2008 分子伴侣综述

Biol. Chem., Vol. 389, pp. 1–11, January 2008 • Copyright � by Walter de Gruyter • Berlin • New York. DOI 10.1515/BC.2008.009 2008/245 Article in press - uncorrected proof Review A counterintuitive approach to treat enzyme deficiencies: use of enzyme inhibitors for restoring mutant enzyme activity Jian-Qiang Fan Department of Human Genetics, Mount Sinai School of Medicine, Fifth Avenue at 100th Street, New York, NY 10029, USA e-mail: jian-qiang.fan@mssm.edu Abstract Pharmacological chaperone therapy is an emerging counterintuitive approach to treat protein deficiencies resulting from mutations causing misfolded protein con- formations. Active-site-specific chaperones (ASSCs) are enzyme active-site directed small molecule pharmaco- logical chaperones that act as a folding template to assist protein folding of mutant proteins in the endoplasmic reticulum (ER). As a result, excessive degradation of mutant proteins in the ER-associated degradation (ERAD) machinery can be prevented, thus restoring enzyme activity. Lysosomal storage disorders (LSDs) are suitable candidates for ASSC treatment, as the levels of enzyme activity needed to prevent substrate storage are relatively low. In addition, ASSCs are orally active small molecules and have potential to gain access to most cell types to treat neuronopathic LSDs. Competitive enzyme inhibitors are effective ASSCs when they are used at sub- inhibitory concentrations. This whole new paradigm pro- vides excellent opportunity for identifying specific drugs to treat a broad range of inherited disorders. This review describes protein misfolding as a pathophysiological cause in LSDs and provides an overview of recent advances in the development of pharmacological chap- erone therapy for the diseases. In addition, a generalized guidance for the design and screening of ASSCs is also presented. Keywords: active-site-specific chaperone; endoplasmic reticulum-associated degradation; enzyme inhibitor; lysosomal storage disorders; pharmacological chaperone; protein misfolding. Introduction Lysosomal storage disorders (LSDs) are a group of dis- orders resulting from the abnormal metabolism of macro- substances, such as glycosphingolipids, glycogen, mucopolysaccharides and glycoproteins (Winchester et al., 2000). Deficiencies in metabolic enzymes or activa- tors required for the degradation of such substances cause massive storages of substrates in lysosomes of various cell types and further introduce secondary inflammations and cell damage that lead to tissue dys- function. More than 50 disorders have been identified, and many are neuronopathic and involve neurological changes, degeneration and retardation in metal devel- opment. The severity of diseases varies depending on the level of residual enzyme activity. In the most severe infantile form, patients are often developmentally or men- tally retarded and do not survive up to the first year of life. In contrast, patients of adult forms of diseases are normally asymptomatic during the infantile period or childhood and are typically not affected in mental devel- opment. Clinical symptoms eventually appear during adulthood and cause shortened life expectation. The overall prevalence of LSDs is estimated as 1 in 7700–13,000 live births (Santavuori, 1988; Meikle et al., 1999; Rider and Rider, 1999). Gaucher disease is the most common LSD, affecting approximately 10,000 patients in developed countries. Over the last 20 years, progress in the development of therapies for LSDs has been dramatic. Enzyme replace- ment therapy (ERT), bone marrow transplantation and substrate reduction therapy (SRT) are currently being used to treat these disorders, and the potential of gene and stem cell therapies is being investigated (Schiffmann and Brady, 2002; Ellinwood et al., 2004; Eto et al., 2004). Since the first ERT for the treatment of Gaucher disease was approved in 1992, ERT has been approved for treat- ing Fabry disease, mucopolysaccharidoses I, II and VI and Pompe disease. Bone marrow transplantation in LSDs was first demonstrated in a muccopolysacchar- dosis I patient (Hobbs et al., 1981) and has been per- formed for mucopolysaccharidoses II and VI, Gaucher disease, Krabbe disease, metachromatic dystrophy and several diseases with varying clinical benefit (Krivit et al., 1999; Schiffmann and Brady, 2002). SRT which uses orally active N-butyl-deoxynojirimycin to partially inhibit the synthesis of glycosphingolipids has been approved and evaluated for type I Gaucher disease (Cox et al., 2000, 2003). However, for most of the LSDs, particularly with neuronopathic symptoms, supportive management remains the only treatment at present. Pharmacological chaperone therapy is a novel and emerging therapeutic strategy using small molecules as potential oral drugs for the treatment of LSDs (Fan, 2003). This therapeutic strategy has the potential to treat diseases caused by mutations that result in the synthesis of improperly folded lysosomal enzymes, although they may remain in full or partial biological activity if they were folded into their native-like proper conformation. Active- site-specific chaperones (ASSCs) are enzyme active-site directed small molecule pharmacological chaperones that increase residual enzyme activity through rescuing misfolded mutant proteins from the endoplasmic reticu- 2 J.-Q. Fan Article in press - uncorrected proof lum-associated degradation (ERAD) by serving as a fold- ing template for the conformational fragile mutant enzymes. ASSC therapy is well suited for LSDs, as a level of enzyme activity needed to prevent substrate storage is relatively low, i.e., ca. 10% of normal level. In addition, small molecule agents are attractive as they can be administered orally and presumably can gain access to most cells types and central neuronal system (CNS). Since the first ASSC study that was demonstrated with Fabry disease (Fan et al., 1999; Asano et al., 2000), ASSCs have been identified for Gaucher disease, Tay- Sachs and Sandhoff diseases, GM1-gangliosidosis and Pompe disease (Sawkar et al., 2002; Matsuda et al., 2003; Tropak et al., 2004; Chang et al., 2006; Okumiya et al., 2007). These findings have been rapidly translated into several Food and Drug Administration (FDA)- approved clinical trials of ASSC therapy. 1-Deoxygalac- tonojirimycin (DGJ, Amigal�) is now in clinical phase II evaluation for Fabry disease (www.amicustherapeutics. com). Phase II clinical trials of isofagomine (IFG, Plice- ra�) for Gaucher disease are being conducted at pres- ent. Separately, an investigational new drug application for treating GM2-gangliosidosis (Tay-Sachs and Sandhoff diseases) using pharmacological chaperone agent (EX- 101) was filed with FDA and a phase I clinical trial is underway (www.exsar.com). This review introduces the broadness of protein mis- folding as a direct consequence of mutations in LSDs and the molecular basis of ASSC therapy, and in addition provides an overview of recent advances in the devel- opment of ASSC therapy for LSDs. Also, a generalized procedure for the design and identification of ASSCs is described. Reviews for small molecule pharmacological chaperones that rescue misfolded/mistargeted mutant proteins in non-lysosomal disease areas are discussed elsewhere (Perlmutter, 2002; Cohen and Kelly, 2003; Bernier et al., 2004; Ulloa-Aguirre et al., 2004). The ther- apeutic strategy of using functional chemicals as phar- macological chaperones has the potential to treat a broad range of genetic metabolic diseases that are caused by misfolding of mutant proteins. Degradation of glycosphingolipids Degradation of glycosphingolipids occurs in lysosomes. Normally, the metabolism of glycosphingolipids is regu- lated in a stepwise fashion, involving various enzymes responsible for the removal of each terminal sugar (Figure 1). A genetic defect in an enzyme that is responsible for the removal of a terminal sugar unit in a particular sub- stance could retard the whole catabolic process. As a result, this causes an abnormal accumulation of the undegraded glycosphingolipid in lysosomes, leading to a LSD. Accumulation of different glycosphingolipids often leads to different LSDs with characteristic clinical symp- toms involving different tissues. For example, the defi- cient lysosomal a-galactosidase A (a-Gal A) leads to the accumulation of globotriaosylceramide, characterized as Fabry disease mainly affecting the kidneys and heart (Brady et al., 1967). Likewise, accumulation of glucosyl- ceramide caused by the deficiency of glucocerebrosi- dase (GCase) results in Gaucher disease (Brady et al., 1965). It has been known that the disease severity in a LSD is well correlated with the level of residual enzyme activ- ity, i.e., severe and infantile-type is often observed in patients with non-detectable residual enzyme activity, whereas patients with measurable residual enzyme activity often result in mild phenotypes. The correlation between residual activity of a lysosomal enzyme and the turnover rate of its substrate has been used to determine the substrate accumulation rate in individual cells and whole organs (Leinekugel et al., 1992). The results indi- cated that degradation of substrate increased steeply with residual enzyme activity when the enzyme activity reached a critical threshold, indicating that a full level of enzyme activity is not required for preventing the accu- mulation of glycosphingolipids. This critical threshold of residual enzyme activity in each LSD varies, typically 5–10% of normal level. For Gaucher disease, rapid accu- mulation of glucosylceramide is observed in patients when GCase activity falls below 11–15% of normal (Schueler et al., 2004), indicating that this could be the critical threshold for the disease. Therefore, from a ther- apeutic aspect, it is an objective to increase the residual enzyme activity to above the critical threshold to effect- ively deplete and prevent the substrate storage in patients. Even for patients whose residual enzyme activ- ity cannot be increased over the critical threshold, any increase in enzyme activity is still considered to be of clinical benefit, as it may substantially modify the clinical phenotype and reduce clinical manifestations that affect quality of life. Protein misfolding as a major pathophysiological cause is responsible for the deficiency of LSD enzymes One of the direct consequences of disease-causing mutations is the misfolding of mutant protein (Ellgaard et al., 1999). Protein initial folding process is a thermo- dynamic equilibrium event. Wild-type proteins gain their conformation largely in folded forms based on their pri- mary amino acid sequences that have been perfected during their evolution process (Figure 2). A single amino acid substitution can easily disrupt the folding equilibrium in favor of misfolded conformations. Such misfolded pro- teins are recognized by the cellular ‘quality control sys- tem’ or the ERAD machinery in which improper folded or misfolded proteins are targeted for degradation to main- tain the integrity of the cellular systems (Hartl and Hayer- Hartl, 2002; Ellgaard and Helenius, 2003), regardless the biological ability of the mutant proteins, leading to an inherited protein deficiency. One well studied example is that the DF508 mutation in CFTR is fully functional if it is properly inserted into the cytoplasm membrane (Pasyk and Foskett, 1995). Retention of the mutant protein by CHIP along with Hsp70 in the ER, and subsequent deg- radation by the ubiquitin proteasome pathway plays a direct role in the depletion of the protein in vivo (Mea- cham et al., 2001). It is estimated that misfolding and aggregated proteins occurs up to 30% of wild-type pro- Use of enzyme inhibitors for restoring mutant enzyme activity 3 Article in press - uncorrected proof Figure 1 Degradation pathway of glycosphingolipids and glycosphingolipidoses. Glycosphingolipids are degraded in lysosomes by various degradation enzymes in a stepwise fashion. A deficiency in one enzyme results in an accumulation of the undegraded substrate, leading to a particular lysosomal storage disease. teins even after interaction with molecular chaperones, and they are rapidly degraded within minutes of their synthesis by the cellular quality control machinery (Yew- dell, 2001). Ron and Horowitz tested GCase protein levels, N-gly- cans processing and intracellular localization in skin fibroblasts derived from patients with Gaucher disease (Ron and Horowitz, 2005). The results strongly suggest that mutant GCase variants present variable levels of ER retention and undergo ERAD in the proteasomes. Bio- synthetic labeling studies combined with immunofluores- cence analyses with fibroblasts from patients with the defined mutations, N370S, L444P, D409H and G202R, unequivocally demonstrate a retarded transport of GCase carrying the mutation N370S and a transport block in the ER of the enzyme with the mutations, G202R, L444P and D409H (Schmitz et al., 2005). These results indicate that the degree of ER retention and pro- teasomal degradation is one of the factors that determine severity of Gaucher disease and the onset of the disease. In Fabry disease, we recently examined molecular basis of deficiency in human mutant a-Gal A enzymes with a variety of disease-causing missense mutations identified in patients who present residual enzyme activ- ity regardless of clinical phenotypes (Ishii et al., 2007). Among them, ten variant mutations (A20P, E66Q, M72V, I91T, R112H, F113L, N215S, Q279E, M296I and M296V), four classic mutations (E59K, A156V, L166V and R356W) and two mutations causing both variant and classic phe- notypes (A97V and R301Q) were expressed in COS-7 cells, respectively, and their proteins were purified from cell lysates. Except for one mutation (E59K), all other mutants appeared to have normal Km and Vmax values, indicating that they retain full or partial catalytic activity. 4 J.-Q. Fan Article in press - uncorrected proof Figure 2 Protein folding in the endoplasmic reticulum (ER) and active-site-specific chaperone (ASSC) therapy. The initial protein folding process in the ER is based on ther- modynamic equilibrium. Much of the folding preference is guid- ed by the primary amino acid sequence coded by each gene. (a) Wild-type proteins tend to gain their folded conformation based on their peptide sequence. These appropriately folded proteins are smoothly transported out of the ER for normal proc- essing. (b) On the other hand, folding preference of a mutant protein can be shifted in favor of a misfolded conformation. These proteins with misfolded/incorrect conformation are retard- ed by molecular chaperones, followed by either entering a refolding process or retro-translocation to the cytosolic protea- some-based degradation pathway. (c) Interaction with ASSCs can dramatically change the folding equilibrium of mutant pro- teins toward the native-like folded conformation, and thus res- cue the proteins from being degraded via ERAD and promoting safe exit from the ER for further processing. Because the binding of ASSC to the active-site of proteins is also thermodynamic, dissociation of ASSC from the mutant proteins can be acceler- ated based on many factors, including changing pH conditions, high concentration of substrates that compete with ASSCs for the active-site, and thereby allowing physiological functionality of the rescued mutant proteins. Figure 3 Effects of ERAD inhibitors on the amount of mutant a-Gal A expressed in COS-7 cells. Wild-type or mutant a-Gal A enzymes were transiently expressed in COS-7 cells. Cells were treated with 2 mM lactacystin (LC) or 0.2 mM kifunensine (KFN) at the 5th h after transfection. Upon harvest, Western blot analyses of cell lysates from transfected COS-7 cells were performed. The 50 kDa band represents the unprocessed ER form of a-Gal A. (C), control. (Reproduced from Ishii et al., 2007.) Western blot analysis of mutant enzymes expressed in transfected COS-7 cells and patient fibroblasts indicated that most of the mutant enzymes had low protein yields, suggesting that excessive degradation of the enzyme protein could be directly responsible for the deficient enzyme activity in these missense mutants. Subcellular fractionations and metabolic labeling studies with the L166V and R301Q mutants indicated that the mutant proteins remained in unprocessed forms within the ER fractions and were eventually degraded without further processing and maturation. We further assessed the involvement of ERAD in the degradation of missense mutant a-Gal A enzymes by inclusion of kifunensine (a selective inhibitor of the ER a-mannosidase I) into the culture medium of transfected COS-7 cells (Ishii et al., 2007). Removal of a mannose residue from Man9 N-linked oligosaccharides by ER a- mannosidase I is a critical luminal event for preventing proteins to reenter the refolding process and serves as a signal for retrotranslocation and degradation of misfolded proteins as a process of ERAD (Helenius and Aebi, 2004). When kifunensine was added to the culture medium, the amount of all mutant proteins (except E59K) appeared to be increased (Figure 3), suggesting that the degradation of mutant proteins was partially inhibited. This result pro- vided clear evidence that degradation of misfolded mutant a-Gal A enzymes occurred by the ERAD, as a result of misfolding of mutant proteins. Protein misfolding has been recognized as an impor- tant cause of protein deficiency in various inherited disorders (Kuznetsov and Nigam, 1998). The results obtained from a large set of Fabry missense mutant pro- teins provide evidence that protein misfolding as a pri- mary pathophysiological cause of protein deficiency is not only limited to a few mutations, but exist rather broadly in many missense mutations. This pathophysio- logical phenomenon could be extendable to other LSDs and other protein deficiencies. Therefore, it is of thera- peutic interest to develop strategies to specifically rescue such misfolded mutant proteins from the ERAD. Active-site-specific chaperones Despite the complexity of the initial protein folding pro- cess, there is ultimately only a small difference in energy that separates the proper functional fold from many of the folding intermediates (Figure 2). Inclusion of a mole- cule that is capable of serving a folding template can dramatically shift the folding dynamics in favor of proper and native-like folding. In such a way, misfolded mutant proteins can be rescued from the degradation within the ERAD machinery and further preceded to the normal processing and trafficking pathways (Fan, 2003). An ASSC is a small molecular chemical which is capable of specific binding to the catalytic domain of an enzyme and effectively promoting its proper folding conformation. Theoretically, any compound that interacts with a mutant Use of enzyme inhibitors for restoring mutant enzyme activity 5 Article in press - uncorrected proof protein and is capable of inducing its proper folding could have chaperone activity. For enzymes, the catalytic site is the preferable domain for ASSC binding. In com- parison with non-functional domains, the active-site is generally well protected by its structure and indeed is often the center of a protein molecule. In addition, a com- pound that targets the functional site of the protein can be rationally designed, as structural information is gen- erally more available for the active-site than for other non-functional domains. A large quantity of information generated from natural and synthetic substrates, analogs and inhibitors can readily be used to design an ASSC. Competitive enzyme inhibitors are expected to be effec- tive ASSCs, because of their high affinity to the catalytic domain. Once the mutant enzyme/inhibitor complex is secreted out of the ER, the inhibitor at sub-inhibito