Small-molecule inhibition of glycogen synthase 1 for the treatment of Pompe disease and other glycogen storage disorders

Abstract

Glycogen synthase 1 (GYS1), the rate-limiting enzyme in muscle glycogen synthesis, plays a central role in energy homeostasis and has been proposed as a therapeutic target in multiple glycogen storage diseases. Despite decades of investigation, there are no known potent, selective small-molecule inhibitors of this enzyme. Here, we report the preclinical characterization of MZ-101, a small molecule that potently inhibits GYS1 in vitro and in vivo without inhibiting GYS2, a related isoform essential for synthesizing liver glycogen. Chronic treatment with MZ-101 depleted muscle glycogen and was well tolerated in mice. Pompe disease, a glycogen storage disease caused by mutations in acid α glucosidase (GAA), results in pathological accumulation of glycogen and consequent autophagolysosomal abnormalities, metabolic dysregulation, and muscle atrophy. Enzyme replacement therapy (ERT) with recombinant GAA is the only approved treatment for Pompe disease, but it requires frequent infusions, and efficacy is limited by suboptimal skeletal muscle distribution. In a mouse model of Pompe disease, chronic oral administration of MZ-101 alone reduced glycogen buildup in skeletal muscle with comparable efficacy to ERT. In addition, treatment with MZ-101 in combination with ERT had an additive effect and could normalize muscle glycogen concentrations. Biochemical, metabolomic, and transcriptomic analyses of muscle tissue demonstrated that lowering of glycogen concentrations with MZ-101, alone or in combination with ERT, corrected the cellular pathology in this mouse model. These data suggest that substrate reduction therapy with GYS1 inhibition may be a promising therapeutic approach for Pompe disease and other glycogen storage diseases.

Fig. 1.. MZ-101 is a potent and selective inhibitor of Glycogen Synthase 1 in vitro.

(A) MZ-101 structure. Inhibition potency of MZ-101 against human recombinant GYS1 (B) and GYS2 (C) in PK-LDH coupled enzyme assay (Exp n=253 GYS1, n=5 GYS2). Dose response of MZ-101 against phosphorylated (D) and fully dephosphorylated (E) human GYS1 across a range of G6P mM (n=4-6 exp). Data shown as mean +/− SEM.

Fig. 2.. MZ-101 inhibits glycogen accumulation in fibroblasts from healthy controls and patients with Pompe.

(A) Schematic of experimental protocol. Potency of MZ-101 GYS1 inhibition EC₅₀ impact on accumulated total glycogen stores over 7 days treatment in healthy donor fibroblasts (B) and Pompe donor fibroblasts (C) with 1.0 corresponding to the average glycogen content in healthy control DMSO-treated cells. Data shown as mean +/− SEM, N=3 experiments.

Fig. 3.. Glycogen accumulation in Pompe (Gaatm1Rabn) mouse gastrocnemius drives pathological upregulation of the GYS1 glycogen biosynthetic pathway.

(A) Gastrocnemius muscle glycogen content in WT and Pompe GAA KO mice. (B) Markers of the glycogen biosynthetic pathway: glucose transporter GLUT1, free glucose, and glucose-6-phosphate (G6P) metabolite. (C) GYS1 de novo glycogen synthesis as quantified from gastrocnemius tissue lysates. (D) Total GYS1 protein abundance and abundance of inhibitory phosphorylation at S641 GYS1. (E) Representative Western blots from gastrocnemius tissue lysates. (F) Key cell biology pathways driving the pathophysiology in Pompe disease. Data are shown as mean +/− SEM. Students t-test ns p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Fig. 4.. MZ-101 inhibits GYS1 but not GYS2 de novo glycogen synthesis in WT & Pompe mice.

(A) Schematic of in vivo de novo glycogen synthesis experiment. (B) De novo glycogen synthesized over a 4-hour time course pulse in male WT (C57BL6) and Pompe (GAA KO) mouse gastrocnemius. (C) Inhibition of glycogen synthesis in gastrocnemius muscle via dose response of MZ-101 in WT mice (n=7-22). MZ-101 dose response in Pompe mice gastrocnemius (D) and liver (E), (n=8-12 mice per treatment group). Data are individual animals (circles) with mean +/− SEM. One way ANOVA with Dunnett’s post hoc analysis vs 0 mg/kg control group, ns p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Fig. 5.. Substrate reduction therapy (SRT) with MZ-101 reduces glycogen levels in WT and Pompe mice over 4-14 weeks.

(A) Glycogen turnover across 12 weeks in WT vs Pompe gastrocnemius. (B and C) 10-12 week old mice provided chow formulated with vehicle (veh.) or MZ-101 (SRT) for 4 or 14 weeks. Glycogen content in WT (B) and Pompe (C) mouse gastrocnemius after 4 and 14 weeks treatment. Data are individual animals (circles) with mean +/− SEM. % reduction calculated at each timepoint for each genotype. Unpaired Students t-Test WT vs Pompe per time point cohort ***p<0.001, ****p<0.0001.

Fig. 6.. SRT, ERT, & combination therapy reduce gastrocnemius glycogen and urine and blood biomarkers after 12 weeks of treatment.

Six to nine-week old mice provided chow formulated with vehicle (veh.), MZ-101 (SRT), or alglucosidase alfa 20mg/kg biweekly (ERT) for 12 weeks. (A) Study design schema. (B) Glycogen content of gastrocnemius tissue lysate assessed via biochemical assay. (C) MALDI analysis of glycogen branch chain length (top) and ratio of phosphorylated glycogen to dephosphorylated (bottom). (E) Urinary tetrasaccharide (uGlc4) glycogen breakdown product. (F) Glycogen in peripheral blood mononuclear cells (PBMC) collected at day +14 after ERT dose. Data are individual animals (circles) with mean +/− SEM. One-way ANOVA with Tukey’s post hoc analysis ns p>0.05, **p<0.01,***p<0.001, ****p<0.0001.

Fig. 7.. SRT, ERT, and combination therapy rescue of Pompe disease pathway biomarkers in gastrocnemius muscle

Six to nine-week old mice provided chow formulated with vehicle (veh.), MZ-101 (SRT), or alglucosidase alfa 20mg/kg biweekly (ERT) for 12 weeks. (A) Western blot quantification of gastrocnemius tissue lysate for LAMP1, p62, and LC3B. (B) Representative IHC of gastrocnemius tissue section for detection of LAMP1 and P62, scale bar 50um. (C) Western blot quantification of gastrocnemius tissue lysate for GYS1, phosphorylated GYS1, and GLUT1. Data are individual animals (circles) with mean +/− SEM. One-way ANOVA with Tukey’s post hoc analysis ns p>0.05, *p<0.05, **p<0.01,***p<0.001, ****p<0.0001.

Fig. 8.. Substrate reduction therapy improves abnormalities in gastrocnemius cellular metabolism and skeletal muscle function in a mouse model of Pompe disease.

Six to nine-week old mice fed chow with vehicle (veh.) or MZ-101 (SRT), or alglucosidase alfa 20mg/kg biweekly (ERT) for 12 wks. Polar metabolite GC/MS and RNA-seq were performed on n=5 mice in each treatment group. (A) Heatmap of cohort average Log2FC vs WT abundance in gastrocnemius. (B) GC/MS glycogen biosynthetic pathway analyte bar graphs n=5, mean, s.e.m. One-way ANOVA Tukey’s post hoc (C) Heatmap average Log2FC expression in each condition vs WT for all genes from the glycogen biosynthetic pathway. Hyper-geometric test (ora) on whole pathway enrichment based on genes significantly differentially expressed (p < 0.05) verses WT. (D) Gastrocnemius glycogen post 7-month treatment. (E) Latency to fall in accelerating rotarod test post 7-months treatment. One-way ANOVA, Tukey’s post hoc ns p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001