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  • Fluorescent probes poised to become part of drug-screening arsenal

Dec 2007
Probes label tumors in living mice with high specificity and reveal protease activity

David L. Shenkenberg

During a suicide mission, a warhead collides with its target. This is not a matter of national security but part of a biological investigation conducted by researchers from Stanford University School of Medicine in California.

The investigators used fluorescent probes designed to elucidate enzyme activity. Once the probes reach an enzyme, their chemically reactive “warhead” binds to it — a suicide mission because they become attached permanently. Enzyme activity modifies them, so they report the activity, not just the location of potential enzymes. Because they bind to enzymes, they enable direct correlation with biochemical data, ensuring that the fluorescence corresponds to the target enzymes and not to nonspecific binding.

Figure 1. Researchers developed fluorescent probes that specifically label cysteine cathepsins, which are potential drug targets. Shown here is a tumor cell labeled with one of the probes and with a commercial fluorophore that stains lysosomes. The top, middle and bottom images show the signal from their fluorophore, the commercial fluorophore and the overlap of the two signals, respectively. Images reprinted withpermission of Nature Chemical Biology.

The researchers applied these probes to monitor tumors in living mice for the activity of cysteine cathepsins, members of a class of protein-degrading enzymes called proteases. According to principal investigator Matthew Bogyo, cysteine cathepsins have been implicated in everything from cancer to Alzheimer’s disease, as well as in hardening of the arteries and inflammation. Therefore, cysteine cathepsins are becoming attractive drug targets, and these probes could serve as labels when screening for drugs that act on them.

According to Bogyo, his group also is investigating the probes for diagnosis of early-stage cancer and for labeling atherosclerotic plaques. He added that, as contrast agents for fluorescence tomography, the probes could be used to monitor the efficacy of tumor drugs in human patients.

Probe design

The investigators designed probes with a warhead made of acyloxymethylketones, which previous work had identified as nontoxic, water-soluble and highly selective for cathepsins B and L, which are expressed in many types of tumors. The scientists attached one warhead to Cy5 from GE Healthcare of Waukesha, Wis., and another to IRDye 800CW, a near-IR fluorophore from Li-Cor Biosciences of Lincoln, Neb. They tested the near-IR probe with and without the QSY21 quencher from GE Healthcare. In addition to functioning as a quencher, QSY21 enabled more favorable binding to cathepsin B. The target enzyme cleaves the quencher from the probe.

During the creation of the probe, they found that the ester chemical group linking the fluorophore and the warhead was being degraded by enzymes present in the serum. Therefore, they changed the ester linker to an alkyl group, which was not degraded.

Tumor grafts

The scientists grafted tumors onto mice, and, after three to four weeks of in vivo tumor growth, they injected the probes into the tail vein. The probes rapidly cleared the bloodstream, leaving only the tumors labeled. The researchers monitored the fluorescence with Xenogen’s Ivis 200 imaging system and used the accompanying software to analyze the images.

To correlate the in vivo images with biochemical data, they harvested proteins from the organs and tumors of the mice and ran the proteins on electrophoretic gels. They captured images of the gels using a Typhoon scanner from GE Healthcare and detected the fluorescently labeled proteins. Bogyo said that the scanner was useful because it enables simultaneous detection of multiple fluorophores — a result of its design with multiple lasers and filter sets.

As detailed in the October issue of Nature Chemical Biology, the fluorescent probes labeled the tumors with high specificity and with signal-to-noise ratios that reached nearly 10-to-1 (Figure 2). The probe containing IRDye 800CW yielded higher signal-to-noise ratios over time than did the one containing Cy5, owing to its longer absorption and emission wavelengths, which enable greater penetration of the excitation source and of the fluorescent signal. The quenched probes reached a high signal-to-noise ratio faster than the unquenched probes.

Figure 2. Quenched near-IR fluorescent probes performed well for imaging live mice bearing grafted tumors.

Biochemical results correlated well with the imaging data, proving that the probes specifically label the cysteine cathepsins. The researchers concluded that the quenched near-IR probes are highly effective for in vivo labeling of cysteine cathepsins B and L.

The investigators also tested the effectiveness of the fluorescent labels for drug screening against K11777, a known inhibitor of cysteine cathepsin activity. The labels reported inhibition by the molecule.

Bogyo said that they plan to determine how stable the probes are in vivo and to move to preclinical testing such as for toxicity. They are developing activity-based probes that target serine proteases as well as cysteine cathepsins and are working on licensing the compounds to pharmaceutical companies.

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