Synucleinopathies are neurodegenerative diseases characterized by the presence of brain inclusions containing the pathologically aggregated protein α-synuclein (αSYN). The development of a positron emission tomography tracer to detect aggregates of misfolded αSYN would revolutionize disease monitoring and the evaluation of therapeutic efficacy. Here we present the development and preclinical in vitro and in vivo validation of [11C]MODAG-005. In vitro binding experiments demonstrate subnanomolar binding affinity to recombinant αSYN fibrils as well as to αSYN inclusions in human brain tissue. Specific binding in multiple system atrophy brain tissue was detected using autoradiography and microautoradiography, and was validated through immunostaining. In vivo, [11C]MODAG-005 shows good brain penetration, rapid clearance from brain tissue and low metabolite formation in rodents and non-human primates. In addition, a pronounced binding and a good signal-to-noise ratio were achieved in an αSYN fibril-injected rat model and in an αSYN(A30P) transgenic mouse model in correlation to the pathological load. To validate its value for therapy development, we show target engagement of the drug candidate anle138b in the brain tissues from αSYN(A30P) mouse and multiple system atrophy as well as in vivo in αSYN fibril-injected rats. Finally, our translational approach in a first-in-human patient with clinically established MSA, revealed a marked tracer binding in regions affected by αSYN pathology, particularly in the striatum, where the pattern corresponded with the neurodegeneration shown by dopamine transporter single-photon emission computed tomography.

The development of positron emission tomography (PET) tracers capable of detecting alpha-synuclein (alpha-syn) aggregates in vivo would represent a breakthrough for advancing the understanding and enabling the early diagnosis of Parkinson's disease and related disorders. It also holds the potential to assess the efficacy of therapeutic interventions. However, this remains challenging due to different structures of alpha-syn aggregates, the need for selectivity over other structurally similar amyloid proteins, like amyloid-beta (A beta), which frequently coexist with alpha-syn pathology, and the low abundance of the target in the brain that requires the development of a high-affinity ligand. To develop a successful PET tracer for the central nervous system (CNS), stringent criteria in terms of polarity and molecular size must also be considered, as the tracer must penetrate the blood-brain barrier and have low nonspecific binding to brain tissue. Here, we report a series of arylpyrazolethiazole (APT) derivatives, rationally designed from a structure-activity relationship study centered on existing ligands for alpha-syn fibrils, with a particular focus on the selectivity toward alpha-syn fibrils and control of physicochemical properties suitable for a CNS PET tracer. In vitro competition binding assays performed against [H-3]MODAG-001 using recombinant alpha-syn and A beta(1-42) fibrils revealed APT-13 with an inhibition constant of 27.8 +/- 9.7 nM and a selectivity of more than 3.3 fold over A beta. Radiolabeled [C-11]APT-13 demonstrated excellent brain penetration in healthy mice with a peak standardized uptake value of 1.94 +/- 0.29 and fast washout from the brain (t(1/2) = 9 +/- 1 min). This study highlights the potential of APT-13 as a lead compound for developing PET tracers to detect alpha-syn aggregates in vivo.

Ziel/Aim PET imaging of alpha-synuclein (αSYN) aggregates would be a game changer for facilitating the diagnosis of synucleinopathies and the development of novel therapies. We previously demonstrated that the structural modification of anle138b, an investigational therapeutic for synucleinopathies [1], led to radiotracer candidates with high-affinity binding to αSYN [2] [3]. Here, we report the preclinical evaluation of MODAG-005 as a promising αSYN PET tracer.

OBJECTIVE:Liver mitochondria adapt to high-calorie intake. We investigated how exercise alters the early compensatory response of mitochondria, thus preventing fatty liver disease as a long-term consequence of overnutrition.METHODS:We compared the effects of a steatogenic high-energy diet (HED) for six weeks on mitochondrial metabolism of sedentary and treadmill-trained C57BL/6N mice. We applied multi-OMICs analyses to study the alterations in the proteome, transcriptome, and lipids in isolated mitochondria of liver and skeletal muscle as well as in whole tissue and examined the functional consequences by high-resolution respirometry.RESULTS:HED increased the respiratory capacity of isolated liver mitochondria, both in sedentary and in trained mice. However, proteomics analysis of the mitochondria and transcriptomics indicated that training modified the adaptation of the hepatic metabolism to HED on the level of respiratory complex I, glucose oxidation, pyruvate and acetyl-CoA metabolism, and lipogenesis. Training also counteracted the HED-induced glucose intolerance, the increase in fasting insulin, and in liver fat by lowering diacylglycerol species and c-Jun N-terminal kinase (JNK) phosphorylation in the livers of trained HED-fed mice, two mechanisms that can reverse hepatic insulin resistance. In skeletal muscle, the combination of HED and training improved the oxidative capacity to a greater extent than training alone by increasing respiration of isolated mitochondria and total mitochondrial protein content.CONCLUSION:We provide a comprehensive insight into the early adaptations of mitochondria in the liver and skeletal muscle to HED and endurance training. Our results suggest that exercise disconnects the HED-induced increase in mitochondrial substrate oxidation from pyruvate and acetyl-CoA-driven lipid synthesis. This could contribute to the prevention of deleterious long-term effects of high fat and sugar intake on hepatic mitochondrial function and insulin sensitivity.

Aspartic proteases are important biomarkers of human disease and interesting targets for modulation of immune response via MHC class II antigen processing inhibition. The lack of inhibitors with sufficient selectivity hampers precise analysis of the role of cathepsin E and napsin A in samples containing the ubiquitous and highly abundant homolog cathepsin D. Grassystatins from marine cyanobacteria show promising selectivity for cathepsin E but contain several ester bonds that make their synthesis cumbersome and thus limit availability of the inhibitors. Herewith, we present grassystatin-derived cathepsin E inhibitors with greatly facilitated synthesis but retained selectivity profile. We demonstrate their affinity and selectivity with both enzyme kinetic assays and streptavidin-based pull-down from cells and mouse organs. Our findings suggest that grassystatin-like inhibitors are useful tools for targeted inhibition of cathepsin E and thus provide a novel approach for cancer and immunology research.

Deposition of misfolded alpha-synuclein (αSYN) aggregates in the human brain is one of the major hallmarks of synucleinopathies. However, a target-specific tracer to detect pathological aggregates of αSYN remains lacking. Here, we report the development of a positron emission tomography (PET) tracer based on anle138b, a compound shown to have therapeutic activity in animal models of neurodegenerative diseases. Specificity and selectivity of [3H]MODAG-001 were tested in in vitro binding assays using recombinant fibrils. After carbon-11 radiolabeling, the pharmacokinetic and metabolic profile was determined in mice. Specific binding was quantified in rats, inoculated with αSYN fibrils and using in vitro autoradiography in human brain sections of Lewy body dementia (LBD) cases provided by the Neurobiobank Munich (NBM). [3H]MODAG-001 revealed a very high affinity towards pure αSYN fibrils (Kd = 0.6 ± 0.1 nM) and only a moderate affinity to hTau46 fibrils (Kd = 19 ± 6.4 nM) as well as amyloid-β1–42 fibrils (Kd = 20 ± 10 nM). [11C]MODAG-001 showed an excellent ability to penetrate the mouse brain. Metabolic degradation was present, but the stability of the parent compound improved after selective deuteration of the precursor. (d3)-[11C]MODAG-001 binding was confirmed in fibril-inoculated rat striata using in vivo PET imaging. In vitro autoradiography showed no detectable binding to aggregated αSYN in human brain sections of LBD cases, most likely, because of the low abundance of aggregated αSYN against background protein. MODAG-001 provides a promising lead structure for future compound development as it combines a high affinity and good selectivity in fibril-binding assays with suitable pharmacokinetics and biodistribution properties.