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Pharmacological Chaperones


Compound V Foto: Jarl Underhaug Copyright:Jarl Underhaug


In drug delivery, what is a “chaperone?“


Many human diseases result from mutations in specific genes. Once translated, the resulting aberrant proteins may be functionally competent and produced at near-normal levels. However, because of the mutations, the proteins are recognized by the quality control system of the endoplasmic reticulum and are not processed or trafficked correctly, ultimately leading to cellular dysfunction and disease. Pharmacological chaperones (PCs) are small molecules designed to mitigate this problem by selectively binding and stabilizing their target protein, thus reducing premature degradation, facilitating intracellular trafficking, and increasing cellular activity. Partial or complete restoration of normal function by PCs has been shown for numerous types of mutant proteins, including secreted proteins, transcription factors, ion channels, G protein-coupled receptors, and, importantly, lysosomal enzymes.


pharmacological chaperone (or pharmacoperone, from “protein chaperone“) is a small 1) ___that enters cells and serves as a molecular scaffolding in order to cause otherwise-misfolded mutant proteins to fold and route correctly within the cell. Mutation of proteins often causes molecular misfolding, which results in protein misrouting within the cell. Accordingly, mutant molecules may retain proper function but end up in parts of the cell where the function is inappropriate, or even deleterious, to cell function. Misfolded proteins are usually recognized by the quality-control system of the 2) ___ and retained (and often destroyed or recycled)in the endoplasmic reticulum.


Pharmacoperones or chaperones, correct the folding of misfolded proteins, allowing them to pass through the cell’s quality-control system and become correctly routed. Since mutations often cause 3) ___ by causing misfolding and misrouting, pharmacoperones are potentially therapeutic agents, since they are able to correct this defect.


Diseases that may be susceptible to such treatments include diabetes, inherited cataracts and cystic fibrosis.


In the context of pharmacology, the term chaperone is borrowed from the name of a class of proteins that function in living cells. Protein molecules are usually only marginally stable under physiological conditions, so some percent of them are often unfolded or misfolded. Such molecules can aggregate with one another, or with properly functioning proteins, with deleterious consequences to the cell. Protein chaperones prevent these unwanted associations by sequestering unfolded and misfolded proteins and providing them with an environment in which they have the opportunity to refold properly. In addition, the interior of a living cell is an extremely crowded environment, in which the concentration of macromolecules may exceed 100 uM. As a protein is being synthesized on the ribosome, 4) ___ chaperones protect the nascent polypeptide chain from undesirable associations in that crowded environment until it can fold properly.


In pharmacology, the role of a chaperone is similar, but instead of being proteins, pharmacological chaperones are small molecules, and instead of assisting in folding, they usually stabilize an already folded macromolecule (usually a protein) by binding to it and stabilizing it against thermal denaturation and proteolytic degradation. A chemical chaperone is subtly different from a pharmacological chaperone. Typical chemical chaperones are molecules such as glycerol and trehalose. Pharmacological chaperones are a special subset of chemical chaperones. Molecules like glycerol and trehalose are nonspecific: they bind to, and stabilize, practically any protein and usually do not have a specific binding site. Pharmacological chaperones, on the other hand, are designed specifically to 5) ___ to their target protein and, ideally, stabilize only that macromolecule. The difference, therefore, is one of specificity: a chemical chaperone usedin vivo would stabilize virtually every macromolecule in the cell. A pharmacological chaperone acts on, at most, only a small number of protein targets. There is a great deal of excitement in the biomedical community these days about pharmacological chaperones because they may be the best approach to treating some serious human diseases, such as cystic fibrosis. Small-molecule chaperones act like molecular glue, holding various parts of the protein structure together through the favorable interactions they make with residues in the binding site. Since specific ligand binding sites are often located at the interfaces between protein domains or subdomains, such ligands can be particularly effective at stabilizing the whole protein structure.


Most proteins in all cell types turn over regularly. If such proteins have a mutation that makes them less stable than normal, they may be degraded more rapidly, thereby lowering their steady-state levels below what is required to maintain the health of the cell. Also, it is possible that the unstable protein may aggregate when it unfolds, and such aggregates may themselves be toxic to the cell. There are many severe human diseases that arise from either mutations that destabilize an essential protein or the age-dependent build-up of toxic misfolded forms of normal proteins. A 6) ___ or pharmacological chaperone can stabilize the native fold of the protein, preventing aggregation and restoring proper steady-state levels. There is a particular need for protein and 7) ___-molecule chaperones in compartments where proteins are subjected to unusual stress. Examples might include the mitochondrion, where large amounts of reactive oxygen species are present, the lysozome, which has a low pH and a high content of degradative enzymes, and the endoplasmic reticulum (ER), where many unstable mutant proteins may misfold during synthesis.


For some diseases, pharmacological chaperones are useful. Obvious examples are the protein-misfolding diseases such as cystic fibrosis, the amyloidoses, Parkinson’s disease, Alzheimer’s disease and Lou Gehrig’s disease. Less obviously, most metabolic disorders involve mutations that destabilize proteins rather than simply inactivating them. For example, Gaucher disease, an autosomal-recessive lysosomal storage disorder, arises from mutations in the gene coding for the lysosomal enzyme acid-?-glucosidase (GCase). More than a hundred such 8) ___ are known, and only a handful are nonsense mutations or involve the replacement of a residue in the active site of GCase. The vast majority of the disease-causing mutations occur randomly throughout the protein and lead to an unstable form that is either degraded in the lysosome (where it normally functions), or never manages to exit the ER in the first place.


Enzyme-replacement therapy using injections of the normal enzyme can alleviate many of the symptoms of Gaucher disease, but the injected enzyme does not reach every affected organ system, and the treatment is onerous and extremely 9) ___. In principle, a pharmacological chaperone could be orally available, relatively inexpensive and might be able to stabilize GCase in every tissue of the body. There are literally hundreds of diseases where the ability to stabilize a specific protein could have similar therapeutic benefits.


Reversible inhibitor is the key to the use of active-site-directed ligands as pharmacological chaperones. An inhibitor that binds irreversibly to a target protein takes that protein out of circulation, activity-wise. But a reversible inhibitor always allows the presence of some equilibrium amount of free enzyme, which is then available for substrate binding (which will also 10) ___ the protein). The affinity of the inhibitor is also important. If it binds too tightly, it may be effectively irreversible. But if it binds too weakly, it may be impossible to supply a high enough concentration of the chaperone to be effective. In practice, inhibitors with a Ki (equilibrium disassociation constant) close to the Km (Michaelis-Menten constant) of the substrate or a bit tighter seem to be useful.


There has been clinical success with pharmacological chaperones. It turns out that an established therapy for a metabolic disorder probably works this way, but no one recognized this until recently: Phenylketonuria (PKU) is caused by mutations in the gene coding for the enzyme phenylalanine hydroxylase. For years, one treatment for a subset of patients with this disease was high doses of tetrahydrobiopterin, the cofactor involved in the reaction catalyzed by PKU. It has since been shown that most of the PKU mutations destabilize the 11) ___ and, in some cases, the increased levels of cofactor enable it to act as a chaperone to stabilize the mutated enzyme. Efforts are under way to apply this technology to cystic fibrosis, the prototypic genetic protein-folding disease, in which many of the mutations, including the most common, lead to a failure to produce enough properly folded protein, called CFTR, in the right place in the cell.


In mouse models of obesity and type 2 diabetes, the development of insulin resistance correlates with elevated levels of ER stress and induction of the ?unfolded protein response’. Ozcan et al. have shown that the chemical chaperones phenylbutyric acid and taurine-conjugated ursodeoxycholic acid, both of which are known to attenuate ER stress, improve glucose tolerance and insulin action in a mouse model of type 2 diabetes. These findings offer a potential new approach to improve insulin action and 12) ___ tolerance in diabetic individuals.


A number of other pharmacological chaperones are in late-stage clinical trials, including drugs for transthyretin-based amyloidosis, the imino sugar isofagomine for Gaucher disease, and 1-deoxygalactonojirimycin for Anderson-Fabry disease. All these compounds are analogs of normal physiological substrates or reaction products that interact with these proteins, and all have shown efficacy in animal models of the disease. They have been shown to be safe, and in some cases to have a therapeutic effect in humans.


Pharmacological chaperones have wider potential. Although most of the work up to now has concerned the chaperoning of unstable 13) ___ proteins, there is no reason, in principle, why the normal form of any protein cannot be stabilized in this way, thereby increasing its steady-state level. Imagine using a small-molecule chaperone to boost, perhaps temporarily, the activity of a tumor suppressor such as p53 or a transcriptional regulator that controls the immune response. Pharmacological chaperoning may be as effective as gene therapy, and much easier to implement, whenever elevated levels of a beneficial protein are desirable. But one of the most exciting applications of small-molecule chaperoning may actually be its use in vitro. Biopharmaceuticals are relatively unstable, both during manufacture and in storage. This is one reason why even injected human proteins often cause an immune 14) ___.


By including a pharmacological chaperone in the manufacture and storage of biopharmaceuticals it may be possible to reduce or eliminate many of these problems. Combining the chaperone with the biopharmaceutical during treatment may also improve in vivo stability, reducing the need for frequent dosing. Joint use of chemical and biological therapeutics could be an exciting new approach to the treatment of disease.


Sources: Journal of Biology, Wikipedia, NIH.gov


ANSWERS: 1) molecule; 2) cell; 3) disease; 4) protein; 5) bind; 6) chemical; 7) small; 8) mutations; 9) expensive; 10) stabilize; 11) enzyme; 12) glucose; 13) mutant; 14) response

Isabella d’Aragona (1470-1524)


(Portrait) National Gallery of Art; Gino Fornaciari; (Graphic), C. Smith/ScienceIsabella


When it comes to reading the signature of disease and foul play in the bones of ancient people, Dr. Gino Fornaciari is the master. A professor in the medical school at the University of Pisa and the father of team member Antonio Fornaciari, he’s famous for investigating the lives and deaths of the ancient nobility of Italy, including the Medici of Florence, who lived just 60 km from Badia Pozzeveri. This particular archeological site, offers Fornaciari and other researchers, a rare chance to examine the health of commoners as well as nobles during the Renaissance. They have already found people of various social classes, buried in area 3000 from 1500 to 1700. One woman was buried with her spectacles – an expensive and treasured accessory – and several skeletons were interred in a costly stone-lined vault inside the ancient church. But most of the bones were buried in wooden coffins outside the churchyard and probably were those of poorer rural people, whose daily lives are less well known than the nobility, from cities.


One July morning, project co-director Gino Fornaciari dropped by the Florentine church site. There he found his former student, Vercellotti, examining teeth. “Why are the teeth so good?“. Fornaciari replied: “They’re young. The teeth are good because the normal age of death was 40.“ He pointed out that tooth wear can also reveal whether someone ate many tough grains such as coarse millet, or a more refined, soft diet.


Commoners’ bones will provide a counterpoint to Fornaciari’s work elsewhere revealing the woeful condition of the well-fed nobility. In Naples, he examined the mummy of Maria d’Aragona, a noblewoman who lived from 1503 to 1568 and was a famed beauty in her youth – but was obese at death. That fits with what he has learned about her fellow nobles’ diet. In 2008, Fornaciari analyzed carbon and nitrogen ratios in bone collagen from other princes of Naples and the Medici of Florence, and found that they had as much nitrogen in their diet as carnivorous mammals. Clearly, Renaissance royalty ate unhealthy quantities of meat at a time when many rural people struggled to get enough calories.


Rank was not a protection against horrific infectious diseases. When Fornaciari cut off a linen bandage from Maria d’Aragona’s arm, he discovered a large ulcer. He examined the tissue with a scanning electron microscope and rinsed it with antibodies that fluoresce in the presence of the bacteria that cause syphilis,Treponema pallidum. The tissue was so well preserved that he could detect the spiral shape of the bacteria; he sent tissue to a lab to confirm the diagnosis. Poor Maria also harbored human papillomavirus in a venereal wart – the first diagnosis of this sexually transmitted, cancer-causing disease in the tissue of a mummy.


Sexually transmitted diseases were common in Renaissance Italy. Syphilis raced through the country in the 1500s, possibly after Spanish sailors brought a new venereal form from the New World. Fornaciari also examined Maria’s distant relative, Isabella d’Aragona, who was also buried in Naples (see graphic, above and below). She was married to the Duke of Milan and is thought by many to be the model for Leonardo da Vinci’s Mona Lisa. When Fornaciari looked closely at this lady’s teeth, he found that they had been abraded to remove most of the enamel. The remaining enamel traces were black, a sign that she had taken mercury, which was then used – ineffectively – to treat syphilis. Lab tests confirmed that the black patina had a high level of mercury and that Isabella d’Aragona was poisoned by her own medicine, dying at age 54 in 1524.


By comparing the teeth and bones of urban nobles with those of Pozzeveri peasants, the team hopes to see how social rank affected health. The teeth of the noblewomen are less worn, because they ate a softer diet with meat, whereas poorer women and children often ate coarse millet. Vercellotti and Larsen expect to see more disruptions in tooth growth caused by lack of food during childhood in the peasants. With the graveyard’s large sample sizes, they hope to compare the men and women of Badia Pozzeveri to see who was better fed.


Isabella of Aragon, Duchess of Milan


A medal depicting Isabella


Isabella of Aragon (Italian: Isabella d’Aragona; 2 October 1470 – February 11, 1524), also known as Isabella of Naples, was the daughter of King Alfonso II of Naples by his wife, Ippolita Maria Sforza. From 1489 to 1494, she was the Duchess consort of Milan, and from 1499 to 1524 the suo jure Duchess of Bari and Princess of Rossano. After her brother Ferdinand II’s death, she was the heir of the Brienne claim to the title King of Jerusalem. She married her first cousin, Gian Galeazzo Sforza, who at the time was the Duke of Milan. However, his uncle Ludovico Sforza was the de facto ruler. With Gian Galeazzo Sforza she had one son and two daughters:


1. Francesco, who was taken to France by the French King Louis XII.in 1499

2. Bona, who married King Sigismund I of Poland

3. Ippolita Maria


Isabella also outlived Francesco, who was killed in 1512 by falling from his horse. Of her three children, only Bona survived her. In 2012, anthropologists excavated the body of Isabella of Aragon, and concluded that she had syphilis. Her teeth had a high level of mercury, which was used (ineffectively) to treat syphilis, and had given a black color to the tooth enamel, most of which had been removed by abrading. They concluded that she was poisoned by her own medicine.

Tobacco, Drug Use in Pregnancy Can Double Risk of Stillbirth


Stillbirth occurs when a fetus dies at or after 20 weeks of gestation. While the U.S. stillbirth rate decreased from 18 per 1,000 births in 1950 to 6 per 1,000 births in 2006, it still remains higher than many other developed countries and affects almost 26,000 U.S. newborns per year.


According to the NIH, with the legalization of marijuana in some states, it is especially important for pregnant women and health care providers to be aware that cannabis use can increase stillbirth risk.


According to an article published on line in Obstetrics & Gynecology (6 December 2013), smoking tobacco or marijuana, taking prescription painkillers, or using illegal drugs during pregnancy is associated with double or even triple the risk of stillbirth. The authors based their findings on measurements of the chemical byproducts of nicotine in maternal blood samples; and cannabis, prescription painkillers and other drugs present in umbilical cords. Taking direct measurements provided more precise information than did previous studies of stillbirth and substance use that relied only on women’s self-reporting.


The study enrolled women between March 2006 and September 2008 in five geographically defined areas delivering at 59 hospitals participating in the Stillbirth Collaborative Research Network. Women who experienced a stillbirth and those who gave birth to a live infant participated in the study. For the study, blood samples were tested at delivery from the two groups of women and the umbilical cords from their deliveries to measure the exposure to the fetus. They also asked participants to self-report smoking and drug use during pregnancy. Participant’s blood was tested for cotinine, a derivative of nicotine, and fetal umbilical cords were tested for evidence of several types of drugs. The authors looked for evidence of the stimulants cocaine and amphetamine; prescription painkillers, such as morphine and codeine, and marijuana. These tests reflect exposure late in pregnancy. Among the women who had experienced a stillbirth, more than 80% showed no traces of cotinine and 93% tested negative for the other drugs. In comparison, about 90% of women who gave birth to a live infant tested tobacco-free and 96% tested negative for other drugs.


Based on the blood test results and women’s own responses, the authors calculated the increased risk of stillbirth for each of the substances they examined:


1. Tobacco use — 1.8 to 2.8 times greater risk of stillbirth, with the highest risk found among the heaviest smokers

2. Marijuana use — 2.3 times greater risk of stillbirth

3. Evidence of any stimulant, marijuana or prescription painkiller use-2.2 times greater risk of stillbirth

4. Passive exposure to tobacco — 2.1 times greater risk of stillbirth


The authors noted that they could not entirely separate the effects of smoking tobacco from those of smoking marijuana.


Only a small number of women tested positive for prescription painkiller use, but there was a trend towards an association of these drugs with an elevated stillbirth risk.

Paradoxical Reduction in HDL-C with Fenofibrate and Thiazolidinedione Therapy In Type 2 Diabetes


As study, published in Diabetes Care (2 December 2013), was performed to determine the occurrence of extremely low HDL-C among participants in the Action to Control Cardiovascular Risk in Diabetes (ACCORD) Lipid Trial and to examine the relationship of this finding with treatment with fenofibrate and thiazolidinedione (TZD).


The ACCORD Lipid Trial was a randomized double blind placebo controlled study conducted in patients with type 2 diabetes at 77 clinical centers across the United States and Canada in a 5,518 patient sub-set of the larger 10,251 ACCORD Glycemia Trial. Patients were enrolled from January 11, 2001 until October 29, 2005 and followed until end of study visits between March 1 and June 30, 2009. Follow-up in ACCORD Lipid was 4 to 8 years (mean 4.7 years). Patients were treated with blinded fenofibrate or placebo on a background of simvastatin therapy. The main outcome measures for these descriptive, post hoc analyses was the occurrence of extremely low HDL-C (defined as <25 mg/dl [0.647 mmol/L]) during the trial.


Results showed that among ACCORD Lipid Trial participants, the occurrence of extremely low HDL-C ever during study follow-up was 106% higher among those randomized to fenofibrate (10.1% fenofibrate vs. 4.9% placebo; P<0.001). The occurrence of low HDL-C was associated with concurrent treatment with fenofibrate and TZD (7.0% for both vs. 2.2% for neither at 48 months post-randomization).


According to the authors, idiosyncratic and marked reduction in HDL-C can occur in some patients treated with both fenofibrate and TZD, and that practitioners should recognize this important potential idiosyncratic reaction and take appropriate corrective action.

TARGET HEALTH excels in Regulatory Affairs. Each week we highlight new information in this challenging area.


FDA Approves Tretten to Treat Rare Genetic Clotting Disorder


Congenital Factor XIII deficiency is an extremely rare genetic disorder. Patients with this deficiency do not make enough Factor XIII, a protein that circulates in the blood and is important for normal clotting. Factor XIII is composed of two subunits, A and B. Factor XIII deficiency is usually caused by a deficiency of the A-subunit.


The FDA approved Tretten, Coagulation Factor XIII A-Subunit (Recombinant), the first recombinant product for use in the routine prevention of bleeding in adults and children who have congenital Factor XIII A-subunit deficiency. Tretten received orphan-drug designation for this use by the FDA because it is intended for treatment of a rare disease or condition.


Tretten is a recombinant analogue of the human Factor XIII A-subunit that is produced in yeast cells and then further purified. It is a sterile freeze-dried-powder to be reconstituted with diluent and injected intravenously. Tretten can be administered by a physician or be self-administered.


The effectiveness of Tretten was studied in 77 patients with congenital Factor XIII A-subunit deficiency, and was effective in preventing bleeding in 90% of the patients when given monthly. Some of the side effects reported in this study were headache, pain in the extremities and pain at injection site. No individual in the trial developed abnormal clots.


Tretten is made by Novo Nordisk A/S, Denmark, and is distributed by Novo Nordisk, Inc., USA.

Smoked Paprika Hummus





Several years ago, our son, Alex, introduced us to Manhattan’s best gourmet vegan restaurant, Candle79; so with him over the holidays, we went for dinner there, once again. We all love it, by the way. Our strategy there is, we have 3 or 4 courses, each ordering something different – and sharing. This way, we all get to sample a wide variety of what this inventive restaurant offers. For the appetizer course, one of the dishes was “Smoked Paprika Hummus.“ This and a few other items on the menu, which we ordered, were so-o delicious, that I bought one of the restaurant’s cookbooks in order to try their recipes out at home. The recipes are not that difficult. We want to share with our readers, Candle79’s “Smoked Paprika Hummus“ recipe, exactly as it appears in their cookbook.



  • 1 cup dried chickpeas, or 2 (15.5-ounce) cans chickpeas, drained and rinsed (I use the canned chickpeas)
  • 2 large cloves garlic, minced
  • 1 teaspoon freshly squeezed lemon juice
  • 1/4 teaspoon cayenne pepper
  • 2 teaspoons smoked paprika, plus more for garnish
  • 1/2 teaspoon sea salt (I don’t use the salt)
  • 1/2 teaspoon freshly ground pepper
  • 2 tablespoons finely chopped fresh flat-leaf parsley (I also like to use cilantro instead of parsley, for a change of pace)
  • 1/3 cup extra-virgin olive oil, plus more for garnish
  • 1/3 cup tahini
  • Optional garnishes: roasted red bell peppers, roasted garlic, lemon slices, olives, mint or parsley sprigs



  1. If using dried chickpeas, put them in a saucepan or bowl and add cold water to cover by about 2 inches. Soak in the refrigerator for at least 6 hours or overnight. Drain and rinse.
  2. Put the chickpeas in a saucepan and add cold water to cover by about 2 inches. Bring to a boil, decrease the heat, cover, and simmer until the chickpeas are tender, 50 to 60 minutes. Drain and let cool, reserving 1/4 to 1/2 cup of the cooking water.
  3. Combine the chickpeas, garlic, lemon juice, cayenne, paprika, salt, pepper, parsley, olive oil, and tahini in a bowl and stir to mix well. Transfer the mixture to a food processor fitted with the metal blade and process until well mixed. Add 1/4 cup of the reserved cooking liquid (or water or vegetable stock if using canned chickpeas) and process until smooth and almost fluffy. Add more liquid if necessary. Scrape down the sides of the bowl once or twice. Transfer to a serving bowl and refrigerate for at least 1 hour. (The hummus can be made up to 3 days ahead and refrigerated. Return to room temperature before serving.)
  4. To serve, drizzle a bit of olive oil over the hummus and sprinkle a bit of paprika. Serve with desired garnishes.



Some of the ingredients — Serve with warm pita bread



In the food processer


Stuffed cucumber with smoked paprika hummus — Try mushrooms stuffed with same



Deviled eggs with smoked paprika hummus


On an informal Sunday night at home, we find that this hummus is so good, we heat up some pita bread, break out a bottle of our favorite white wine, and just sit at our kitchen table, with raw carrot, celery, and cucumber sticks (and a few olives) and eat this delicious repast with total satisfaction. Sorta like a picnic at home.