A recent breakthrough means that for the first time, some people with cystic fibrosis (CF) can lead their lives with greatly reduced symptoms of the disease. CF is an inherited disease affecting numerous organs throughout the body, including the lungs, pancreas, and intestines. Thick mucus in the lungs of people with CF promotes infections by Pseudomonas aeruginosa bacteria, which thrive in the mucus and gradually damage lung tissue. Digestive and pancreatic manifestations of the disease lead to delayed growth and malnutrition. While research has led to enormous strides in CF treatment during the last few decades, dramatically increasing life expectancy for those with the disease, treatments can be arduous and time consuming, and those with CF remain highly susceptible to dangerous infections and other serious complications. The breakthrough—development of a new medication that can overcome the fundamental molecular flaw in people with a particular mutation of the CF gene—stems from decades of research.
A Big Advance from Sweating the Small Stuff: The Genetic Cause of CF
The new treatment has its roots in some of the earliest characterization of the disease. One characteristic of CF that may at first glance seem strangely unconnected to its profound lung, pancreatic, and other consequences is that people with the disease have particularly salty perspiration. Indeed, for many years CF was typically (and quite accurately) diagnosed with a sweat test. Salt is a key ingredient in perspiration, enabling sweat glands to release water in response to increases in body temperature. Evaporation of the water helps cool the body, but the salt—after it has done its job of helping move water out of sweat glands—is no longer needed on the skin. In fact, because the body needs salt (for many purposes in addition to producing more sweat), sweat glands normally reabsorb a portion of what they have released. The first clue to the function of the CF gene came in 1983, with the discovery that the salty skin of people with CF is caused by a defect in this reabsorption process. Specifically, the scientists found that sweat glands from CF patients are much less capable of absorbing chloride—one of the chemical components of salt—than are normal sweat glands. This finding suggested that the genetic mutation underlying CF results in the inactivation of a chloride transport protein.
But more dramatic progress was not possible until the gene encoding that chloride transporter was discovered. Through painstaking genetic analysis, NIDDK-funded investigator Dr. Lap-Chee Tsui and colleagues had identified a region of human chromosome 7 as the likely location of the CF gene. Using the standard gene discovery methods of the era, it might have taken decades to find the CF gene within this region, but Tsui recruited Dr. Francis Collins and his group to join in the effort. Dr. Collins—now the director of the NIH—had recently published a method for making jumps across difficult-to-analyze sections of DNA. This “chromosome jumping” approach dramatically hastened the search. Although it was still a mammoth undertaking, the two groups of investigators announced in 1989 that working together they had succeeded in identifying the gene that is mutated in CF. Confirmation might have been simpler if DNA samples from some people with CF had a large deletion, chromosome rearrangement, or at least a mutation that would radically change or eliminate the gene, but that did not turn out to be the case. Instead, combing through the genetic information of the gene, they found that just over two-thirds of the chromosome 7s they tested from people with CF (each of whom has two chromosome 7s, one from each parent) were missing the code for just one of the protein’s predicted 1,480 amino acid building blocks. Subtle though this tiny deletion seemed to be, they found that most people with CF had two copies of the gene with the deletion, and most of their parents had one copy with the deletion and one without it. Significantly, none of the parents had two copies with the deletion. This was very strong evidence that the researchers did indeed have the correct CF gene, and that the single amino acid deletion was the most common disease-causing mutation.
This signal discovery made it possible to deduce some basic characteristics of the encoded protein, including tell-tale motifs that suggest it functions in cell membranes, and binds a molecule called ATP, which is key for many cellular processes. The arrangement of these motifs was similar to those of a previously characterized protein involved in the transport of substances out of cells. Putting this together with the observations about salty sweat, the researchers correctly guessed that they were characterizing a protein involved in the transport of salt. The gene’s discoverers dubbed it the “cystic fibrosis transmembrane conductance regulator” (CFTR). We now know that when the protein binds ATP, it opens a pore on the cell surface through which negatively charged chloride ions (one of the two chemical components of table salt) can travel. The positively charged sodium ions of salt then follow in other ways, keeping the electrical charge balanced in cells. Importantly, this ion flow has the key effect of drawing water along via osmosis, to balance salt concentrations. The flow of water out of lung cells, made possible by movement of chloride ions through healthy CFTR proteins, hydrates the thin, protective layer of mucus on their surface. In people with CF, neither salt nor water flow—so what should be thin, protective mucus becomes thick, sticky, and an ideal habitat for lung damaging bacteria.
Progress from Studying CFTR Mutations
The mutation the researchers discovered that causes elimination of a single amino acid was dubbed ΔF508 (because it deletes the 508th amino acid in CFTR, a phenylalanine, designated F), and was later shown to represent about two-thirds of CFTR mutations worldwide. Thus, about 90 percent of people with CF have at least one copy of ΔF508. (Among people of European descent, about 1 person in 30 has a single copy of ΔF508, along with a normal working copy of the CFTR gene, which is enough to avoid CF. Some evidence suggests that CFTR mutations are as common as they are because people with one mutated and one normal version were historically less likely to succumb to diseases like typhoid fever, tuberculosis, or cholera.) Although ΔF508 is by far the most common mutation, about half of people with CF have at least one copy of one of the thousands of other known CFTR mutations. By studying these various CF-causing mutations, researchers have discovered a great deal about the CFTR protein, its function, the disease physiology of CF, and—ultimately—medicinal approaches to address those problems at the molecular level. For example, they found that ΔF508 results in a protein that is unstable, and that is degraded before it gets to the cell membrane—which helped clarify the maturation process by which the cell prepares the protein for its role on the cell surface. Other mutations, such as one designated G551D, result in CFTR proteins that reach the cell surface in adequate quantities, but which fail to open and therefore do not allow the flow of chloride through the CFTR “gate.” CF researchers therefore designated mutations like G551D that yield stable but non-functional CFTR proteins to be “gating” mutations. “Conductance” mutations gate normally, but contain a defective pore through which chloride cannot travel. The different types of mutations provide key information about the way the parts of CFTR work together to regulate the flow of chloride.
Intriguingly, researchers found that ΔF508-CFTR proteins could actually reach the cell surface—and even transport chloride—if made by cells grown in cool conditions (well below body temperature) in the laboratory. This suggested the tantalizing possibility that if they could somehow identify medicinal compounds that stabilize ΔF508 at body temperature, or that allow CFTR proteins made with gating or conductance mutations to allow chloride flow, they might effectively be able to treat the fundamental molecular problem in CF.
Progress from Technological Innovation
CF researchers therefore sought to identify candidate medicines that could promote CFTR function. A key step in that discovery process was the creation of stable cell lines bearing various human CFTR mutations that could be grown in the laboratory. In principle, investigators could then expose the cells to different candidate drugs, and ask which allowed the transport of chloride. Checking for ion transport, however, is no easy task if it has to be performed one chemical at a time for hundreds of thousands of potential medicines. The work was therefore greatly advanced by the development of fluorescent markers of CFTR activity. For example, one NIDDK-supported group created a protein that fluoresces when exposed to ultraviolet light, but dims substantially when bound to ions like chloride. Another group employed chloride-sensitive-dyes. These innovations allowed the medicine hunters to create large arrays of test chambers containing CFTR-mutant cells, accompanied by a different candidate drug in each chamber, and then use fluorescence to detect ion flow in many chambers at once. Screening hundreds of thousands of compounds, in this fashion, the searchers were able to identify a few that had properties they were looking for—helping different mutant CFTR proteins function better. The promising candidates they discovered were then chemically tweaked in various ways, to try to improve on their ability to promote chloride flow. Of course, these compounds might work beautifully in the laboratory, but to be useful as CF drugs, the compounds would have to be proven safe, and capable of reaching cells where they are needed. Thus, laboratory animals such as mice that have been engineered to have the same CFTR mutations found in humans were another critical resource for preclinical testing.
Among the compounds found to have the most promising qualities was one designated VX-770 by the pharmaceutical company where it was identified. Following animal testing and a preliminary dosage trial in humans, it was eventually shown in trials of increasing size and length to significantly improve function of the lungs, pancreas, and other affected organs in people with at least one copy of the G551D gating mutation. Based on these strong safety and efficacy results, the U.S. Food and Drug Administration approved VX-770 (as “ivacaftor,” marketed as “Kalydeco™”) in January 2012. This is great news for people with CF in the United States who have at least one copy of the G551D mutation. It accounts for about 2 percent of CF-causing mutations in the United States, (meaning about 4 percent of U.S. residents with CF have a copy of the mutation), but is somewhat more common in Ireland, Scotland, Brittany (in France), and the Czech Republic. Several other, rarer gating mutations are also known, and it is hoped the drug may also prove valuable in patients with some of these forms of CFTR as well.
Although initial characterization of VX-770 and of a few other compounds identified through similar screening approaches suggested they may be of benefit to anyone with the much more common ΔF508 mutation, this has unfortunately not proved to be the case. A drug does not have to restore complete function to CFTR to be of substantial benefit to people with CF—achieving 10 percent or more of chloride transport activity is expected to have a real impact on symptoms. So why have such efforts so far been unsuccessful in the case of ΔF508? Two recent papers are helping provide an explanation: ΔF508 not only interferes with folding of the first ATP binding domain of CFTR, it also disrupts interaction of this domain with a neighboring part of the protein. Because there are two physiological problems with the same protein, it is not enough to correct one of them. In fact, it may be necessary to improve the protein folding, the domain interaction, or both, by quite a bit more than 10 percent to achieve a net restoration of 10 percent of normal channel function. This is a substantially higher hurdle to cross, but the search may be facilitated by this better understanding of what such a drug (or combination of drugs) must achieve. Thus, it may be productive to search for a compound that promotes a significant increase in chloride transport in cells with the ΔF508 mutation in the presence of VX-770 or another compound, which by itself confers only a modest improvement.
Other drugs are in development for other types of CFTR mutations. For example, the CFTR gene can be thought of as an instruction list for the cell, indicating the order and identity of each of the CFTR protein’s 1,480 amino acids. Some mutations change the list, so that the code for a particular amino acid is changed to a code instructing the cell’s protein production machinery to stop adding amino acids to that protein. Drugs that can induce the cell to “read through” such “stop” instructions are also in development. Importantly, these ongoing CF drug searches are made possible by the same fundamental advances that enabled the development of VX-770 for people with CF and the G551D mutation: discovery of the CFTR gene, characterization of the protein it encodes, analysis of the CF mutations, and the creation of CF cell lines, animal models, and fluorescent ion sensors. Thus, the quest continues for medicines to help more people with CF lead lives that are longer, healthier, and less burdened by the disease.