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.

Fig 1. Schematic representation of NDST1 gene.

The human NDST1 gene is located on chromosome 5 and encompasses 15 exons. The first exon and a part of the second are untranslated regions. NDST1 contains four alternative transcription starting sites indicated as P_A, P_B, P_C, and P_D. (A) The parametric clustering of CAGE sequences (green peaks) and DPI clusters (red and yellow arrows) indicates high tag expression level for P_A region. (B) Transcription associated features such as CpG island, Pol II binding site and histone methylation/acetylation profile identifies P_A region as the most usable promoter for NDST1 gene transcription. Data is filtered for K562 cell line. Screenshots for NDST1 promoter analysis were obtained from ZENBU genome browser (FANTOM5 Project) (http://fantom.gsc.riken.jp/zenbu/) and from UCSC Genome Database browser (http://genome.ucsc.edu/) using Human Feb. 2009 (GRCh37/hg19) Assembly.

Fig 2. Confirmation of alternative NDST1 transcripts by RT-PCR and assessment of NDST1 promoter (P_A region)—luciferase construct activity.

(A) Reverse transcription PCR for alternative NDST1 transcripts. Complementary DNA reversely transcribed from total extracted RNA of normal human fibroblast was used in the PCR to identify expression levels of each of the possible NDST1 transcripts. Primers flanked the sequence of 5’ UTR (exon 1) and coding region (exon 2) were expected to produce 637, 700, 688 bp PCR fragments for NDST1 mRNA transcribed from P_A, P_B and P_C respectively. PCRs with these primers amplified only NDST1 mRNA transcribed from P_A region. 18S rRNA was used as an internal control. (B) Activity of NDST1 promoter—renilla luciferase reporter construct. Renilla luciferase expression vector containing either: i) two random DNA sequences, RO1 and RO2 (negative controls); ii) NDST1 promoter (P_A region); or iii) GAPDH promoter (positive control) were transiently transfected into HeLa cells. DNA fragments activity is shown as a normalized value of luminescence (produced by renilla luciferase) to fluorescence (produced by Hex). Experiments were conducted in triplicates. Ren—renilla luciferase activity, l.u.—luminescence units, f.u.—fluorescence units.

Fig 3. High-content screening of 1,200 Prestwick Chemical Library compounds.

HeLa cells stably expressing firefly luciferase under control of NDST1 promoter were treated with compounds from the Prestwick Chemical Library. The reporter activity of the tested compounds is expressed relative to the mean activity of DMSO treated cells. Drugs showing cytotoxic effects, as tested in parallel by the Hexosaminidase assay, were removed from the graph. Thirty eight compounds resulting in the luminescence decrease by >75% relative to the mean of DMSO control were considered as potentially strong NDST1 inhibitors (shown in red). Seven compounds (shown in green) increased luminescence by ≥ 30% over the mean DMS control. These compounds could be considered as potential inducers of NDST1 expression. Eight potentially strong inhibitors and 2 average inhibitors (indicated by blue circles) were selected for the dose-response assessment (Fig 4A). Experiments were performed in triplicates. Abbreviation: stdev–standard deviation.

Fig 4. Secondary screening and ‘hit’ validation.

(A) Dose-dependent inhibition of the NDST1 promoter activity. HeLa cells stably expressing NDST1 promoter were treated with serially diluted potential NDST1 repressors. Dose-response curves were produced from 9 out of 10 drug candidates. Experiments were performed in singles. (B) Western blot densitometry analysis of NDST1 protein expression after treatment with selected potential NDST1 repressors. Normal human fibroblast cells were treated with potential NDST1 inhibitors at their calculated 1x IC₅₀ or 2x IC₅₀. The effect of the compound on the NDST1 expression was evaluated using densitometry analysis of NDST1 band normalized to its corresponding GAPDH band. Distinct decrease in NDST1 expression was observed for 2x IC₅₀ SAHA treatment. (C) ELISA assay of total NDST activity in normal human fibroblast cells. Lysates of cells treated with either SAHA or DMSO were assessed for total NDST activity. Treatment with 1.2 μM of SAHA resulted in a ~ 40% decrease in NDST enzyme activity. Experiments were conducted in triplicates. (D) The effect of SAHA on NDST1 mRNA expression in normal human fibroblast cells. Normal human fibroblast cells were treated with 2x IC₅₀ (2.4 μM) of SAHA for 5 days. The level of NDST1 mRNA was reduced by 60% after the treatment. Expression of NDST1 mRNA level was normalized to 18S rRNA level. Experiments were performed in duplicates. MEC–meclocycline, THIO–thiostrepton, ENTA–enthacapone, MIF–mifepristone, SAHA–SAHA, GRIS–griseofulvin, ANT–antralin, PENT–penthamidine, PYR–pyrimethamine. r.u.–relative units, l.u.–luminescence units.

Fig 5. ³⁵S Incorporation into newly synthesized GAGs.

After five days of SAHA treatment, incorporation of ³⁵S into GAGs was measured at 24, 48 and 72 hours of pulse. All patient cells showed reduced level of ³⁵S labeled GAGs synthesis. Experiments were conducted in triplicates. cpm–counts per million, mg–milligrams of protein. * p < 0.05, *** p < 0.001.

Fig 6. Histone deacetylase (HDAC) inhibitor assessment.

(A) Dose-response of HDAC inhibitors on NDST1 promoter. HeLa cells stably expressing the NDST1 promoter—luciferase reporter gene were treated with serially diluted selected HDAC inhibitors. ITF 2357, an HDAC inhibitor which is structurally and functionally similar to SAHA, produced a dose-response curve with IC₅₀ of 0.2 μM. Experiments were performed in duplicates. (B) NDST1 mRNA level after treatment with ITF 2357. Normal human fibroblast cells were treated with 2x IC₅₀ (0.4 μM) of ITF 2357 for 5 days. Level of NDST1 mRNA was reduced by 40% after the treatment. Expression of NDST1 mRNA level was normalized to 18S rRNA level. Experiments were conducted in duplicates. (C) Western blot densitometry analysis of NDST1 protein expression after treatment with ITF 2357. Normal human fibroblast, MPS I patient, and MPS IIIA patient cells were treated with ITF 2357 at its calculated 2x IC₅₀. The effect of the compound on the NDST1 expression was evaluated using densitometry analysis of NDST1 band normalized to its corresponding actin band. Significant decrease in NDST1 expression was observed in all treated cells. l.u.–luminescence units. r.u.–relative units.