Invitro Profiling of Chondroitin/Dermatan Trisulphate Disaccharide Profiling as pharmacological chaperone for Mucopolysaccharidoses Type II

Introduction Mucopolysaccharidosis type II (MPS II, OMIM 309900) also known as Hunter syndrome, is an X-linked recessive disorder caused by a deficiency of iduronate-2-sulphatase (IDS) (1). • MPS II is characterized by progressive lysosomal accumulation of glycosaminoglycans (GAGs) comprised of dermatan sulphate and heparan sulphate, and presents systemic manifestations such as skeletal deformities, mental retardation, valvular heart disease, hepatosplenomegaly and skin abnormality (2). • Enzyme replacement therapy (ERT) is currently the standard treatment for MPS II patients while hematopoietic stem cell transplantation (HSCT) is a potential option for MPS II treatment. • However, these therapies have several limitations; no or little effects on brain, bone and heart valves, need of weekly intravenous administration of ERT, and risk of mortality due to the conditioning regiment using chemotherapy agents in HSCT. • The use of small molecules as pharmacological chaperone in therapeutic alternative to restore the defective IDS has been extensively explored. • Here, we demonstrate the profiling of chondroitin/dermatan trisulphate dissacharide (CD3S) using invitro recombinant human iduronate-2-sulphatase (rhIDS).

Materials and Methods Our study revealed that chondroitin dermatan trisulphate (CD3S), heparin tetrasaccharide (H4Sac), heparin octasaccharide (H8Sac) and heparin octadecasaccharide (H18Sac) showed low IC50 and high Ki (Table 1). • After incubation with H4Sac, H8Sac and H18Sac, the activities of rhIDS were totally suppressed and denatured when reaching 67oC (Figure 1). • However, there was still ~20% activity of rhIDS when incubated with CD3S at 67oC.

Results [Refer to Poster].

Discussion From the inhibition assay, only four small molecules showed the lowest IC50 with the highest Ki . IC50 referring to concentration required to produce 50% inhibition of enzyme activity while Ki is an equilibrium constant of a reversible inhibitor for complexation with its target enzyme. The thermal stability of rhIDS experiment was conducted to determine which small molecules have protective effect on the enzyme from heat-induced inactivation. • A stable conformation of a protein resists denaturation as compared to fragile conformational structure which often intolerant to heat denaturation. It has been postulated that N-linked oligosaccharides played an important role in the folding, function and stability of glycoprotein. We hypothesized that the N-terminus in the structure of CD007 may interact with any of the eight N-linked glycosylation sites in the rhIDS to stabilise the folding of the enzyme during the heat-induced activation process. The importance of N-glycosylation for folding, catalytic activity and processing of IDS has been demonstrated.

Conclusion Overall, our experiments discovered that CD3S was able to bind, inhibit and chaperone rhIDS and may serve as potential pharmacological chaperone for MPS II.