Anle138b: a novel oligomer modulator for disease-modifying therapy of neurodegenerative diseases such as prion and Parkinson's disease

Abstract

In neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD) and prion diseases, deposits of aggregated disease-specific proteins are found. Oligomeric aggregates are presumed to be the key neurotoxic agent. Here we describe the novel oligomer modulator anle138b [3-(1,3-benzodioxol-5-yl)-5-(3-bromophenyl)-1H-pyrazole], an aggregation inhibitor we developed based on a systematic high-throughput screening campaign combined with medicinal chemistry optimization. In vitro, anle138b blocked the formation of pathological aggregates of prion protein (PrP(Sc)) and of α-synuclein (α-syn), which is deposited in PD and other synucleinopathies such as dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Notably, anle138b strongly inhibited all prion strains tested including BSE-derived and human prions. Anle138b showed structure-dependent binding to pathological aggregates and strongly inhibited formation of pathological oligomers in vitro and in vivo both for prion protein and α-synuclein. Both in mouse models of prion disease and in three different PD mouse models, anle138b strongly inhibited oligomer accumulation, neuronal degeneration, and disease progression in vivo. Anle138b had no detectable toxicity at therapeutic doses and an excellent oral bioavailability and blood-brain-barrier penetration. Our findings indicate that oligomer modulators provide a new approach for disease-modifying therapy in these diseases, for which only symptomatic treatment is available so far. Moreover, our findings suggest that pathological oligomers in neurodegenerative diseases share structural features, although the main protein component is disease-specific, indicating that compounds such as anle138b that modulate oligomer formation by targeting structure-dependent epitopes can have a broad spectrum of activity in the treatment of different protein aggregation diseases.

Figures

Fig. 1

Summary of experimental strategy. In a first project phase, we tested a library of 10,000 chemically diverse drug-like compounds in regard to inhibition of prion protein aggregation in a molecular SIFT screening assay. Based on this data in combination with testing of primary hits in a cellular anti-prion assay, we identified N-benzylidene-benzohydrazide (NBB) derivatives as a new lead structure with anti-prion activity providing a proof of principle of the experimental strategy. Notably, the same compounds also inhibit Poly-Q and α-syn aggregation both in vitro and in vivo. However, NBB’s contain a Schiff’s base like =N–NH–CO– structure that can result in rapid metabolism in vivo. In order to identify further lead structures with favourable medicinal chemical properties, we continued by screening of 10,000 additional compounds and performed a parallel analysis of all these 20,000 compounds in a microtiter plate based high-throughput anti-prion cell culture assay followed by structure–activity and cluster analysis of the data obtained with these two independent approaches (Suppl. Fig. 1). This analysis identified a cluster of highly active compounds belonging to the chemical compound class of 3,5-diphenyl-pyrazole (DPP) derivatives. A pilot experiment with one DPP compound from the initial screening library showed a significant effect on survival in prion-infected mice (Suppl. Fig. 2). Therefore, we synthesized a focused library of ~150 DPP-related compounds (Suppl. “Compound Synthesis”) for further testing in vitro, in cell culture, and in vivo in regard to therapeutic effects on prion and α-syn aggregation and disease progression in animal models. Based on results from the SIFT and cell culture anti-prion assays, 38 compounds were selected for in vivo testing in prion-infected mice (Suppl. Fig. 3). In these experiments, the compound anle138b [3-(1,3-benzodioxol-5-yl)-5-(3-bromophenyl)-1H-pyrazole] showed the highest anti-prion activity. In addition, anle138b showed efficacy in in vitro and in vivo models for synucleinopathies such as PD

Fig. 2

Correlation between anti-prion activity in vitro and in vivo. For compounds shown in Table 1 that reach brain levels ≥15 nmol/g (indicated by dark blue dots), there is a strong linear correlation (R = 0.951) between anti-prion activity in vitro and in vivo. Those compounds that reach lower brain levels (light blue dots) result in lower in vivo activities. No compounds with low anti-prion activity in the PMCA assay in vitro are active in vivo. Anle138b is the most active compound in both assays. Anti-prion activity is provided as % inhibition

Fig. 3

Influence of daily anle138b treatment on PrPSc accumulation and prion pathology of mice infected with RML scrapie. a Survival curves of mice treated orally with anle138b beginning at 0, 80 or 120 days post i.c. prion infection. Treatment resulted in prolonged survival, even when started at an advanced disease stage at 120 dpi. For detailed statistics see Suppl. Fig. 6, n = 7–9. b Control mice showed progressive weight loss starting after 100 dpi. Treatment with anle138b from 80 dpi onwards prevents weight loss for ~100 days. Treatment from 120 dpi inhibits further weight loss for ~70 days. c Immunohistochemistry (upper row cortex/hippocampus, middle row cerebellum) and dot blot analysis (lower row) showed that anle138b treatment inhibits PrPSc accumulation in comparison to DMSO-treated control animals. d Quantification of brain PrPSc levels at different time points shows highly significant inhibition of PrPSc accumulation in mice treated from 80 dpi onwards. PrPSc accumulation is also reduced in animals treated from 120 dpi. e Histological analysis reveals a significantly reduced number of pyknotic nuclei both in mice treated from 80 and 120 dpi onwards. Inset shows an example of a pyknotic granule cell nucleus (arrow). Error bars in (d) and (e) indicate standard error (n = 4), **p < 0.01, ***p < 0.001. The legend shown in (a) also applies to (b–e)

Fig. 4

Size modulation of PrPSc oligomers by anle138b and inhibition of various human and non-human prion strains in PMCA. (a, b) Size distribution of PrPSc aggregates was analyzed by sucrose-gradient centrifugation. Mice treated with anle138b show a strong reduction of high molecular weight species (fractions 7–12). Also small molecular weight oligomers (fractions 3–4) are reduced and show a shift towards smaller size (fraction 2) indicating that anle138b blocks aggregation at the level of small oligomers. DMSO-treated mice are indistinguishable from terminally ill untreated mice. (c, d) For PMCA assay, infected brain homogenates were diluted 100-fold by appropriate normal brain homogenates containing compounds (1 μl of 10 mM solutions in DMSO) or 1 μl of DMSO as control. c Mouse-adapted scrapie strains (RML, ME7) and Mouse-adapted BSE (301C) were used as seed in C57/BL6 mouse normal brain homogenates. d sCJD and vCJD samples were propagated in non-CJD human brain homogenates. In all gels, 0.5 % (w/v) normal brain homogenates from C57/BL6 and human brain, respectively, were loaded directly in the last lane without proteolysis as a reference (indicated as PrPC on the top). All other samples were treated with 50 μg/ml PK. “Start” (lane 1 of each gel) indicates samples containing infected brain homogenate before PMCA. Molecular weight markers are indicated on the right. PrP migrates in three different bands due to the presence of non-glycosylated, mono-glycosylated, and di-glycosylated forms, and digestion with Proteinase K results in a shift to lower molecular weights with the non-glycosylated band migrating at ~20 kD. Anle138b shows a strong inhibitory activity in all prion strains tested. As an additional control, the inactive isomer anle234b (see Table 1, Suppl. Fig. 4) was used. Quantitative PMCA results are provided in Table 1 and Suppl. Fig. 8

Fig. 5

Pharmacokinetic analysis of anle138b. (a, b) A single dose of anle138b was applied to non-infected C57/BL6 mice as indicated in the figure legends. In detail, intraperitoneal application was performed using 1 mg compound in 25 μl DMSO, oral application by gavage was done with 1 mg compound in 10 μl DMSO + 40 μl vegetable oil, and oral application of 1 mg as well as 5 mg compound in peanut butter was done by providing the compound dissolved in 10 μl DMSO mixed with 200 μl peanut butter. At different time points after application (0.5, 1, 2, 4, 8, and 24 h) two animals per group and time point were sacrificed and the amount of compound in the serum (a) and brain (b) was measured by LC-MSMS. c From the results obtained following i.p. application, half-life of anle138b in serum and brain was calculated. d Calculation of AUC values. To facilitate comparison with the groups receiving a dose of 1 mg, for the group receiving 5 mg anle138b (green columns) we also provide the calculated AUC values per 1 mg (column: “(5 mg)/5”)

Fig. 6

Effect of anle138b on α-syn oligomer formation. a Iron-induced oligomer formation of α-syn analyzed by SIFT assay [38] is blocked by anle138b in a dose-dependent manner, the apparent EC50 is 2.8 μM. Error bars indicate standard error. b Pore formation in lipid bilayers by α-syn oligomers is significantly reduced in presence of 25 μM anle138b. Similar results are obtained for 50 μM baicalein. Raw data for panels a and b are shown in Suppl. Fig. 13. For statistical analysis, we used student’s t test (a) and Fisher exact test (b). Statistical significance is indicated by *p < 0.05; **p < 0.01; ***p < 0.001

Fig. 7

Effect of anle138b in animal models of Parkinson’s disease: low-dose intragastric rotenone model. a A significant decrease in motor performance (Rotarod) can be observed in rotenone-treated mice (normal feed and placebo feed) compared to the non rotenone-treated control group both after 3 (p < 0.01) and 4 (p < 0.001) months of rotenone treatment. This effect is ameliorated by treatment with anle138b. Both anle138b and hemivagotomized mice present a significantly improved motor performance after 3 (p < 0.01) and 4 months (p < 0.01) when compared to rotenone-treated (normal feed and placebo feed) mice. Interestingly, no significant differences (p > 0.20) could be observed between hemivagotomized or anle138b rotenone-treated mice at any time point, indicating an effect size of anle138b similar to the hemivagotomy which has recently been shown to partially block the spread of α-syn pathology from the enteric nervous system to the CNS [49]. b 1-h stool collection gut motility test. Decreases in stool deposition have been previously shown to correlate with the presence of α-syn pathology in the enteric nervous system as a result of rotenone treatment. Co-treatment with anle138b results in a complete rescue of enteric function. Whereas all rotenone-treated mice (including hemivagotomized mice) present significant alterations (p < 0.05) in stool deposition, anle138b-rotenone-treated mice show no significant difference in stool deposition when compared to controls. Error bars in (a, b) indicate standard error. For statistical analysis 2-way ANOVA with Bonferroni post hoc test was used

Fig. 8

Effect of anle138b in animal models of Parkinson’s disease: A30P-hum-α-syn transgenic mice. Effect of treatment with anle138b in a transgenic mouse Parkinson model based on neuronal overexpression of A30P-hum-α-syn [47]. a In the prodromal phase of the disease (300–450 days of age), placebo-treated transgenic animals exhibit significantly increased fluctuations in rotarod performance. This phenotype can be rescued by treatment with anle138b. b In this time span, nearly normal development of body weight can be observed in the anle138b treated group, whereas the placebo-treated group shows reduced gain of weight. Mouse weights were normalized to the mean weights obtained at measurements between 250 and 315 days of age. c Kaplan–Meier evaluation of treatment with anle138b shows a significant increase in disease-free survival. The median disease-free survival is prolonged by 10 weeks. d Representative sections from the brain stem show a reduction of pathological α-syn deposits in 69-week-old anle138-treated mice in comparison to placebo-treated age- and sex-matched littermates (see also Suppl. Fig. 15). (e, f) Sucrose-gradient centrifugation shows that in young transgenic mice α-syn is found in the same fractions (1–2) as monomeric recombinant α-syn. In 69-week-old placebo-treated mice, α-syn oligomers can be found that show the same size distribution as oligomers derived from recombinant α-syn by treatment with DMSO/Fe3+. Oligomer formation in transgenic mice is inhibited by treatment with anle138b. For the corresponding graph shown in (f), four mice per group were analyzed (Suppl. Fig. 16). Error bars in (a, b, f) indicate standard error. For statistical analysis, we used student’s t test, Statistical significance is indicated by **p < 0.01 (a), log-rank test (Mantel-Cox) and Gehan-Breslow-Wilcoxon test (c), and ANOVA and multiple comparison post-test (f)

Fig. 9

Structure-dependent binding of anle138b. Intrinsic Fluorescence of 250nM anle138b was measured with excitation at 300 nm. a Addition of α-syn monomers does not significantly change the weak fluorescence of anle138b. b Addition of pre-aggregated α-syn results in a dramatic change in the fluorescence properties with a strong increase of fluorescence at ~340 nm by more than a factor of 30. This points to specific binding of anle138b to a structure-dependent binding site