ADVANCES FROM THE RECENT LITERATURE CONCERNING CFTR
October 2, 2001
July 28, 2001
CFTR is not the only chloride channel in the lung. In fact, it isn't even the most efficient when it comes to transporting chloride. Unfortunately, CFTR does seem to be the most important when it comes to regulating what some of the other chloride channels do. There has been evidence for several years supporting the idea that CFTR activates the chloride channel ORCC which also sits in the same lung cell CFTR does. Some researchers believe ORCC and other ion channels are what really is responsible for the "nuts and bolts" of fluid hydration of the mucus, so understanding how they are regulated will no doubt be important. Interestingly, before CFTR was discovered, it was believed by many in the field that the gene that codes for ORCC would turn out to be the cystic fibrosis gene. It didn't, we now know, but perhaps the field is coming full circle again because of the realization that activation of ORCC or other chloride channels has the potential for solving the problem of sticky mucus that leads to so much trouble in the lungs of CF patients. In other words, it might be possible to bypass CFTR someday if one could activate a chloride channel which could function in place of CFTR. Importantly, a newer group of chloride ion channels, the CLC channels, have been discovered in several tissues and open up a possibility for using this type of strategy to activate them as well. That is, if they turn out to be present in lung epithelial cells like CFTR and ORCC are.
This month, Cuppoletti et al., published a paper describing
their efforts at answering some of these questions. They apparently have
found two new ways of activating CLC-2 channels which were placed into a cell
line called HEK-293. They also discovered that cell lines derived
from different human lung epithelia contained messages to code for ClC-2 (i.e.
mRNA), and they were able to increase chloride transport out of these cells
using their small molecule activators for CLC-2. This demonstrated
not only that CLC channels are present in the lung, but that it can be activated by
artificial means, which is significant. These investigators also
obtained similar results using cells from normal tissues as well as those from cystic
fibrosis patients. They concluded by suggesting that the ClC-2 chloride ion
channel is a potential target for therapy in cystic fibrosis. As a
cautionary note, it should be remembered that there is often a large amount of work that needs to
be done whenever transferring results obtained in cell cultures (as in this
study) to a practical therapy, not least of which involve large scale clinical
trials. Am J Physiol Cell Physiol 2001
Jul;281(1):C46-54
June 14, 2001
Those interested in finding a cure for CF are often on the lookout for ways of increasing the amount of CFTR at the surface of the cell membrane in the lung. This is because, even when mutated, CFTR (such as the common deltaF508 mutant) has been shown to work somewhat if enough of it can find its way to the cell surface. Zaman et al (from the University of Virginia, Charlottesville) believe they may have found one possible way to increase expression of CFTR where it belongs. A molecule (specifically, a naturally occurring biologically modified peptide) called GSNO (short for S-nitrosoglutathione) appears to increase expression and maturation of CFTR deltaF508. These researchers grew lung cells in cell culture (i.e. in vitro) and exposed them to GSNO and saw a dose- and time-dependent increase in CFTR expression. Strong evidence for GSNO influence on the production of CFTR. They concluded in their article published this month in the following way: "....because endogenous levels of GSNO are low in the cystic fibrosis (CF) airway, these results raise the possibility that GSNO replacement therapy could be an effective treatment for CF." Zaman K, McPherson M, Vaughan J, Hunt J, Mendes F, Gaston B, Palmer LA. Biochem Biophys Res Commun 2001 Jun 1;284(1):65-70
May 12, 2001
"Gene Therapy" has been of interest for researchers and physicians for several reasons. One is that gene therapy provides for a means of correcting the underlying problem in genetic diseases (like CF) directly. Gene therapy provides for an entirely new type of intervention which was unthinkable as late as the 1980s, correction of an inherited defect by the addition of the "correct" gene. Gene therapy also is a means of intervening early in the process of disease progression and may therefore function as a preventative; as opposed to many traditional "supportive care" treatments, which have often involved making the patient feel more comfortable after the disease has already done much of the damage. The problem to date is that gene therapy is still relatively unproven in the clinical situation. As of this writing, a rare genetic blood disorder (ADA) is the only known long-term success of gene therapy, however several studies are still in progress (some involving CF) and await analysis. Bottom Line: it is still too early to know what kind of an impact gene therapy will have on most genetic diseases like CF.
Initial interest in the possibility of the use of gene therapy in the treatment of CF was mainly due to the unique environment in which the lungs provide. Being one of the most accessible areas of the human body compared to other organ systems, the lumen of the lungs can be reached from the outside without the need to introduce vectors (and the genes they carry) into the bloodstream. This provides at least two significant advantages. First, it circumvents the need for special targeting molecules on the gene vector in order to reach an organ of interest, and it also eliminates the need for increasing the stability of the vector in order to survive in the bloodstream (note: vector is a generic term for the vehicle used to carry the new gene. A vector can be a virus, a liposome, or a piece of "naked DNA" such as a plasmid). In effect, the lungs provide researchers the opportunity to add genes (for example, the gene encoding the protein CFTR) and gene vectors directly to the problem area in CF, i.e. the lungs, making gene therapy much easier in principle. During the time since these initial gene therapy trials began, however, more recent research has revealed that there are many processes occurring in the lungs (for example, phagocytosis on the airway surface of the lung lumen) which were not previously anticipated. This is important because some of these processes could have been causing the ambiguous results obtained from previous gene therapy trials involving CF patients.
In February, 2001 Baatz and coworkers reported that they discovered a molecule (normally used by the immune system to cause inflammation) which has the ability to inhibit a type of liposome vector called "cationic Lipofectin" in model lung airway cells. This is important because Lipofectin has been used previously in CF gene therapy trials with somewhat disappointing results. This immune system protein, which seem to inhibit Lipofectin, is known as TNF-alpha, and when these workers knocked TNF-alpha out of commission by using an antibody specific for TNF-alpha, it greatly increased Lipofectin's ability to deliver genes to the airway cells that were being cultured in dishes. They were also able to conclude that the uptake of the gene-carrying liposomes by theses cells (a process called "endocytosis") is somehow directly inhibited by TNF-alpha. In the future, it might be possible to add this specific antibody to TNF-alpha during gene therapy treatments to enhance the effects of liposome vectors like Lipofectin. There are currently several precedents for combination treatments in other diseases where a second drug is used to enhance the effects of the first drug, for example in chemotherapy for certain types of cancers, and in many vaccines where an adjuvent is co-administered along with the antigen. Baatz JE, Zou Y, Korfhagen TR. Biochim Biophys Acta 2001 Feb 14;1535(2):100-9
April 1, 2001
It is known that the viscosity of mucus in the lungs can be affected by its surroundings. For example, if the pH of the mucus is too low, the mucus can become overly sticky and difficult to remove, providing an environment conducive for bacterial colonization. Consequently, any treatment that can effect the elastic properties of the mucus in the lungs holds out promise as a way to manage CF in the future. One of the more interesting features of CFTR is its apparent ability to let the bicarbonate anions thru its pore in addition to chloride anions. This information may be useful because bicarbonate anions are known to effect pH: raising it when they are present and lowering it when they are not.
In the March 3rd issue of the journal Nature, Joo Choi, et al. (U.Texas Southwestern Medical Center, Dallas) report that they were able to show that certain mutations in CFTR can affect bicarbonate transport (thru CFTR's pore?) without significantly changing its ability to pass chloride anions. This is important because it means that CFTR may be affecting the pH of the mucus in the lungs and not just the water content of the mucus, as has previously been believed. This could account for the unusually viscous mucus known to exist inside the lungs of CF patients, because in theory, if no bicarbonate anions are released into the lungs, the mucus could become too acidic and therefore too viscous. Future treatments may involve changing not only the water content, but the pH in the lung as well to make it less acidic. The authors also point out that it is yet to be resolved exactly how the bicarbonate anions are transported thru the lung and pancreas. It could be accomplished directly by CFTR itself, or by another transporter protein which is controlled by CFTR. This question is an important one and will need to be addressed in the future because insight into bicarbonate anion transport mechanisms could allow for an entirely new treatment direction. Nature 3/1/01 410: 94-97
April 1, 2001
One of the problems with the CFTR protein is that there isn't very much of it in the location is should be: the cell surface of the lung epithelia. This doesn't seem to matter with the normal protein, but with a common mutant version of the protein (found in the majority of CF patients), it is a significant problem. Therefore, any treatment which can increase the availability of CFTR to the surface of the cell could help out many CF patients. It has been shown in the past that the common mutant version of CFTR called deltaF508 gets stuck in the interior of the cell and very few copies of it ever make it to the surface of the cell where it belongs. One theory has been that the CFTR protein gets stuck simply because it doesn't fold fast enough. And these mis-folded proteins are gotten rid of before they have a chance to get to their destinations (i.e. the surface of the cell in the case of CFTR). It is perhaps equally likely that CFTR gets stuck because it is held there too long by other protein inside the cell, for example BAP31.
In an article entitled "Control of CFTR: Expression by
BAP31", Lambert et al. found that by inhibiting this molecular
chaperone protein BAP31, they could get significantly more CFTR delivered to the
cell membrane. They concluded that association of BAP31 with CFTR which
may control "maturation" (folding) or "trafficking"
(movement) of CFTR and thus expression to the plasma membrane (i.e. the cell
surface). These results were obtained by using CHO
cells, which are not the same cells as are found in the lung, but the results
are none-the-less interesting, in part because these investigators were also
able to obtain increased presence of the common deltaF508 mutant CFTR protein to
the cell surface apparently in similar amounts as the normal (wild-type) CFTR
protein. J Biol Chem. 2001 Jun
8;276(23):20340-5.
March 28, 2001
One possible treatment for cystic fibrosis involves the discovery and use of small molecules capable of helping the CFTR protein "do its job better". A kind of "molecular crutch" for a protein. Patients with the deltaF508 version of CFTR have small amounts of the deltaF508 version of the CFTR protein in the proper place in the cell membrane in the lungs, which is good news. However not very much of it makes it there. And what little makes it there is somewhat less active than normal CFTR is. As approximately 90% of cystic fibrosis patients have a version of this deltaF508 CFTR protein, if a treatment existed to improve its function, this potential treatment could help a considerable number of patients. Interestingly, it has been known for several years that a small drug molecule called genistein is able to bind CFTR directly and make it more active.
Galietta, et al. (University of California, San Francisco) recently has established an efficient screening procedure to help identify new activators and inhibitors of CFTR. His group found that compounds called 7,8-benzoflavones and pyrazolo derivatives are a new class of activators. In fact, when compared to genistein, the most active compounds they discovered had up to 10 times greater potency in activating wildtype and/or G551D-CFTR. In addition, these new activators had a low cellular toxicity (i.e. were relatively harmless) and the way they activate CFTR may involve a direct interaction with CFTR itself.
A problem with any drug discovery initiative always involves showing that the drug works in the patient the same way that it works in the "test tube". As a cautionary warning, it has been estimated that over 90% of compounds found by drug discovery companies in the past which work in the "test tube" either don't work in the patient, or have too many dangerous side effects. In fact, drug companies spend billions of dollars each year on large-scale clinical trials in an attempt to figure out what actually happens when a drug is given to a large number of people. It's the only way to obtain this kind of information. It is time-consuming as well, and accounts for the average 10 years it takes to get a drug from its discovery to the pharmacist. J Biol Chem 2001 Jun 8;276(23):19723-8