Scientists at The Scripps Research Institute have reported a light-activated “warhead” molecule that, when tethered to protein-binding small molecules, greatly increases their ability to inactivate target proteins. Using this molecule with chromophore-assisted light inactivation – or CALI – could contribute to the development of research tools that could advance cancer treatments and other therapies. The warhead came about because the researchers wanted to assemble a large collection of specific, potent antagonists for proteins. “Our screening methodology is capable of quite high throughput,” said Thomas Kodadek, who led the study, “but the protein ligands that come out of the primary screen are not very potent antagonists, though they are quite specific.” Investigators have described a light-activated “warhead” molecule that, when appended to protein-binding small molecules, contributes to significant increases in the latter molecules’ ability to inactivate target proteins. KD = dissociation constant. The potency can be improved by a classical medicinal chemistry approach, he added – by making structural analogues and testing them for improved inhibition of protein function – but this method is far too slow to use in developing hundreds of protein inhibitors, as the researchers wanted to do. They needed an “instant” way to increase the potency of the hits from the library screens. To achieve this, the investigators appended to the protein-binding molecules a second, “warhead” molecule capable of delivering a reactive intermediate to the protein target that would result in its irreversible inactivation. Doing so, they reasoned, would give them a higher apparent potency: Once a protein is destroyed, even if the inhibitor dissociates, the protein is still inactive. They wanted to limit inactivation of proteins to the immediate vicinity of the warhead, so they focused on singlet oxygen. “Singlet oxygen is perfect for this since it has a diffusion radius of only 40 to 60 Å, about the size of a protein,” Kodadek said. To this end, they used derivatives of ruthenium, an unusually efficient photocatalyst for the generation of singlet oxygen. In the April 2010 issue of Nature Chemical Biology, Kodadek and colleagues reported significant increases in protein-killing potency when using the warhead with CALI. Others have developed CALI reagents using singlet oxygen-generating molecules, they noted, but this represents an important technical advance, especially as it allows researchers to target both extracellular and intracellular proteins. “The upshot,” Kodadek said, “is that we now believe that we have the ability to indeed ‘instantly’ transform primary screening hits into extremely potent photoactivated protein inhibitors. This solves a major problem in chemical biology because, as stated above, primary hits from screens are otherwise simply not potent enough to be really useful, and standard methods of improving potency are far too slow to allow for proteome-wide projects to be carried out.” Using the warhead, the researchers are beginning to generate a large stable of CALI reagents to target a variety of different proteins. At the same time, they would like to develop alternative chromophores enabling use of red or infrared light to generate singlet oxygen. The ruthenium complex described in the present study has an absorption max of 450 nm – that is, in the blue range of the spectrum – and for this reason cannot penetrate into a living organism, “making our current technology great for cell culture work but not for experiments with whole animals,” Kodadek said. “An infrared-absorbing chromophore capable of mediating this chemistry would allow us to target proteins in live animals with light.”