Effect Of Chemical Chaperones Research Articles

Designing Protein Therapies: Lessons Learned from Cerezyme® for Gaucher Disease

The development of carbohydrate-modified human glucocerebrosidase (Ceredase® and Cerezyme® [Genzyme Corporation, Cambridge, Massachusetts]) for the treatment of Gaucher disease has transformed the lives of patients and led to the development of enzyme replacement therapies (ERTs) for many other lysosomal storage disorders (LSDs). Gaucher disease and the use of Cerezyme as an ERT serve as a useful model for the design of protein and other therapies for the treatment of LSDs. Like other LSDs, Gaucher disease is a progressive and lethal metabolic disorder that results in irreversible endorgan damage when untreated. Early intervention is key to preventing irreversible complications. Several hundred different mutations have been identified that give rise to Gaucher disease,1 but there is still no reliable correlation between genotype and phenotype. Response to therapy is organ specific; that is, liver and spleen demonstrate a rapid response at a relatively low dose2 but a higher dose, earlier intervention, and long-term therapy are necessary to demonstrate benefit for the skeletal disease. Gaucher disease differs from other LSDs in the low percentage of patients that develop antibodies3; this reflects the fact that many patients produce residual, though inactive or partially active, protein. Cerezyme has an excellent safety and efficacy record, demonstrated over 15 years of clinical use.4 Nevertheless, we have evaluated alternative production methods in an attempt to improve mannose receptor targeting and enzyme delivery to target tissues. To evaluate the effect of increased amounts of terminal mannose residues on the uptake and biodistribution of human glucocerebrosidase, we produced human glucocerebrosidase with mannose chain lengths of 3 to 9 residues.5 Because Cerezyme differs from the human placental enzyme used in the production of Ceredase by 1 amino acid, we also evaluated the effect of that amino acid difference on uptake and biodistribution (even though no clinical difference has been observed between Cerezyme and Ceredase).6 Although terminal mannose is essential for efficient uptake into target cells, more mannose does not alter the uptake or biodistribution of human glucocerebrosidase. More mannose did, however, increase binding to the mannose-binding lectin in vitro, which could have negative consequences on clearance and uptake in vivo. The single amino acid difference between Cerezyme and placental glucocerebrosidase had no effect on half-life, uptake, or tissue targeting.5 In addition to engineering the carbohydrate residues on glucocerebrosidase, it is also possible to engineer the protein backbone to improve biological properties. Although the crystal structure of glucocerebrosidase has been determined,7 it is of little use for predicting how to improve the protein. It does not explain why most mutations cause disease or how the protein is activated by saposin C or phospholipids and is inconsistent with biochemical data. This most likely reflects the fact that the crystal structure is just a static image of a dynamic molecule. The use of competitive inhibitors as chemical chaperones has recently been proposed as a therapeutic strategy.8 This is based on the hypothesis that mutations which cause LSDs result in misfolded proteins, which are degraded by the proteasome, an effect that is likely to be mutation specific. We have studied the effect of chemical chaperones on the uptake and stabilization of Cerezyme as well as recombinantly produced N370S mutant glucocerebrosidase.9 These studies demonstrated that the increased enzyme levels observed are due to lysosomal stabilization. Chaperones, which are competitive inhibitors, remain bound in the lysosome and inhibit enzyme activity, as also demonstrated by Steet et al.10 Because these competitive inhibitors tend to have the same pH optima as the natural substrate—that is, they bind better at low pH—there is likely to be a narrow (if any) therapeutic window. As our results demonstrate, the effects of chaperones on protein levels reported in the literature can be accounted for by reduced lysosomal degradation. Gaucher disease is the first and most effectively treated of the LSDs, and Cerezyme is the gold standard against which to measure future LSD therapies. Alternative manufacturing platforms are unlikely to offer any improvement over Cerezyme. Small molecule chaperones are at best likely to be limited to a few of the several hundred disease-causing mutations. More promising approaches for the future include the development of a secondgeneration substrate inhibition therapy with improved specificity and reduced adverse effects, as well as gene therapy for LSDs with central nervous system involvement. Clinical Therapeutics/Volume 29, Supplement C, 2007

Effects of chemical chaperones on oxidative stress and detergent-insoluble species formation following conditional expression of amyloid precursor protein carboxy-terminal fragment

Oxidative stress, protein misfolding, protein complex formation, and detergent insolubility are biochemical features of Alzheimer's disease (AD). We tested the cause-and-effect relationships among these using MC65 human neuroblastoma cells that exhibit toxicity upon conditional expression of carboxy-terminal fragments (CTFs) of the human amyloid precursor protein (APP). Treatments with three different antioxidants (alpha-tocopherol, N-acetyl cysteine, and alpha-lipoic acid) or three different compounds (glycerol, trimethylamine-N-oxide, and 4-phenylbutyric acid) that have been described to have a "chemical chaperone" function in promoting protein folding all had a protective effect on MC65 cells and decreased markers of oxidative damage and accumulation of high molecular weight amyloid (A) beta-immunoreactive (IR) species. However, chaperones partially reduced detergent insolubility of the remaining Abeta-IR species, while antioxidants did not. These results suggest that protein misfolding associated with overexpression of APP CTFs promotes oxidative stress and cytotoxicity and contributes to formation of detergent-insoluble species that appear unrelated to cytotoxicity.

Effects of chemical chaperones on partially retarded NaCl cotransporter mutants associated with Gitelman's syndrome in a mouse cortical collecting duct cell line.

Epithelial cells lining the distal convoluted tubule express the thiazide-sensitive Na-Cl cotransporter (NCC) that is responsible for the reabsorption of 5-10% of the filtered load of Na(+) and Cl(-). Mutations in NCC cause the autosomal recessive renal disorder Gitelman's syndrome (GS). GS mutations give rise to mutant transporters that are either fully (class I) or partially (class II) retarded. Recent evidence indicates that class II mutations do not alter the intrinsic transport activity of NCC. These findings suggest that in GS caused by class II NCC mutations, pharmacological chaperones may be useful in treatment. Initial attempts using 4-phenylbutyrate and glycerol to increase Na(+) uptake in Xenopus laevis oocytes expressing the class II mutant L215P were unsuccessful. To study the effect of the chaperones in a more physiological setting, we next expressed hNCC in the polarized epithelial cell line of distal tubular origin, mpkCCD. mpkCCD cells readily expressed the class II mutant R955Q, but not the class I mutant G741R. Wild-type hNCC was predominantly present in the approximately 120-1403 kD complex glycosylated form. In contrast, the R955Q mutant was predominantly present in a lower molecular weight form of approximately 100 kD. Pretreatment of R955Q expressing cells with 4-phenylbutyrate (5 mM, 16 h), but not thapsigargin (1 microM, 90 min), dimethyl sulfoxide (1%, 16 h) or glycerol (4%, 16 h), increased the expression of the complex glycosylated form and in parallel the number of hNCC positive cells. Taken together, the data indicate that 4-phenylbutyrate is a promising candidate for rescuing partially retarded but otherwise functional class II GS mutants.

Structural Control of Endoplasmic Reticulum-associated Degradation: EFFECT OF CHEMICAL CHAPERONES ON 3-HYDROXY-3-METHYLGLUTARYL-CoA REDUCTASE

The endoplasmic reticulum (ER) quality control pathway destroys misfolded and unassembled proteins in the ER. Most substrates of this ER-associated degradation (ERAD) pathway are constitutively targeted for destruction through recognition of poorly understood structural hallmarks of misfolding. However, the normal yeast ER membrane protein 3-hydroxy-3-methylglutaryl-CoA reductase (Hmg2p) undergoes ERAD that is physiologically regulated by sterol pathway signals. We have proposed that Hmg2p ERAD occurs by a regulated transition to an ERAD quality control substrate. Consistent with this, we had previously shown that Hmg2p is strongly stabilized by chemical chaperones such as glycerol, which stabilize misfolded proteins. To understand the features of Hmg2p that permit regulated ERAD, we have thoroughly characterized the effects of chemical chaperones on Hmg2p. These agents caused a reversible, immediate, direct change in Hmg2p degradation consistent with an effect on Hmg2p structure. We devised an in vitro limited proteolysis assay of Hmg2p in its native membranes. In vitro, chemical chaperones caused a dramatic, rapid change in Hmg2p structure to a less accessible form. As in the living cell, the in vitro action of chemical chaperones was highly specific for Hmg2p and completely reversible. To evaluate the physiological relevance of this model behavior, we used the limited proteolysis assay to examine the effects of changing in vivo degradation signals on Hmg2p structure. We found that changes similar to those observed with chemical chaperones were brought about by alteration of natural degradation signal. Thus, Hmg2p can undergo significant, reversible structural changes that are relevant to the physiological control of Hmg2p ERAD. These findings support the idea that Hmg2p regulation is brought about by regulated alteration of folding state. Considering the ubiquitous nature of quality control pathways in biology, it may be that this strategy of regulation is widespread.