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Small molecules that bind to oligomeric protein species such as membrane proteins and fibrils are of clinical interest for development of therapeutics and diagnostics. Definition of the binding site at atomic resolution via NMR is often challenging due to low binding stoichiometry of the small molecule. For fibrils and aggregation intermediates grown in the presence of lipids, we report atomic-resolution contacts to the small molecule at sub nm distance via solid-state NMR using dynamic nuclear polarization (DNP) and orthogonally labelled samples of the protein and the small molecule. We apply this approach to α-synuclein (αS) aggregates in complex with the small molecule anle138b, which is a clinical drug candidate for disease modifying therapy. The small central pyrazole moiety of anle138b is detected in close proximity to the protein backbone and differences in the contacts between fibrils and early intermediates are observed. For intermediate species, the 100 K condition for DNP helps to preserve the aggregation state, while for both fibrils and oligomers, the DNP enhancement is essential to obtain sufficient sensitivity.

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.

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.

Aggregation of islet amyloid polypeptide (IAPP) in the human pancreas is linked to the pathogenesis of type-II diabetes. Interactions of IAPP with membranes may play an important role in the disease, because membranes may catalyze IAPP aggregation and because amyloid formation of IAPP results in membrane permeabilization, and hence cytotoxicity [1]. In order to control this cytotoxicity, there is an intense focus on the development of amyloid inhibitors. Here, we studied the interaction of a novel amyloid inhibitor “anle145c” [2,3,4] on IAPP aggregation in the absence and presence of model membranes using a range of biophysical techniques. Our results show that in both cases the compound acts as an efficient inhibitor, but that it has a distinctly different mode of action in the presence of membranes. Importantly, similar results were obtained with the structurally related natural inhibitor EGCG but with less efficiency. Our complementary experimental approaches reveal that the inhibitors target a different species in the absence and presence of membranes. We propose a model to explain our findings and we discuss its implications for in-vivo studies on these inhibitors.[1] Engel M et al., PNAS. (2008) - 105:6033.[2] Wagner J et al., Acta Neuropathologica (2013) - 125:795.[3] Levin J et al., Acta Neuropathologica (2014) - 127:779.[4] Wagner J et al., Acta Neuropathologica in press (2015) “Reducing tau aggregates with anle138b delays disease progression in a mouse model of tauopathies”.

Amyloid formation in the pancreas by islet amyloid polypeptide (IAPP) is closely associated with type-2 diabetes. Compelling evidence indicates that membranes play a crucial role in contributing to IAPP amyloid formation and that IAPP amyloid formation leads to cell membrane disruption [1]. Since both IAPP amyloid formation and membrane damage are considered perilous to the pancreatic beta-cells, their inhibition may be an effective strategy for the prevention and/ or treatment of the disease. Here, we studied the interaction between a novel amyloid inhibitor “anle145c” [2] and IAPP in the absence and presence of model membranes. Our results suggest that addition of anle145c to IAPP inhibits IAPP fibril growth even at sub-stoichiometric concentrations, both in the absence and presence of membranes. Anle145c also reduces membrane damage induced by IAPP but does not induce membrane leakage by itself. Most notably, anle145c shows a strong membrane interaction, resulting in a distinctly different mode of inhibition than in the absence of membranes. We present a model in which anle145c interacts with oligomeric IAPP species in solution, but with monomeric or early oligomeric IAPP species in the presence of membranes.[1] Engel M et al., PNAS. (2008) - 105 :6033[2] Wagner J et al., Acta Neuropathologica (2013) - 125: 795