NDST1 Preferred Promoter Confirmation and Identification of Corresponding Transcriptional Inhibitors as Substrate Reduction Agents for Multiple Mucopolysaccharidosis Disorders

Abstract

The stepwise degradation of glycosaminoglycans (GAGs) is accomplished by twelve lysosomal enzymes. Deficiency in any of these enzymes will result in the accumulation of the intermediate substrates on the pathway to the complete turnover of GAGs. The accumulation of these undegraded substrates in almost any tissue is a hallmark of all Mucopolysaccharidoses (MPS). Present therapeutics based on enzyme replacement therapy and bone marrow transplantation have low effectiveness for the treatment of MPS with neurological complications since enzymes used in these therapies are unable to cross the blood brain barrier. Small molecule-based approaches are more promising in addressing neurological manifestations. In this report we identify a target for developing a substrate reduction therapy (SRT) for six MPS resulting from the abnormal degradation of heparan sulfate (HS). Using the minimal promoter of NDST1, one of the first modifying enzymes of HS precursors, we established a luciferase based reporter gene assay capable of identifying small molecules that could potentially reduce HS maturation and therefore lessen HS accumulation in certain MPS. From the screen of 1,200 compounds comprising the Prestwick Chemical library we identified SAHA, a histone deacetylase inhibitor, as the drug that produced the highest inhibitory effects in the reporter assay. More importantly SAHA treated fibroblasts expressed lower levels of endogenous NDST1 and accumulated less 35S GAGs in patient cells. Thus, by using our simple reporter gene assay we have demonstrated that by inhibiting the transcription of NDST1 with small molecules, identified by high throughput screening, we can also reduce the level of sulfated HS substrate in MPS patient cells, potentially leading to SRT.

Plant chemical genetics reveals colistin sulphate as a SA and NPR1-independent PR1 inducer functioning via a p38-like kinase pathway

“…The PCL is comprised of well-annotated compounds used already in different model systems 61–63 but plants. We reasoned the knowledge gained from previous PCL-based screens, and specifically with CS , could therefore be useful to unravel the molecular mechanisms of CS in plants.…”

Substrate reduction therapy in aDrosophila melanogastermodel of Sanfilippo syndrome

“…Suberanilohydroxamic acid (SAHA or Vorinostat) is a pan-histone deacetylase inhibitor that was identified by screening of a chemical library for inhibition of the HS modifying enzyme N-deacetylase/N-sulfotransferase (NDST1) (29). Treatment of MPS IIIA and IIIC patient fibroblasts with SAHA inhibited glycosaminoglycan biosynthesis (29). Compounds rhodamine B and genistein also slow HS/glycosaminoglycan accumulation and improve MPS IIIA or IIIB mouse behaviour (30)(31)(32).…”

Substrate reduction therapy for inborn errors of metabolism

“…Other medium/high-throughput screening efforts for SRT inhibitors have applied target-specific cell-based assays. In one study [73] for MPS, a reporter gene assay was used to screen 1200 compounds for transcriptional inhibition of NDST1, an enzyme involved in the early stage of HS synthesis. Because of its role, hits identified for this enzyme could have therapeutic potential for a subset of six MPS subtypes due to defective HS degradation.…”

“…Despite the advances in uncovering the genetic, biochemical, and pathological mechanisms for IEMs, the number of approved therapies remains very small (circa [70][71][72][73][74][75][76][77][78][79][80]. This gap is partly attributable to the conceptual challenge underlying the drug development for LOF disorders.…”