Key to New Treatments: Discovering What Turns a Hedgehog Off

hedgehog proteins

Two research papers recently published by collaborating groups at Binghamton University, Binghamton New York, and the Rensselaer Institute in Troy New York have revealed evidence for a new pharmacologic strategy to inhibit the activity of hedgehog (Hh) proteins, and of a novel mechanistic link between zinc and the Hh signaling pathway.^1,2

“Pharmaceutical companies have been after Hh for years,” said Brian Callahan, PhD, assistant professor of biological chemistry at Binghamton University in a press release. Callahan explained that his group is not trying to compete with Big Pharma, but using a new strategy and going after Hh directly.³

“Hh is critical to normal development, but in adults, the pathway, if reactivated, may lead to uncontrolled cell growth and proliferation in cancer,” said Chunyu Wang, MD, PhD, associate professor in biological sciences at Rensselaer, member of the Center for Biotechnology and Interdisciplinary studies, in a press release. “Our paper suggests a link between zinc deficiency and activation of the Hh pathway in many diseases where zinc deficiency and Hh activation coexist.”⁴Both Callahan and Wang are authors on each paper.

What Makes a Hedgehog?

The Hh precursor protein contains three functional regions; an amino terminal domain, a central autocatalytic Hedgehog Intein region (“HINT” domain), and a carboxy terminal subdomain. An autocatalytic reaction reconfigures an internal peptide bond into a thioester and this is then displaced by cholesterol to produce HhN, the amino terminal, to which the cholesterol is attached. This step is known as cholesterolysis and is essential for HhN biological activity. Callahan et al note that it is specific to Hh proteins and reason that any small-molecule modulators of this step will be of interest.^1,2

Inhibiting a Hedgehog

Using a conventional gel-based cholesterolysis assay and an engineered Hh precursor protein, Callahan et al showed that the trivalent arsenical compound phenylarsine oxide (PhA) markedly inhibited Hh precursor cholesterolysis. The PhA was bound covalently and stoichiometrically to the precursor. That cholesterolysis was inhibited strongly suggested that the binding occurred in the HINT domain, and mapping the sites of the interaction was investigated using two nuclear magnetic resonance techniques. They identified ten residues as comprising the PhA binding site, and importantly that these residues in their engineered Hh are present in the HINT domains of human Hh proteins. In their paper Callahan et al also claim that the approach and techniques they used constitute a practical platform for discovering inhibitors of Hh cholesterolysis.¹

The Role of Zinc

hedgehog pathways

Introducing this work carried out jointly with Callahan, Wang states that chronic Hh pathway stimulation has been shown in prostate, lung, and ovarian cancers and that zinc levels decrease early in prostate cancer continuing to castration-resistant disease.^2,5-8The team considered that zinc may suppress the autocatalysis in the Hh precursor and inhibit production of biologically active Hh ligand.²

An in vitro approach established zinc inhibition of cholesterolysis monitored by SDS-Page. Increasing concentrations of zinc resulted in detection of more of the unprocessed precursor remaining, and that the effect was reversed on the addition of ethylene diamine tetra acetic acid (EDTA), a divalent metal chelating agent. Cell culture studies using astrocytes, again showed a concentration-dependent effect of zinc on Hh autocatalysis. Again, because autocatalysis was being inhibited by zinc, NMR was used to map zinc binding sites in the HINT domain. They detected reductions in NMR signals on the addition of zinc and these signals recovered following the addition of the chelator EDTA, and identified three key residues bound by zinc. When these three residues are bound, catalytic activity is impaired.²

Where Next?

“We show that zinc inhibits this autoprocessing reaction from the precursor to the ligand, providing an additional mechanism of how zinc deficiency may promote cancer development. This is something that nobody else has shown before,” said Wang. “Zinc and Hedgehog are essential and extremely versatile biomolecules; linking these two will have profound implications for normal physiology and disease.”⁴

Turning to the prospect of a new targeted therapy, Callahan mentioned that he would be collaborating with Michelle Arkin, PhD, associate professor in the school of pharmacy at the University of San Francisco. Arkin intends to search for molecules that inhibit the functions that Callahan described. “The best candidates will be tested in lab animals, probably sometime in 2017,” continued Callahan commenting that, “We want to get a molecularly targeted therapy, in essence, a magic bullet that kills the cancer and not the patient.”³

References

1. Owen TS, Xie XJ, Laraway B, et al. Active site targeting of hedgehog precursor protein with phenylarsine oxide.Chembiochem. 2015;16:55-58
2. Xie J, Owen T, Xia K, et al. Zinc inhibits Hedgehog autoprocessing: linking zinc deficiency with Hedgehog activation.J Biol Chem. 2015;290:11591-11600
3. Newswise. Binghamton University professor fights cancer with Hedgehogs.http://www.newswise.com/articles/binghamton-university-professor-fights-cancer-with-hedgehogs. Accessed October 2015.
4. Rensselaer. RPI News. Zinc deficiency linked to activation of Hedgehog signaling pathway.http://news.rpi.edu/content/2015/04/16/zinc-deficiency-linked-activation-hedgehog-signaling-pathway. Accessed October 10, 2015
5. Karhadkar SS, Bova GS, Abdallah N, et al. Hedgehog signalling in prostate regeneration, neoplasia and metastasis.Nature. 2004;431(7009):707-712.
6. Watkins DN, Berman DM, Burkholder SG, et al. Hedgehog signaling within airway epithelial progenitors and in small-cell lung cancer.Nature. 2003;422(6929):313-317.
7. Liao X, Siu MK, Au CW, et al. Aberrant activation of hedgehog signaling pathway in ovarian cancers: effect on prognosis, cell invasion and differentiation.Carcinogenesis. 2009;30(1):131-140.
8. Kolenko V, Teper E, Kutikov A, et al. Zinc and zinc transporters in prostate carcinogenesis.Nat Rev Urol. 2013;10:219-226.