Brigitte Rigat

Identification and Characterization of Ambroxol as an Enzyme Enhancement Agent for Gaucher Disease

Gaucher disease (GD), the most prevalent lysosomal storage disease, is caused by a deficiency of glucocerebrosidase (GCase). The identification of small molecules acting as agents for enzyme enhancement therapy is an attractive approach for treating different forms of GD. A thermal denaturation assay utilizing wild type GCase was developed to screen a library of 1,040 Food and Drug Administration-approved drugs. Ambroxol (ABX), a drug used to treat airway mucus hypersecretion and hyaline membrane disease in newborns, was identified and found to be a pH-dependent, mixed-type inhibitor of GCase. Its inhibitory activity was maximal at neutral pH, found in the endoplasmic reticulum, and undetectable at the acidic pH of lysosomes. The pH dependence of ABX to bind and stabilize the enzyme was confirmed by monitoring the rate of hydrogen/deuterium exchange at increasing guanidine hydrochloride concentrations. ABX treatment significantly increased N370S and F213I mutant GCase activity and protein levels in GD fibroblasts. These increases were primarily confined to the lysosome-enriched fraction of treated cells, a finding confirmed by confocal immunofluorescence microscopy. Additionally, enhancement of GCase activity and a reduction in glucosylceramide storage was verified in ABX-treated GD lymphoblasts (N370S/N370S). Hydrogen/deuterium exchange mass spectrometry revealed that upon binding of ABX, amino acid segments 243–249, 310–312, and 386–400 near the active site of GCase are stabilized. Consistent with its mixed-type inhibition of GCase, modeling studies indicated that ABX interacts with both active and non-active site residues. Thus, ABX has the biochemical characteristics of a safe and effective enzyme enhancement therapy agent for the treatment of patients with the most common GD genotypes.

The pharmacological chaperone isofagomine increases the activity of the Gaucher disease L444P mutant form of β-glucosidase

Gaucher disease is caused by mutations in the gene that encodes the lysosomal enzyme acid β‐glucosidase (GCase). We have shown previously that the small molecule pharmacological chaperone isofagomine (IFG) binds and stabilizes N370S GCase, resulting in increased lysosomal trafficking and cellular activity. In this study, we investigated the effect of IFG on L444P GCase. Incubation of Gaucher patient‐derived lymphoblastoid cell lines (LCLs) or fibroblasts with IFG led to approximately 3.5‐ and 1.3‐fold increases in L444P GCase activity, respectively, as measured in cell lysates. The effect in fibroblasts was increased approximately 2‐fold using glycoprotein‐enrichment, GCase‐immunocapture, or by incubating cells overnight in IFG‐free media prior to assay, methods designed to maximize GCase activity by reducing IFG carryover and inhibition in the enzymatic assay. IFG incubation also increased the lysosomal trafficking and in situ activity of L444P GCase in intact cells, as measured by reduction in endogenous glucosylceramide levels. Importantly, this reduction was seen only following three‐day incubation in IFG‐free media, underscoring the importance of IFG removal to restore lysosomal GCase activity. In mice expressing murine L444P GCase, oral administration of IFG resulted in significant increases (2‐ to 5‐fold) in GCase activity in disease‐relevant tissues, including brain. Additionally, eight‐week IFG administration significantly lowered plasma chitin III and IgG levels, and 24‐week administration significantly reduced spleen and liver weights. Taken together, these data suggest that IFG can increase the lysosomal activity of L444P GCase in cells and tissues. Moreover, IFG is orally available and distributes into multiple tissues, including brain, and may thus merit therapeutic evaluation for patients with neuronopathic and non‐neuronopathic Gaucher disease.

An open-label Phase I/II clinical trial of pyrimethamine for the treatment of patients affected with chronic GM2 gangliosidosis (Tay-Sachs or Sandhoff variants)

Late-onset GM2 gangliosidosis is an autosomal recessive, neurodegenerative, lysosomal storage disease, caused by deficiency of ß-hexosaminidase A (Hex A), resulting from mutations in the HEXA (Tay-Sachs variant) or the HEXB (Sandhoff variant) genes. The enzyme deficiency in many patients with juvenile or adult onset forms of the disease results from the production of an unstable protein, which becomes targeted for premature degradation by the quality control system of the smooth endoplasmic reticulum and is not transported to lysosomes. In vitro studies have shown that many mutations in either the α or β subunit of Hex A can be partially rescued, i.e. enhanced levels of both enzyme protein and activity in lysosomes, following the growth of patient cells in the presence of the drug, pyrimethamine. The objectives of the present clinical trial were to establish the tolerability and efficacy of the treatment of late-onset GM2 gangliosidosis patients with escalating doses of pyrimethamine, to a maximum of 100 mg per day, administered orally in a single daily dose, over a 16-week period . The primary objective, tolerability, was assessed by regular clinical examinations, along with a panel of hematologic and biochemical studies. Although clinical efficacy could not be assessed in this short trial, treatment efficacy was evaluated by repeated measurements of leukocyte Hex A activity, expressed relative to the activity of lysosomal ß-glucuronidase. A total of 11 patients were enrolled, 8 males and 3 females, aged 23 to 50 years. One subject failed the initial screen, another was omitted from analysis because of the large number of protocol violations, and a third was withdrawn very early as a result of adverse events which were not drug-related. For the remaining 8 subjects, up to a 4-fold enhancement of Hex A activity at doses of 50 mg per day or less was observed. Additionally marked individual variations in the pharmacokinetics of the drug among the patients were noted. However, the study also found that significant side effects were experienced by most patients at or above 75 mg pyrimethamine per day. We concluded that pyrimethamine treatment enhances leukocyte Hex A activity in patients with late-onset GM2 gangliosidosis at doses lower than those associated with unacceptable side effects. Further plans are underway to extend these trials and to develop methods to assess clinical efficacy.

Identification of Pharmacological Chaperones for Gaucher Disease and Characterization of Their Effects on β-Glucocerebrosidase by Hydrogen/Deuterium Exchange Mass Spectrometry

Point mutations in β‐glucocerebrosidase (GCase) can result in a deficiency of both GCase activity and protein in lysosomes thereby causing Gaucher Disease (GD). Enzyme inhibitors such as isofagomine, acting as pharmacological chaperones (PCs), increase these levels by binding and stabilizing the native form of the enzyme in the endoplasmic reticulum (ER), and allow increased lysosomal transport of the enzyme. A high‐throughput screen of the 50 000‐compound Maybridge library identified two, non‐carbohydrate‐based inhibitory molecules, a 2,4‐diamino‐5‐substituted quinazoline (IC50 5 μM) and a 5‐substituted pyridinyl‐2‐furamide (IC50 8 μM). They raised the levels of functional GCase 1.5–2.5‐fold in N370S or F213I GD fibroblasts. Immunofluorescence confirmed that treated GD fibroblasts had decreased levels of GCase in their ER and increased levels in lysosomes. Changes in protein dynamics, monitored by hydrogen/deuterium‐exchange mass spectrometry, identified a domain III active‐site loop (residues 243–249) as being significantly stabilized upon binding of isofagomine or either of these two new compounds; this suggests a common mechanism for PC enhancement of intracellular transport.

Fluorous iminoalditols: a new family of glycosidase inhibitors and pharmacological chaperones.

A collection of new reversible glycosidase inhibitors of the iminoalditol type featuring N‐substituents containing perfluorinated regions has been prepared for evaluation of physicochemical, biochemical and diagnostic properties. The vast variety of feasible oligofluoro moieties allows for modular approaches to customised structures according to the intended applications, which are influenced by the fluorine content as well as the distance of the fluorous moiety from the ring nitrogen. The first examples, in particular in the D‐galacto series, exhibited excellent inhibitory activities. A preliminary screen with two human cell lines showed that, at subinhibitory concentrations, they are powerful pharmacological chaperones enhancing the activities of the catalytically handicapped lysosomal D‐galactosidase mutants associated with GM1 gangliosidosis and Morquio B disease.

Amerindian pyruvate carboxylase deficiency is associated with two distinct missense mutations.

We characterized the pyruvate carboxylase (PC) gene by PCR amplification, subcloning, and sequencing. The coding region has 19 exons and 18 introns spanning approximately 16 kb of genomic DNA. Screening both the cDNA and the gene of individuals with the simple A form of PC deficiency revealed an 1828G-->A missense mutation in 11 Ojibwa and 2 Cree patients and a 2229G-->T transversion mutation in 2 brothers of Micmac origin. Carrier frequency may be as high as 1/10 in some groupings. The two point mutations are located in a region of homology conserved among yeast, rat, and human PC, in the vicinity of the carboxylation domain of the enzyme. These data provide the first characterization of the human PC gene structure, the identification of common pathogenic mutations, and the demonstration of a founder effect in the Ojibwa and Cree patients.

Diltiazem, a L-type Ca2+ channel blocker, also acts as a pharmacological chaperone in Gaucher patient cells

Recently, inhibition of L-type Ca(2+) channels, using either Diltiazem or Verapamil, has been reported to partially restore mutant glucocerebrosidase activity in cells from patients with Gaucher disease homozygous for the N370S or L444P alleles, as well as cells from patients with two other lysosomal storage diseases. It was hypothesized that these drugs act on the endoplasmic reticulum, increasing its folding efficiency, inhibited due to altered calcium homeostasis. Several other laboratories have reported that cells carrying either the N370S or the F213I alleles are amenable to enzyme enhancement therapy with pharmacological chaperones, whereas cells homozygous for L444P respond poorly. We found that Verapamil treatment does not enhance mutant enzyme activity in any of the cell lines tested, while Diltiazem moderately increases activity in normal cells, and in N370S/N370S and F213I/L444P, but not in L444P/L444P Gaucher cells, or in either of two adult Tay-Sachs disease cell lines. Since the mode of action of pharmacological chaperones and Diltiazem are believed to be different, we examined the possibility that they could act in concert. Diltiazem co-administered with known chaperones failed to increase enzyme activities above that reached by chaperone-treatment alone in any of the patient cell lines. Thus, we re-examined the possibility that Diltiazem acts as a pharmacological chaperone. We found that, at the acidic pH of lysosomes, Diltiazem was not an inhibitor, nor did its presence increase the heat stability of glucocerebrosidase. However, at neutral pH, found in the endoplasmic reticulum, Diltiazem exhibited both of these properties. Thus Diltiazem exhibits the biochemical characteristics of a glucocerebrosidase pharmacological chaperone.

Characterization of the affinity of the GM2 activator protein for glycolipids by a fluorescence dequenching assay

The G(M2) activator protein is a substrate specific cofactor for degradation of G(M2) ganglioside by lysosomal beta-hexosaminidase A. Mutations in the gene encoding the activator result in the AB-variant form of G(M2) gangliosidosis. The activator protein contains at least three functional elements; a hydrophobic binding pocket, an oligosaccharide binding site(s), and an area that interacts with hexosaminidase A. In this report a fluorescence dequenching assay specific for only the hydrophobic binding pocket is evaluated and optimized. It is shown that various glycolipids inhibit the transport between liposomes of a self-quenching fluorescent lipid probe, octadecylrhodamine, by the activator protein. The level of inhibition produced by each glycolipid is then used to characterize the oligosaccharide-binding specificity of the activator. The fluorescence dequenching assay is also used to evaluate the functionality of a truncated form of the activator protein. Our results indicate that this simple assay can be used to determine structure-function relationships within the normal or mutant forms of the activator. The data suggest that the C-terminus of the activator is required to produce a functional hydrophobic binding pocket.

Molecular consequences of the pathogenic mutation in feline GM1 gangliosidosis

G(M1) gangliosidosis is an inherited, fatal neurodegenerative disease caused by deficiency of lysosomal beta-d-galactosidase (EC 3.2.1.23) and consequent storage of undegraded G(M1) ganglioside. To characterize the genetic mutation responsible for feline G(M1) gangliosidosis, the normal sequence of feline beta-galactosidase cDNA first was defined. The feline beta-galactosidase open reading frame is 2010 base pairs, producing a protein of 669 amino acids. The putative signal sequence consists of amino acids 1-24 of the beta-galactosidase precursor protein, which contains seven potential N-linked glycosylation sites, as in the human protein. Overall sequence homology between feline and human beta-galactosidase is 74% for the open reading frame and 82% for the amino acid sequence. After normal beta-galactosidase was sequenced, the mutation responsible for feline G(M1) gangliosidosis was defined as a G to C substitution at position 1448 of the open reading frame, resulting in an amino acid substitution at arginine 483, known to cause G(M1) gangliosidosis in humans. Feline beta-galactosidase messenger RNA levels were normal in cerebral cortex, as determined by quantitative RT-PCR assays. Although enzymatic activity is severely reduced by the mutation, a full-length feline beta-galactosidase cDNA restored activity in transfected G(M1) fibroblasts to 18-times normal. beta-Galactosidase protein levels in G(M1) tissues were normal on Western blots, but immunofluorescence analysis demonstrated that the majority of mutant beta-galactosidase protein did not reach the lysosome. Additionally, G(M1) cat fibroblasts demonstrated increased expression of glucose-related protein 78/BiP and protein disulfide isomerase, suggesting that the unfolded protein response plays a role in pathogenesis of feline G(M1) gangliosidosis.

Evaluation of N-nonyl-deoxygalactonojirimycin as a pharmacological chaperone for human GM1 gangliosidosis leads to identification of a feline model suitable for testing enzyme enhancement therapy

Deficiencies of lysosomal β-D-galactosidase can result in GM1 gangliosidosis, a severe neurodegenerative disease characterized by massive neuronal storage of GM1 ganglioside in the brain. Currently there are no available therapies that can even slow the progression of this disease. Enzyme enhancement therapy utilizes small molecules that can often cross the blood brain barrier, but are also often competitive inhibitors of their target enzyme. It is a promising new approach for treating diseases, often caused by missense mutations, associated with dramatically reduced levels of functionally folded enzyme. Despite a number of positive reports based on assays performed with patient cells, skepticism persists that an inhibitor-based treatment can increase mutant enzyme activity in vivo. To date no appropriate animal model, i.e., one that recapitulates a responsive human genotype and clinical phenotype, has been reported that could be used to validate enzyme enhancement therapy. In this report, we identify a novel enzyme enhancement-agent, N-nonyl-deoxygalactonojirimycin, that enhances the mutant β-galactosidase activity in the lysosomes of a number of patient cell lines containing a variety of missense mutations. We then demonstrate that treatment of cells from a previously described, naturally occurring feline model (that biochemically, clinically and molecularly closely mimics GM1 gangliosidosis in humans) with this molecule, results in a robust enhancement of their mutant lysosomal β-galactosidase activity. These data indicate that the feline model could be used to validate this therapeutic approach and determine the relationship between the disease stage at which this therapy is initiated and the maximum clinical benefits obtainable.