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Plexxikon Combines Cellular, Structural Assays to ID Specific, Potent Inhibitors

Researchers at Plexxikon and the Wistar Institute have used the company’s internally developed scaffold-based drug-discovery platform with cell-based assays and animal models to show that a cancer candidate developed by Plexxicon can “potently and selectively” destroy certain skin cancer cells.
The researchers claim that in vitro and in vivo studies show that the compound, known as PLX4720, can kill tumor cells that contain an activating mis-sense valine-to-glutamate mutation of the BRAF oncogene, a mutation that occurs only in tumor cells.
Gideon Bollag, Plexxikon’s vice president of discovery biology, told CBA News this week that the company has used the same combination of methodologies to discover an entire portfolio of selective B-RafV600E inhibitors.
The cell-based assays used in the study, which is published online in the Feb. 26 issue of the Proceedings of the National Academy of Sciences, serve two purposes: to show that the candidate compound hit the target — in this case the mutant B-Raf protein — and to show that by hitting that target it triggered an appropriate physiological response — in this case, inhibition of cellular proliferation.
Bollag said that the melanoma cell biology, three-dimensional tissue culture experiments, and in vivo melanoma xenograft studies presented in the paper were performed at the Wistar Institute.
According to Bollag, the company has “assembled a library of small molecules that are quite different from other libraries in that they conform to a very specific set of physical properties,” such as smaller molecular weight, fewer hydrogen-bond interactions, and fewer rotatable bonds.
Bollag pointed out that the compounds are not going to be very potent because they lack all of these extra moieties. “The first step was to take this library of compounds and screen it against [the mutant B-Raf protein] at a very high concentration,” he said. “We then identified compounds that were clearly bound to the protein based on the biochemical assay, and then took quite a few of them and set up co-crystallization reactions.”

“We are collaborating with Roche to demonstrate that PLX4032/R7204, which works nicely on paper … can be translated to patients.”

Bollag said that this was made possible after the company developed an automated high-content co-crystallography platform that allows “us to work with large numbers of very weak compounds by co-crystallizing them with the protein.”
“Now we have a weak compound, bound to the active sight, in this case, of B-Raf,” he said. “The chemists, the computational chemists, and the structural biologists sit down and look at how the compound binds.”
Bollag said that it soon became clear which sites one would focus on to build a more potent and selective inhibitor. “We specifically selected the melanoma lines used in the paper so that we would have the V600E B-Raf mutation or not,” Bollag explained. The researchers treated them with the compounds and determined if they hit their target by measuring the phosphorylation events that are downstream of the B-Raf protein, namely the phosphorylation of the oncogenes ERK or MEK.
Bollag reported that the investigators found that inhibition of ERK or MEK phosphrylation by PLX4720 only happens in cells with the V600E mutation and not in those with wild-type B-Raf.
They then found that PLX4720 inhibited the proliferation of only those cells with the V600E B-Raf mutation.
“Based on this technology and our ability to develop potent compounds, we select compounds for clinical trials,” said Bollag.
The company also has a B-RafV600E inhibitor, called PLX4032/R7204, that it is developing with Roche in a phase 1 clinical trial in cancer patients. Enrollment is complete for the dose-escalation phase of the trial. The next phase of the trial will test efficacy of the drug only in melanoma patients with the B-Raf mutation.
“We are collaborating with Roche … to demonstrate that PLX4032/R7204, which works nicely on paper in preclinical cellular and in vivo assays, can be translated to patients,” Bollag said.
The collaboration with Plexxikon on PLX4032 and a research program to identify back-up and follow-on compounds started in September 2006, and is a portfolio investment in oncology, Peter Singer, global alliance director at Roche, told CBA News in an e-mail.
“PLX4032 offered a promising pre-clinical profile as a first-in-class oral cancer drug,” Singer said. “In pursuit of our strategy to develop personalized medicines, this collaboration also offered the potential to develop a diagnostic test.”
“What makes this particular alliance so powerful is the added collaboration of Roche Diagnostics to develop an in vitro diagnostic test … to identify the patients who may have the greatest therapeutic benefit from treatment, increase the probability of success, and further accelerate drug development,” Singer said.
Terms of the partnership call for Plexxikon to be responsible for IND and Phase 1 studies, and for Roche to complete further development, Singer explained.
Singer added that Plexxikon could receive bonus milestones and a royalty step-up if accelerated development is achieved.

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