Pharmacological Chaperone Therapy Research Articles

Exploring the efficacy and safety of Ambroxol in Gaucher disease: an overview of clinical studies.

Gaucher disease (GD) is mainly caused by glucocerebrosidase (GCase) enzyme deficiency due to genetic variations in the GBA1 gene leading to the toxic accumulation of sphingolipids in various organs, which causes symptoms such as anemia, thrombocytopenia, hepatosplenomegaly, and neurological manifestations. GD is clinically classified into the non-neuronopathic type 1, and the acute and chronic neuronopathic forms, types 2 and 3, respectively. In addition to the current approved GD medications, the repurposing of Ambroxol (ABX) has emerged as a prospective enzyme enhancement therapy option showing its potential to enhance mutated GCase activity and reduce glucosylceramide accumulation in GD-affected tissues of different GBA1 genotypes. The variability in response to ABX varies across different variants, highlighting the diversity in patients' therapeutic outcomes. Its oral availability and safety profile make it an attractive option, particularly for patients with neurological manifestations. Clinical trials are essential to explore further ABX's potential as a therapeutic medication for GD to encourage pharmaceutical companies' investment in its development. This review highlights the potential of ABX as a pharmacological chaperone therapy for GD and stresses the importance of addressing response variability in clinical studies to improve the management of this rare and complex disorder.

Treatment of Fabry Nephropathy: A Literature Review.

Fabry disease is an X-linked inherited lysosomal storage disorder with a deficiency of α-galactosidase A activity, which results in the intracellular accumulation of globotriaosylceramide (Gb3) and related glycosphingolipids in various organs. Fabry nephropathy is one of the major complications of Fabry disease, and kidney damage is often related to cardiovascular disease and mortality. The treatment of Fabry nephropathy thus helps prolong life expectancy. Two treatment options for Fabry nephropathy and cardiopathy are now commercially available: enzyme replacement therapy (agalsidase α agalsidase β, and a biosimilar of agalsidase β) and pharmacological chaperone therapy (migalastat). In this review, we summarize the efficacy of these treatment options for Fabry nephropathy with respect to renal function, proteinuria, and renal pathological findings. We also describe the importance of adjunctive therapy for Fabry nephropathy.

Gene therapy for lysosomal storage diseases: Current clinical trial prospects.

Lysosomal storage diseases (LSDs) are a group of metabolic inborn errors caused by defective enzymes in the lysosome, resulting in the accumulation of undegraded substrates. LSDs are progressive diseases that exhibit variable rates of progression depending on the disease and the patient. The availability of effective treatment options, including substrate reduction therapy, pharmacological chaperone therapy, enzyme replacement therapy, and bone marrow transplantation, has increased survival time and improved the quality of life in many patients with LSDs. However, these therapies are not sufficiently effective, especially against central nerve system abnormalities and corresponding neurological and psychiatric symptoms because of the blood-brain barrier that prevents the entry of drugs into the brain or limiting features of specific treatments. Gene therapy is a promising tool for the treatment of neurological pathologies associated with LSDs. Here, we review the current state of gene therapy for several LSDs for which clinical trials have been conducted or are planned. Several clinical trials using gene therapy for LSDs are underway as phase 1/2 studies; no adverse events have not been reported in most of these studies. The administration of viral vectors has achieved good therapeutic outcomes in animal models of LSDs, and subsequent human clinical trials are expected to promote the practical application of gene therapy for LSDs.

A CRISPR/Cas9-engineered avatar mouse model of monocarboxylate transporter 8 deficiency displays distinct neurological alterations

Inactivating mutations in the specific thyroid hormone transporter monocarboxylate transporter 8 (MCT8) lead to an X-linked rare disease named MCT8 deficiency or Allan-Herndon-Dudley Syndrome. Patients exhibit a plethora of severe endocrine and neurological alterations, with no effective treatment for the neurological symptoms. An optimal mammalian model is essential to explore the pathological mechanisms and potential therapeutic approaches. Here we have generated by CRISPR/Cas9 an avatar mouse model for MCT8 deficiency with a point mutation found in two MCT8-deficient patients (P253L mice). We have predicted by in silico studies that this mutation alters the substrate binding pocket being the probable cause for impairing thyroid hormone transport. We have characterized the phenotype of MCT8-P253L mice and found endocrine alterations similar to those described in patients and in MCT8-deficient mice. Importantly, we detected brain hypothyroidism, structural and functional neurological alterations resembling the patient's neurological impairments. Thus, the P253L mouse provides a valuable model for studying the pathophysiology of MCT8 deficiency and in the future will allow to test therapeutic alternatives such as in vivo gene therapy and pharmacological chaperone therapy to improve the neurological impairments in MCT8 deficiency.

Cardiac involvement in Fabry disease - A non-invasive assessment and the role of specific therapies.

Fabry disease is an X-linked inherited metabolic disorder due to the pathogenic mutation of the GLA gene, which codes lysosomal enzyme alpha-galactosidase A. The resultant accumulation of glycosphingolipids causes various systemic symptoms in childhood and adolescence, and major organ damage in adulthood. Cardiac involvement is important as the most frequent cause of death in Fabry disease patients. Progressive left ventricular hypertrophy with varying degrees of contractile dysfunction as well as conduction abnormalities and arrhythmias are typical cardiac features, and these findings can be evaluated in detail via non-invasive modalities, such as an electrocardiogram, echocardiography and cardiac magnetic resonance. In addition, specific therapies of enzyme replacement therapy and pharmacological chaperone therapy are available, and their beneficial effects on cardiac involvement have been reported. This minireview highlights recent evidence concerning non-invasive modalities for assessing cardiac involvement in Fabry disease and the effects of enzyme replacement therapy and pharmacological chaperone therapy on the findings of those modalities.

5-C-Branched Deoxynojirimycin: Strategy for Designing a 1-Deoxynojirimycin-Based Pharmacological Chaperone with a Nanomolar Affinity for Pompe Disease

In recent years, the function of pharmacological chaperones as a "thermodynamic stabilizer" has been attracting attention in combination therapy. The coadministration of a pharmacological chaperone and recombinant human acid α-glucosidase (rhGAA) leads to improved stability and maturation by binding to the folded state of the rhGAA and thereby promotes enzyme delivery. This study provides the first example of a strategy to design a high-affinity ligand toward lysosomal acid α-glucosidase (GAA) focusing on alkyl branches on 1-deoxynojirimycin (DNJ); 5-C-heptyl-DNJ produced a nanomolar affinity for GAA with a Ki value of 0.0047 μM, which is 13-fold more potent than DNJ. The protein thermal shift assay revealed that 10 μM 5-C-heptyl-DNJ increased the midpoint of the protein denaturation temperature (Tm) to 73.6 °C from 58.6 °C in the absence of the ligand, significantly improving the thermal stability of rhGAA. Furthermore, 5-C-heptyl-DNJ dose dependency increased intracellular GAA activities in Pompe patient's fibroblasts with the M519V mutation. The introduction of C5 alkyl branches on DNJ provides a new molecular strategy for pharmacological chaperone therapy for Pompe disease, which may lead to the development of higher-affinity and practically useful chaperones.

Pharmacological Chaperone Therapy for Pompe Disease.

Pompe disease (PD), a lysosomal storage disease, is caused by mutations of the GAA gene, inducing deficiency in the acid alpha-glucosidase (GAA). This enzymatic impairment causes glycogen burden in lysosomes and triggers cell malfunctions, especially in cardiac, smooth and skeletal muscle cells and motor neurons. To date, the only approved treatment available for PD is enzyme replacement therapy (ERT) consisting of intravenous administration of rhGAA. The limitations of ERT have motivated the investigation of new therapies. Pharmacological chaperone (PC) therapy aims at restoring enzymatic activity through protein stabilization by ligand binding. PCs are divided into two classes: active site-specific chaperones (ASSCs) and the non-inhibitory PCs. In this review, we summarize the different pharmacological chaperones reported against PD by specifying their PC class and activity. An emphasis is placed on the recent use of these chaperones in combination with ERT.

Congenital metabolic diseases. Lysosomal storage diseases

The classification and epidemiology of hereditary metabolic disorders are presented. That is a large group consisting from more them 800 monogenic diseases, each of which caused by inherited deficiency of certain metabolic fate. Many of these disorders are extremely rare, but their total incidence in the population is close to 1:10005000. Lysosomal storage diseases (LSD) resulting from inherited deficiency in lysosomal functions occupy a special place among hereditary metabolic disorders. The defects of catabolism cause the accumulation of undigested or partially digested macromolecules in lysosomes (that is, storage), which can result in cellular damage. About 60 diseases take part in this group with total incidence of about 1:70008000. LSDs typically present in infancy and childhood, although adult-onset forms also occur. Most of them have a progressive neurodegenerative clinical course, although symptoms in other organ systems are frequent. The etiology and pathogenetic aspects of their main clinical entities: mucopolysaccharidosis, glycolipidosis, mucolipidosis, glycoproteinosis, etc, are presented. Mucopolysaccharidoses caused by malfunctioning of lysosomal enzymes needed to break down glycosaminoglycans are more frequent among LSD. Sphingolipidoses caused by defects of lipid catabolism are second for frequency group of LSD. The state-of-art in field of newborn screening. clinical, biochemical and molecular diagnostics of these grave diseases are discussed. The main directions of modern lysosomal storage diseases therapy are characterized: transplantation of hematopoietic stem cells; enzyme replacement therapy; therapy with limitation of substrate synthesis (substrate-reducing therapy); pharmacological chaperone therapy. Perspective directions for LSD therapy are gene therapy and genome editing which are at advanced preclinical stages.

Considerations for Home-Based Treatment of Fabry Disease in Poland during the COVID-19 Pandemic and Beyond.

Current therapy for Anderson–Fabry disease in Poland includes hospital or clinic-based intravenous enzyme replacement therapy with recombinant agalsidase alpha or beta, or oral pharmacological chaperone therapy with migalastat. Some countries around the world offer such treatment to patients in the comfort of their own homes. The 2020–2021 COVID-19 pandemic has pushed global healthcare providers to evolve their services so as to minimize the risk of COVID-19 exposure to both patients and providers; this has led to advances in telemedicine services and the increasing availability of at-home treatment for various procedures including parenteral drug administration. A total of 80% of surveyed Anderson–Fabry disease patients in Poland would prefer home-based treatment, which would be a safe and convenient alternative to clinic-based treatment if patient selection is based on our proposed algorithm. Our recommendations for home-based treatments appear feasible for the long term care of Anderson–Fabry disease patients during the COVID-19 pandemic and beyond. This may also serve as a basis for home-based treatment programs in other rare and ultra-rare genetic diseases.

Human α-Galactosidase A Mutants: Priceless Tools to Develop Novel Therapies for Fabry Disease.

Fabry disease (FD) is a lysosomal storage disease caused by mutations in the gene for the α-galactosidase A (GLA) enzyme. The absence of the enzyme or its activity results in the accumulation of glycosphingolipids, mainly globotriaosylceramide (Gb3), in different tissues, leading to a wide range of clinical manifestations. More than 1000 natural variants have been described in the GLA gene, most of them affecting proper protein folding and enzymatic activity. Currently, FD is treated by enzyme replacement therapy (ERT) or pharmacological chaperone therapy (PCT). However, as both approaches show specific drawbacks, new strategies (such as new forms of ERT, organ/cell transplant, substrate reduction therapy, or gene therapy) are under extensive study. In this review, we summarize GLA mutants described so far and discuss their putative application for the development of novel drugs for the treatment of FD. Unfavorable mutants with lower activities and stabilities than wild-type enzymes could serve as tools for the development of new pharmacological chaperones. On the other hand, GLA mutants showing improved enzymatic activity have been identified and produced in vitro. Such mutants could overcome several complications associated with current ERT, as lower-dose infusions of these mutants could achieve a therapeutic effect equivalent to that of the wild-type enzyme.

Fabry Disease: Molecular Basis, Pathophysiology, Diagnostics and Potential Therapeutic Directions.

Fabry disease (FD) is a lysosomal storage disorder (LSD) characterized by the deficiency of α-galactosidase A (α-GalA) and the consequent accumulation of toxic metabolites such as globotriaosylceramide (Gb3) and globotriaosylsphingosine (lysoGb3). Early diagnosis and appropriate timely treatment of FD patients are crucial to prevent tissue damage and organ failure which no treatment can reverse. LSDs might profit from four main therapeutic strategies, but hitherto there is no cure. Among the therapeutic possibilities are intravenous administered enzyme replacement therapy (ERT), oral pharmacological chaperone therapy (PCT) or enzyme stabilizers, substrate reduction therapy (SRT) and the more recent gene/RNA therapy. Unfortunately, FD patients can only benefit from ERT and, since 2016, PCT, both always combined with supportive adjunctive and preventive therapies to clinically manage FD-related chronic renal, cardiac and neurological complications. Gene therapy for FD is currently studied and further strategies such as substrate reduction therapy (SRT) and novel PCTs are under investigation. In this review, we discuss the molecular basis of FD, the pathophysiology and diagnostic procedures, together with the current treatments and potential therapeutic avenues that FD patients could benefit from in the future.

A charitable access program for patients with lysosomal storage disorders in underserved communities worldwide

BackgroundLysosomal storage disorders (LSDs) are rare genetic disorders, with heterogeneous clinical manifestations and severity. Treatment options, such as enzyme replacement therapy (ERT), substrate replacement therapy, and pharmacological chaperone therapy, are available for several LSDs, including Gaucher disease (GD), Fabry disease (FD), and Hunter syndrome (mucopolysaccharidosis type II [MPS II]). However, patients in some countries face challenges accessing treatments owing to limited availability of locally licensed, approved drugs.MethodsThe Takeda LSD Charitable access program aims to meet the needs of individuals with GD, FD or MPS II with the greatest overall likelihood of benefit, in selected countries, through donation of ERT to nonprofit organizations, and support for medical capacity-building as well as family support via independent grants. Long-term aims of the program are to establish sustainable healthcare services delivered by local healthcare providers for patients with rare metabolic diseases. Patients receiving treatment through the program are monitored regularly, and their clinical data and progress are reviewed annually by an independent medical expert committee (MEC). The MEC also selects patients for enrollment completely independent from the sponsoring company.ResultsAs of 31 August, 2019, 199 patients from 13 countries were enrolled in the program; 142 with GD, 41 with MPS II, and 16 with FD. Physicians reported improvements in clinical condition for 147 (95%) of 155 patients with follow-up data at 1 year.ConclusionsThe response rate for follow-up data at 1 year was high, with data collected for > 90% of patients who received ERT through the program showing clinical improvements in the majority of patients. These findings suggest that the program can benefit selected patients previously unable to access disease-specific treatments. Further innovative solutions and efforts are needed to address the challenges and unmet needs of patients with LSDs and other rare diseases around the world.

Carnitine is a pharmacological allosteric chaperone of the human lysosomal α-glucosidase

Pompe disease is an inherited metabolic disorder due to the deficiency of the lysosomal acid α-glucosidase (GAA). The only approved treatment is enzyme replacement therapy with the recombinant enzyme (rhGAA). Further approaches like pharmacological chaperone therapy, based on the stabilising effect induced by small molecules on the target enzyme, could be a promising strategy. However, most known chaperones could be limited by their potential inhibitory effects on patient’s enzymes. Here we report on the discovery of novel chaperones for rhGAA, L- and D-carnitine, and the related compound acetyl-D-carnitine. These drugs stabilise the enzyme at pH and temperature without inhibiting the activity and acted synergistically with active-site directed pharmacological chaperones. Remarkably, they enhanced by 4-fold the acid α-glucosidase activity in fibroblasts from three Pompe patients with added rhGAA. This synergistic effect of L-carnitine and rhGAA has the potential to be translated into improved therapeutic efficacy of ERT in Pompe disease.

The Bicyclic Form of galacto-Noeurostegine Is a Potent Inhibitor of β-Galactocerebrosidase.

Competitive inhibitors of galactocerebrosidase (GALC) could be candidates for pharmacological chaperone therapy of patients with Krabbe disease. The known and selective nortropane-type iminosugar galacto-noeurostegine has been found to competitively inhibit GALC with K i = 7 μM at pH 4.6, which is 330-fold more potent than the analogous deoxynoeurostegine. It was shown through X-ray protein crystallography that galacto-noeurostegine binds to the active site of GALC in its bicyclic form.

Hormonal dysfunction in adult patients affected with inherited metabolic disorders.

Inherited metabolic disorders (IMDs ) are a rare and diverse group of metabolic conditions mainly caused by enzyme deficiencies, and in some of these, hormonal dysfunction is a relatively common complication. It may present in childhood and subsequently hormonal replacement is required throughout their life. Endocrinopathies can be a presenting symptom of an IMD in adulthood, which should be suspected when associated with multiorgan involvement (neurological, musculoskeletal or liver, etc.). A single IMD can affect any gland with hypogonadism, adrenal insufficiency, diabetes mellitus and thyroid dysfunction being the most common. In some cases, however, it is diagnosed later in their adult life as a secondary complication of previous therapies such as chemotherapy used during Haematopoietic Stem Cell Transplantation (HSCT) in childhood.The mechanisms of endocrine dysfunction in this group of conditions are not well understood. Regardless, patients require ongoing clinical support from the endocrine, metabolic, bone metabolism and fertility specialists throughout their life.Hormonal profiling should be part of the routine blood test panel to diagnose asymptomatic endocrine disorders with delayed manifestations. It is also worth considering screening for common hormonal dysfunction when patients exhibit atypical non-IMD related symptoms. In some adult-onset cases presenting with multiple endocrinopathies, the diagnosis of an IMD should be suspected.Given that new therapies are in development (e.g. gene therapies, stem cell therapies, pharmacological chaperone and substrate reduction therapies), clinicians should be aware of their potential long-term effect on the endocrine system.

Pathological Aspects of COVID-19 as a Conformational Disease and the Use of Pharmacological Chaperones as a Potential Therapeutic Strategy.

Coronavirus disease 2019 (COVID-19), the seventh human coronavirus infectious disease, was first reported in Wuhan, China, in December 2019, followed by its rapid spread globally (251,059 deaths, on May 5, 2020, by Johns Hopkins University). An early clinical report showed that fever, cough, fatigue, sputum production, and myalgia were initial symptoms, with the development of pneumonia as the disease progressed. Increases in the level of serum liver enzymes, D-dimer, cardiac troponin I, and creatinine have been observed in severely ill patients, indicating that multiple organ failure had occurred in these cases. Lymphopenia and an increase in interleukin-6 (IL-6) were also observed. Although COVID-19 patients are administered glucocorticoid therapy to treat the excessive immune response to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection, the efficacy of this form of therapy is unclear. Viremia is observed in severe cases, suggesting that in addition to type II alveolar epithelial cells, many cell types, such as vascular endothelial cells, cardiomyocytes, renal tubular cells, neuronal cells, and lymphocytes, may be damaged. The improvement of survival rates requires elucidation of the mechanism by which cellular damage occurs during viral infection. Cellular therapy, along with organ support systems such as oxygen therapy, artificial ventilation, extra corporeal membrane oxygenation and dialysis, as well as antiviral therapy, are required. Viral replication in infected host cells may perturb protein folding in the endoplasmic reticulum (ER), causing ER stress. Although an adaptive cellular response, i.e. the unfolded protein response, can compensate for the misfolded protein burden to some extent, continued viral proliferation may induce inflammation and cell death. Therefore, we propose that proteostasis dysfunction may cause conformational disorders in COVID-19. The application of pharmacological chaperone therapy to treat COVID-19 patients is additionally discussed.

Second-Generation Pharmacological Chaperones: Beyond Inhibitors.

Protein misfolding induced by missense mutations is the source of hundreds of conformational diseases. The cell quality control may eliminate nascent misfolded proteins, such as enzymes, and a pathological loss-of-function may result from their early degradation. Since the proof of concept in the 2000s, the bioinspired pharmacological chaperone therapy became a relevant low-molecular-weight compound strategy against conformational diseases. The first-generation pharmacological chaperones were competitive inhibitors of mutant enzymes. Counterintuitively, in binding to the active site, these inhibitors stabilize the proper folding of the mutated protein and partially rescue its cellular function. The main limitation of the first-generation pharmacological chaperones lies in the balance between enzyme activity enhancement and inhibition. Recent research efforts were directed towards the development of promising second-generation pharmacological chaperones. These non-inhibitory ligands, targeting previously unknown binding pockets, limit the risk of adverse enzymatic inhibition. Their pharmacophore identification is however challenging and likely requires a massive screening-based approach. This review focuses on second-generation chaperones designed to restore the cellular activity of misfolded enzymes. It intends to highlight, for a selected set of rare inherited metabolic disorders, the strategies implemented to identify and develop these pharmacologically relevant small organic molecules as potential drug candidates.

Development of Specific Fluorogenic Substrates for Human β-N-Acetyl-D-hexosaminidase A for Cell-Based Assays.

Inhibitors of human β-N-acetyl-D-hexosaminidase (hHEX) A and human O-GlcNAcase (hOGA) reportedly play roles in multiple diseases, suggesting their potential for pharmacological chaperone (PC) therapy of Sandhoff disease (SD) and Tay-Sachs disease (TSD), as lysosomal storage diseases, and Alzheimer's disease and progressive supranuclear palsy, respectively. In particular, hHEXA inhibitors as PCs have been shown to successfully enhance hHEXA levels, leading to the chronic form of SD and TSD. In the diagnosis of enzyme deficiencies in SD and TSD, artificial hHEXA substrates based on 4-methylumbelliferone as a fluorophore are available and generally used; however, they do not have sufficient performance to screen for potential inhibitors for a PC therapy from compound libraries. Further, there are currently few fluorogenic substrates for hHEXA suitable for such requirements and there are no substrates ideal for cell-based inhibitor screening. Here, we clarified the difference in enzyme active site structure between hHEXA and hOGA from their tertiary structures. To develop lysosome-localized hHEXA-specific fluorogenic substrates based on the difference in their active site structures, our developed quinone methide cleavage substrate design platform was applied for the molecular design of substrates. Thereafter, we synthesized via the shortest route and evaluated novel three-color fluorogenic substrates for hHEXA that exhibited excellent specificity and sensitivity in three human cell lines. The designed substrates represent the first-in-a class of new substrates that can be utilized to screen hHEXA inhibitors in adherent human cultured cells.

Imino- and Azasugar Protonation Inside Human Acid β-Glucosidase, the Enzyme that is Defective in Gaucher Disease.

Gaucher disease is caused by mutations in human acid β-glucosidase or glucocerebrosidase (GCase), the enzyme responsible for hydrolysis of glucosyl ceramide in the lysosomes. Imino- and azasugars such as 1-deoxynojirimycin and isofagomine are strong inhibitors of the enzyme and are of interest in pharmacological chaperone therapy of the disease. Despite several crystal structures of the enzyme with the imino- and azasugars bound in the active site having been resolved, the actual acid-base chemistry of the binding is not known. In this study we show, using photoinduced electron transfer (PET), that 1-deoxynojirimycin and isofagomine derivatives are protonated by human acid β-glucosidase when bound, even if they are completely unprotonated outside the enzyme. While isofagomine derivative protonation to some degree was foreshadowed by earlier crystal structures, 1-deoxynojirimycin derivatives were not believed to act as basic amines in the enzyme.

Imino‐ and Azasugar Protonation Inside Human Acid β‐Glucosidase, the Enzyme that is Defective in Gaucher Disease

AbstractGaucher disease is caused by mutations in human acid β‐glucosidase or glucocerebrosidase (GCase), the enzyme responsible for hydrolysis of glucosyl ceramide in the lysosomes. Imino‐ and azasugars such as 1‐deoxynojirimycin and isofagomine are strong inhibitors of the enzyme and are of interest in pharmacological chaperone therapy of the disease. Despite several crystal structures of the enzyme with the imino‐ and azasugars bound in the active site having been resolved, the actual acid–base chemistry of the binding is not known. In this study we show, using photoinduced electron transfer (PET), that 1‐deoxynojirimycin and isofagomine derivatives are protonated by human acid β‐glucosidase when bound, even if they are completely unprotonated outside the enzyme. While isofagomine derivative protonation to some degree was foreshadowed by earlier crystal structures, 1‐deoxynojirimycin derivatives were not believed to act as basic amines in the enzyme.