Sergey Ryazanov

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 (PrPSc) 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.

The oligomer modulator anle138b inhibits disease progression in a Parkinson mouse model even with treatment started after disease onset

]. parkinson’s disease (pD) is characterized by the deposition of aggregated alpha-synuclein (asyn). recent evidence suggests that oligomers formed in the aggregation process constitute the main toxic species causing neurodegeneration. recently, we reported that the oligomer modulator “anle138b” [3-(1,3-benzodioxol-5-yl)-5-(3-bromophenyl)-1H-pyrazole] is capable of prolonging the survival of prion-infected mice and of various animal models of pD [5blocked formation and accumulation of asyn oligomers in the brain, reduced disease-associated motor deficits, and led to prolonged disease-free survival [5 ]. These findings support the following

Reducing tau aggregates with anle138b delays disease progression in a mouse model of tauopathies.

Pathological tau aggregation leads to filamentous tau inclusions and characterizes neurodegenerative tauopathies such as Alzheimer’s disease and frontotemporal dementia and parkinsonism linked to chromosome 17. Tau aggregation coincides with clinical symptoms and is thought to mediate neurodegeneration. Transgenic mice overexpressing mutant human P301S tau exhibit many neuropathological features of human tauopathies including behavioral deficits and increased mortality. Here, we show that the di-phenyl-pyrazole anle138b binds to aggregated tau and inhibits tau aggregation in vitro and in vivo. Furthermore, anle138b treatment effectively ameliorates disease symptoms, increases survival time and improves cognition of tau transgenic PS19 mice. In addition, we found decreased synapse and neuron loss accompanied by a decreased gliosis in the hippocampus. Our results suggest that reducing tau aggregates with anle138b may represent an effective and promising approach for the treatment of human tauopathies.

The bacterial SRP receptor, FtsY, is activated on binding to the translocon.

Proteins are inserted into the bacterial plasma membrane cotranslationally after translating ribosomes are targeted to the translocon in the membrane via the signal recognition particle (SRP) pathway. The targeting pathway involves an interaction between SRP and the SRP receptor, FtsY. Here we focus on the role of FtsY and its interaction with the translocon in controlling targeting. We show that in unbound FtsY the NG and A domains interact with one another. The interaction involves the membrane‐targeting region at the junction between A and N domain. The closed form of FtsY is impaired in binding to SRP. Upon binding to the phospholipid‐embedded translocon the domains of FtsY move apart. This enhances the docking of the FtsY NG domain to the homologous NG domain of the SRP protein Ffh. Thus, FtsY binding to the translocon has a central role in orchestrating the formation of a quaternary transfer complex in which the nascent peptide is transferred to the translocon. We propose that FtsY activation at the translocon ensures that ribosome–SRP complexes are directed to available translocons. This way sequestering SRP in futile complexes with unbound FtsY can be avoided and efficient targeting to the translocon achieved.

Anle138b and related compounds are aggregation specific fluorescence markers and reveal high affinity binding to α-synuclein aggregates.

BACKGROUND Special diphenyl-pyrazole compounds and in particular anle138b were found to reduce the progression of prion and Parkinsons disease in animal models. The therapeutic impact of these compounds was attributed to the modulation of α-synuclein and prion-protein aggregation related to these diseases. METHODS Photophysical and photochemical properties of the diphenyl-pyrazole compounds anle138b, anle186b and sery313b and their interaction with monomeric and aggregated α-synuclein were studied by fluorescence techniques. RESULTS The fluorescence emission of diphenyl-pyrazole is strongly increased upon incubation with α-synuclein fibrils, while no change in fluorescence emission is found when brought in contact with monomeric α-synuclein. This points to a distinct interaction between diphenyl-pyrazole and the fibrillar structure with a high binding affinity (Kd=190±120nM) for anle138b. Several α-synuclein proteins form a hydrophobic binding pocket for the diphenyl-pyrazole compound. A UV-induced dehalogenation reaction was observed for anle138b which is modulated by the hydrophobic environment of the fibrils. CONCLUSION Fluorescence of the investigated diphenyl-pyrazole compounds strongly increases upon binding to fibrillar α-synuclein structures. Binding at high affinity occurs to hydrophobic pockets in the fibrils. GENERAL SIGNIFICANCE The observed particular fluorescence properties of the diphenyl-pyrazole molecules open new possibilities for the investigation of the mode of action of these compounds in neurodegenerative diseases. The high binding affinity to aggregates and the strong increase in fluorescence upon binding make the compounds promising fluorescence markers for the analysis of aggregation-dependent epitopes.

The diphenylpyrazole compound anle138b blocks Aβ channels and rescues disease phenotypes in a mouse model for amyloid pathology

Alzheimers disease is a devastating neurodegenerative disease eventually leading to dementia. An effective treatment does not yet exist. Here we show that oral application of the compound anle138b restores hippocampal synaptic and transcriptional plasticity as well as spatial memory in a mouse model for Alzheimers disease, when given orally before or after the onset of pathology. At the mechanistic level, we provide evidence that anle138b blocks the activity of conducting Aβ pores without changing the membrane embedded Aβ‐oligomer structure. In conclusion, our data suggest that anle138b is a novel and promising compound to treat AD‐related pathology that should be investigated further.

Anle138b Partly Ameliorates Motor Deficits Despite Failure of Neuroprotection in a Model of Advanced Multiple System Atrophy

The neurodegenerative disorder multiple system atrophy (MSA) is characterized by autonomic failure, cerebellar ataxia and parkinsonism in any combination associated with predominantly oligodendroglial α-synuclein (α-syn) aggregates (glial cytoplasmic inclusions = GCIs). To date, there is no effective disease modifying therapy. Previous experiments have shown that the aggregation inhibitor anle138b reduces neurodegeneration, as well as behavioral deficits in both transgenic and toxin mouse models of Parkinsons disease (PD). Here we analyzed whether anle138b improves motor skills and reduces neuronal loss, as well as oligodendroglial α-syn aggregation in the PLP-α-syn transgenic mouse challenged with the mitochondrial toxin 3-nitropropionic acid (3-NP) to model full-blown MSA. Following 1 month of treatment with anle138b, MSA mice showed signs of motor improvement affecting stride length, but not pole, grip strength, and beam test performance. Loss of dopaminergic nigral neurons and Purkinje cells was not attenuated and GCI density remained unchanged. These data suggest that the pathology in transgenic PLP-α-syn mice receiving 3-NP might be too advanced to detect significant effects of anle138b treatment on neuronal loss and intracytoplasmic α-syn inclusion bodies. However, the partial motor amelioration may indicate potential efficacy of anle138b treatment that may be mediated by its actions on α-syn oligomers or may reflect improvement of neuronal dysfunction in neural at risk populations. Further studies are required to address the efficacy of anle138b in transgenic α-syn models of early-stage MSA and in the absence of additional toxin application.

Quantitative Real-Time Quaking-Induced Conversion Allows Monitoring of Disease-Modifying Therapy in the Urine of Prion-Infected Mice

Abstract Prion diseases are fatal neurodegenerative diseases characterized by accumulation of the pathogenic prion protein PrPSc in the brain. We established quantitative real-time quaking-induced conversion for the measurement of minute amounts of PrPSc in body fluids such as urine. Using this approach, we monitored the efficacy of antiprion therapy by quantifying the seeding activity of PrPSc from the brain and urine of mice after prion infection. We found that the aggregation inhibitor anle138b decreased the levels of PrPSc in the brain and urine. Importantly, variations of PrPSc levels in the urine closely corresponded to those in the brain. Our findings indicate that quantification of urinary PrPSc enables measurement of prion disease progression in body fluids and can substitute for immunodetection in brain tissue. We expect PrPSc quantification biologic fluids (such as urine and cerebrospinal fluid) with quantitative real-time quaking-induced conversion to emerge as a valuable noninvasive diagnostic tool for monitoring disease progression and the efficacy of therapeutic approaches in animal studies and human clinical trials of prion diseases. Moreover, highly sensitive methods for quantifying pathologic aggregate seeds might provide novel molecular biomarkers for other neurodegenerative diseases that may involve prion-like mechanisms (protein aggregation and spreading), such as Alzheimer disease and Parkinson disease.

Treatment with diphenyl–pyrazole compound anle138b/c reveals that α-synuclein protects melanoma cells from autophagic cell death

Significance People with Parkinson’s disease, the second most common neurodegenerative disorder, have a lower risk and decreased incidence of cancer with the one exception being melanoma. The fact that, compared with other malignancies, melanoma occurs more frequently in patients with Parkinson’s disease and vice versa and that there is an association between a history of melanoma and an increased prevalence of prodromal markers of Parkinson’s disease prompted us to explore the possibility of an inverse biological link between these two diseases. The findings of our study suggest that α-synuclein, one of the key regulators in Parkinson’s disease, although toxic to dopaminergic neurons, is protective for advanced melanoma cells. Recent epidemiological and clinical studies have reported a significantly increased risk for melanoma in people with Parkinson’s disease. Because no evidence could be obtained that genetic factors are the reason for the association between these two diseases, we hypothesized that of the three major Parkinson’s disease-related proteins—α-synuclein, LRRK2, and Parkin—α-synuclein might be a major link. Our data, presented here, demonstrate that α-synuclein promotes the survival of primary and metastatic melanoma cells, which is the exact opposite of the effect that α-synuclein has on dopaminergic neurons, where its accumulation causes neuronal dysfunction and death. Because this detrimental effect of α-synuclein on neurons can be rescued by the small molecule anle138b, we explored its effect on melanoma cells. We found that treatment with anle138b leads to massive melanoma cell death due to a major dysregulation of autophagy, suggesting that α-synuclein is highly beneficial to advanced melanoma because it ensures that autophagy is maintained at a homeostatic level that promotes and ensures the cell’s survival.

Photophysics of diphenyl-pyrazole compounds in solutions and α-synuclein aggregates

BACKGROUND Recently diphenyl-pyrazole (DPP) compounds and especially anle138b were found to reduce the aggregation of α-synuclein or Tau protein in vitro as well as in a mouse model of neurodegenerative diseases [1,2]. Direct interaction of the DPPs with the fibrillar structure was identified by fluorescence spectroscopy. Thereby a strong dependence of the fluorescence on the surroundings could be identified [3]. METHODS Stationary and time-resolved emission experiments were performed on DPP compounds substituted by different halogens. RESULTS The compounds reveal a pronounced dependence of the fluorescence on the surrounding solvent. In non-polar solvents they show strong emission in the blue part of the spectrum while in polar and proton donating solvents, such as water or acetic acid a dual fluorescence can be observed where a red-shifted emission points to a charge transfer in the excited state with large dipole moment. Non-radiative processes including photochemical reactions are observed for DPP substituted with heavy halogens. Upon binding of anle138b and its derivatives to protein fibrils in aqueous buffer, strong enhancement of the fluorescence at short wavelengths is found. CONCLUSION The investigations of the DPPs in different surroundings lead to a detailed model of the fluorescence characteristics. We propose a model for the binding in fibrils of different proteins, where the DPP is located in a hydrophobic groove independent of the specific sequence of the amino acids. GENERAL SIGNIFICANCE These investigations characterize the binding site of the DPP anle138b in protein aggregates and contribute to the understanding of the therapeutic mode of action of this compound.