University of Pennsylvania School of Medicine scientists have developed a high-throughput functional cell-based screen using bioluminescence imaging to identify small molecules that modulate p53 transcriptional activity or p53-related proteins in cancer cells, and exhibit anti-cancer activity in human colon cancer xenografts.
The study underscores the importance of p53-related pathways and downstream molecules as potential drug targets, and could help establish bioluminescent cell-based assays as an effective way to screen modulators of such targets.
In addition, the research, which used an imaging system from California biotech Xenogen, provides an example of how the planned acquisition of that company by Caliper fits into Caliper's overall drug-discovery technology business.
As detailed in the July 11 online edition of the Proceedings of the National Academy of Sciences, the scientists, led by Wafik El-Deiry, professor of medicine, genetics, and pharmacology at UPenn, expressed a firefly luciferase gene under the control of p53-responsive elements in p53-deficient human colon cancer cells.
The idea, El-Deiry told CBA News this week, was to build a construct that could report on the downstream activation of molecules or transcriptional responses to p53 in cancer cells that didn't actually have a functional p53 molecule. This is typically the case in actual human tumor cells, but not in normal cells where p53 is crucial for arresting the cell cycle.
"The day it came to the lab I realized we could do this type of screening."
"We're looking for surrogate ways to activate that program," El-Deiry said. "It's a huge problem, because cancers lose their p53 function. We've only known about the p53 family in the recent past, and what's interesting is that you almost never have mutations in the p53 family members. So they're just sitting there waiting to be activated and exploited in drug development."
According to El-Deiry, many efforts have been made to restore p53 function in tumor cells, but almost no one has targeted the downstream molecules that p53 is supposed to activate in normal cells. The hope is that small molecules that can trigger these downstream responses could prove to be promising leads for anti-cancer drugs.
In their study, the UPenn scientists screened approximately 2,000 chemical compounds from the National Cancer Institute's Developmental Therapeutics Program's small-molecule library against the luciferase reporter cell lines. To do this, they plated the cells in 96-well plates, added various concentrations of compounds, and imaged for p53 transcriptional activity using an IVIS multimode imaging system from Xenogen.
Primary and secondary screens with this method uncovered 33 compounds that appeared to induce p53-responsive reporter activation at low drug doses, but induced cell death at higher drug concentrations or later time points.
They subsequently tested a select number of these compounds for their anti-tumor activity in colon-tumor xenograft-bearing mice by measuring the reduction of tumor weight over time, as well as changes in bioluminescence of xenografted reporter cells — again using the IVIS system for the latter measurement.
Overall, the researchers identified a handful of compounds that both stimulated p53 transcriptional activity in vitro and shrunk mouse tumors in vivo. El-Deiry said that the group intends to further investigate the pathways or downstream p53 family proteins on which these molecules might be acting.
In addition, the group, in conjunction with a startup El-Deiry founded as well as with other academic, government, and commercial drug-discovery operations, hopes to ramp up the scale and throughput of the bioluminescent cell-based screening method to conduct additional small-molecule screens (see below).
The Caliper Fit
The use of a Xenogen IVIS imaging system by El-Deiry and colleagues to conduct both cell-based assays in well plates and small-animal imaging provides a clue about how Xenogen's technology might fit into the drug-discovery business of Caliper, which announced it would acquire Xenogen in February (see CBA News, 2/17/2006).
"You almost never have mutations in the p53 family members. So they're just sitting there waiting to be activated and exploited in drug development."
Xenogen's imaging systems were originally designed almost exclusively with small-animal imaging in mind. The IVIS and other Xenogen platforms typically consist of a stage area large enough to accommodate mice, or perhaps large well plates or slides containing zebrafish or nematodes. The company has in the past touted the usefulness of its systems for cellular imaging in well plates, but the majority of the company's customers use the platform for small-animal imaging.
So when Caliper announced the intended acquisition, it seemed like an odd fit for a company whose drug-discovery business focuses primarily on microfluidics-based technology platforms.
Caliper's CEO Kevin Hrusovsky told CBA News at the time that Xenogen's capabilities fit into a larger strategy: to offer drug-discovery products and services spanning the entire continuum from in vitro biochemical assays to in vivo small-animal testing.
In addition, Caliper has said that the integration of its microfluidics technology with Xenogen's imaging technology is feasible in the future. Plus, Caliper now has a drug-discovery services arm through its addition of NovaScreen (see CBA News, 9/15/2005), which will likely incorporate Xenogen's imaging platforms into its offerings.
"The day I bought the IVIS — it was the first one in Philadelphia, I believe — and the day it came to the lab I realized we could do this type of screening," El-Deiry said. "I wasn't even thinking about that, necessarily. But it took me about two seconds of knowing what it could do to know that we could use it to do this."
Furthermore, the IVIS platform is capable of fluorescence and near-IR imaging in addition to bioluminescence, so other reporting methods are feasible. El-Deiry said that his group chose bioluminescence reporting for the p53 screening experiments because of its outstanding sensitivity, but that fluorescence-based assays or molecular beacon reporter mechanisms could also be used.
— Ben Butkus ([email protected])
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You Can Always Build It in Academia
The research conducted by Wafik El-Deiry and colleagues at the University of Pennsylvania's School of Medicine is another recent example of how academic labs in the US are increasingly building high-throughput drug-discovery operations.
But this particular case also happens to be the product of El-Deiry's difficulties obtaining venture capital financing for his startup company, OncoCeutics, which he founded two years ago to conduct the p53 pathway-based development of cancer therapeutics on a much larger scale. It is now on temporary hiatus due to its inability to raise funds.
"Funding is a problem for this type of work," El-Deiry said. "Everybody assumes that someone else is paying for this type of work."
El-Deiry eventually received funding for his work from the National Cancer Institute's Network for Translational Research and Optical Imaging program. Now he is taking a route frequented by many of his colleagues in academia — to set up high-throughput drug-discovery operations under the university umbrella.
El-Deiry said that his group is currently pursuing interactions and collaborations with other entities — including the National Cancer Institute Developmental Therapeutics Program and the Broad Institute — to scale up its efforts. The group has also recently hired a medicinal chemist to help with efforts.
"I've been inspired by many of my colleagues, and we think that we have something important to contribute, not the least of which is that we're not necessarily driven by the bottom line of profit," El-Deiry said. "On the other hand, I think it wouldn't take much for people to realize the potential impact of this type of screen for agents that could target drug-resistant cancer — which is a huge problem.
"It's unfortunate that many of the very large companies don't support this kind of work at its earliest stages," he added. "It's taken us a long time to get to this point. I could be exactly where I am right now but have saved two years of my life if I had better support."