Discovery of a novel noniminosugar acid α glucosidase chaperone series

Abstract

Pompe disease is an autosomal recessive lysosomal storage disorder (LSD) caused by deficiency of the lysosomal enzyme acid α-glucosidase (GAA). Many disease-causing mutated GAA retain enzymatic activity but are not translocated from endoplasmic reticulum (ER) to lysosomes. Enzyme replacement therapy (ERT) is the only treatment for Pompe disease but remains expensive, inconvenient, and does not reverse all disease manifestations. It was postulated that small molecules which aid in protein folding and translocation to lysosomes could provide an alternate to ERT. Previously, several iminosugars have been proposed as small-molecule chaperones for specific LSDs. Here we identified a novel series of noniminosugar chaperones for GAA. These moderate GAA inhibitors are shown to bind and thermostabilize GAA and increase GAA translocation to lysosomes in both wild-type and Pompe fibroblasts. AMDE and physical properties studies indicate that this series is a promising lead for further pharmacokinetic evaluation and testing in Pompe disease models.

Figure 1

Known iminosugars as inhibitors of alpha glucosidase.

Figure 2

Hydrolytic reactions of the red and blue dyes.

Figure 3

(A) Chemical structure of compound 1 identified from qHTS. (B) The GAA inhibitory activity of compound 1 as demonstrated by the hydrolysis of red and blue substrates using isolated GAA enzyme or tissue homogenates.

Figure 4

Thermostabilization of GAA functional activity. Time indicates length of incubation at 66 °C. Ratio is the ratio of enzymatic activity after incubation at 66 °C compared to room temperature incubation. Black: DMSO (duplicated); Red: DNJ (2.5 μM); Green: qHTS sample of compound 1 (50 μM); Blue: re-synthesized compound 1 (50 μM).

Figure 5

Capacity of inhibitors at 50 μM to maintain the function of GAA after incubation at 66 °C for 60 minutes.

Figure 6

(A). ΔT_m versus concentrations for analogs 1 and 22. (B). Microscale thermophoresis study of analogs 1 and 22 with GAA.

Figure 7

Evaluation of compound 22 in GAA translocation assays using wild type and Pompe fibroblasts. (A) Western blot analysis of protein extracts from HEK cells over-expressing GAA (lane 1), HEK cells (lane 2) and wild type primary fibroblasts (lane 3). Tubulin was used as a loading control. (B, a) DMSO-treated Primary wild type fibroblasts. (B, b) DMSO-treated primary wild type fibroblasts stained with mouse Alexa-488 and goat Alexa-555(red) as a negative staining control. (B, c, d) Primary wild type fibroblasts treated with 15 μM (c) and 5 μM (d) of compound 22 for 6 days. (C, a) DMSO-treated primary Pompe fibroblasts. (C, b) Pompe fibroblasts (F2845) treated with 5 μM of compound 22 for 5 days. All stainings were performed with the anti-GAA mouse monoclonal antibody (green) and the anti-Cathepsin D goat polyclonal antibody (red); a DAPI stain was performed to visualize the nucleus (blue). Scale bar = 20 μm (B) and 50 μm (C).

Figure 8

Relative inhibition of GAA in WT (A) and Pompe F2845 fibroblasts (B) in a whole cell assay with treatment of compound 22 and compound 1 at pH 4 with no wash-out and after 18 and 40 hours wash out. Data points have been normalized to GAA activity in vehicle treated cells (100%)

Scheme 1

Synthesis of analogs with modifications of phenylpiperazine moiety of the molecule.

Scheme 2

Synthesis of analogs with modifications of indolinone ring or sulfonamide portion of the molecule.