Researchers at Columbia University and Prolexys Pharmaceuticals have used a pull-down assay to identify a target for the experimental small molecule anti-tumor agent erastin.
Prolexys is also using erastin as an experimental probe to identify the protein target and better understand its mechanism of action, with the goal of creating a drug-discovery franchise around the biology of the protein target, Sudhir Sahasrabudhe, Prolexys’ CSO and co-founder, told CBA News this week.
Brent Stockwell, an associate professor of biology and chemistry at Columbia, said that he and his team are taking a novel approach to screening potential anti-cancer compounds. They want to do what they call a “synthetic lethal” screen in which they look for compounds that have a genetic selectivity similar to erastin.
Sahasrabudhe said Prolexys is using a similar strategy for two additional programs: the heat shock protein-20 pathway and the beta-catenin pathway.
For the erastin research, the investigators discovered that the compound bound to two isoforms of the voltage-dependant anionic channel protein. The researchers used a pull-down assay to identify VDAC as erastin’s target.
The team synthesized active and inactive analogs of erastin and linked them to a solid-phase resin, according to Stockwell.
The researchers incubated the resin with a lysate of engineered tumor cells expressing multiple combinations of oncogenes, such as hTERT, the SV40 large and small T oncoproteins, the human papillomavirus type 16 E6 and E7 oncoproteins, and oncogenic HRAS.
Stockwell said that the authors found all three VDAC isoforms on the resin containing the active analog, and they found VDAC1 on the resin with the inactive analog as well.
The results, which appear in the June 14 issue of Nature (see CBA News, 6/15/07), suggest that VDAC1 may or may not be relevant to the mechanism of erastin, but because VDAC2 and VDAC3 seemed to be specifically pulled down by the active analog of erastin, they were more likely to be relevant to its mechanism of action, said Stockwell, a co-author on the Nature paper.
To demonstrate that VDAC2 and VDAC3 in some way pertained to erastin’s activity, Stockwell said the researchers tried to knock down VDAC using a lentiviral short hairpin RNA system. They found they could knock down VDAC2 and VDAC3 and get resistance to erastin, but if they knocked down VDAC1, they did not see any resistance.
The researchers also found that when cells express oncogenic RAS they express increased levels of VDAC proteins by about four-fold.
Sahasrabudhe said that these findings are significant to Prolexys because, although researchers have known for some time that VDAC influences cell death, its role in cancer is poorly understood. He said that Prolexys decided to license erastin from Columbia and MIT-Whitehead only after it obtained the protein-binding data.
What interested Sahasrabudhe was the fact that erastin displayed robust toxicity against the RAS mutated cell line but showed no toxicity towards the isogenic cell line that expressed normal RAS. He said it seemed like an ideal setting to directly screen and identify anti-cancer compounds that may be selectively toxic to cells with an aberrantly active RAS pathway.
However, erastin is not very potent against VDAC, and it exhibits some undesirable pharmaceutical properties, said Sahasrabudhe. He said that the Prolexys team decided to see if they could identify a protein target that would specifically bind to the compound, and begin to better understand the mechanism by which erastin selectively targets cancer cells with activated RAS.
The initial assay was a simple, multi-well cell viability screen using Alamar blue to determine the viability of the cells in the presence or absence of erastin, said Sahasrabudhe. Prolexys researchers seeded the cells at a density of 6,000 cells per well and incubated for 24 hours with the test compound. After incubating the cells for an additional 16 hours with Alamar blue, the team used a Packard Fusion plate reader to measure fluorescence intensity.
At Prolexys, the initial screen was performed using the same engineered fibroblast cell lines used by Stockwell, said Sahasrabudhe. The researchers immediately followed it up with a cell-line panel of 35 tumor cell lines, many of which have aberrantly active RAS pathways.
Sahasrabudhe said the initial screening was done in cells with an HRAS activating mutation. However, the two RAS isoforms that seem to be most prevalent in many tumor types are KRAS and NRAS — NRAS in melanoma cells and KRAS mutations in a variety of cancers, including pancreatic, colon, and ovarian cancer.
The Prolexys investigators primarily used the pancreatic cell line PANC-1 (KRAS mutation) and the fibrosarcoma cell-line HT-1080 (NRAS mutation) to test erastin analogs. Sahasrabudhe said that the analog dubbed PRLX 93936 is active in a large number of tumor cell lines, including many with an aberrantly active RAS pathway.
PRLX 93936 is also highly potent in non-small cell lung cancer cells resistant to EGFR inhibitors and in NSCLC cells with activated KRAS mutations, said Sahasrabudhe. Significant activity of 93936 was also found in colon cancer, ovarian cancer, renal cell cancer, and melanoma cell lines.
Sahasrabudhe said the researchers selected 93936 because they began to heavily depend on their panel of colon cancer cell lines, particularly PANC-1, HT-1080, DLD-1, OVCAR-5, and SK-Mel-28.
The team used Sytox to measure the cytotoxicity of 93936, and employed an additional measurement by lysing the cells with saponin, Sahasrabudhe said.
He said that the Prolexys team’s next step was to implant nude mice with xenografts derived from the HT-1080, PANC-1, DLD-1, OVCAR-5, and SK-Mel-28 melanoma cell lines. Once the tumor grew to a particular size, the animals were treated with 93936.
Regardless of the route of administration, higher doses of 93936 yielded complete tumor regression, Sahasrabudhe said. Based on the xenograft data, they started to optimize the compound and began to select and prioritize those compounds that had, in addition to improved potency in the tumor cell lines, desirable pharmacokinetic profiles and solubility. The compounds also had an acceptable toxicology profile in rodent and non-rodent animal species.
Sahasrabudhe reported that the compound maintained selectivity in vivo with no myelosuppression and no toxicity in normally dividing cells at the dose levels required for anti-tumor activity. The selective toxicity of PRLX 93936 for tumor cells with an aberrantly active RAS pathway may be because of the disruption of VDAC function in tumor cells, Sahasrabudhe pointed out. However, the molecular mechanism of action of PRLX 93936 is not fully understood.
Prolexys, formerly Myriad Proteomics, was founded in 2001 in Salt Lake City as a proteomics technology platform company. The goal was to develop a protein-protein interaction database looking at interactions one tissue at a time in both diseased and healthy human tissues.
“As a result of our medicinal chemistry effort, erastin’s solubility was improved by about 3 log cycles and its potency by about 2 log cycles.”
However, it became clear by late 2002 and early 2003 that a market no longer existed for biotech companies that claimed to produce data sets for faster and more efficient drug discovery, said Sahasrabudhe. Prolexys decided to look inward and ask if it could do something with the technology suite that it had already assembled to ensure that the information was used for drug discovery.
Sahasrabudhe said Prolexys decided it couldn’t do conventional target identification and validation-based drug discovery because it had only two to three years worth of cash left. So it took erastin and used it as a template to create analogs and identify a small-molecule clinical candidate with desirable pharmaceutical properties.
“Within about two years of licensing erastin, we have the clinical candidate pinned down, which is totally impossible if you are using conventional means of finding and validating a novel target,” Sahasrabudhe said. “In addition, we are learning more about the biology of VDAC proteins and their potential role in cancer cell death.”
Prolexys is also looking at the HSP-20 pathway, said Sahasrabudhe. The company’s scientists use a fluorescence polarization-based, protein-based screen where they have a phosphorylated HSP-20 peptide that binds to its protein target, which at Prolexys is protein 14-3-3 gamma.
He said the researchers look for small-molecule mimetics of phosphorylated HSP-20. These compounds are applicable in the therapeutic areas of asthma and pulmonary hypertension. He reported that the company has a number of hits; specifically it has identified three scaffolds, each supported by a number of analogs.
However, because Prolexys is a small company, it needs to focus its resources on PRLX 93936, said Sahasrabudhe. Although the company has temporarily put aside the HSP-20 program, it plans to revive it as soon as more resources become available.
Sahasrabudhe said the third program under way at Prolexys involves colon cancer. The company has extensive data on beta-catenin protein-protein interactions, he said.
The beta-catenin pathway increasingly appears to be important in the area of cancer stem cells, said Sahasrabudhe. The company considers the pathway a franchise, in which it has collected a lot of biochemical information regarding what proteins bind to it, and has assembled what it calls the beta-catenin interactome. Using the information provided in the interactome, researchers could test several potential disease targets that are druggable, Sahasrabudhe said.
While Prolexys translates the 93936 erastin analog into a potential therapeutic, Stockwell and his team are working to better define erastin’s novel mechanism for killing RAS-expressing tumor cells.
Much is still not understood about why the binding of erastin to VDACs 2 and 3 triggers cell death, Stockwell said. The other question is, “How are these cells actually dying?” Stockwell said.
Stockwell said that the results presented in this paper suggest erastin-treated cells do not die through apoptosis. They instead appear to die through some novel form of oxidative cell death. “We want to understand on a mechanistic level how binding to VDAC causes oxidative cell death,” said Stockwell.
Stockwell said that he and his team have identified additional compounds that selectively target RAS-expressing cells, and have done some follow-up studies on the mechanism of action of those compounds. Some of this work relates to erastin’s mechanism of action. That manuscript has already been submitted to Chemistry & Biology, said Stockwell, and the authors are waiting for it to be reviewed.
In other words, they are not generally lethal, but become lethal in the presence of the right oncogene. Then they want to find the mechanism of action of that compound that allows for its selective lethality.
The advantage of this approach is that researchers can discover novel targets which they may not have thought of and whole new pathways, such as the one involving VDAC-mediated cell death.