Molecular docking analysis of α-Synuclein aggregation with Anle138b

α-Synuclein aggregation into toxic oligomeric species is central to Parkinson's disease pathogenesis. Anle138b is a recently identified inhibitor of α-synuclein oligomerization showing promise in preclinical studies. This study employed computational approaches to elucidate Anle138b's mechanism of oligomer-specific action. The inhibitory potential of Anle138b against α-synuclein oligomers was evaluated by performing molecular docking studies using AutoDock Tools, followed by their binding pocket analysis. Further, protein-protein docking studies were performed using Hex8.0 to validate the aggregation inhibitory potential of Anle138b. Molecular docking revealed increasing binding affinity of Anle138b against higher order α-synuclein oligomers (dimer to decamer). Anle138b occupied oligomeric cavity and interacted with residues Thr54, Gly73, Val74 and Thr75 across several oligomers. Protein-protein docking showed that Anle138b interferes with α-synuclein decamer formation. These results highlight the oligomer-directed inhibitory mechanism of Anle138b, without hindering the monomeric forms and provide molecular insights to advance its therapeutic development for Parkinson's and related synucleinopathies.


Background:
α-Synuclein oligomerization is considered a crucial event in the pathogenesis of Parkinson's disease (PD).α-Synuclein is a small, presynaptic protein whose physiological function is not entirely understood, but it is believed to play a role in neurotransmitter release and synaptic function [1].However, under pathological conditions, α-synuclein can misfold and aggregate, forming oligomers, protofibrils, and eventually insoluble fibrils that accumulate in Lewy bodies, the pathological hallmark of PD and other synucleinopathies [2].Several studies have highlighted the importance of α-synuclein oligomers, rather than the larger fibrillar aggregates, as the primary toxic species responsible for neurodegeneration in PD [3].Oligomeric α-synuclein has been shown to disrupt cellular functions, induce oxidative stress, impair protein degradation pathways, and promote neuroinflammation [4].Additionally, α-synuclein oligomers can interact with cellular membranes, leading to increased permeability and disruption of cellular homeostasis [5].The process of α-synuclein oligomerization is thought to be a critical step in the disease cascade, as it precedes the formation of larger, insoluble aggregates [6].Factors such as oxidative stress, posttranslational modifications, and genetic mutations can influence the propensity of α-synuclein to oligomerize and aggregate [7].Moreover, the oligomerization process can be self-propagating, as oligomers can seed the conversion of monomeric α-synuclein into additional oligomers, facilitating the spread of pathology throughout the brain [8].Targeting α-synuclein oligomerization has emerged as a promising therapeutic strategy for PD and related synucleinopathies [9].Several approaches have been explored, including the development of small molecules, antibodies, and peptide inhibitors that can interfere with the oligomerization process or promote the clearance of existing oligomers [10].Additionally, modulating cellular pathways involved in protein quality control, such as the ubiquitinproteasome system and autophagy, may help reduce the accumulation of toxic α-synuclein species [11].In conclusion, the oligomerization of α-synuclein is widely recognized as a pivotal event in the pathogenesis of Parkinson's disease [12].Targeting this process through various therapeutic approaches holds great promise for developing effective treatments that can slow or halt the progression of PD.To inhibit the α-synuclein aggregation various therapeutics are in preclinical trials, including molecular chaperons (Hsp70 and Hsp104), molecular tweezer (CRL01), prolyl oligopeptidase inhibitor (KYP-2047) and oligomer modulator (Anle138b), showing promising neuroprotection and decreased α-synuclein pathology [13].Anle138b was first identified using high-throughput screening for small-molecule inhibitors of α-synuclein oligomerisation.In vitro studies demonstrated Anle138b's ability to inhibit the oligomerization of pathogenic proteins like prion protein (PrP) and α-synuclein while allowing the formation of less harmful amyloid fibrils.In vivo experiments using transgenic mouse models showed that Anle138b treatment reduced the accumulation of toxic oligomers, improved pathological phenotypes like motor impairments and neuronal loss, and suggested a favorable safety profile and bioavailability [14].Therefore, it is of interest to investigate the possible mechanisms of action and potential interactions of Anle138b with various oligomeric conformations of α-synuclein.

Methodology: Molecular docking studies:
Molecular docking experiments were undertaken in a stepwise, systematic manner to evaluate Anle138b's inhibitory activity against the in-house generated α-synuclein oligomeric conformations of dimer (I-I), trimer (II-I), tetramer (II-II), pentamer (IV-I), hexamer (V-I), heptamer (VI-I), octamer (IV-IV), nonamer (V-IV) and decamer (IV-VI) using AutoDock 4.2 [15].All the forms of target receptor protein α-synuclein (monomer to decamer) and the ligand (Anle138b), were prepared for molecular docking studies using AutoDock Tools [16].Polar hydrogens, Gasteiger and Kollman charges were added at each preparation (monomer to decamer) of the receptor protein αsynuclein.The various docking parameters used for the grid parameter file generation for the target receptor proteins (monomeric to decameric α-synuclein) are listed in Table 1.
Molecular docking was performed using 100 runs of the Lamarckian genetic algorithm at a mutation rate of 0.02 and a crossover rate of 0.8, with a population size of 300.The output files (.dlg) of molecular docking studies of the target receptors (monomeric to decameric α-synuclein) with Anle138b were analysed for various binding poses and the binding poses with maximum cluster size and minimum binding energy were selected for further studies of their binding pocket interactions.

Conclusion:
We evaluated the inhibitory mechanism of the compound Anle138b against α-synuclein aggregation at various oligomeric stages, from dimer to decamer, using computational approaches.Molecular docking analyses revealed that Anle138b displayed increasing binding affinity and inhibition potential against higher order α-synuclein oligomers, with the most favourable binding observed against nonameric and decameric conformations.Anle138b was found to occupy the oligomeric cavity and interact with key hydrophobic and hydrogen bonding residues like Thr54, Gly73, Val74 and Thr75 across several αsynuclein oligomers.Protein-protein docking experiments also demonstrated that Anle138b could interfere with and decrease the affinity for α-synuclein decamer formation, which is a critical event preceding fibrillization.Taken together, these computational findings provide valuable insights into the mechanism of Anle138b as an oligomerization inhibitor -by directly binding to oligomeric intermediates with high affinity, blocking their aggregation into toxic, fibrillar end-products.The oligomer-specific action and increased inhibitory potency of Anle138b against later stage oligomers provide complementary in-silico support to its promising therapeutic potential for Parkinson's and other synucleinopathy diseases characterized by pathogenic α-synuclein aggregation.

Table 1 :
Grid parameter files dimensions for the target protein receptors (Monomeric to decametric α-synuclein) α