Using zebrafish, researchers at the Institute of Genomics and Integrative Biology (CSIR-IGIB) have successfully discovered a pathway that regulates hepcidin hormone production. The hepcidin hormone, released by the liver, is a central regulator of iron in the body. Dysregulation of the hormone leads to anaemia on one hand and excess iron accumulation in organs such as liver and heart leading to multi-organ failure.
Hemochromatosis is a rare hereditary disease that is characterised by iron accumulation or overload in various tissues. The symptoms are non-specific and hence difficult to diagnose. Current options only manage the disease by removing excess iron. “One method is to bleed the patients and the other is to absorb iron using iron chelation, which is toxic to liver and kidney and may also cause hearing problems. There is no therapy currently available,” says Sandeep Basu from CSIR-IGIB and first author of a paper published in ACS Chemical Biology.
Gene mutations
Mutations in about six genes are known to cause reduction in hepcidin hormone production thereby causing excess iron accumulation. But for the study, the research team led by Chetana Sachidanandan created a disease model in zebrafish (a fish exhibiting similar characteristics of the human disease) by mutating one of these genes (TFR2). Mutations in the TFR2 gene cause a severe form of the disease.
The zebrafish with the mutant gene showed excess iron accumulation in organs, quite similar to what is seen in humans.
In the conventional drug discovery approach, the target protein and even the pathway are already known and molecules that would either inhibit or overproduce the target protein are screened for. But the researchers adopted a different approach for this study.
“Hepcidin gene is controlled by many factors, many of them not well understood. So, instead of picking one target for our drug screen, we focused on the symptoms of the disease — excess accumulation of iron,” says Dr. Sachidanandan. “We knew that hepcidin hormone is low in the hemochromatosis patients, and that this causes iron overload.”
Since the researchers knew hepcidin production is regulated by many signalling pathways, they selected 80 compounds that specifically target signalling pathways in zebrafish. Of the 80 compounds tested, eight were found to induce the production of hepcidin hormone in the fish. “One of these compounds is a blocker of NFkB signalling pathway. This pathway was not known to be important in hepicidin regulation in the liver.” This is the first time that researchers have been able to identify and tell that the NFkB pathway regulates liver hepcidin production.
Unbiased strategy
“It was not a blind screening, but rather we used our existing knowledge about hepcidin regulation. When we don’t have a deep understanding of a disease, but need to discover drug candidates, this kind of unbiased strategy is more efficient,” she says.
The researchers tested the ability of this compound to regulate hepcidin production. “The small molecule targets and inhibits the NFkB pathway signaling thus increasing hepcidin production and reducing iron overload,” says Basu. They tested three more compounds that target the same pathway to confirm that the NFkB pathway is indeed a key to regulating hepcidin production. “There are other signalling pathways that regulate hepcidin production but our discovery places NFkB pathway as an important negative hepcidin hormone production,” Basu says.
When the pathway in the zebrafish model of Hemochromatosis was inhibited using the four compounds, the hepcidin production was restored and iron overload was reduced, thus reducing the severity of the disease.
To further reconfirm the role of the NFkB pathway, the researchers removed it in zebrafish. This led to overproduction of hepcidin hormone in the fish, confirming that the pathway negatively regulates the production of the hormone.
None of the four compounds tested in zebrafish is approved for use in humans; one compound tested is approved for use in poultry. “The target pathway has been identified even though the tested compounds cannot be used as drugs. What is important is that we have found a strategy for therapy,” says Dr. Sachidanandan. “Now that we know the pathway to be targeted, it’s easier to discover drug molecules.”
“In India, Hemochromatosis is still not commonly seen, perhaps because of our underlying iron deficiency. But Thalassemia, is a serious problem where iron overload is very common. We are now working to see if we can apply this therapeutic strategy for Thalassemia patients,” Basu says.
