A new drug design targeting the adenosinergic system for Huntington's disease

Abstract

Background: Huntington's disease (HD) is a neurodegenerative disease caused by a CAG trinucleotide expansion in the Huntingtin (Htt) gene. The expanded CAG repeats are translated into polyglutamine (polyQ), causing aberrant functions as well as aggregate formation of mutant Htt. Effective treatments for HD are yet to be developed.

Methodology/principal findings: Here, we report a novel dual-function compound, N(6)-(4-hydroxybenzyl)adenine riboside (designated T1-11) which activates the A(2A)R and a major adenosine transporter (ENT1). T1-11 was originally isolated from a Chinese medicinal herb. Molecular modeling analyses showed that T1-11 binds to the adenosine pockets of the A(2A)R and ENT1. Introduction of T1-11 into the striatum significantly enhanced the level of striatal adenosine as determined by a microdialysis technique, demonstrating that T1-11 inhibited adenosine uptake in vivo. A single intraperitoneal injection of T1-11 in wildtype mice, but not in A(2A)R knockout mice, increased cAMP level in the brain. Thus, T1-11 enters the brain and elevates cAMP via activation of the A(2A)R in vivo. Most importantly, addition of T1-11 (0.05 mg/ml) to the drinking water of a transgenic mouse model of HD (R6/2) ameliorated the progressive deterioration in motor coordination, reduced the formation of striatal Htt aggregates, elevated proteasome activity, and increased the level of an important neurotrophic factor (brain derived neurotrophic factor) in the brain. These results demonstrate the therapeutic potential of T1-11 for treating HD.

Conclusions/significance: The dual functions of T1-11 enable T1-11 to effectively activate the adenosinergic system and subsequently delay the progression of HD. This is a novel therapeutic strategy for HD. Similar dual-function drugs aimed at a particular neurotransmitter system as proposed herein may be applicable to other neurotransmitter systems (e.g., the dopamine receptor/dopamine transporter and the serotonin receptor/serotonin transporter) and may facilitate the development of new drugs for other neurodegenerative diseases.

Figure 1. A fraction of the GE extract prevents serum-deprived PC12 cell apoptosis.

(A, C, E) Serum-deprived PC12 cells were treated with or without the indicated reagent(s) for 24 h. Cell viability was expressed as a percentage of the MTT activity measured in the serum-containing group. Data points represent the mean ± s.e.m. of at least three independent experiments. *p<0.05, versus the corresponding serum-deprived group. ^a p<0.05, versus the corresponding serum-deprived/T1-11 treated group. (B) Chromatogram of active fractions of GE conducted by HPLC on a Merck RP-18e (250×4.6 mm) column. The position of T1-11 is indicated by an arrow. The structure of T1-11 is shown in the upper right corner. (D) Serum-deprived PC12 cells were treated with serum or T1-11 (10 µM) as indicated for 24 h, stained with annexin V-FITC, and analyzed using microscope and flow cytometry. The median values of FITC fluorescence intensities were collected using an FL-1 channel (bottom panel). Representative pictures of cells in each condition are shown. Bars:10 µm. Data points represent the mean ± s.e.m. of at least three independent experiments.

Figure 2. T1-11 is an agonist of the A_2AR.

(A) PC12 cells were treated with T1-11 (closed circles) and CGS21680 (open circles) at the indicated concentration for 20 min at room temperature (RT). (B) PC12 cells were stimulated with T1-11 (10 µM) in the absence or presence of an A_2AR antagonist (SCH, 1 µM) for 20 min at RT. (C) Wildtype and A_2AR knockout (KO) mice were intraperitoneally administrated with T1-11 (5 mg/kg body weight, n = 4) or vehicle for 60 min to measure the cAMP level in the brain.

Figure 3. T1-11 inhibited the uptake of adenosine.

(A) Adenosine uptake by PC12 cells was analyzed in the presence of T1-11 at the indicated concentration. (B) Adenosine uptake by PC12 cells was evaluated in the presence of T1-11 (30 µM) or NBTI (0.1 µM) as indicated for 10 min. (C) T1-11 (100 µM) was perfused throughout the dialysis probe. The collected perfusates were analyzed for striatal adenosine levels. Data points represent the mean ± s.e.m.. * p<0.05, compared to the basal level.

Figure 4. Interactions of the agonists with the ligand binding sites of the A_2AR.

(A) The binding pose of CGS21680 (a selective agonist) on the human A_2AR, as predicted by combined homology modeling and docking analysis. The three-dimensional structure of the activated-state A_2AR was constructed based on the inactive-state structure of the A_2AR and the opsin structure. (B) Similar to (A), the binding pose of T1-11 on the human A_2AR.

Figure 5. Interactions of inhibitors with ligand binding sites of ENT1.

(A) The binding pose of NBTI (a selective inhibitor of ENT1) on human ENT1, as predicted using threading-based ab inito modeling of this transporter. The three-dimensional structure of ENT1 was constructed based on the lactose permease (GlpT) structure. (B) Similar to (A), the binding pose of T1-11 on human ENT1.

Figure 6. T1-11 exhibited beneficial effects in a mouse model of HD.

R6/2 mice were given the vehicle (1% DMSO; CON, n = 53) or T1-11 (0.05 mg/ml, n = 27)-containing drinking water from the age of 7 weeks. (A) Rotarod performance was conducted as described in “Methods”. (B) Striatal lysates (50∼100 µg) collected from the indicated mice at the age of 12 weeks old were subjected to a filter retardation assay. The insoluble Htt aggregates retained on the filter were detected using an anti-Htt antibody (upper panel). The amount of protein in each corresponding lysate was independently assessed by Western blot analyses using an anti-actin antibody (middle panel). The relative aggregate formation was quantified by dividing the Htt signals in filter assays with those of the corresponding actin signals in Western blots (bottom panel). Data are presented as the mean ± s.e.m. values from three independent experiments. * p<0.05, versus R6/2 mice with no treatment (CON, Student's t test). (C) Brain sections of 12-week-old animals [vehicle (CON)-treated WT mice (n = 5), vehicle-treated R6/2 mice (n = 5), and T1-11-treated R6/2 mice (n = 5)] were stained with an anti-Htt antibody. Mutant Htt aggregates were visualized using Alexa Flour 568 (red). Nuclei were visualized using H33258 (blue). Representative pictures are shown. The scale bar is 10 µm. Data are presented as the mean ± SEM in each group. *** p<0.001, versus R6/2 mice treated with vehicle (CON, Student's t test). (D) The chymotrypsin-like activity of proteasomes in striatal synaptosomes of the indicated 12-week-old mice (n = 3) was assessed as described in “Methods”. (E) Cortical tissues (n = 3) were collected to determine the transcript level of BDNF using a quantitative RT-PCR technique. The expression levels of BDNF were normalized to that of GAPDH. * P<0.05, versus R6/2 mice with no treatment (CON, Student's t test).