While about one-third of all proteins across all species are estimated to be attached to or associated with a cell’s membrane, excruciatingly little is known about them and the roles they play in cellular processes.
A new project backed by the European Commission and 27 universities and companies in Europe, however, is aiming to change that by expanding the knowledgebase of membrane proteins, hopefully leading to the development of new drugs.
Called the European Drug Initiative for Channels and Transporters, or EDICT, the €15.7 million [$24.2 million], four-year project includes researchers from the Max Planck Institute, the Karolinska Institute, Oxford University, and the University of Zurich. Drug makers AstraZeneca and Xention, which develops ion-channel drugs, are also participants.
A total of 37 individual principal investigators are involved, including Nobel Laureates John Walker, who shared the Nobel Prize for chemistry in 1997, and is chairing the consortium, and Hartmut Michel, who shared the Nobel, also in chemistry, in 1988.
Coordinating the effort will be Peter Henderson, professor of biochemistry and molecular biology at the University of Leeds, UK.
The EC is providing €11.9 million to the effort with the remaining funding coming from EDICT’s participants. The goal of the project is to gain more knowledge about the structure of membrane proteins, and then identify compounds that can be developed to treat diseases for which such proteins have been implicated.
EDICT started when the EC put out a call for researchers for a project to develop new drugs in relation to ion channels and transport proteins. From his own work in purification and amplification of membrane proteins in bacteria, Henderson knew other members of the community he felt would be interested in answering EC’s call.
Indeed, the interest level was high enough that some parties had to be turned away, Henderson told ProteoMonitor, and there are no plans to add more participants. EDICT draws upon researchers from all fields connected with membrane protein research, including experts in X-ray crystallography and other technologies, experts in the pharmacology of specific proteins, structural biologists, and chemists “who understand about docking putative drugs into protein targets,” he said.
“What the European [Commission] has given is financial support to bring together these experts who’ve worked, more or less, isolated from each other in the past, and who have complementary skills in order to devise new generations of drugs,” Henderson said.
AstraZeneca spokeswoman Heather Derrick said in an e-mail to ProteoMonitor that a number of drugs currently on the market work by interacting directly with membrane proteins. The company, she said, has made “considerable investments to drive the discovery and development of compounds targeting these membrane proteins to battle a wide range of human diseases, and fundamental understanding of function and structure of these proteins is a valuable aid in this process with high strategic relevance for R&D.”
The consortium will be targeting about 80 membrane targets including sodium potassium ATPase, which has been implicated in diabetes; proton ATPase, implicated in osteoporosis; sodium proton exchanger, implicated in infectious diseases, such as pneumonia; antibiotic proteins; and nucleotide transport proteins, implicated in zoonosis.
‘Huge Gap’ in Understanding
While membrane proteins are suspected as major players in a range of diseases such as diabetes, heart disease, epilepsy, osteoporosis, stomach ulcers, and cataracts, little is known about them. Indeed, while the three-dimensional structure of about 40,000 soluble proteins are known, the 3D structure of between only 100 and 150 membrane proteins are known, Henderson said, even though about 70 percent of the documented potential pharmaceutical targets for humans are membrane proteins.
“So there’s a huge gap in [the] understanding of the three-dimensional structure of membrane proteins, including transport proteins and ion channels,” Henderson said. “And yet they constitute a high proportion of the notional targets for pharmaceutical intervention in the future.”
“What the European [Commission] has given is financial support to bring together these experts who’ve worked, more or less, isolated from each other in the past, and who have complementary skills in order to devise new generations of drugs.”
One reason for the paucity of information is that membrane proteins are notoriously difficult to detect and identify. Because they are hydrophobic, extracting and purifying them from cellular material for imaging purposes have proven to be a significant challenge. Methods to do so have included X-ray crystallography, electron microscopy, and nuclear magnetic resonance.
During the past 20 years, however, a number of detergents have been created that can make the proteins water-soluble without denaturing them. Tags have also been developed allowing for more efficient purification of membrane proteins. New techniques have been developed in which cells are genetically engineered to express in large numbers such proteins with slight changes to them. These altered proteins have a chemical tag giving them a strong affinity to nickel. With the membrane proteins attached to nickel, the remaining proteins are washed off, and the isolated proteins are then identified by imaging.
The other major obstacle has been the low abundance of membrane proteins in the cells. More recently, “amplification of expression … using vectors in suitable hosts has been another major factor that has enabled the field to lurch forward,” Henderson said, though “the amplified expression of un-denatured mammalian membrane proteins, that’s the biggest problem that remains.”
In addition to new-drug development, EDICT is directed at improving existing technologies for the study of membrane protein structure, Henderson said. In particular, he said, there is interest in further improving the technology for expressing mammalian proteins, which Henderson said, is the least well-developed.
“The technology, one would say, has largely been created. X-ray crystallography is becoming very successful,” he said. “Electron microscopy and NMR have got a ways to go.”
Interest from NIH
While much about membrane proteins may be a mystery, it isn’t due to a lack of effort. According to a keyword search on PubMed, work on such proteins has resulted in the publication of a total of more than 117,000 papers, nearly half of which have appeared since 2000.
The NIH also has initiatives targeted at membrane proteins. As part of its Protein Structure Initiative for the large-scale determination of protein structures, the National Institute of General Medical Sciences has two specialized centers focused on researching such proteins: one at the New York Structural Biology Center and the other at the University of California, San Francisco.
The two centers share about $5 million per year in funding from NIH.
As part of the NIH Roadmap, two centers are also doing work in the area, one at UCSF, the other at the Scripps Research Institute. They also share about $5 million annually in funding from the NIH.
While EDICT is specifically researching mammalian membrane proteins, the NIH initiatives are doing work across multiple species in prokaryotic and eukaryotic systems, said Peter Preusch, chief of the biophysics branch in the Cell Biology and Biophysics division at NIGMS.
The NIH efforts also have more of a technology development component than EDICT. At Scripps, for example, researchers are looking at ways of reducing sample volumes and designing new detergents that will enable membrane proteins to be solubilized from the membrane while maintaining activity, said Jean Chin, program director in cell biology in the Cell Biology and Biophysics division at NIGMS,
And at UCSF and the Salk Institute, researchers are studying screens for expression, and the Mistic homologs, a chaperone protein that improves how membrane proteins are expressed.
“One of the philosophies of the PSI had to do with ‘We’re going to do lots of structures,’” Preusch said. “And these specialized centers of PSI have to do with enabling membrane protein crystallography and other structure determination methods to begin to approach the high-throughput levels that we’re capable of generating for soluble proteins now.”
“The Roadmap really started at a much more basic level with the premise that producing membrane proteins, just producing the proteins itself in a form that is stable, homogeneous, and representative of the functional state that we want to study is the challenging problem. And its investment is primarily on that end of the problem,” he said. “The money and the emphasis [are] really on the development of methods.”
While there is potential for overlap with the work of the PSI, Henderson said that because so much remains unknown about membrane protein structures, there is room for projects such as his and the NIH initiatives to co-exist.
“There are thousands of proteins that need studying out there. Of course some of them will be very high-profile targets engaged by more than one research group [but] quite often these studies complement,” he said. “If the investigators are really unlucky, then they both discover the same thing, and one scoops the other. On the other hand, there’s a good chance in X-ray crystallography [for example], that one will produce a structure that will be significantly different from the other because the protein undergoes structural changes during its catalytic cycle. We all expect that. So then, we’ve got a complementary illumination of the protein’s mechanism.”