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The drug Rimantadine binds to two different sites in the M2 protein from influenza A, a peripheral site and a pore site that is the primary site of efficacy. It remained enigmatic that pore binding did not occur in certain detergent micelles, and in particular incomplete binding was observed in a mixture of lipids selected to match the viral membrane. Here we show that two effects are responsible, namely changes in the protein upon pore binding that prevented detergent solubilization, and slow binding kinetics in the lipid samples. Using 55–100 kHz magic-angle spinning NMR, we characterize kinetics of drug binding in three different lipid environments: DPhPC, DPhPC with cholesterol and viral mimetic membrane lipid bilayers. Slow pharmacological binding kinetics allowed the characterization of spectral changes associated with non-specific binding to the protein periphery in the kinetically trapped pore-apo state. Resonance assignments were determined from a set of proton-detected 3D spectra. Chemical shift changes associated with functional binding in the pore of M2 were tracked in real time in order to estimate the activation energy. The binding kinetics are affected by pH and the lipid environment and in particular cholesterol. We found that the imidazole-imidazole hydrogen bond at residue histidine 37 is a stable feature of the protein across several lipid compositions. Pore binding breaks the imidazole-imidazole hydrogen bond and limits solubilization in DHPC detergent.

Understanding how small molecules bind to pathological aggregates is of importance for therapeutic and diagnostic development in diseases such as Parkinson’s Disease. Here, the authors reveal a binding site of anle138b to lipid-induced α-synuclein fibrils.

Summary: Background: Synucleinopathies such as Parkinson ́s disease (PD), Dementia with Lewy bodies (DLB) and Multiple System Atrophy (MSA) are characterized by deposition of misfolded and aggregated α-synuclein. Small aggregates (oligomers) of α-synuclein have been shown to be the most relevant neurotoxic species and are targeted by anle138b, an orally bioavailable small molecule compound which shows strong disease-modifying effects in animal models of synucleinopathies. Methods: Anle138b was studied in a single-centre, double-blind, randomised, placebo-controlled single ascending dose (SAD) and multiple ascending dose (MAD) study in healthy subjects. Eligible participants were randomly assigned (1:1 for sentinel subjects and 1:5 for main group) to placebo or anle138b (dose range 50 mg to 300 mg per day), respectively. In addition, the effect of food on the pharmakokinetics of anle138b in healthy subjects was examined in doses of 150 mg per day. Participants were randomized to treatment sequence (fed→fasted) or (fasted→fed). Treatment was administered orally in hard gelatine capsules containing either 10 mg or 30 mg of anle138b or excipient only. The primary endpoints were safety and tolerability, the secondary endpoint was pharmakokinetics. Data from all randomized individuals were evaluated. Clinicaltrials.gov-identifier: NCT04208152. EudraCT-number: 2019-004218-33. Findings: Between December 17th, 2019 and June 27th, 2020 196 healthy volunteers were screened and 68 participants were enrolled. Of these, all completed the study per protocol. There were no major protocol deviations. Adverse events in this healthy volunteer trial were mostly mild and all fully recovered or resolved prior to discharge. From baseline to completion of the trial no medically significant individual changes were observed in any system organ class. Already at multiple doses of 200 mg, exposure levels above the fully effective exposure in the MI2 mouse Parkinson model were observed. Interpretation: The favourable safety and PK profile of anle138b in doses resulting in exposures above the fully effective plasma level in a mouse Parkinson model warrant further clinical trials in patients with synucleinopathies. Funding: This study was funded by MODAG GmbH and by the Michael J. Fox foundation for Parkinson's Research.

Abstract Background Misfolded oligomeric α-synuclein plays a pivotal role in the pathogenesis of α-synucleinopathies including Parkinson’s disease and multiple system atrophy, and its detection parallels activation of microglia and a loss of neurons in the substantia nigra pars compacta. Here we aimed to analyze the therapeutic efficacy of PD03, a new AFFITOPE® immunotherapy approach, either alone or in combination with Anle138b, in a PLP-α-syn mouse model. Methods The PLP-α-syn mice were treated with PD03 immunotherapy, Anle138b, or a combination of two. Five months after study initiation, the mice underwent behavioral testing and were sacrificed for neuropathological analysis. The treatment groups were compared to the vehicle group with regard to motor performance, nigral neuronal loss, microglial activation and α-synuclein pathology. Results The PLP-α-syn mice receiving the PD03 or Anle138b single therapy showed improvement of gait deficits and preservation of nigral dopaminergic neurons associated with the reduced α-synuclein oligomer levels and decreased microglial activation. The combined therapy with Anle138b and PD03 resulted in lower IgG binding in the brain as compared to the single immunotherapy with PD03. Conclusions PD03 and Anle138b can selectively target oligomeric α-synuclein, resulting in attenuation of neurodegeneration in the PLP-α-syn mice. Both approaches are potential therapies that should be developed further for disease modification in α-synucleinopathies.

In this work, we studied single-layer ZrN and CrN coatings, as well as multi-layer ZrN/CrN coatings deposited by the vacuum-arc method on WC-8 wt.% Co substrates. The sliding friction parameters were preset to simulate different operating conditions for coatings, i.e., severe and zero wear regimes. During the tests, the friction coefficient, acoustic emission (AE) and vibration signals were recorded. After testing, the worn surfaces of the samples were studied using confocal laser scanning and scanning electron microscopy, elemental microanalysis and synchrotron XRD. Estimation of vibration accelerations and AE energy turned out to be very effective means of monitoring the wear of coatings, while median AE frequency turned out to be a less informative one. With the increase in the normal load applied on the samples after testing at zero wear regime, the coefficient of friction increased and wear transition to severe wear regime occurred but vibration acceleration decreased. The multi-layer ZrN/CrN coatings demonstrated much higher wear resistance as compared to those of single-layer ZrN and CrN.

Multilayer nitride coatings provide some of the best performance when in their use for the production of metalworking tools. In this work, vacuum-arc plasma-assisted deposited multilayer ZrN/CrN coatings with different numbers of constituent layers were characterized for high-temperature oxidization in air using weighing, confocal and scanning electron microscopy and synchrotron XRD. Oxidizing at 300 °C did not deteriorate the coating surfaces, while higher temperatures caused surface deterioration and oxidation accompanied by cracking, delamination and considerable mass gains. The coating with higher number of thinner layers showed higher oxidation resistance due to more prominent oxygen barrier effect.

Abstract Background Augmenting the brain clearance of toxic oligomers with small molecule modulators constitutes a promising therapeutic concept against tau deposition. However, there has been no test of this concept in animal models of Alzheimer’s disease (AD) with initiation at a late disease stage. Thus, we aimed to investigate the effects of interventional late-stage Anle138b treatment, which previously indicated great potential to inhibit oligomer accumulation by binding of pathological aggregates, on the metabolic decline in transgenic mice with established tauopathy in a longitudinal 18F-fluorodeoxyglucose positron emission tomography (FDG-PET) study. Methods Twelve transgenic mice expressing all six human tau isoforms (hTau) and ten controls were imaged by FDG-PET at baseline (14.5 months), followed by randomization into Anle138b treatment and vehicle groups for 3 months. FDG-PET was repeated after treatment for 3 months, and brains were analyzed by tau immunohistochemistry. Longitudinal changes of glucose metabolism were compared between study groups, and the end point tau load was correlated with individual FDG-PET findings. Results Tau pathology was significantly ameliorated by late-stage Anle138b treatment when compared to vehicle (frontal cortex − 53%, p

Abstract Alzheimer's 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 Alzheimer's 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.

The outer mitochondrial membrane translocator protein (TSPO) mediates several mitochondrial functions and binds cholesterol with a high affinity. Here the authors use solid-state NMR to show that cholesterol binding to TSPO results in allosteric changes that modulate TSPO oligomerization.

A DNP set-up is described where a liquid sample is hyperpolarized by the electron-nucleus Overhauser effect in a field of 0.34 T and transferred to a field of 14.09 T for NMR detection. In contrast to a previous set-up, using two dedicated magnets for polarization and detection, a dedicated ferroshim system was inserted into the bore of a 14.09 T shielded cryomagnet to provide a homogeneous low-field region in the stray field above the magnetic center. After polarization in the low-field the sample is transferred to the high-field magnetic center within 40 ms by a pneumatic shuttle system. In our set-up a standard high-resolution inverse 1H/13C selective probe was used for NMR detection and a homebuilt EPR cavity, operating in the TM110mode was used for polarisation. First experimental data are presented. We observed a maximum proton Overhauser enhancement of up to HF= −3.7 in the high-field position for a 5 mM 4-Oxo-TEMPO-D,15N (TEMPONE)/H2O sample. While this reproduces the DNP enhancement observed also in the old set-up, with the new set-up we observe enhancement on larger molecules that were impossible to enhance in the old set-up. Therefore, we can demonstrate for the first time Overhauser enhanced high resolution proton spectra of glucose and 2,2-dimethyl-2-silapentane-5-sulfonic acid sodium salt (DSS) in D2O, where the high resolution spectrum was acquired in the high-field position after polarizing the sample in the low-field. [ABSTRACT FROM AUTHOR]