There are more ways to study protein interactions than to skin a cat, speakers at IBC Life Sciences’ “Protein-Protein Interactions” meeting demonstrated last week with their presentations.
Against a noisy, busy backdrop of highways and runways, about 70 conferees gathered at the Hilton Newark Airport hotel to take in one technology after another. The techniques included everything from high-end mass spectrometry, capillary electrophoresis, and microfluidics to measuring heat released during a reaction. Following are some highlights from the presentations during the first two days of the conference:
Linda Engle, senior scientist of Marlborough, Mass.-based Cetek, presented her company’s approach, capillary electrophoresis to separate a target protein mixed with a library of compounds. If the protein forms a complex with one of the compounds, it changes its mobility in the electric field, which a laser can detect. Engle cited an example in which Cetek screened a protein against a library of nearly 50,000 compounds for a pharmaceutical company and found that three were binding with nanomolar affinity: One is now at the lead optimization stage. In another project, Cetek determined which of three different forms of a single protein a small ligand bound to. For its own drug discovery program, Cetek has chosen to screen natural products, which “hold greater promise to inhibit protein-protein interactions than synthetic molecules,” according to Engle. It focuses currently on 12 therapeutic targets. In a screen of microbial extracts, the company found novel compound that — like cyclosporin — disrupts a complex between a human and an HIV protein.
Moving from capillaries to microchannels, Bernhard Weigl, director of microfluidic applications at Micronics, talked about measuring protein interactions using two or more liquids that flow next to each other in the same microchannel without mixing, like a liquid version of striped toothpaste. At the interface of the fluids, tagged proteins interact in a narrow zone of diffusion, which can be measured optically. Moreover, by taking measurements along the channel, researchers can determine the rate of the complex-forming reaction. A different channel setup allows for diffusion-based separation of protein complexes from unbound molecules, without the need of a filter. Micronics, which is based in Redmond, Wash., manufactures chips with complex microfluidic structures by laminating 15-20 layers of plastic on top of each other.
But why stay with micro when nano is within reach? NanoInk of Chicago reported that it is using dip pen nanolithography (DPN) to print nanostructures, such as protein spots of less than 100 nanometers in diameter, onto substrates. The technology, which is based on delivering liquids via the tip of an atomic force microscope, comes out of Chad Mirkin’s lab at Northwestern University. According to Guy della Cioppa, NanoInk’s executive vice president for business development, new methods of screening become available at the single molecule level. “Proteins, after all, are very small nanomachines — they have dimensions in the sweet spot of nanotechnololgy,” he said. The company is planning to come out with a DPN writer as well as with a multi-pen system next month, followed soon by a catalog of packaged “inks.”
Zooming in on individual molecules, Xencor of Monrovia, Calif., aims to engineer proteins, using a combined computational and screening approach. For example, it modified a subunit of tumor necrosis factor such that it would still form trimers with wildtype proteins but no longer bind to its receptor. Animal studies to assess in vivo effects are underway, according to John Desjarlais, Xencor’s director of computational biology.
Targeting interfaces of protein-protein interactions, on the other hand, which often lack the “nooks and crannies” a drug can grab onto, is no easy feat. In his keynote lecture, James Wells of Sunesis Pharmaceuticals pointed to a possible way: screening for two weakly binding drug fragments and connecting them to a tightly binding ligand. In order to catch the weak binders, his company, based in South San Francisco, engineers both its library fragments and protein targets to contain cysteines that can form covalent bonds.
While most approaches presented at the meeting aim to study proteins in vitro, several companies focused on methods to follow protein-protein interactions inside mammalian cells. Applied Biosystems fuses part of an enzyme to one protein and another part of the same enzyme to a second protein. If the two proteins, after expressing them in a cell, interact — for example in a signaling pathway — the enzyme parts come together and its activity as a whole can be measured. ABI’s technology is available to researchers in various modes, including custom cell line development and technology transfer.
Odyssey Pharma’s technology uses a strategy similar to ABI’s, fusing proteins to two protein fragments that, when combined, become active. The company has been studying the effects of drugs that promote or inhibit protein-protein interactions, focusing on a small number of about 20 disease pathways. It is hoping to dissect every step in these pathways.
Rigel of South San Francisco doesn’t rely on reporter genes but looks for functional changes in response to retrovirally introduced peptides that disturb protein-protein interactions inside a cell. Esteban Masuda, the company’s associate director of biology, showed how specific peptides were able to inhibit a signaling pathway induced by interleukin-4. These peptides can then be used to look for the intracellular target. Rigel also uses libraries of cyclic peptides in its screens, which may become drugs directly.