Researchers from Abbott Laboratories and the Northwestern University School of Medicine have used a cell-based screen to suggest that mitochondria play an essential role in regulating the hypoxia-inducible factor-1 (HIF-1) pathway.
They also found that production of mitochondrial reactive oxygen species, or ROS, is a potential druggable target against tumor development and growth.
In the study, the researchers first screened an siRNA library against the entire druggable genome and failed to identify any targets.
“From my perspective, good traditional druggable targets covered by our siRNA library do not exist along the HIF-1 pathway,” Yu Shen, a group leader in Abbott’s cancer cell biology group, told CBA News this week. “When you look at mitochondrial ROS production, the pathway that our compounds hit, the compounds do not hit any traditional druggable target.”
In addition, the difficult part of the siRNA library screen is the off-target effect, said Shen. “Even if you get one or two real hits, because the number of off-target hits is so overwhelming, it is easy to miss the real ones.”
Writing in the Jan. 8 issue of the Proceedings of the National Academy of Sciences, the investigators noted that a “critical homolog of a critical target may need to be inhibited simultaneously to block hypoxia-dependent HIF-1 activation.”
In a second experiment, the researchers screened a small library consisting of 691,200 compounds using a HIF-1 reporter cell line, dubbed H1299-HRE, that was derived from a non-small-cell lung carcinoma cell line.
That screen identified a class of alkyliminophenylacetate compounds that inhibited hypoxia-induced HIF-1 reporter activity at single-digit nanomolar concentrations.
“It was pretty surprising that we got a number of very good, potent inhibitors,” said Shen, who added that, typically, in cell-based compound screens, once an inhibitor is identified, it is not always easy to discern the compound’s exact target.
In this particular HIF-1 study, the researchers glimpsed a number of “hints” that led them to identify a target site of those compounds “relatively easily,” said Shen, who was also corresponding author on the PNAS paper.
To arrive at the results of the second experiment, the group identified 250 HIF-1 reporter inhibitors from the screen and found that 18 of them shared the core structure of alkyliminophenylacetate, which was similar to the mitochondrial inhibitors described in previous publications.
“When we characterized some of the good inhibitors, we noticed that they behaved like some of the inhibitors for the HIF-1 pathway previously described in the literature,” said Shen.
For example, they inhibited the hypoxia-induced HIF-1α protein accumulation, but only moderately inhibited deferoximine (DFO)-induced HIF-1α accumulation. Shen explained that this differential impact of alkyliminophenylacetate compounds on hypoxia- and DFO-induced HIF-1α accumulation is characteristic of ROS scavengers and mitochondrial electron transport chain inhibitors.
“We identified the target relatively easily and then tested this hypothesis of whether the inhibitors were actually mitochondrial inhibitors,” Shen said. According to the researchers, cells are highly sensitive to mitochondrial inhibitors when cultured in media that contain galactose but not glucose.
“It was pretty surprising that we got a number of very good, potent inhibitors.”
Cells were cultured in either glucose- or galactose-containing media and then exposed to alkyliminophenylacetate compounds selected from Abbott’s internal library. Many of the compounds were found to induce cell death in the galactose-containing media at very low concentrations, but none induced cell death in glucose-containing media.
In the parallel experiment, the ability of each compound to inhibit the HIF-1 reporter was found to directly correlate with its ability to induce cell death in the galactose-containing media.
“We found further evidence that alkyliminophenylacetate compounds specifically inhibit mitochondrial ROS production,” said Shen. In control cells, hypoxia triggered a 3-fold increase of 2,7-dichlorodihydrofluorescein diacetate, or DCF, a cell-permeable, fluorescent indicator of ROS levels.
Treating the cells with alkyliminophenylacetate compounds, however, significantly reduced hypoxia-dependent ROS production.
Shen said his team was surprised to find that nonalkyliminophenylacetate inhibitors induced robust cell death in galactose-containing media at very low concentrations, which supports the hypothesis that inhibiting mitochondrial ROS production is a mechanism common to structurally diverse HIF-1 inhibitors.
“We were pretty surprised” by this finding, Shen said, adding that he felt the likeliest explanation for this result is that the ROS pathway is the most sensitive way to inhibit HIF-1.
Although Shen declined to comment on Abbott’s future work in this area, the researchers pointed out in this paper that it remains to be seen whether the alkyliminophenylacetate compounds are tolerated in animal models in doses that inhibit HIF-1.
“Instead of inhibiting the electron-transport chain, the use of antioxidants is an attractive alternative for inhibiting hypoxia-dependent ROS production,” they wrote.