Pharmacological Chaperone Therapy: Preclinical Development, Clinical Translation, and Prospects for the Treatment of Lysosomal Storage Disorders

Transcription

1 review Pharmacological Chaperone Therapy: Preclinical Development, Clinical Translation, and Prospects for the Treatment of Lysosomal Storage Disorders Giancarlo Parenti, 1,2 Generoso Andria 1 and Kenneth J Valenzano 3 1 Department of Translational Medical Sciences, Section of Pediatrics, Federico II University, Naples, Italy; 2 Telethon Institute of Genetics and Medicine, Pozzuoli, Italy; 3 Amicus Therapeutics, Cranbury, New Jersey, USA Lysosomal storage disorders (LSDs) are a group of inborn metabolic diseases caused by mutations in genes that encode proteins involved in different lysosomal functions, in most instances acidic hydrolases. Different therapeutic approaches have been developed to treat these disorders. Pharmacological chaperone therapy (PCT) is an emerging approach based on small-molecule ligands that selectively bind and stabilize mutant enzymes, increase their cellular levels, and improve lysosomal trafficking and activity. Compared to other approaches, PCT shows advantages, particularly in terms of oral administration, broad biodistribution, and positive impact on patients quality of life. After preclinical in vitro and in vivo studies, PCT is now being translated in the first clinical trials, either as monotherapy or in combination with enzyme replacement therapy, for some of the most prevalent LSDs. For some LSDs, the results of the first clinical trials are encouraging and warrant further development. Future research in the field of PCT will be directed toward the identification of novel chaperones, including new allosteric drugs, and the exploitation of synergies between chaperone treatment and other therapeutic approaches. Received 19 February 2015; accepted 1 April 2015; advance online publication 19 May doi: /mt LYSOSOMAL STORAGE DISORDERS Lysosomal storage disorders (LSDs) represent a heterogeneous group of more than 50 distinct diseases, each of which results from functional deficiency of a particular lysosomal protein. For the majority of LSDs, the defective protein is a soluble acidic hydrolase, while several others are caused by deficiency of integral membrane, activator, transporter, or nonlysosomal proteins that are necessary for lysosomal function. Deficiency of a lysosomal function invariably leads to the accumulation of a wide range of complex substrates, which may include various glycosphingolipids, glycosaminoglycans, glycogen, oligosaccharides, cholesterol, peptides, and/or glycoproteins, 1,2 and in secondary impairment of lysosome-related pathways. 3 Storage of different substrates in multiple organs and systems results in the variable association of visceral, ocular, hematologic, skeletal, and neurological manifestations. Severity of clinical manifestations, age at onset, and disease course often vary among individuals with the same LSD, resulting in broad clinical presentation. In general, LSDs substantially impact patients health, quality of life, life expectancy, and physical and intellectual performances. While each LSD is quite rare, collectively they affect a large number of individuals, with an estimated incidence rate of 1:5,000 to 1:7,000 live births. 4 For their impact on patients health and for their cumulative frequency, LSDs represent a heavy burden in terms of public health and economical costs. TREATMENT OF LSDs Even though LSDs are rare, their biological and clinical interest is high. These diseases represent models to understand lysosomal function and its role in cellular biology. Furthermore, over the past 25 years, intensive and continuous advancements have been made to develop therapies that are specifically aimed at correcting the metabolic defect(s) of LSDs. 5 The majority of these therapeutic approaches are directed toward increasing the cellular activity or level of the defective enzyme or protein, with the ultimate goal of lowering the accumulated substrate in key cell types and tissues. Given that the underlying cause of most LSDs is deficiency of a particular enzyme activity, the primary therapeutic approach that has been most successful and broadly reached to date is enzyme replacement therapy (ERT). ERT is based on the periodic intravenous administration of a manufactured enzyme that can be taken up into cells, be delivered to lysosomes, and reduce substrate storage. 6 To date, seven LSDs have marketed ERT products, and in some cases, multiple products exist for a single LSD. Similar to ERT, normal enzyme also may be provided as a precursor that is secreted into the circulation by allograft of transplanted cells (hematopoietic stem cell transplantation (HSCT)) 7 or by a patient s own engineered cells. 8 Similarly, the gene mutation may potentially be corrected by delivering a wild-type copy that will direct the synthesis of the normal enzyme in the patient s Correspondence: Giancarlo Parenti, Department of Translational Medical Sciences, Section of Pediatrics, Federico II University, Via S. Pansini 5, Naples, Italy. parenti@unina.it vol. 23 no. 7, jul. 2015

2 Chaperone Therapy for Lysosomal Storage Disorders cells. 9 Alternative strategies also exist and are directed toward reducing the synthesis of substrates (i.e., substrate reduction therapy, for which two drugs currently exist), 10 by enhancing clearance of substrates from cells and tissues, or by manipulating specific cellular pathways (such as those involved in vesicle trafficking). 11 The focus of this paper, however, is pharmacological chaperones, small molecules that can selectively bind and stabilize mutant enzymes, protecting them from premature denaturation and degradation, and ultimately increasing their lysosomal levels and activity Recently, pharmacological chaperone therapy (PCT) for a number of LSDs has been evaluated in the clinic, with some molecules showing therapeutic promise. THE CONCEPT OF PCT The balance between protein synthesis, folding, and degradation is a closely monitored and highly dynamic process that is critical to maintain cellular protein homeostasis, i.e., proteostasis (Figure 1). 15,16 Cells have evolved complex quality control systems that function in various organelles, including the endoplasmic reticulum (ER), to aid in these processes. 16,17 These mechanisms rely on multiple classes of proteinaceous molecular chaperones and folding factors (e.g., heat-shock proteins) that recognize and cotranslationally interact with various partially folded, aggregation-prone structural motifs of nascent proteins during synthesis, such as exposed hydrophobic regions, regions of unstructured polypeptide sequence, or unpaired cysteine residues, to distinguish stable, native conformations from unstable, misfolded conformations. 16 Upon recognition and binding to the nascent polypeptide, molecular chaperones can stabilize protein conformation, inhibit premature misfolding, and prevent aggregation Only those proteins that are correctly folded and stable can exit the ER efficiently and traffic to the lysosome. If folding of the nascent protein fails despite the action of molecular chaperones, the protein may be recognized by the ER quality control system as aberrant and targeted for degradation either by the lysosome or by the proteasome. 21,22 For the latter, ER-associated degradation involves polyubiquination, translocation to the cytosol, and proteasomal degradation. In the case of LSDs, the ER quality control often recognizes mutant forms of lysosomal enzymes that retain catalytic activity or that have only modestly compromised function, due to slight modifications in their stability or conformation. This recognition may prevent trafficking through the secretory pathway and result in loss of function due to premature degradation or ER aggregation. Based on an evolving understanding of these mechanisms, several small-molecule approaches to correct deficiencies that result from mutations in lysosomal enzymes have emerged over the past 15 years. 16 One of the more promising and advanced Nucleus Endoplasmic reticulum (ER) Golgi apparatus & trans-golgi network Co-translational interaction with molecular chaperones Folding into native conformation Exit from ER Glycosylation and trafficking Folding intermediate Nascent protein Folding Unfolding Misfolded state (mutant) QC Native protein ERAD Aggregation Degradation ER retention Defective glycosylation and trafficking Late endosomes - lysosomes QC Quality control (QC) Molecular chaperones ERAD Defective activity in lysosomes ER associated degradation (ERAD) system Figure 1 The cellular pathways that control folding of lysosomal enzymes. During synthesis, proteins (in this case, lysosomal enzymes or proteins) are cotranslationally assisted by molecular chaperones and folding factors (e.g., heat-shock proteins) that interact with partially folded, aggregation-prone structural motifs of the nascent protein. Upon recognition and binding to the nascent polypeptide, molecular chaperones can stabilize protein conformation, inhibit premature misfolding, and prevent aggregation. Enzymes that are correctly folded and stable pass the quality control (QC) of the endoplasmic reticulum (ER), exit the ER efficiently, and traffic to lysosomes. Mutant, misfolded enzymes may undergo ER retention or be recognized by the ER QC, retro-translocated to the cytosol, and degraded by ER-associated degradation systems (ERAD). Molecular Therapy vol. 23 no. 7 jul

3 Chaperone Therapy for Lysosomal Storage Disorders approaches utilizes pharmacological chaperones: small-molecule ligands that selectively bind and stabilize otherwise unstable enzymes to increase total cellular levels and improve lysosomal trafficking and activity (Figure 2). Lysosomal enzyme synthesis occurs in the neutral ph environment of the ER. As these enzymes are acidic hydrolases, many tend to be thermodynamically less stable at neutral ph compared to the low-ph environment of lysosomes. 23 For some mutant lysosomal enzymes, this thermodynamic instability can be exacerbated, with consequently even less of the properly folded enzyme able to exit the ER. In this context, pharmacological chaperones selectively bind and stabilize a specific target enzyme, resulting in increased total cellular levels, and passage through the quality control mechanisms of the ER, with subsequent delivery to lysosomes. 12,13 WHY USE PCT TO TREAT LSDs? For a number of reasons, LSDs can be considered excellent candidates for PCT. LSDs are often caused by mutations that are associated with protein misfolding While many types of mutations have been identified in LSDs, including large deletions, insertions, premature stop codons, and splicing mutations, missense mutations tend to be more common. 13 In most cases, missense mutations occur outside the enzyme s active site and have negative effects on protein folding efficiency, thermodynamic stability, and lysosomal trafficking, although the mutant enzymes retain their catalytic properties. 16 This concept has been characterized in detail for several LSDs. For example, Gaucher disease, generally thought to be the most prevalent LSD, is caused by mutations in the GBA1 gene that encodes glucocerebrosidase (GCase; EC ), 24 and is characterized by progressive accumulation of glucosylceramide primarily within macrophages of the liver, bone marrow, and spleen. Among more than 200 mutations identified in GBA1, the 2 most prevalent missense mutations are N370S and L444P, 24 retaining ~30 and 10 12% residual cellular activity, respectively. 25 These mutations result in deleterious changes in the three-dimensional structure and in variable levels of ER retention and ER-associated degradation. 26 Evidence that mutations in the genes encoding different lysosomal enzymes result in protein misfolding, retention in the ER/ Golgi, degradation, lack of appropriate protein processing, and/ or defective transport to lysosomes has also been reported for many other LSDs. 27 Since the mutations that cause misfolding are relatively prevalent in some LSDs, like Gaucher disease and Fabry disease, PCT has the potential to be a suitable strategy for the treatment of a substantial fraction of affected patients. Minimal increases in activity may be sufficient to positively impact phenotype For most LSDs, it has been speculated that substrate storage occurs if residual enzyme activity falls below a certain threshold Nucleus Misfolded protein Failure of molecular chaperones to promote folding Synthesis of A mutant protein Endoplasmic reticulum (ER) STOP QC QC GO PC Rescue of folding Golgi apparatus & trans-golgi network ER retention Aggregation Degradation Defective Glycosylation and trafficking Improved glycosylation and trafficking Late endosomes - lysosomes Defective activity in lysosomes Molecular chaperones Increased residual activity in lysosomes Pharmacological chaperone (PC) Figure 2 Mechanism of action of pharmacological chaperones. Pharmacological chaperones (hexagons) are small-molecule ligands that selectively bind and stabilize otherwise unstable enzymes and enhance or partially restore their folding and stability. Enzymes that are rescued by pharmacological chaperones can be normally trafficked, thus increasing residual activity in lysosomes vol. 23 no. 7 jul. 2015

4 Chaperone Therapy for Lysosomal Storage Disorders Threshold High Residual enzime activity Low Phenotype Normal Mild Intermediate Severe Figure 3 Correlations between residual activity and LSD phenotype. For most LSDs, correlations have been observed between residual enzyme activity and disease severity. It has been speculated that substrate storage occurs if residual activity falls below a certain threshold. For several LSDs, it has been assumed that a threshold activity of approximately 10% is sufficient to prevent storage, while 3 5% residual activity is associated with attenuated phenotypes. LSD, lysosomal storage disorder. (Figure 3) and that above a certain level, accumulation of clinically relevant quantities of substrate would require a duration that exceeds the human life span. 28 It has been assumed that a threshold activity of ~10% is sufficient to prevent storage in several LSDs, with restoration of 3 5% activity often cited as sufficient to slow down the disease progress This suggests that enhancement of the residual cellular activity by PCT may attenuate disease progression and translate into benefit for patients. However, the residual activity known to be associated with attenuated phenotypes and required to prevent massive storage seems to vary among different disorders and may depend, in part, on various factors such as the severity of the enzymatic defect, the rate of substrate accumulation/turnover, the stage of disease progression, sex, etc. For example, while 1 2% of normal GCase and α-iduronidase activity have been reported for some mild cases of Gaucher disease and mucopolysaccharidosis I, respectively, higher activities (6%) have been reported in late-onset GM1 gangliosidosis. 13 Existing therapies for LSDs show major limitations ERT is currently considered the standard of care for several LSDs. While some of the underlying pathologies and affected organ systems are treated well by ERT, others are not. This is often due to poor tissue access, as well as inefficient cellular uptake and lysosomal delivery. In particular, the ability to target the central nervous system has not been achieved, given the inability of large proteins to cross the blood brain barrier. 32,33 Furthermore, most of the current ERTs are immunogenic, eliciting immune responses that can limit tolerability and efficacy In addition, ERT requires lifelong intravenous infusion, with frequent hospital admissions, need for central venous devices (and related risk of infections), and high costs. 38 The other therapeutic approaches mentioned above (e.g., substrate reduction therapy, HSCT, gene therapy) are either restricted to a few LSDs or are in early stages of clinical development. In principle, PCT has the potential to address at least some of the limitations of existing therapies. Pharmacological chaperones are small molecules with published data indicating, as a class, good oral bioavailability, broad tissue distribution to key cell types and tissues including the brain, and the ability to diffuse across membranes, achieving therapeutic concentrations in specific cellular compartments. Enhancement of enzyme activity by PCT also may lead to sustained and stable enzyme levels, more closely mimicking the natural production of these endogenous enzymes as compared to weekly or biweekly ERT administration that leads to intermittent and fluctuating cellular activity. In addition, unlike the manufactured enzymes used for ERT, pharmacological chaperones are nonimmunogenic and would not be expected to have tolerability issues similar to those described for a number of different ERTs. In fact, the safety profiles of some pharmacological chaperones have been evaluated clinically and appear to be acceptable. PRECLINICAL DEVELOPMENT OF PCT FOR LSDs Since 1999, PCT has been evaluated in preclinical studies for a number of LSDs. Table 1 provides a list of selected chaperones that have shown the greatest potential for clinical studies. The first studies on PCT in an LSD were done for Fabry disease, an X-linked disorder caused by mutations in the GLA gene that encodes α-galactosidase A (α-gal A; EC ). 39 Deficiency of α-gal A activity results in accumulation of glycosphingolipids, primarily globotriaosylceramide (GL-3) and globotriaosylsphingosine (lyso-gb 3 ), in various organs and tissues. 39 A natural substrate mimetic, 1-deoxygalactonojirimycin (DGJ, AT1001, migalastat), was described as a pharmacological chaperone for α-gal A. 40,41 DGJ binds reversibly and selectively to the active site of α-gal A with high affinity 42 and can increase in vitro the cellular activity of different missense mutant forms of α-gal A with concomitant reductions in GL In vivo studies using a transgenic mouse model that expresses the human R301Q transgene on a Gla knockout (KO) background (hr301q α-gal A Tg/ KO mice) showed increased α-gal A activity and reduced GL-3 levels in various disease-relevant tissues following oral administration of DGJ. 46 PCT has also been proposed as a potential therapy for Gaucher disease. 47,48 A wide variety of compounds that increase the cellular activity of various mutant forms of GCase in cell lines derived from Gaucher patients have been evaluated. These molecules can be categorized as carbohydrate mimetics (iminosugars, azasugars, and carbasugars) or noncarbohydrate compounds identified by high-throughput screening initiatives. 13,14 From this collection, two appeared to be particularly promising and have advanced through preclinical studies and into early-stage clinical development, namely, isofagomine (IFG, AT2101, afegostat tartrate) and Ambroxol. IFG is an azasugar that binds both wild-type and mutant forms of GCase, resulting in stabilization and increased cellular and lysosomal levels, 49,50 particularly for the N370S variant. 50,51 Similarly, the cellular levels of a number of other missense mutant forms of GCase, including L444P, are also increased in response to incubation with IFG. 50,52,53 Preclinical in vivo studies evaluated the effects of IFG in different transgenic mouse models of Gaucher disease homozygous for the mutations L444P, V394L, N370S, D409H, or D409V. 50,53,54 Overall, IFG administration (at different doses) led to Molecular Therapy vol. 23 no. 7 jul

5 Chaperone Therapy for Lysosomal Storage Disorders Table 1 Current stage of development of select pharmacological chaperones Disease Therapeutic agent (route of administration, as applicable) Orphan drug designation Development status/ clinical trial study ID Fabry Galactose (intravenous) N/A Monotherapy Pilot clinical study Company, sponsor Università Cattolica del Sacro Cuore, Roma, Italy References 84 Migalastat HCl/AT1001/1- deoxygalactonojirimycin/dgj (oral) US: 02/25/2004 EU: 05/22/2006 Monotherapy phase 2: NCT (FAB-CL-201) NCT (FAB-CL-202) NCT (FAB-CL-203) NCT (FAB-CL-204) NCT (FAB-CL-205) phase 3: NCT (AT ) NCT (AT ) NCT (AT ) NCT (AT ) Amicus Therapeutics 88,91 Combination therapy (phase 2) + intravenous agalsidase alfa or beta: NCT (AT ) Amicus Therapeutics 109 Gaucher, type I Afegostat tartrate/at2101/ isofagomine/ifg (oral) US: 01/10/2006 EU: 10/23/2007 Monotherapy (phase 2) NCT (GAU-CL-104) NCT (GAU-CL-201) NCT (GAU-CL-202) NCT (GAU-CL-202X) Amicus Therapeutics Ambroxol (oral) US: 06/29/2011 EU: N/A Monotherapy (phase 1/2) NCT ExSAR Corp. 103 Pompe Duvoglustat HCl/AT2220/1- deoxynojirimycin/dnj (oral) US: 06/18/2007 EU: N/A Monotherapy (phase 2) NCT (POM-CL-201) Amicus Therapeutics Combination therapy (phase 2) Amicus Therapeutics intravenous alglucosidase alfa: NCT (AT ) Miglustat (oral) N/A Combination therapy (phase 2) + intravenous alglucosidase alfa: EudraCT n Combination therapy (phase 1/2) + intravenous alglucosidase alfa: NCT Università degli Studi di Napoli Federico II, Italy University of Florida/ Amicus Therapeutics 106 GM1 gangliosidosis NOEV (oral) N/A Monotherapy N/A 64 Preclinical cell-based and in vivo studies GM2 gangliosidosis N-acetylglucosamine thiazoline (NGT) N/A Monotherapy Preclinical cell-based studies only N/A 110,111 AdNDJ/OT1001 (oral) N/A Monotherapy N/A 110,112 Preclinical cell-based and in vivo studies Pyrimethamine (oral) US: 08/16/2011 EU: N/A Monotherapy (phase 1/2) NCT The Hospital for Sick Children, Canada 105,112 MPS IIIC Glucosamine N/A Monotherapy N/A 113 Preclinical cell-based studies only Batten CS38 N/A Monotherapy N/A 114 Preclinical cell-based studies only EU, European Union; N/A, not applicable; US, United States vol. 23 no. 7 jul. 2015

6 Chaperone Therapy for Lysosomal Storage Disorders statistically significant increases in GCase activity in liver, spleen, lung, bone, and brain, as well as in liver macrophages. In the L444P and N370S models, IFG administration resulted in reduced liver and spleen weights, providing evidence of in vivo efficacy. In the V394L model, which to some extent mimics neuronopathic forms of the disease, increases in GCase protein levels and activity in brain, reduced neuroinflammation, delayed onset of neurological disease, and increased life spans were demonstrated. 50 Ambroxol, an approved expectorant, was identified from a screen of 1,040 Food and Drug Administration approved drugs by its ability to stabilize wild-type GCase against thermal denaturation. 55 Ambroxol also showed ph-dependent affinity for GCase, with decreasing inhibition at lysosomal ph values. In in vitro studies, Ambroxol elevated the cellular and lysosomal levels of multiple GCase mutants in patient-derived cells expressing R120W, R131C, N188S, G193W, F213I, N370S, L444P, and/ or P415R. 56,57 In vivo, in N370S or L444P transgenic mice, daily subcutaneous injections of Ambroxol for 14 days resulted in consistently elevated GCase levels in spleen and liver. 54 Pompe disease (acid maltase deficiency, glycogen storage disease type II) is an LSD caused by mutations in the GAA gene that encodes acid α-glucosidase (GAA; EC ), 58 an enzyme involved in lysosomal glycogen catalysis. Deficiency in GAA activity results in generalized glycogen accumulation in heart, skeletal muscle, and other tissues. 58 Currently, ERT with recombinant human GAA (rhgaa; alglucosidase alfa) is the only approved treatment for Pompe disease. However, ERT shows limitations, particularly in terms of restricted bioavailability, insufficient correction of disease pathology in some muscles, tolerability issues, and immunogenicity. Small-molecule chaperones have been proposed as a potential alternative to ERT for the treatment of Pompe disease. Some of the ~150 mutant forms of GAA are responsive to 1-deoxynojirimycin (DNJ, AT2220, duvoglustat) and N-butyldeoxynojirimycin (NB-DNJ, miglustat) Mechanistically, DNJ has multiple modes of action during the synthesis and maturation of mutant GAA, including increased specific activity prior to proteolytic processing in lysosomes, facilitated export from the ER with subsequent trafficking and processing through the secretory pathway to lysosomes, and stabilization of mature isoforms in lysosomes. 62 The in vivo effects of DNJ were tested in a mouse model of Pompe disease that expresses the human mutant GAA transgene P545L on a Gaa KO background. 62 Daily oral administration of DNJ to the transgenic mice resulted in significant and dose-dependent increases in GAA activity with concomitant reduction in tissue glycogen levels. Pyrimethamine, an approved antimalarial drug, was identified as a potential chaperone of β-hexosaminidase (β-hex; EC ), a lysosomal hydrolase that cleaves N-acetylgalactosamine from glycosphingolipids, various oligosaccharides, and glycoproteins. 63 β-hex deficiency results in two progressive neurodegenerative GM2 gangliosidoses (Tay-Sachs disease and Sandhoff disease), for which there are currently no effective treatment options. In another severe neurodegenerative LSD that results from deficiency of β-galactosidase (EC ), GM1 gangliosidosis, the small molecules N-octyl-4-epi-β-valienamine (NOEV) and 5N,6S- (N -butyliminomethylidene)-6-thio-1-deoxygalactonojirimycin were shown to significantly enhance β-galactosidase activity both in vitro and in transgenic animal models of the disease. 64,65 Interestingly, NOEV administration starting at the early stage of disease resulted in a reduced rate of disease progression, arrest of neurological involvement, and prolonged survival of treated animals. THE OTHER SIDE OF THE COIN: LIMITATIONS OF PCT Although PCT has several potential advantages over existing therapies and has already shown clinical efficacy in one LSD (i.e., Fabry disease), there are some challenges to be addressed by future research. In most cases, chaperones are reversible competitive inhibitors of target enzymes. To date, most pharmacological chaperones that have been identified bind to the active site of their target enzyme, thus acting as inhibitors. Initially, the idea of using small-molecule, active-site inhibitors to increase total cellular enzyme activity was counterintuitive. However, in many cases, if used appropriately, net gains in in situ lysosomal activity and reduced substrate levels can be realized. 12,13 In fact, some pharmacological chaperones bind with high affinity to their target enzyme at neutral ph but show lower binding affinity at acidic ph, thus favoring dissociation and substrate turnover in the lysosome. In addition, once delivered to the lysosome, high concentrations of accumulated substrate can compete with the chaperone for binding to the target enzyme, thus facilitating substrate turnover rather than enzyme inhibition. Increases in residual activity may be too low for significant benefit. Preclinical studies have shown variable increases in activity in the presence of chaperones. While some enzyme variants appear to respond quite well, others show only minor net increases in activity (i.e., enhancement), lysosomal trafficking, and enzyme processing/maturation. For some diseases and for some specific mutations, the enhancement in residual activity obtained with PCT may not be sufficient to translate into significant benefit for patients, particularly if tissue pathology is already fully established. However, insufficient correction of the enzymatic defect and pathology in specific tissues is also a challenge with ERT and other approaches (see above). Only a fraction of mutations are responsive to chaperone therapy. Not all LSD patients have missense mutations and not all missense mutations are responsive to chaperones. The determinants of chaperone response have been analyzed and depend both on the type of mutation and on the specific chaperone molecule tested. 42,66 68 In silico characterization of the molecular interactions between enzymes and chaperones may allow prediction of responsiveness of mutant enzymes to chaperones. 42,66 It is reasonable to expect that mutations that lead to major reductions in physical stability, that prevent folding, or that affect catalytic activity may not be responsive to active-site pharmacological chaperones. Molecules that interact with different domains of the protein (including noncatalytic domains) may have the potential to expand the spectrum of responsive mutations. 69 The rate of responsive mutations varies in the different LSDs. For Fabry disease, in which most mutations are private, with none showing high prevalence, 70 the fraction of missense Molecular Therapy vol. 23 no. 7 jul

7 Chaperone Therapy for Lysosomal Storage Disorders mutations that are potentially responsive is estimated to be 30 50%. For Gaucher disease, more than 70% of patients within the Ashkenazi Jewish population carry at least one N370S allele, while 38% of non-jewish patients carry the L444P allele. 71,72 Both mutations are, in principle, responsive to PCT. For Pompe disease, it is possible that 10 15% of patients would be amenable to PCT. 61 However, to estimate the true percent of LSD patients that are amenable to PCT, it should be considered that only a fraction of mutations cause premature termination of translation, gene deletions, rearrangements, splicing mutations, or are unknown. Interestingly, in some cases, endogenous wild-type enzymes also show a response to chaperones, while in other instances, they do not. The reasons for this are not clear, with several factors possibly playing roles, including the overall folding efficiency for the enzyme/protein of interest, which may be different across cell types and species. For example, DNJ enhances wild-type Gaa in mice and rats very robustly, but less so in monkeys; virtually no enhancement of wild-type GAA was seen in white blood cells (WBCs) of healthy human volunteers (unpublished results). As mentioned above, how well the chaperone interacts and coordinates key amino acid side chains to confer enhanced stability may also play a role. The molecular interactions that lead to the desirable chaperone properties of DGJ for α-gal A have been described and are different than the interactions between α-gal A and galactose, a low-affinity ligand with generally poorer chaperone properties. 42 HOW TO CIRCUMVENT THE LIMITATIONS OF PCT? Different strategies have been devised to address the potential limitations of pharmacological chaperones. Discontinuous administration of chaperones The lysosomal half-life of the target enzyme (typically days) relative to that of the pharmacological chaperone (typically hours) can be used to develop optimal dose and administration regimens that allow maximal substrate turnover, thereby addressing concerns on potential inhibition. For instance, in hr301q α-gal A Tg/KO mice, discontinuous oral administration of the chaperone DGJ (cycles of 4 days with DGJ followed by 3 days without DGJ to allow for tissue clearance of the chaperone) resulted in greater substrate reduction compared to daily administration. The shorter tissue half-life of DGJ compared to that of α-gal A could be exploited to stabilize α-gal A in the ER and promote lysosomal trafficking during DGJ administration and to maximize lysosomal α-gal A activity and substrate turnover during periods in the absence of DGJ, thus producing a larger net gain in lysosomal enzyme activity. 46 Pharmacological chaperones that target allosteric sites Current research is focused on the identification of pharmacological chaperones that bind allosteric sites of target enzymes. The stabilization mechanism of these allosteric compounds would be, in principle, similar to the active site-directed compounds and require a sufficient binding affinity to increase the stability of the mutant enzyme structure. However, allosteric chaperones would have the additional advantage that they can remain bound to the enzyme during catalysis without risk of inhibition. Examples of allosteric pharmacological chaperones are now being reported. α-gal A contains an allosteric site that selectively binds the β-anomer of d-galactose. 73 For GCase, compounds that enhance enzymatic activity, possibly by binding to sites other than the catalytic site, also have been described. 74 An in silico study identified regions on the surface of GCase that may host allosteric ligands. 75 Allosteric pharmacological chaperones also have been documented for GAA. A high-throughput screen of more than 200,000 compounds identified 1-(3,4-dimethoxybenzyl)-6-propyl- 2-thioxo-2,3,5,6,7,8-hexahydropyrimido[4,5-d]pyrimidin-4 (1H)-one (ML247). 76 In addition, Porto et al. 69 showed that N-acetyl-cysteine, and the related compounds N-acetyl-serine and N-acetyl-glycine, increased the physical stability of the enzyme as a function of ph and temperature and enhanced the activity of the enzyme in cultured Pompe fibroblasts. Combination therapies Although PCT has been designed to rescue endogenous mutant misfolded proteins, studies suggest that chaperones are able to increase the stability of the wild-type enzymes that are commonly used for ERT. This effect has the potential to translate into enhanced efficacy of ERT and to address some of the limitations of chaperones. To this end, preclinical studies demonstrated that pharmacological chaperones enhance enzyme stability, lysosomal trafficking, and/or processing in Pompe, Fabry, and Gaucher cultured cells incubated with the respective recombinant enzymes, as well as in Pompe and Fabry animal models. 69,77 82 In cultured fibroblasts from Pompe patients, coincubation of the chaperone NB-DNJ with rhgaa ERT resulted in greater correction of enzyme activity and increased amounts of the recombinant enzyme in lysosomal compartments. 78 Coadministration of DNJ with rhgaa ERT to Gaa KO mice resulted in significantly greater rhgaa levels in plasma, and greater enzyme uptake and glycogen reduction in heart and skeletal muscles, compared to administration of rhgaa alone, indicating enhanced efficacy. 81 Similarly, coincubation with DGJ significantly increased the physical stability of α-gal A ERT at neutral ph and prevented loss of activity in human whole blood ex vivo relative to incubation without DGJ. 80 Furthermore, coincubation of Fabry patient-derived fibroblasts with DGJ and α-gal A ERT resulted in greater cellular enzyme levels, and greater GL-3 reduction, compared to incubation with α-gal A alone. 78,79,80,82 In vivo, coadministration of DGJ and α-gal A ERT to rats and Gla KO mice resulted in significantly greater plasma exposure of active enzyme, higher tissue levels of active enzyme, and greater reduction of tissue GL-3, when compared to administration of α-gal A alone. 80 Similar effects were seen when DGJ was coformulated with Fabry ERT and infused into rats and Gla KO mice, offering a potential alternative route of administration for the chaperone. 83 The synergy between chaperones and ERT may help broaden the use of PCT beyond the rescue of mutant enzymes. With the combination of PCT and ERT, the effect of chaperones is directed toward the ERT and is mutation independent. Thus, in principle, vol. 23 no. 7 jul. 2015

8 Chaperone Therapy for Lysosomal Storage Disorders this approach may be beneficial to any patient on ERT, if treated with the specific chaperone. In addition, the administration of the chaperone could be restricted only to the time of ERT infusions, thus reducing the risk of any potentially undesired events that may be related to the chronic use of the chaperone. TRANSLATION OF PCT INTO CLINICAL STUDIES Monotherapy After preclinical studies, pharmacological chaperone research is in a phase of clinical translation, with five chaperones for four LSDs now evaluated in clinical studies, and others showing promise (Table 1). Galactose was the first pharmacological chaperone investigated for Fabry disease. Every-other-day intravenous infusion of 1 g/kg galactose improved cardiac function in a male patient who presented with severe cardiomyopathy. 84 After 2 years, cardiac transplantation was unnecessary, offering the first proof-of-concept for PCT in an LSD. DGJ, the active component of the investigational drug migalastat hydrochloride (Amicus Therapeutics, Cranbury, NJ) is being developed as a treatment for Fabry disease. After successfully completing phase 1 studies, 85 two phase 2 studies enrolled nine male Fabry subjects to investigate the safety and efficacy of 150 mg migalastat every other day Increased α-gal A activity and decreased GL-3 were seen in WBCs, skin, urine, and/or kidney in the six subjects that expressed mutant forms of α-gal A that were responsive to migalastat in an in vitro cell-based assay (i.e., amenable); the other three subjects had nonamenable mutant forms. 88,89 In another phase 2 study conducted in females, 90,91 subjects with migalastat-amenable mutant forms showed greater responses in vivo based on reduced urine GL-3 and reduced renal peritubular capillary inclusions. Across all phase 2 studies, migalastat was generally safe and well tolerated. 92,93 As such, phase 3 studies were initiated to further evaluate the effect of 150 mg migalastat every other day. To date, data presented at scientific conferences have shown the efficacy of migalastat in stabilizing renal function, reducing left ventricular mass, and improving gastrointestinal symptoms in Fabry disease patients with amenable mutations. Long-term open-label extension studies of migalastat are ongoing As discussed, two pharmacological chaperones have advanced to the clinic as potential therapies for Gaucher disease: IFG and Ambroxol. In phase 1 studies, IFG showed dose-related elevations of up to 3.5-fold in WBC GCase levels. Subsequently, two phase 2 studies were initiated. In a 4-week study conducted in subjects with 12 different GBA1 mutations (including N370S and L444P) previously treated with ERT, 101 WBC GCase activity increased in 20 of 26 subjects. As expected for a short-term study, disease markers including platelet, hemoglobin, glucosylceramide, and chitotriosidase levels were unchanged. In another 6-month phase 2 study in patients who had never received ERT, 102 all subjects showed an increase in WBC GCase levels, but only one attained improvements in clinical measures. The tolerability and efficacy of once-daily 150 mg Ambroxol in 12 Gaucher patients were evaluated in an investigator-sponsored pilot study. 103 These subjects were ERT naive, with 11 being N370S homozygotes. Of the nine subjects who completed the 6-month study, none showed deterioration of Gaucher-related parameters (e.g., weight, hemoglobin, platelet count, liver/spleen volume, and chitotriosidase activity), and three continued therapy for an additional 6 months. These three also showed mean reductions of ~15 and ~40% in spleen volume and chitotriosidase activity, respectively. In one subject, platelet counts increased over 50%. Pharmacological chaperones also have been proposed as a treatment for Pompe disease To this end, phase 1 studies with DNJ showed that the drug was generally safe and well tolerated. A phase 2 study 104 conducted in adult Pompe subjects enrolled into one of three dose cohorts (2.5 to 5 g, with discontinuous administration protocols) was terminated due to severe adverse events in two subjects (muscle weakness) that resolved following DNJ withdrawal. These adverse events were deemed due to DNJ-mediated inhibition of endogenous GAA activity. A follow-up phase 1 study was conducted to evaluate the muscle pharmacokinetics of DNJ after a single 1,000 mg oral dose. DNJ rapidly appeared and remained in muscle for over 1 week at concentrations that were in excess of its IC 50 value for inhibition of GAA. The muscle profile of DNJ suggests that the doses selected for the phase 2 study were too high and that an appropriate balance between chaperoning and inhibition was not achieved. The potential benefits of pyrimethamine on endogenous levels of mutant β-hex were investigated in a 16-week phase 1/2 study in late-onset GM2 gangliosidosis patients. 105 Eight of 11 subjects completed the study and showed up to fourfold increases in β-hex activity in WBCs at doses 50 mg per day. Increased ataxia, lack of coordination, and seizures were observed in most subjects at higher doses. These data indicate that pyrimethamine can enhance β-hex activity in peripheral cells of late-onset GM2 gangliosidosis patients at doses lower than those associated with adverse effects. Combination therapy (ERT + chaperones) Given the promising preclinical results, the potential of therapeutic protocols based on the combination of ERT and chaperones has also been evaluated in clinical studies. The first published clinical study on the combination of rhgaa ERT and the chaperone NB-DNJ (miglustat) in Pompe disease reported on the results of a collaborative trial in 13 patients with different presentations (3 infantile onset and 10 late onset). 106 All patients had been previously treated with ERT for variable periods (1 8 years). The primary endpoint of the study was to obtain higher levels of blood GAA activity with the combination of rhgaa and the chaperone, compared to the activities obtained with ERT alone. GAA activity was measured by tandem-mass spectrometry in dried blood spots. In 11 patients, the combination treatment resulted in GAA activities greater than 1.85-fold the activities seen with ERT alone. In the whole patient population, GAA activity was significantly increased at 12, 24, and 36 hours. In another phase 2 study conducted by Amicus Therapeutics, Pompe subjects were orally administered a single dose of 50, 100, 250, or 600 mg DNJ 1 hour prior to ERT infusion. Dose-dependent increases in plasma GAA activity were observed for all subjects, attaining 1.5- to 2.8-fold greater exposures compared to ERT alone. In muscle biopsy samples taken 3 or 7 days after administration, increases in total GAA activity were observed in 16 of 24 subjects with evaluable data. 107 Molecular Therapy vol. 23 no. 7 jul

9 Chaperone Therapy for Lysosomal Storage Disorders These results indicate consistency with preclinical data obtained in rats and Gaa KO mice that showed a longer circulating half-life of GAA when coadministered with chaperone. 81 Lastly, a phase 2 study 108 was initiated by Amicus Therapeutics to investigate the effects of 150 and 450 mg migalastat HCl when administered 2 hours prior to infusion of α-gal A ERT. As seen in preclinical studies, plasma exposures of active α-gal A were 1.2- to 5.0-fold greater in 22 of the 23 male Fabry subjects following coadministration compared to levels seen following administration of ERT alone. Migalastat also led to greater total α-gal A activity in the skin of 19 of the 23 subjects relative to ERT alone, as measured in biopsies collected 24 hours postinfusion. CONCLUSIONS The complexity of LSD pathophysiology has made the development of therapies a major challenge. Presently, none of the therapeutic approaches that are already approved for clinical use have proven suitable to treat all LSDs or all patients with a specific disorder. PCT appears to have the potential to address some of the medical needs posed by LSDs and the limitations of currently available therapies. Although for some LSDs PCT has gone through remarkable progress and has shown efficacy and safety in one LSD, Fabry disease, further development and innovation is expected. Future research will be directed toward the identification of novel drugs with different chaperoning profiles to target a larger number of mutations. In addition, it will be important to identify new allosteric chaperones to exploit the full potential of chaperones and obtain the greatest clinical efficacy across LSDs. 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