Protein Trafficking Diseases, Small Molecule Approaches to - CHEMICAL BIOLOGY


Protein Trafficking Diseases, Small Molecule Approaches to

Heidi M. Sampson and David Y. Thomas, McGill University, Quebec, Canada

doi: 10.1002/9780470048672.wecb667

Many diseases are caused by defects in protein trafficking. Protein trafficking diseases occur when a mutant protein is recognized by the endoplasmic reticulum (ER) quality control system (ERQC), retained in the ER, and degraded in the cytosol by the proteasome rather than being trafficked to its correct site of action. Among these diseases are cystic fibrosis, lysosomal storage diseases (Fabry, Gaucher, and Tay-Sachs), nephrogenic diabetes insipidus, oculocutaneous albinism, protein C deficiency, and many others. A characteristic of many of these diseases is that the mutant protein remains functional, but it cannot escape the stringent ER quality-control machinery, and it is retained in the ER. This characteristic suggests that pharmacological interventions that promote the correct folding of the mutant protein would enable its escape from the ER and ameliorate the symptoms of the disease. In this review, we focus on specific examples of protein trafficking diseases in pharmacological or chemical chaperones have been shown to rescue trafficking of the mutant protein.

The etiology of several diseases can be traced to defects in protein trafficking. Recent studies on several different disease mutants have shown that chemicals and small molecules can correct trafficking of these mutants. As these compounds promote correct folding in a fashion analogous to the action of molecular chaperone proteins, these compounds have been termed “correctors” or “chaperones.” Several different classes of chaperones can be designated based on their mechanisms of action. Chemical chaperones are the least specific and the least potent, and which often require millimolar concentrations to function. Pharmacological chaperones are small-molecule correctors that can be subdivided into two classes based on their specificities. Specialized pharmacological chaperones are protein- or mutation-specific small molecules that interact directly with the mutant protein to provide a folding template, such as enzyme active-site inhibitors. Generalized correctors are less specific and are likely to function on the endoplasmic reticulum (ER) retention machinery, ER-associated degradation (ERAD), or other signaling pathways involved in trafficking rather than through a direct interaction with mutant proteins. We will begin with a brief overview of ER quality control, followed by specific examples of protein trafficking diseases and then discuss the different classes of correctors below.

Quality Control in the ER

Proteins that travel along the secretory pathway are subject to many quality-control checkpoints. Inside the ER, proteins must fold into their proper conformation before being sorted into vesicles destined for the Golgi apparatus. Once proteins have translocated into the ER through the translocon (Fig. 1), molecular chaperones, which include the heat shock chaperone family (e.g., Hsp70, BiP), the lectins calnexin and calreticulin, and the oxidoreductases (e.g., PDI), act on the nascent chain to promote the correctly folded conformation. Together with the oxidoreductase ERp57, the lectin chaperones act on the nascent protein in cycles of binding and release through the recognition of a monoglucosylated N-glycan. These cycles are controlled by N-glycan-modifying proteins including UDP-glucose:glycoprotein glucosyltransferase (UGGT) and glucosidases I and II (Fig. 1). Once the protein is correctly folded, the N-glycan is further modified by glucosidase II and proceeds along the secretory pathway. If several cycles of lectin binding do not result in a correctly folded protein, then the α(1-2)-ER mannosidase I (ManI) modifies the N-glycan, which prevents it from re-entering the lectin-binding cycle and targets it for degradation through ERAD in a process that involves the ER degradation-enhancing a-mannosidase-like proteins (EDEMs), Derlin and the p97/valosin-containing protein (VCP) (1). In some cases, the misfolded protein aggregates and accumulates in the ER, which triggers the unfolded protein response.

Three different classes of ERAD-targeting components function depending on the location of the lesion: a cytosolic group, a lumenal group, and a transmembrane group. These proteins somehow sense the folding status of their substrates and triage those with defects for degradation. However, the mechanisms that regulate the decision to undergo ERAD instead of forward transport in the secretory pathway are not known. It is known that to be recruited to coat protomer II (COPII) vesicles that leave the ER, proteins must be recognized by the Sec24 machinery, either directly through an ER exit code or through another receptor that can then interact with Sec24. Several different ER exit codes have been identified, including cytosolic diacidic codes such as the one found in the cystic fibrosis transmembrane conductance regulator (CFTR) protein (2), dihydrophobic motifs like that in ER-Golgi intermediate compartment protein 53 (3), and various hydrophobic signals that are found in G-protein coupled receptors (GPCRs) (4). In yeast, simply inhibiting ERAD genetically does not permit ER-retained proteins to enter COPII vesicles, which suggests that some other level of recognition by the ERQC exists. However, even when folding of substrates has improved as judged by trypsin-sensitivity assays, inhibition of ERAD still does not permit entry into COPII vesicles (5). This discrepancy could be explained by the masking of the ER exit signal in the misfolded protein. In yeast, car- boxypeptidase Y mutants whose ER exit signal is not obscured can escape ERAD even though other parts of the protein are misfolded (6). For many ER-retained disease mutant proteins, the ERQC performs its function too well and retains otherwise functional proteins. Small molecules that function in any process described above could potentially correct the trafficking of these ER-retained proteins and would be useful therapeutics.

Figure 1. Protein folding in the ER is schematicized. Folding pathways that lead to secretion are depicted in the upper half of the ER. Nonproductive folding and its consequences are depicted in the lower half of the ER. For simplicity, some pathways have been omitted, and some proteins are indicated by text alone. Once a protein enters the ER through the translocon, it undergoes N-glycosylation and is acted on by several chaperones including BiP. Glucosidases I and II (GI/GII) remove the terminal glucoses on the N-glycan moiety which enables recognition of the protein by calnexin (CNX) or calreticulin (not shown). The oxidoreductase ERp57 is bound to CNX and also acts on the protein. If the resulting protein has a non-native fold, then UGGT recognizes it and adds a terminal glucose to enable rebinding with CNX. Once the protein has folded properly, the N-glycan is modified even more by GII and can be recognized by the Sec24 protein for COPII-dependent secretion. If the protein cannot be folded natively after several cycles of CNX binding, then ManI removes the terminal mannose on the N-glycan which prevents UGGT from reglycosylating it and targets it for destruction through ERAD in a process that involves the EDEMs, Derlin, and VCP. The mutant protein is retrotranslocated out of the ER, ubiquitinated, and degraded by the 26S proteasome. In some cases, misfolded proteins have a propensity to aggregate, which overwhelms the ER folding capacity and triggers the unfolded protein response (UPR) through ER stress-sensor kinases. Correction of ER-retained mutant protein trafficking can occur through several steps of the folding pathway as indicated by numbered stars and described in the text. Pharmacological chaperones interact directly with the mutant protein (1) to stabilize its fold. Inhibitors of glucosidases (2) alter the processing of the N-glycan moiety to prevent the recognition of mutant proteins by CNX and calreticulin. Proteasomal inhibitors (3) can directly increase levels of mutant proteins and in some cases this permits some of the protein to escape ERQC. Inhibitors of SERCA calcium channels (4) correct trafficking for many different proteins by lowering the calcium levels in the ER.

Protein Trafficking Diseases

As many ER-retained mutant proteins are functional, rerouting them to their appropriate subcellular localization would restore the phenotype caused by their mislocalization. In some cases, it is estimated that only a small amount of functional protein (10-15%) is necessary to maintain health. Indeed, for some diseases a critical threshold seems to exist (7). Patients who express functional protein above this threshold have mild or no symptoms of the disease (7, 8), which suggests that even a modest increase in protein rerouting would improve the quality of life for many patients with these diseases. This finding provides the impetus for identifying correctors, even those with modest effects.

Properties of correctable protein trafficking disease mutants

Mutations that destroy protein activity such as those that abolish enzyme active sites are not amenable to rescue with correctors. However, mutations that alter the stability or folding of the protein, but that do not abrogate its activity, are good candidates for rescue by correctors. Most ER-retained missense mutations identified to date fall in this class. Frequently, these mutations are temperature sensitive and show some degree of correction at permissive temperatures (usually <30 °C). Another important feature of diseases that are amenable to rescue with correctors is the degradability of accumulated substrates or byproducts. For example, the lysosomal storage diseases Fabry, Gaucher, and Tay-Sachs all result in substrate accumulation in the lysosome; however, after treatment with pharmacological chaperones and rerouting of the enzymes, the corrected proteins reduce these substrate stores (9-12). In contrast, ER-retention diseases that result in the deposit of toxic or non-native proteins, such as fibrils associated with amyloidoses, likely would not be amenable to rescue with correctors because of the cell’s inability to degrade the non-native deposits, unless treatment with the corrector begins before irreversible damage is done. Several examples of trafficking diseases and their correctors are described below.

CFTR delta F508

One of the best-studied examples of a mutation that is amenable to rescue is the deltaF508 mutation in the chloride channel CFTR, which is the gene responsible for cystic fibrosis (13). Cystic fibrosis (CF) is the most common autosomal recessive genetic disease that affects the Caucasian population. It is a lethal disease characterized by severe dehydration of the cells lining the lung, intestine, and exocrine tissues (13). Most cases of CF can be attributed to a single mutation resulting in the deletion of a phenylalanine codon at position 508 in the protein, which is known as deltaF508. DeltaF508 CFTR can be rescued by incubation at permissive temperature (i.e., 27-30 °C), by the addition of chemical chaperones such as sodium 4-phenylbutyrate, or other osmolytes such as glycerol, dimethyl sulfoxide, and trimethylamine N-oxide (Fig. 2a) (14-16). Modulation of ER calcium levels with curcumin and thapsigargin (Fig. 2b) has also been shown to correct deltaF508 CFTR trafficking; however, these results remain controversial (17, 18). Recent high-throughput screens have identified several classes of small-molecule correctors of protein trafficking, which include aminobenzothiazoles, aminoarylthiazoles, quinazolinylaminopyrimidinones, bisaminomethylbithiazoles, quinazolinones, khivorins and substituted 1-phenylsulfonylpiperazines (19-22).

Three different assays were used to screen for correctors. One assay measured directly the amount of protein that trafficked to the cell membrane using immunofluorescence against an extracellular epitope tag in deltaF508 CFTR (19). The second assay monitored deltaF508 CFTR function by iodide influx with a halide-sensitive-YFP construct as readout (20). The third assay also monitored function but through fluorescence energy transfer between a membrane-soluble voltage-sensitive dye bis-(1,2-dibutylbarbituic acid)trimethine oxonol [DiSBAC2(3)] and a plasma membrane localized fluorescent coumarin-linked phospholipid CC2-DMPE (22). The compounds identified in these screens are likely to function at different steps of the deltaF508 CFTR folding pathway. Corr4a, which is a bisaminomethylthiazole, was shown to increase the folding efficiency of deltaF508 CFTR (20). Both Corr4a and the quinazolinone VRT-325 were shown to delay ERAD of deltaF508 CFTR (20, 22). Compounds of the aminoarylthiazole class were found to act after ER folding as no increase in folding efficiency was detected, but the stability of deltaF508 was increased at the cell surface (20). VRT-325 also increased the stability of deltaF508 CFTR at the cell surface (22). This compound was shown to correct other CFTR mutants and even a mutant in the human ether-a-go-go-related gene (hERG) (see below). Derivatives of some of these compounds are now in clinical trials.

Figure 2. Structures of selected chemical (a) and pharmacological (b) chaperones are shown.

Lysosomal storage diseases

The lysosomal storage diseases, such as Tay-Sachs, Sand- hoff, Gaucher, and Fabry disease, are autosomal recessive loss-of-function disorders. The mutant enzymes fail to degrade their respective lysosomal substrates because of their retention in the ER and subsequent degradation. This result leads to an accumulation of substrates and a variety of phenotypes including enlargement of affected organs, skeletal lesions, neurological abnormalities, and premature death. The current therapies for these diseases include inhibition of substrate production and enzyme-replacement therapy. However, enzyme-replacement therapy is not suitable for neurological phenotypes associated with some types of these diseases because of the impermeability of the blood-brain barrier; hence, other treatments are necessary. Recently, pharmacological chaperones for these diseases have been identified. As these proteins are enzymes, the pharmacological chaperones identified tend to be competitive active-site inhibitors. This finding may seem counterintuitive, but inhibitors are frequently trafficking correctors at subinhibitory doses (see below). The rationalization is that inhibitors stabilize the fold of the mutant proteins at the neutral pH of the ER and allow them to evade the ERQC. When the proteins reach the lysosome, the high concentration of the substrates successfully compete away inhibitor binding thereby enabling degradation of the substrates to occur. Some examples of correctors for each of these diseases are discussed below.

Several active-site specific chaperones have been identified for beta-hexosaminidase A, which is the multisubunit enzyme responsible for Tay-Sachs and Sandhoff diseases (9, 11, 23). The screening assays monitor inhibition of purified enzyme activity fluorometrically using the fluorescent artificial substrate 4-methylumbelliferyl-β-N-acetylglucosamine. These assays are then followed by cell-based assays to determine the amount of functional protein that traffics to the lysosome. Several distinct structural classes of pharmacological chaperones have been identified for these diseases including aza-sugars, pyrimethamine (Fig. 2b), and substituted bicyclic and tricyclic nitrogen-containing heterocycles (9, 11, 23).

Gaucher disease is caused by a deficiency in lysosomal beta-glucosidase activity. Deoxynojirimycins are known inhibitors of several enzymes, which include beta-glucosidase. Several alkylated deoxynojirimycins were screened for correction of N370S Gaucher mutant lysosomal beta-glucosidase activity in an intact cell assay (10). N-(n-nonyl)deoxynojirimycin (Fig. 2b) was found to be the most potent corrector, but consistent with its activity as a competitive inhibitor, it inhibited beta-glucosidase activity at higher concentrations. Because this deoxynojirimycin was shown to inhibit alpha-glucosidase at the effective concentrations for beta-glucosidase correction, other structural variants were tested and alpha-1-C-octyl-deoxynojiri- mycin was shown to have the most promise (24). Miglustat, which is the deoxynorijimycin derivative N-(n-butyl)deoxynojirimycin (Fig. 2b), corrected trafficking of several mutants including N370S in one cell type, but not in another (25). Several alkylated iminosugars, which include morpholine, piperazine, isofagomine, and 2,5-dideoxy-2,5-imino-D-glucitol, were correctors for both N370S and G202R mutants in cell culture (26). The addition of an adamantyl cap on the alkyl chain also increased the amount of correction achieved with the de- oxynojirimycin compounds and improved the activity of the other iminosugar derivatives as well (26-28). Interestingly, the carbohydrate portion of these compounds was shown to be sufficient for chaperone activity (29, 30). Mutation-specific correctors have also been identified. The synthetic carbohydrate mimic N-octylvalienamine specifically corrects the trafficking of the F213I mutant, but not the N370S mutant, which suggests that subtle differences may exist between these two mutants (31). Recently, a high-throughput screen for inhibitors of beta-glucosidase identified three classes of corrector compounds: aminoquinolines, sulfonamides, and triazines (32). These compounds were shown to be potent inhibitors and correct trafficking of the N370S mutant. In addition to active-site inhibitors, the hydroxymethylglutaryl (HMG)-CoA reductase inhibitor mevastatin (Fig. 2b) was recently shown to correct beta-glucosidase trafficking by altering the levels of free unesterified cholesterol in severe Gaucher mutant cells (33). Recent work on three lysosomal storage disease proteins, including beta-glucosidase, has shown that altering the intracellular calcium levels with the voltage-gated calcium channel inhibitors diltiazem and verapamil can upregulate the ER folding capacity and correct the trafficking of these mutants (34).

Fabry disease is caused by mutations in the gene-encoding lysosomal alpha galactosidase A. Treatment of R301Q Fabry mutant cells with subinhibitory concentrations of the potent competitive inhibitor 1-deoxygalactonojirimycin results in efficient folding and stabilization of the mutant protein (35). Several derivatives of this compound were also shown to correct trafficking of Fabry mutants, but they were less effective than 1-deoxygalactonojirimycin (36). Several other mutations, including A97V, R112H, R112C, A143T, and L300P, were also corrected by treatment with 1-deoxygalactonojirimycin (37). No high-throughput screens for other correctors of Fabry mutants have been performed.


hERG encodes the pore-forming subunit of the rapidly activating delayed rectifier potassium channel. Mutations or drug treatments that cause retention of hERG in the ER result in cardiac arrhythmias that can lead to sudden death. Hence, drugs are now routinely screened for their effects on hERG function before being pursued in clinical trials. Several ER-retained hERG mutants can be rescued by chemical and pharmacological chaperones. The G601S and N470D mutations can be rescued by growth at permissive temperature or incubation with known hERG channel blockers E-4031, cisapride, and astemizole (Fig. 2b) (38, 39). hERG G601S has recently been used in a small molecule screen to identify correctors of hERG trafficking (40). This assay measured the amount of hERG G601S that trafficked to the cell surface by chemiluminescent detection of an extracellular epitope tag. Several different hERG blockers were found to rescue the ER retained mutant, which is consistent with their action as pharmacological chaperones. Other compounds have also been shown to rescue the trafficking of certain hERG mutants selectively. For example, the sarcoplas- mic/ER calcium ATPase (SERCA) inhibitor thapsigargin can rescue the trafficking of G601S and F805C mutants, but not N470D, whereas E-4031 rescues G601S and N470D mutants but not F805C mutants (41).

G protein-coupled receptors

Many diseases caused by mutations in GPCRs are also the result of ER retention. Mutations in the vasopressin 2 receptor result in nephrogenic diabetes insipidus, which is a disease characterized by the kidney’s inability to concentrate urine. Trafficking of several different ER-retained mutants can be corrected by small nonpeptide V2R and V1R antagonists (42-46). The V206D mutation can also be corrected with glycerol, DMSO, SERCA inhibitors thapsigargin and curcumin, and the calcium ionophore ionomycin (44). This mutation cannot be corrected by growth at permissive temperature or with the addition of 4-phenylbutyrate (44). In contrast, the A98P, L274P, and R113W mutations can be corrected with the osmolytes trimethylamine N-oxide and DMSO, as well as with growth at permissive temperature (47). However, glycerol treatment does not correct the trafficking of these mutations.

Mutations in another GPCR, which is the gonadotropinreleasing hormone receptor, result in hypogonadotropic hypogonadism. Seventeen mutations in this gene have been characterized as misfolding or misrouting mutants, and most of these can be rescued by incubation with peptidomimetic antagonists (48). These pharmacological chaperones are indoles, quinolones, and erythromycin macrolides. Another example of a GPCR with ER-retained disease mutations is rhodopsin, which when mutated causes retinitis pigmentosa (49). A mild rhodopsin mutant P23H associated with night blindness can be rescued by treatment with 11-cis -retinal, which is its covalently bound chromophore (49).

Alpha-1-antitrypsin Z variant

Alpha-1-antitrypsin mutations are associated with early-onset emphysema and liver disease that results in early death (50). The Z-variant of the disease is the most common and is present in over 95% of cases. This variant has been shown to be rescued in vitro by treatment with proteasome inhibitors (50). Several chemical and pharmacological chaperones have also been shown to correct the secretion of the mutant protein, which include the glucosidase inhibitor castanospermine, as well as the mannosidase inhibitors kifunensine (Fig. 2b) and 1,4-dideoxy-1,4-imino-D-mannitol hydrochloride (51). Incubation with the chemical chaperone 4-phenylbutyrate also results in increased trafficking. However, growth at permissive temperature does not result in trafficking correction, although it does decrease the amount of mutant protein that becomes degraded (52).

Correctors of Protein Trafficking

Chemical chaperones

Several different classes of chaperones can be designated based on their mechanisms of action. Chemical chaperones are those that act nonspecifically. Although these chaperones are nonspecific, their use as therapeutics may be limited because of the high concentrations required to achieve correction. The mechanisms of correction employed by chemical chaperones may include the induction of molecular chaperone transcription or the nonspecific stabilization of proteins through masking of hydrophobic domains. For example, 4-phenylbutyrate (4-PBA) is a histone deacetylase inhibitor that activates transcription of different genes including the heat shock proteins (53). Studies on deltaF508 CFTR suggest that 4-PBA reduces the protein levels of the constitutive Hsc70 chaperone (54). This in turn reduces the amount of deltaF508 CFTR that interacts with Hsc70. The decrease in Hsc70 protein levels induces an increase in Hsp70 levels, which has been proposed to be a more effective chaperone, thus helping deltaF508 CFTR to fold and enabling it to escape from the ERQC (55). Interestingly, other phenyl-fatty acids have also been shown to correct trafficking, and their efficacy is not correlated with their efficacy as histone deacetylase inhibitors, which suggests that they function through another mechanism (56), perhaps through their ability to bind and mask hydrophobic regions. Although 4-PBA corrects trafficking of many misfolded proteins, its use as a therapeutic may be limited. For instance, although it corrects trafficking of Fabry disease mutants, the mutant protein is not functional (57).

Specialized pharmacological chaperones

Pharmacological chaperones can be divided into two subclasses depending on their mechanisms of action. Specialized pharmacological chaperones are correctors that interact directly with the mutant protein to stabilize correct folding and enable it to escape ER retention mechanisms. This subclass of chaperones is the most specific and is mainly composed of inhibitors. Specialized pharmacological chaperones may also show mutation-specific profiles. For example, two common mutations in Gaucher disease N370S and G202R are both localized to the catalytic domain of the protein and are both corrected by active-site directed inhibitors. However, the L444P mutation, which is located in another domain, cannot be corrected with these chaperones. However, it can be corrected by incubation at permissive temperature, which suggests that simply another class of pharmacological chaperone is necessary to achieve correction (26).

Examples of specialized pharmacological chaperones include the competitive inhibitors of the lysosomal storage disease enzymes, the potent hERG channel blockers, and the

cell-permeable nonpeptide antagonists of the vasopressin receptor described above. Enzyme substrates can also act as pharmacological chaperones by providing a scaffold on which the native fold can be formed. For example, the R402Q mutant of tyrosinase, which causes oculocutaneous albinism, can be rescued by the addition of its substrate (L-tyrosine) or its sub- strate/cofactor L-Dopa (58). Some specialized pharmacological chaperones are not inhibitors, such as the VRT-325 and Corr4a correctors of deltaF508 CFTR (20). VRT-325 is an interesting compound as it has also been shown to correct trafficking of P-glycoprotein, a protein related to CFTR, and the structurally unrelated G601S mutant of the hERG potassium channel. This compound has not yet been demonstrated to interact directly with hERG.

Generalized pharmacological chaperones

Another subclass of pharmacological chaperones includes compounds that act through other mechanisms. This subclass includes generalized chaperones—those that rescue the trafficking defects of many or all ER-retained proteins through a universal mechanism. Many different mechanisms of correction are possible. Examples of this subclass of chaperones are described below.

One might expect that simply inhibiting ERAD with pro- teasome inhibitors would be the most generalized form of correction (Fig- 1, star 3); however, this strategy gives inconsistent results with different ER-retained mutants. The hERG Y611H mutant is not corrected, deltaF508 CFTR is only weakly corrected and alpha-1-antitrypsin Z variant is corrected as visualized by confocal microscopy (50, 59, 60).

As discussed above, for many ER-retained proteins, altering the intracellular calcium levels with SERCA inhibitors or with calcium ionophores corrects trafficking of the mutant proteins, presumably by altering the ER’s capacity for folding (Fig. 1, star 4), because several ER chaperones require calcium for function. Interestingly, for lysosomal storage diseases, trafficking correction occurs through changing the levels of calcium in the cytosol and not in the ER. These two different mechanisms of correction highlight the importance of calcium signaling in protein trafficking.

Another common mechanism of correction is ER glucosidase inhibition (Fig. 1, star 2), which prevents the recognition of misfolded proteins by calnexin and calreticulin and presumably allows them to escape the ERQC. Inhibition of glucosidase with castanospermine and miglustat were shown to correct the trafficking of deltaF508 CFTR and alpha-1-antitrypsin (51, 61). Whether this mechanism also works on other ER-retained mutants remains to be determined.

For other small molecules, the mechanism of action is still unclear. For example, sildenafil and structural analogs have recently been shown to correct trafficking of deltaF508 CFTR mutants (19, 21). Sildenafil is an inhibitor of phosphodiesterase activity; however, the link between this function and CFTR trafficking correction remains to be elucidated.


The etiology of many genetic diseases can be traced to defects in protein trafficking. Several mutants are functional but are retained in the ER because of the overly stringent ERQC. Corrector compounds that permit the escape of ER-retained mutant proteins from the ERQC have great potential as therapeutics. Several types of trafficking correctors have been identified. Chemical chaperones are nonspecific and require high concentrations to be effective, which thereby limits their potential as therapeutics. Pharmacological chaperones function at much lower concentrations and show greater promise for drug development. Pharmacological chaperones can be classified into specialized chaperones that are protein or mutation-specific or generalized chaperones that can correct the trafficking of many ER-retained mutants. The identification of a generalized pharmacological chaperone that can correct all ER-retained disease mutants without grossly affecting normal proteins would enable treatment of many different protein trafficking diseases. Defining the mechanisms of action for this class of pharmacological chaperones will be the next milestone in trafficking disease research.

Additive effects of correctors have recently been shown for the deltaF508 CFTR mutant (62), which suggests that treatments that combine two or more chaperones may be a good option if no single potent corrector can be identified for a particular trafficking disease. Indeed combinatorial studies are likely to be the next key area of screening for correctors of protein trafficking diseases.


Research on CFTR trafficking in the Thomas lab is supported by grants from Cystic Fibrosis Foundation Therapeutics, Inc., and the Canadian Institutes of Health Research. H.M.S. is supported by a fellowship from the Canadian Cystic Fibrosis Foundation.


1. Nakatsukasa K, Brodsky JL. The recognition and retrotransloca- tion of misfolded proteins from the endoplasmic reticulum. Traffic. In press.

2. Wang X, Matteson J, An Y, Moyer B, Yoo JS, Bannykh S, Wilson IA, Riordan JR, Balch WE. COPII-dependent export of cystic fibrosis transmembrane conductance regulator from the ER uses a di-acidic exit code. J. Cell Biol. 2004; 167:65-74.

3. Kappeler F, Klopfenstein DR, Foguet M, Paccaud JP, Hauri HP. The recycling of ERGIC-53 in the early secretory pathway. ERGIC-53 carries a cytosolic endoplasmic reticulum-exit determinant interacting with COPII. J. Biol. Chem. 1997; 272:31801- 31808.

4. Dong C, Filipeanu CM, Duvernay MT, Wu G. Regulation of G protein-coupled receptor export trafficking. Biochim. Biophys. Acta 2007; 1768:853-870.

5. Pagant S, Kung L, Dorrington M, Lee MC, Miller EA. Inhibiting endoplasmic reticulum (ER)-associated degradation of misfolded Yor1p does not permit ER export despite the presence of a diacidic sorting signal. Mol. Biol. Cell 2007; 18:3398-3413.

6. Kincaid MM, Cooper AA. Misfolded proteins traffic from the endoplasmic reticulum (ER) due to ER export signals. Mol. Biol. Cell 2007; 18:455-463.

7. Amaral MD. Processing of CFTR: traversing the cellular mazehow much CFTR needs to go through to avoid cystic fibrosis? Pediatr. Pulmonol. 2005; 39:479-491.

8. Mahuran DJ. Biochemical consequences of mutations causing the GM2 gangliosidoses. Biochim. Biophys. Acta 1999; 1455:105-138.

9. Maegawa GH, Tropak M, Buttner J, Stockley T, Kok F, Clarke JT, Mahuran DJ. Pyrimethamine as a potential pharmacological chaperone for late-onset forms of GM2 gangliosidosis. J. Biol. Chem. 2007; 282:9150-9161.

10. Sawkar AR, Cheng WC, Beutler E, Wong CH, Balch WE, Kelly JW. Chemical chaperones increase the cellular activity of N370S beta -glucosidase: a therapeutic strategy for Gaucher disease. Proc. Natl. Acad. Sci. U.S.A. 2002; 99:15428-15433.

11. Tropak MB, Reid SP, Guiral M, Withers SG, Mahuran D. Pharmacological enhancement of beta-hexosaminidase activity in fibroblasts from adult Tay-Sachs and Sandhoff Patients. J. Biol. Chem. 2004; 279:13478-13487.

12. Yam GH, Zuber C, Roth J. A synthetic chaperone corrects the trafficking defect and disease phenotype in a protein misfolding disorder. FASEB J. 2005; 19:12-18.

13. Ratjen F, Doring G. Cystic fibrosis. Lancet 2003; 361:681-689.

14. Brown CR, Hong-Brown LQ, Biwersi J, Verkman AS, Welch WJ. Chemical chaperones correct the mutant phenotype of the delta F508 cystic fibrosis transmembrane conductance regulator protein. Cell Stress Chaperones. 1996; 1:117-125.

15. Denning GM, Anderson MP, Amara JF, Marshall J, Smith AE, Welsh MJ. Processing of mutant cystic fibrosis transmembrane conductance regulator is temperature-sensitive. Nature 1992; 358:761-764.

16. Sato S, Ward CL, Krouse ME, Wine JJ, Kopito RR. Glycerol reverses the misfolding phenotype of the most common cystic fibrosis mutation. J. Biol. Chem. 1996; 271:635-638.

17. Egan ME, Glockner-Pagel J, Ambrose C, Cahill PA, Pappoe L, Balamuth N, Cho E, Canny S, Wagner CA, Geibel J, Caplan MJ. Calcium-pump inhibitors induce functional surface expression of Delta F508-CFTR protein in cystic fibrosis epithelial cells. Nat. Med. 2002; 8:485-492.

18. Song Y, Sonawane ND, Salinas D, Qian L, Pedemonte N, Galietta LJ, Verkman AS. Evidence against the rescue of defective DeltaF508-CFTR cellular processing by curcumin in cell culture and mouse models. J Biol Chem. 2004; 279:40629-40633.

19. Carlile GW, Robert R, Zhang D, Teske KA, Luo Y, Hanrahan JW, Thomas DY. Correctors of protein trafficking defects identified by a novel high-throughput screening assay. ChemBioChem. 2007; 8:1012-1020.

20. Pedemonte N, Lukacs GL, Du K, Caci E, Zegarra-Moran O, Galietta LJ, Verkman AS. Small-molecule correctors of defective DeltaF508-CFTR cellular processing identified by high-throughput screening. J. Clin. Invest. 2005; 115:2564-2571.

21. Robert R, Carlile GW, Pavel C, Liu N, Anjos SM, Liao J, Luo Y, Zhang D, Thomas DY, Hanrahan JW. Structural analogue of sildenafil identified as a novel corrector of the F508del-CFTR trafficking defect. Mol. Pharmacol. 2007.

22. Van Goor F, Straley KS, Cao D, Gonzalez J, Hadida S, Hazlewood A, Joubran J, Knapp T, Makings LR, Miller M, Neuberger T, Olson E, Panchenko V, Rader J, Singh A, Stack JH, Tung R, Grootenhuis PD, Negulescu P. Rescue of DeltaF508-CFTR trafficking and gating in human cystic fibrosis airway primary cultures by small molecules. Am. J. Physiol. Lung Cell Mol. Physiol. 2006; 290:L1117-1130.

23. Tropak MB, Blanchard JE, Withers SG, Brown ED, Mahuran D. High-throughput screening for human lysosomal beta-N-Acetyl hexosaminidase inhibitors acting as pharmacological chaperones. Chem. Biol. 2007; 14:153-164.

24. Yu L, Ikeda K, Kato A, Adachi I, Godin G, Compain P, Martin O, Asano N. Alpha-1-C-octyl-1-deoxynojirimycin as a pharmacological chaperone for Gaucher disease. Bioorg. Med. Chem. 2006; 14:7736-7744.

25. Alfonso P, Pampin S, Estrada J, Rodriguez-Rey JC, Giraldo P, Sancho J, Pocovi M. Miglustat (NB-DNJ) works as a chaperone for mutated acid beta-glucosidase in cells transfected with several Gaucher disease mutations. Blood Cells Mol. Dis. 2005; 35:268-276.

26. Sawkar AR, Adamski-Werner SL, Cheng WC, Wong CH, Beutler E, Zimmer, KP, and Kelly, JW. Gaucher disease-associated glucocerebrosidases show mutation-dependent chemical chaperoning profiles. Chem. Biol. 2005; 12:1235-1244.

27. Sawkar AR, Schmitz M, Zimmer KP, Reczek D, Edmunds T, Balch WE, Kelly JW. Chemical chaperones and permissive temperatures alter localization of Gaucher disease associated glucocerebrosidase variants. ACS Chem. Biol. 2006; 1:235-251.

28. Yu Z, Sawkar AR, Whalen LJ, Wong CH, Kelly JW. Isofagomine- and 2,5-anhydro-2,5-imino-D-glucitol-based glucocerebrosidase pharmacological chaperones for Gaucher disease intervention. J. Med. Chem. 2007; 50:94-100.

29. Chang HH, Asano N, Ishii S, Ichikawa Y, Fan JQ. Hydrophilic iminosugar active-site-specific chaperones increase residual gluco- cerebrosidase activity in fibroblasts from Gaucher patients. FEBS J. 2006; 273:4082-4092.

30. Steet RA, Chung S, Wustman B, Powe A, Do H, Kornfeld SA. The iminosugar isofagomine increases the activity of N370S mutant acid beta-glucosidase in Gaucher fibroblasts by several mechanisms. Proc. Natl. Acad. Sci. U.S.A. 2006; 103:13813-13818.

31. Lin H, Sugimoto Y, Ohsaki Y, Ninomiya H, Oka A, Taniguchi M, Ida H, Eto Y, Ogawa S, Matsuzaki Y, Sawa M, Inoue T, Hi- gaki K, Nanba E, Ohno K, Suzuki Y. N-octyl-beta-valienamine up-regulates activity of F213I mutant beta-glucosidase in cultured cells: a potential chemical chaperone therapy for Gaucher disease. Biochim. Biophys. Acta 2004; 1689:219-228.

32. Zheng W, Padia J, Urban DJ, Jadhav A, Goker-Alpan O, Simeonov A, Goldin E, Auld D, LaMarca ME, Inglese J, Austin CP, Sidransky E. Three classes of glucocerebrosidase inhibitors identified by quantitative high-throughput screening are chaperone leads for Gaucher disease. Proc. Natl. Acad. Sci. U.S.A. 2007; 104:13192-13197.

33. Ron I, Horowitz M. Intracellular cholesterol modifies the ERAD of glucocerebrosidase in Gaucher disease patients. Mol. Genet. Metab. 2008; 93:426-436.

34. Mu TW, Fowler DM, Kelly JW. Partial restoration of mutant enzyme homeostasis in three distinct lysosomal storage disease cell lines by altering calcium homeostasis. PLoS Biol. 2008; 6:e26.

35. Fan JQ, Ishii S, Asano N, Suzuki Y. Accelerated transport and maturation of lysosomal alpha-galactosidase A in Fabry lymphoblasts by an enzyme inhibitor. Nat. Med. 1999; 5:112-115.

36. Asano N, Ishii S, Kizu H, Ikeda K, Yasuda K, Kato A, Martin OR, Fan JQ. In vitro inhibition and intracellular enhancement of lysosomal alpha-galactosidase A activity in Fabry lymphoblasts by 1-deoxygalactonojirimycin and its derivatives. Eur. J. Biochem. 2000; 267:4179-4186.

37. Shin SH, Murray GJ, Kluepfel-Stahl S, Cooney AM, Quirk JM, Schiffmann R, Brady RO, Kaneski CR. Screening for pharmacological chaperones in Fabry disease. Biochem. Biophys. Res. Commun. 2007; 359:168-173.

38. Gong Q, Anderson CL, January CT, Zhou Z. Pharmacological rescue of trafficking defective HERG channels formed by coassembly of wild-type and long QT mutant N470D subunits. Am. J. Physiol. Heart Circ. Physiol. 2004; 287:H652-658.

39. Zhou Z, Gong Q, January CT. Correction of defective protein trafficking of a mutant HERG potassium channel in human long QT syndrome. Pharmacological and temperature effects. J. Biol. Chem. 1999; 274:31123-31126.

40. Wible BA, Hawryluk P, Ficker E, Kuryshev YA, Kirsch G, Brown AM. HERG-Lite: a novel comprehensive high-throughput screen for drug-induced hERG risk. J. Pharmacol. Toxicol. Methods 2005; 52:136-145.

41. Delisle BP, Anderson CL, Balijepalli RC, Anson BD, Kamp TJ, January CT. Thapsigargin selectively rescues the trafficking defective LQT2 channels G601S and F805C. J. Biol Chem. 2003; 278:35749-35754.

42. Bernier V, Lagace M, Lonergan M, Arthus MF, Bichet DG, Bouvier M. Functional rescue of the constitutively internalized V2 vasopressin receptor mutant R137H by the pharmacological chaperone action of SR49059. Mol Endocrinol. 2004; 18:2074-2084.

43. Morello JP, Salahpour A, Laperriere A, Bernier V, Arthus MF, Lonergan M, Petaja-Repo U, Angers S, Morin D, Bichet DG, Bouvier M. Pharmacological chaperones rescue cell-surface expression and function of misfolded V2 vasopressin receptor mutants. J. Clin. Invest. 2000; 105:887-895.

44. Robben JH, Sze M, Knoers NV, Deen PM. Rescue of vasopressin V2 receptor mutants by chemical chaperones: specificity and mechanism. Mol. Biol. Cell 2006; 17:379-386.

45. Robben JH, Sze M, Knoers NV, Deen PM. Functional rescue of vasopressin V2 receptor mutants in MDCK cells by pharmacochaperones: relevance to therapy of nephrogenic diabetes insipidus. Am. J. Physiol. Renal Physiol. 2007; 292:F253-260.

46. Wuller S, Wiesner B, Loffler A, Furkert J, Krause G, Hermosilla R, Schaefer M, Schulein R, Rosenthal W, Oksche A. Pharmacochaperones post-translationally enhance cell surface expression by increasing conformational stability of wild-type and mutant vasopressin V2 receptors. J. Biol. Chem. 2004; 279:47254-47263.

47. Cheong HI, Cho HY, Park HW, Ha IS, Choi Y. Molecular genetic study of congenital nephrogenic diabetes insipidus and rescue of mutant vasopressin V2 receptor by chemical chaperones. Nephrology (Carlton). 2007; 12:113-117.

48. Janovick JA, Brothers SP, Cornea A, Bush E, Goulet MT, Ashton WT, Sauer DR, Haviv F, Greer J, Conn PM. Refolding of misfolded mutant GPCR: post-translational pharmacoperone action in vitro. Mol. Cell Endocrinol. 2007; 272:77-85.

49. Noorwez SM, Kuksa V, Imanishi Y, Zhu L, Filipek S, Palczewski K, Kaushal S. Pharmacological chaperone-mediated in vivo folding and stabilization of the P23H-opsin mutant associated with autosomal dominant retinitis pigmentosa. J. Biol. Chem. 2003; 278:14442-14450.

50. Novoradovskaya N, Lee J, Yu ZX, Ferrans VJ, Brantly M. Inhibition of intracellular degradation increases secretion of a mutant form of alpha1-antitrypsin associated with profound deficiency. J. Clin. Invest. 1998; 101:2693-2701.

51. Marcus NY, Perlmutter DH. Glucosidase and mannosidase inhibitors mediate increased secretion of mutant alpha1 antitrypsin Z. J. Biol. Chem. 2000; 275:1987-1992.

52. Burrows JA, Willis LK, Perlmutter DH. Chemical chaperones mediate increased secretion of mutant alpha 1-antitrypsin (alpha 1-AT) Z: A potential pharmacological strategy for prevention of liver injury and emphysema in alpha 1-AT deficiency. Proc. Natl. Acad. Sci. U.S.A. 2000; 97:1796-1801.

53. Wright JM, Zeitlin PL, Cebotaru L, Guggino SE, Guggino WB. Gene expression profile analysis of 4-phenylbutyrate treatment of IB3-1 bronchial epithelial cell line demonstrates a major influence on heat-shock proteins. Physiol. Genomics. 2004; 16:204-211.

54. Rubenstein RC, Zeitlin, PL. Sodium 4-phenylbutyrate downregulates Hsc70: implications for intracellular trafficking of DeltaF508-CFTR. Am. J. Physiol. Cell Physiol. 2000; 278:C259-267.

55. Choo-Kang LR, Zeitlin PL. Induction of HSP70 promotes DeltaF508 CFTR trafficking. Am. J. Physiol. Lung Cell Mol. Physiol. 2001; 281:L58-68.

56. Tveten K, Holla OL, Ranheim T, Berge KE, Leren TP, Kulseth MA. 4-Phenylbutyrate restores the functionality of a misfolded mutant low-density lipoprotein receptor. FEBS J. 2007; 274:1881- 1893.

57. Yam GH, Roth J, Zuber C. 4-Phenylbutyrate rescues trafficking incompetent mutant alpha-galactosidase A without restoring its functionality. Biochem. Biophys. Res. Commun. 2007; 360:375-380.

58. Halaban R, Patton RS, Cheng E, Svedine S, Trombetta ES, Wahl ML, Ariyan S, Hebert DN. Abnormal acidification of melanoma cells induces tyrosinase retention in the early secretory pathway. J. Biol. Chem. 2002; 277:14821-14828.

59. Gong Q, Keeney DR, Molinari M, Zhou Z. Degradation of trafficking-defective long QT syndrome type II mutant channels by the ubiquitin-proteasome pathway. J. Biol. Chem. 2005; 280: 19419-19425.

60. Vij N, Fang S, Zeitlin PL. Selective inhibition of endoplasmic reticulum-associated degradation rescues DeltaF508-cystic fibrosis transmembrane regulator and suppresses interleukin-8 levels: therapeutic implications. J. Biol. Chem. 2006; 281:17369-17378.

61. Norez C, Noel S, Wilke M, Bijvelds M, Jorna H, Melin P, DeJonge H, Becq F. Rescue of functional delF508-CFTR channels in cystic fibrosis epithelial cells by the alpha-glucosidase inhibitor miglustat. FEBS Lett. 2006; 580:2081-2086.

62. Wang Y, Loo TW, Bartlett MC, Clarke DM. Additive effect of multiple pharmacological chaperones on maturation of CFTR processing mutants. Biochem. J. 2007; 406:257-263.

Further Reading

Aridor M, Hannan LA. Traffic jam: a compendium of human diseases that affect intracellular transport processes. Traffic 2000; 1:836-851.

Aridor M, Hannan LA. Traffic jams II: an update of diseases of intracellular transport. Traffic 2002; 3:781-790.

Balch WE, Morimoto RI, Dillin A, Kelly JW. Adapting proteostasis for disease intervention. Science 2008; 319:916-919.

Bernier V, Lagace M, Bichet DG, Bouvier M. Pharmacological chaperones: potential treatment for conformational diseases. Trends Endocrinol. Metab. 2004; 15:222-228.

Conn PM, Ulloa-Aguirre A, Ito J, Janovick JA. G protein-coupled receptor trafficking in health and disease: lessons learned to prepare for therapeutic mutant rescue in vivo. Pharmacol. Rev. 2007; 59:225-250.

Fan JQ, Ishii S. Active-site-specific chaperone therapy for Fabry disease. Yin and Yang of enzyme inhibitors. FEBS J. 2007; 274:4962-4971.

Hebert DN, Molinari M. In and out of the ER: protein folding, quality control, degradation, and related human diseases. Physiol. Rev. 2007; 87:1377-1408.

Tropak MB, Mahuran D. Lending a helping hand, screening chemical libraries for compounds that enhance beta-hexosaminidase A activity in GM2 gangliosidosis cells. FEBS J. 2007; 274:4951-4961.

Yu Z, Sawkar AR, Kelly JW. Pharmacologic chaperoning as a strategy to treat Gaucher disease. FEBS J. 2007; 274:4944-4950.

See Also

Chaperones, Molecular

Chemical Libraries: Screening for Biologically Active Small Molecules

Protein Misfolding and Disease, Chemical Biology of

Lysosomal Disorders

Endoplasmic Reticulum (ER): Topics in Chemical Biology