Parkinson's disease (PD) is characterized by the loss of dopaminergic neurons in the substantia nigra and the presence of abnormal inclusions-Lewy bodies (LB) and Lewy neurites (LN) in the brain [1,2]. The primary component of these inclusions is fibrils formed by the protein α-Synuclein (α-Syn), consisting of 140 amino acids, typically adopting an intrinsically disordered conformation [[3], [4], [5]]. Under pathological conditions, α-Syn undergoes a significant conformational transition to a β-sheet-rich structure, forming pathological amyloid fibrils. The resulting toxic α-Syn aggregates propagate between neurons in a prion-like manner, promoting the misfolding and aggregation of host α-Syn [[6], [7], [8]]. These aggregates, including fibrils, contribute significantly to LB formation by recruiting other proteins, membranous structures, and organelles [9,10], thereby accelerating dysfunction and death of dopaminergic neurons. Despite the complexity of PD pathogenesis, it is evident that the α-Syn aggregation plays a pivotal role in PD development [11]. Focusing on maintaining α-Syn proteostasis through anti-α-Syn aggregation and disaggregation of formed aggregates is considered an efficient approach to PD prevention and treatment.

The modulation of α-Syn aggregation is influenced by various factors, both endogenous and exogenous, such as environmental and genetic factors, post-translational modifications, and protein-protein interactions [4]. The pathological α-Syn aggregation progresses through three phases from lag and elongation to the stationary phase [12]. The lag phase, a critical step, involves the gradual accumulation of misfolded α-Syn monomers, forming initial β-sheet-rich oligomeric nuclei that convert into more stable, compact, and toxic nuclei [13]. A certain number of such species trigger the fast elongation of aggregates to form fibrils via nucleation-dependent polymerization and the secondary nucleation process [14,15], eventually leading to the formation of complex fibers in the stationary phase, contributing to LB formation.

Traditionally, α-Syn is divided into three main domains: 1) the N-terminus (1–60) with amphipathic and α-helical propensity involved in membrane binding. The fragments 36–42 and 45–57 are found to be more aggregation-prone [16]; 2) the hydrophobic central region (61–95), known as the non-amyloid-beta component (NAC), abundant in β-sheet structure and responsible for aggregation. In the NAC domain, the amino acid fragments 68–78 [17] and 71–82 [18] contribute more to aggregation. Recent research has identified the aggregation-prone regions also include the cross-domain of N-terminus and NAC (fragments 15–65 [19] and 58–79 [20]); 3) the C-terminus (96–140) with highly negatively charged and unstructured characteristics, which play an anti-aggregation effect. Recent reports show that the C-terminally truncated α-Syn form (α-Syn(1–121)) aggregates more rapidly than full-length α-Syn [21], and its fibril structure has been determined by Cryo-electron microscopy. Residues 38 to 95, including the fibril core and the non-amyloid component region, form the backbone of the mono-protofilament with eight β-stands. Two protofilaments form a polar fibril composed of staggered β-strands, with residues 50–57 contributing to fibril stability through intermolecular interaction including salt-bridges between amino acids K45, E57, or E46 within the two protofilaments [22,23].

Therefore, identifying inhibitors that act reversibly on the aggregation-prone fragments or their adjacent sites can interfere with and prevent protein aggregation, thereby maintaining α-Syn proteostasis and function. In turn, this can reduce the stability of aggregates and decompose them into monomer protein molecules to achieve the purpose of treating PD.

In recent years, researchers have reported various natural polyphenols and their derivatives with excellent neuroprotective properties for PD through multiple interrelated mechanisms [[24], [25], [26], [27], [28], [29], [30]]. Our group has also developed various polyphenolic hybrids that exhibit excellent in vitro anti-aggregation of α-Syn, disaggregation of preformed α-Syn fibrils, high performance in reducing inclusions in neuron cells, and good in vivo neuroprotection, repairing damaged neurons, and improving PD-like symptoms [[31], [32], [33], [34], [35], [36], [37]]. However, the discovery of novel inhibitors with improved performance remains a challenging endeavor.

Here we report a series of N-aryl-3-aryl-pyrazole-5-carboxamide derivatives, evaluating their anti-aggregation of α-Syn, disaggregation against α-Syn fibers, and the structure-activity relationship (SAR) via Thioflavin T (ThT) fluorescence assay. The initial mechanisms of anti-aggregation and disaggregation are revealed through inhibitory kinetics, molecular docking, disaggregation kinetics, morphology, and circular dichroism spectroscopy. The inhibitory activities of some candidate compounds on inclusions in neural cells were also evaluated, yielding positive results and laying a solid foundation for the prevention and treatment of PD targeting α-Synucleinopathy and LB formation.