Sarcoglycanopathies are rare autosomal recessive pathologies, a subtype of the Limb-Girdle Muscular Dystrophies (LGMDs), a subfamily of a large group called muscular dystrophy (MD). The sarcoglycanopathies are caused by mutations in sarcoglycans, α-, β, γ- and δ-, that are transmembrane proteins coded respectively by the SGCA, SGCB, SGCD and SGCG genes. Depending on the protein involved, the LGMD can be divided in 2D, 2E, 2F and 2C, and symptomatology and severity can vary. Generally, the disease is characterized by muscular weakness of skeletal muscles, specifically proximal muscles of the shoulder and the pelvic area. Respiratory complication can rise along the way, and cardiomyopathies are less frequent. Variants in sarcoglycan genes are mainly missense mutations consisting of a residue change leading to an incorrect folding, without compromising the functionality of the sarcoglycan complex. This is corroborated by the fact that in same patients a low amount of proteins can be localized at the sarcolemma, representing the milder form of the disease. The problem resides in the trafficking of the sarcoglycans to the membrane. The maturation of these proteins occurs in the endoplasmic reticulum (ER), in which the quality control system recognizes the defective sarcoglycans and degrades them via the ER-associated protein degradation (ERAD) pathway. The inhibition of the ubiquitin-proteasome system allows the delivery of the mutated sarcoglycan at the sarcolemma. The sarcoglycan complex is an essential component of the dystrophin-glycoprotein complex (DGC), which acts as shock absorber for the mechanic muscular stress under contraction. The four sarcoglycans form a tetrameric complex, and the absence of one often compromise the presence of the entire sarcoglycan complex in the membrane. The inhibition of a degradative pathway isn’t a suitable treatment, since the elimination of such a control system means that the possible accumulation of a large amount of incorrect folded proteins. A possible pharmacological strategy is to assist the folding of the proteins by the use of CFTR correctors, a family of small molecules used in the treatment of cystic fibrosis. In vitro tests showed the efficacy of the CFTR correctors in heterologous cellular models and in primary LGMD2D patient-derived myotubes. CFTR correctors help the mutated sarcoglycan to pass the quality control system and reach the sarcolemma. The most promising identified compound was the C17. This CFTR corrector was tested in a mouse model of alpha-sarcoglycanopathy with “humanized hind-limbs”. The α-sarcoglycan knock-out (KO) mouse was injected in hind-limbs with an adeno-associated virus vector with the α-sarcoglycan carrying the human R98H mutation. Mice were subjected to chronical treatment with C17 and tissues were analyzed. The treatment resulted in a significative localization of mutated protein at the level of sarcolemma, the entire sarcoglycan complex relocated with the modify α-sarcoglycan and the dystrophic muscles recovered from the tissue damage. The compound didn’t give toxic effect during the treatments and the animal didn’t exhibited any side effect directly related to the drug administration. The C17 was also investigated in term of biodistribution and biotransformation. C17 was found in many organs, in all of them the compound didn’t appear to be metabolize. Biotransformation study were performed through the use of human and mouse hepatic microsomes and S9-fractions. These analyses improve the confidence about the stability of C17. Same metabolites have been found in the feces of the treated mouse. The efficacy of the C17 was proven, but the compound requires further investigation about the pharmacokinetics and pharmacodynamic to completely understand its properties.
Sarcoglycanopathies are rare autosomal recessive pathologies, a subtype of the Limb-Girdle Muscular Dystrophies (LGMDs), a subfamily of a large group called muscular dystrophy (MD). The sarcoglycanopathies are caused by mutations in sarcoglycans, α-, β, γ- and δ-, that are transmembrane proteins coded respectively by the SGCA, SGCB, SGCD and SGCG genes. Depending on the protein involved, the LGMD can be divided in 2D, 2E, 2F and 2C, and symptomatology and severity can vary. Generally, the disease is characterized by muscular weakness of skeletal muscles, specifically proximal muscles of the shoulder and the pelvic area. Respiratory complication can rise along the way, and cardiomyopathies are less frequent. Variants in sarcoglycan genes are mainly missense mutations consisting of a residue change leading to an incorrect folding, without compromising the functionality of the sarcoglycan complex. This is corroborated by the fact that in same patients a low amount of proteins can be localized at the sarcolemma, representing the milder form of the disease. The problem resides in the trafficking of the sarcoglycans to the membrane. The maturation of these proteins occurs in the endoplasmic reticulum (ER), in which the quality control system recognizes the defective sarcoglycans and degrades them via the ER-associated protein degradation (ERAD) pathway. The inhibition of the ubiquitin-proteasome system allows the delivery of the mutated sarcoglycan at the sarcolemma. The sarcoglycan complex is an essential component of the dystrophin-glycoprotein complex (DGC), which acts as shock absorber for the mechanic muscular stress under contraction. The four sarcoglycans form a tetrameric complex, and the absence of one often compromise the presence of the entire sarcoglycan complex in the membrane. The inhibition of a degradative pathway isn’t a suitable treatment, since the elimination of such a control system means that the possible accumulation of a large amount of incorrect folded proteins. A possible pharmacological strategy is to assist the folding of the proteins by the use of CFTR correctors, a family of small molecules used in the treatment of cystic fibrosis. In vitro tests showed the efficacy of the CFTR correctors in heterologous cellular models and in primary LGMD2D patient-derived myotubes. CFTR correctors help the mutated sarcoglycan to pass the quality control system and reach the sarcolemma. The most promising identified compound was the C17. This CFTR corrector was tested in a mouse model of alpha-sarcoglycanopathy with “humanized hind-limbs”. The α-sarcoglycan knock-out (KO) mouse was injected in hind-limbs with an adeno-associated virus vector with the α-sarcoglycan carrying the human R98H mutation. Mice were subjected to chronical treatment with C17 and tissues were analyzed. The treatment resulted in a significative localization of mutated protein at the level of sarcolemma, the entire sarcoglycan complex relocated with the modify α-sarcoglycan and the dystrophic muscles recovered from the tissue damage. The compound didn’t give toxic effect during the treatments and the animal didn’t exhibited any side effect directly related to the drug administration. The C17 was also investigated in term of biodistribution and biotransformation. C17 was found in many organs, in all of them the compound didn’t appear to be metabolize. Biotransformation study were performed through the use of human and mouse hepatic microsomes and S9-fractions. These analyses improve the confidence about the stability of C17. Same metabolites have been found in the feces of the treated mouse. The efficacy of the C17 was proven, but the compound requires further investigation about the pharmacokinetics and pharmacodynamic to completely understand its properties.
Preliminary pharmacological study on C17 CFTR corrector related to sarcoglycanopathies
PAROLIN, ALESSANDRO
2022/2023
Abstract
Sarcoglycanopathies are rare autosomal recessive pathologies, a subtype of the Limb-Girdle Muscular Dystrophies (LGMDs), a subfamily of a large group called muscular dystrophy (MD). The sarcoglycanopathies are caused by mutations in sarcoglycans, α-, β, γ- and δ-, that are transmembrane proteins coded respectively by the SGCA, SGCB, SGCD and SGCG genes. Depending on the protein involved, the LGMD can be divided in 2D, 2E, 2F and 2C, and symptomatology and severity can vary. Generally, the disease is characterized by muscular weakness of skeletal muscles, specifically proximal muscles of the shoulder and the pelvic area. Respiratory complication can rise along the way, and cardiomyopathies are less frequent. Variants in sarcoglycan genes are mainly missense mutations consisting of a residue change leading to an incorrect folding, without compromising the functionality of the sarcoglycan complex. This is corroborated by the fact that in same patients a low amount of proteins can be localized at the sarcolemma, representing the milder form of the disease. The problem resides in the trafficking of the sarcoglycans to the membrane. The maturation of these proteins occurs in the endoplasmic reticulum (ER), in which the quality control system recognizes the defective sarcoglycans and degrades them via the ER-associated protein degradation (ERAD) pathway. The inhibition of the ubiquitin-proteasome system allows the delivery of the mutated sarcoglycan at the sarcolemma. The sarcoglycan complex is an essential component of the dystrophin-glycoprotein complex (DGC), which acts as shock absorber for the mechanic muscular stress under contraction. The four sarcoglycans form a tetrameric complex, and the absence of one often compromise the presence of the entire sarcoglycan complex in the membrane. The inhibition of a degradative pathway isn’t a suitable treatment, since the elimination of such a control system means that the possible accumulation of a large amount of incorrect folded proteins. A possible pharmacological strategy is to assist the folding of the proteins by the use of CFTR correctors, a family of small molecules used in the treatment of cystic fibrosis. In vitro tests showed the efficacy of the CFTR correctors in heterologous cellular models and in primary LGMD2D patient-derived myotubes. CFTR correctors help the mutated sarcoglycan to pass the quality control system and reach the sarcolemma. The most promising identified compound was the C17. This CFTR corrector was tested in a mouse model of alpha-sarcoglycanopathy with “humanized hind-limbs”. The α-sarcoglycan knock-out (KO) mouse was injected in hind-limbs with an adeno-associated virus vector with the α-sarcoglycan carrying the human R98H mutation. Mice were subjected to chronical treatment with C17 and tissues were analyzed. The treatment resulted in a significative localization of mutated protein at the level of sarcolemma, the entire sarcoglycan complex relocated with the modify α-sarcoglycan and the dystrophic muscles recovered from the tissue damage. The compound didn’t give toxic effect during the treatments and the animal didn’t exhibited any side effect directly related to the drug administration. The C17 was also investigated in term of biodistribution and biotransformation. C17 was found in many organs, in all of them the compound didn’t appear to be metabolize. Biotransformation study were performed through the use of human and mouse hepatic microsomes and S9-fractions. These analyses improve the confidence about the stability of C17. Same metabolites have been found in the feces of the treated mouse. The efficacy of the C17 was proven, but the compound requires further investigation about the pharmacokinetics and pharmacodynamic to completely understand its properties.File | Dimensione | Formato | |
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https://hdl.handle.net/20.500.12608/43072