Smart Probes Offer Non-invasive In Vivo Imaging

Bioscience Technology

100 Enterprise Drive
Rockaway, NJ, 07866

Smart Probes Offer Non-invasive In Vivo Imaging

Cell biologists today are not content with molecular imaging tools that just provide images of cells. They are looking for tools that can provide more information on what's going on inside the cell. Activity-based proteomics is a developing field that uses small molecule probes as reporters to study the function of specific target proteins and their role in disease pathogenesis.

Matthew Bogyo, Ph.D., assistant professor in the department of pathology at Stanford University in CA, is very involved in this field. His group has synthesized a series of 'smart' probes to specifically study the papain family of lysosomal cysteine proteases, also called cysteine cathepsins, which are known to play an important role in tumorigenesis. These 'smart' probes are small drug-like molecules that covalently bind the active site of a protease. "They act as what's called a suicide substrate," says Bogyo. The probe molecule consists of three parts a reactive 'warhead' that binds to the active site, a group that confers specificity and directs binding to the target protein, and a reporter tag that allows the probe-labeled protein to be visualized. The probes that Bogyo has designed have near-infrared fluorescent reporter tags that enable non-invasive imaging by allowing the emitted light to pass through the skin.

Optical imaging of tumors in live mice using quenched near-infrared fluorescent activity-based probes. The image shows a nude mouse bearing two xenografted tumors of human breast cancer cells. The tumor on the left is derived from a cell line that has low cathepsin activity and the tumor on the right is derived from a cell line with high cathepsin activity. The probe was administered by tail vein injection (hence the background in the tail). The image of the live mouse was taken using the IVIS 200 imaging system (Xengoen, Alameda,CA) 24 hours after probe injection. (Source: Matthew Bogyo)

Bogyo's group recently published a paper in Nature Chemical Biology showing that the probes worked well for non-invasive imaging of tumors in mouse xenograft models. Many groups in the past have published studies using near infra-red fluorescent (NIRF) probes for in vivo animal imaging. "The difference from what's been published previously is that most methods used either proteins or antibodies as a way to deliver these fluorophors," says Bogyo. "Those are really large molecules that can't get into cells, are pretty slow to distribute and turn over pretty rapidly. Ours are small molecules that have drug-like properties, so it circulates throughout the animal rather rapidly. We can detect specific signals after 30 minutes, which is much faster than with any other technique out there."

This rapid distribution and detection of signal using the probes has led to an interesting collaboration with a group at Case Western Reserve University in Cleveland, Ohio. The probes are now being applied topically to mice with brain cancers, prior to surgery, to help distinguish gliomas which are known to have high protease activity, from normal tissue. "It's the kind of thing where during surgery, it works fast enough that a surgeon could actually have a hand-held device to light up the tumors and it allows them to determine if they are getting all of the tumor out," says Bogyo. The probes have exciting applications in basic research as well. "We're starting to look at the role of various proteases during development in zebrafish. We're also thinking of using these probes in some genetically tractable organisms like Drosophila," says Bogyo.

The group is also looking at other applications where protease activation has been determined such as imaging atherosclerotic plaques in blood vessels and gastrointestinal imaging in colon cancer. Currently the main limitation to using this technique is that it works only in tissues that show elevated protease activity. "Clearly in tissues which normally have high levels of these kinds of [cysteine] proteases, like the liver, kidney or spleen, it would be hard to pick out small tumors in the background." However, there are other types of head, neck and brain tumors where it could work successfully. "We are also starting to get some evidence that we can see metastasis in the lung," says Bogyo. "That would be huge because the current technique for detecting lung tumors is standard chest x-rays and by the time you can detect the cancer on chest x-rays, you are out of luck for being treated."

Bogyo is hoping that if they can get past safety assessment in animals and get it into FDA-approved clinical trials, then eventually the technique could be used systemically for routine types of examinations. The group is also looking to broaden the types and classes of probes used by designing ones that target other proteins, like caspases. "By using different combinations of probes we might actually get signatures that are more specific to a tumor and would allow us to look at tumors in tissues with high background from other proteases. We have to do a lot more chemistry and design additional classes of probes to try and expand on this technique," says Bogyo.

Utilizing Inter-Disciplinary Skills

Matthew Bogyo did his undergraduate studies in chemistry at Bates College, a private school in Maine, and then joined the doctoral program in organic Chemistry at MIT. Along the way, an immunology course that he took got him very interested in biology. "I ended up doing my Ph.D. working in chemistry but in an immunology lab," says Bogyo. "I got interested in proteases and started using my chemistry skills to study them." After finishing his Ph.D., Bogyo established his own independent lab as a faculty fellow at the University of California in San Francisco. But he later left academia to pursue small molecule drug discovery at Celera Genomics. "I eventually decided I liked the academic setting better and moved to Stanford about four years ago." Today his group in the department of pathology at Stanford is split between biologists and chemists and they all work in both areas the biologists do a some chemistry and the chemists do some biology. "I'm trained as a chemist but I've always worked more in a biological setting. So I'm always trying to apply chemical methods and tools to solve biological problems," says Bogyo.

Utilizing Inter-Disciplinary Skills

Matthew Bogyo did his undergraduate studies in chemistry at Bates College, in Lewiston, Maine, and later joined the doctoral program in organic Chemistry at MIT. Along the way, an immunology course got him interested in biology. “I ended up doing my Ph.D. working in chemistry but in an immunology lab,” says Bogyo. “I got interested in proteases and started using my chemistry skills to study them.” After finishing his Ph.D., Bogyo established his own independent lab as a faculty fellow at the University of California in San Francisco. He left academia to pursue small molecule drug discovery at Celera Genomics, but “...eventually decided I liked the academic setting better and moved to Stanford about four years ago.” His group in the department of pathology at Stanford is split between biologists and chemists and they all work in both areas. Says Bogyo, “I’m trained as a chemist but I’ve always worked more in a biological setting. I’m always trying to apply chemical methods and tools to solve biological problems.”