Breathing life into signalling pathways
Understanding how the body senses oxygen leads to major spin-out success.
1 May 2012
People are able to live at a variety of altitudes, from below sea level to up on mountains high. This is because the body can sense changes in atmospheric oxygen levels and adjust its metabolic activities to suit the local environment. Who'd have thought that a close look at how the body's cells sense oxygen could breathe new life into drug discovery, lead to new compounds that could combat diseases ranging from anaemia to cancer, as well as promote healthy ageing.
Using a variety of funding sources from BBSRC and other organisations, Professor Chris Schofield, Peter Ratcliffe and colleagues at the University of Oxford teased apart the complex molecular biology of how cells detect, and respond to, oxygen levels. The mechanism involves a cascade of proteins and metabolites that can lead to increased red blood cell and blood vessel formation.
Realising knowledge of this fundamental mechanism could lead to new drug targets for conditions including diabetes and heart disease, spin-out company ReOx was formed which catalysed further work by major pharmaceutical companies. "It opened up a whole new field of research," says ReOx co-founder Schofield. (See 'Timeline').
The work gathered pace a decade ago when Schofield teamed up with Ratcliffe's group, also at the University of Oxford, to try to unravel the molecular mechanism by which humans and other animals react to changes in atmospheric oxygen levels.
Low oxygen levels are important because they are associated with biological processes applicable to many diseases. For instance, cancer tumours are very often hypoxic (meaning using lower than normal oxygen levels) because they are rapidly dividing. Similarly, ischaemic heart disease, such as occurs after a heart attack, can result from lowered oxygen levels in heart tissues, and diabetes sufferers can be forced to suffer limb amputation due to impaired oxygen delivery to tissues.
A 'master regulator' of the hypoxic response had already been identified as a transcription factor – a protein that binds to DNA to regulate what portions of the genetic code are 'read'. This master regulator, called hypoxia inducible factor (HIF), activates a cell's stress response to low oxygen levels in organisms ranging from the simplest animal, Trichoplax adhaerens, all the way to humans (ref 1). Schofield says that their big discovery was identifying a set of enzymes that regulate both the levels and activity of HIF – the HIF hydroxylases (ref 2, ref 3, ref 4).
"Because the HIF enzymes have a requirement for atmospheric oxygen they are able to act as oxygen sensors," says Schofield. "Basic research funded by BBSRC in terms of structural and mechanistic studies was absolutely crucial in identifying the HIF hydroxylases."
The team went on to produce recombinant enzymes [inserting the enzyme DNA into bacteria] that led to crystal structures for HIF-hydroxylases being deduced, along with details of their mechanisms of action (ref 5). "This work had widespread medical interest because of the wide role of HIF in many diseases," he explains. "It was the first time that post-translational hydroxylation [how the protein-making activity of DNA is modified] had been shown to play a role in signalling inside in cells." (See 'How it works').
Measure, signal, mimic
Follow up proof-of-principle analyses using funding for postdoctoral workers from the BBSRC-EPSRC Selective Chemical Intervention in Biological Systems programme, as well as a BBSRC Training Grant, developed research on selective inhibitors – most importantly that inhibition of the HIF hydroxylases turns on HIF-target genes that would boost blood vessel and red blood cell formation (ref 6, ref 7). This hit a biomedical bullseye: a standard treatment for ischaemic heart disease is the natural hormone erythropoietin (EPO) which is banned in sports use because of its ability to boost blood cell formation.
"My raison d'être is not to start up companies but to do basic research" Schofield explains. "But we were aware that as oxygen sensors these enzymes could be useful drug targets." Inhibiting the HIF hydroxylases would have a noteable effect on human physiology because the HIF system is a core regulator for changes in oxygen levels.
However, the researchers were aware that developing the enzymes as genuine pharmaceutical targets would require a significant financial investment. "I was very keen to see the project taken on by major pharma companies, ideally more than one," says Schofield.
But despite Schofield and his colleague Ratcliffe (funded by the Wellcome Trust) visiting a number pharmaceutical companies and saying that this was the most important thing they'd ever worked on, the general response was along the lines of: "... gentlemen you're doing great science but the project is too early for us."
By this stage, patents had been filed with Isis Innovations, a commercial branch of the University of Oxford founded to exploit intellectual property arising from research, and in due course the spin-out company ReOx was founded which duly set about developing selective inhibitors of the for HIF hydroxylases for use in animal models or cellular studies.
After ReOx had been running for a couple of years it became clear that big pharma companies were genuinely interested. The company chose to partner with Amgen, a US California-based company, as their major product was EPO which is also regulated by the HIF hydroxylases. "So you could see their interest in what we were doing," says Schofield. "EPO is an important treatment for anaemia with annual sales of about US$12Bn."
Amgen paid a fee to gain a license to take the technology in-house and try to develop a new generation of EPO-like drugs and inhibitors that will safely do the same job – EPO is a natural hormone in the body after all. Hence, the Oxford academics and ReOx scientists worked with Amgen to help them develop potent and selective HIF hydroxylase inhibitors.
Back to the bench
And that is very much where this part of the story ends, for Schofield and his colleagues who founded ReOx at least. Rather than forming a company aimed at developing a drug, technique or formulation all the way to commercial sales, they were happy that they'd persuaded big pharma to take on their discovery. To develop a new small molecule based therapy to market may take hundreds of millions of pounds; in most cases it's not credible for a small company to do it alone.
"For us it was job done. Rather than engaging in more pharmaceutical development, we wanted to move on to continue basic research," says Schofield. "ReOx made a profit, and if a company's drug comes to market there will be milestone payments."
Leaving the costly development – and not to mention testing – to the experts, this left Schofield able to get back to what he loved best – basic science involving interesting chemistry and biology. "So we've gone back to our labs – ideally we'll find some new targets!"
More articles about the impact of University of Oxford science can be found on the Oxford Impacts webpage, including a shorter summary of this work.
- 1980s: BBSRC-funded basic research in Oxford reveals the mechanism by which the beta-lactam antibiotics, including the famous penicillins and cephalosporins are synthesized. The structure reveals that related enzymes are widely distributed in nature.
- Mid-1990s: Schofield refocuses his research group to look at the role of some oxygenases in human biology, originally using BBSRC funding.
- 1997-2000: BBSRC-funded work on small molecules (chlorophyll metabolism) provides template for later work on other human enzymes.
- 1999: Schofield teams up with Peter Ratcliffe and colleagues to work on the mechanism by which levels of the HIF sub-alpha unit are degraded in the presence of oxygen.
- 2000: Work shows that a key event in the oxygen-dependent degradation of HIF-alpha is hydroxylation in one of two proline residues. Paper published in Science; analogous discoveries by Bill Kaelin's group at Harvard University around the same time.
- 2003: ReOx founded with funding from venture capitalists.
- 2004: Pharmaceutical companies express interest in ReOx's technology.
- 2005: ReOx signs a deal to license HIF hydroxylase inhibitor technology to Amgen.
As oxygen levels drop, activity of the HIF-hydroxylases drops. The HIF hydroxylase enzymes add an oxygen atom (hydroxylation) to the transcription factor hypoxia inducible factor (HIF) which causes it to degrade. Hence, the HIF-hydroxylases act as oxygen sensors because as oxygen levels drop the extent of hydroxylation drops, HIF doesn't degrade so rapidly, but instead accumulates and switches on a set of genes that work to counteract the effects of hypoxia. (Another HIF hydroxylase switches off HIF activity)
And the genes to counteract hypoxia include ones that regulate red blood cell blood vessel production. Hence, inhibiting HIF hydroxylation increases (or upregulates) HIF levels, which switch on red blood cell formation or can induce blood vessel formation, thus presenting a target for drugs in the treatment of anaemia.
- The hypoxia-inducible transcription factor pathway regulates oxygen sensing in the simplest animal, Trichoplax adhaerens
- Targeting of HIF-a to the von Hippel-Lindau Ubiquitylation Complex by O2-Regulated Prolyl Hydroxylation
- C.elegans EGL-9 and mammalian SM-20 define a conserved family of dioxygenases that regulate hypoxia inducible factor (HIF) through prolyl hydroxylation
- Structural basis for the recognition of hydroxyproline in HIF-1[alpha] by pVHL
- Cellular oxygen sensing: Crystal structure of hypoxia-inducible factor prolyl hydroxylase (PHD2)
- Structure of factor-inhibiting hypoxia-inducible factor (HIF) reveals mechanism of oxidative modification of HIF-1 alpha
- Selective inhibition of factor inhibiting hypoxia-inducible factor hydroxylases