphorylation breaks down.
By creating ATP analogues we are attempting to provide a means of disrupting these processes within a cell. But, in order to do this, we need to design ATP analogues that:
1) bind kinases as well or better than ATP.
2) are not so bulky so that a kinase can transfer the ruthenated phosphate to a protein substrate.
3) are bulky enough so that a phosphatase cannot remove the ruthenated phosphate from the protein substrate.
4) Are stable enough that the ruthenated phosphate doesn’t isn’t hydrolized from the full ATP molecule in the reducing environment of the cell.
Kinases and Phosphatases
Kinases are proteins that bind ATP along with a substrate-protein. The kinase transfers the terminal phosphate group from ATP onto either a serine, threonine, tyrosine, or histidine residue in that substrate-protein. The image on the left (pdb code 3PP1) shows an ATP molecule (green and organge) bound to a kinase (white with binding pocket highlighted in red). Notably, you can see the space where extra chemical bulk might be tolerated. And, there are several reports in the literature already of modified ATPs that will bind to a kinase and have their modified phosphate groups transferred to a protein-substrate.
Phosphatases are proteins that will bind phosphorylated proteins and remove the phosphate group. The image on the right (pdb code 1PTY) shows where a phosphorylated tyrosine (green and red) binds this particular phosphatase (white with binding pocket highlighted in blue). It is obvious from this picture that there is not a lot of room for extra bulk inside of the phosphatase binding pocket.
This same synthetic strategy has been used previously to add organic appendages to the terminal phosphate in ATP. And, similar methodology has been used to add ferrocene molecules to ATP as well. The chemistry described here is not very demanding and should be suitable for the excellent undergraduates here at American.
The image above shows some of the targets we are making. On the left are shown the different ruthenium complexes we are working with. The complex on top is very luminescent and should be an excellent probe to enable visualization of phosphorylation events as they occur. The complexes on the bottom have shown therapeutic anticancer properties. We plan on studying if covalent attachment of these kinds of molecules to proteins in cancerous cells can increase their efficiency as drugs.
The different linkers (joining ATP with the ruthenium complex) we are using are shown on the right of this image. We will test different linker lengths in an attempt to find the optimal size for both kinase binding and enabling phosphate transfer.