alpha-synuclein and fasudil

Intrinsically disordered proteins (IDPs) are implicated in many human diseases. They have generally not been amenable to conventional structure-based drug design, however, because their intrinsic conformational variability has precluded an atomic-level understanding of their binding to small molecules. Here we present long-time-scale, atomic-level molecular dynamics (MD) simulations of monomeric α-synuclein (an IDP whose aggregation is associated with Parkinson's disease) binding the small-molecule drug fasudil in which the observed protein-ligand interactions were found to be in good agreement with previously reported NMR chemical shift data. In our simulations, fasudil, when bound, favored certain charge-charge and π-stacking interactions near the C terminus of α-synuclein but tended not to form these interactions simultaneously, rather breaking one of these interactions and forming another nearby (a mechanism we term

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; alpha-Synuclein; Amino Acid Sequence; Hydrogen Bonding; Intrinsically Disordered Proteins; Ligands; Molecular Conformation; Molecular Dynamics Simulation; Protein Binding; Small Molecule Libraries

Microglia activation and inflammatory factors in brain microenvironment are associated with degeneration of neurons in the substantia nigra (SN) of Parkinson's disease (PD) patients and various PD models. There is increasing evidence that the Rho/ROCK (Rho kinase) signalling pathway may play a critical role in the inflammatory response, and ROCK inhibitor has been reported to have neuroprotective effects. In this study, we examined the neuroprotective potential and possible mechanism of ROCK inhibitor Fasudil in an intranasal lipopolysaccharide (LPS)-induced PD model. ROCK was activated with LPS stimulation and inhibited by Fasudil treatment in this PD model. Behavioural tests demonstrated a clear improvement in motor performance after Fasudil treatment. Furthermore, Fasudil resulted in a significant attenuation of dopamine cell loss, α-synuclein accumulation and inflammatory response with the reversion of inflammatory M1 to anti-inflammatory M2 microglia, decreased NF-кB activation, and IL-12 and TNF-α generation in the SN and olfactory bulb in this model. This study establishes a role for Fasudil in protecting against LPS-mediated dopamine degeneration and provides a therapeutic strategy for the treatment of PD.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Administration, Intranasal; alpha-Synuclein; Animals; Dopaminergic Neurons; Encephalitis; Female; Inflammation Mediators; Lipopolysaccharides; Mice, Inbred C57BL; Microglia; Motor Activity; Neuroprotective Agents; NF-kappa B; Olfactory Bulb; Parkinsonian Disorders; rho-Associated Kinases; Signal Transduction; Substantia Nigra

Accumulation of α-synuclein (α-syn) is pivotally implicated in the pathogenesis of Parkinson׳s disease (PD), and enhancing its clearance might be a promising strategy in PD treatment. It has recently been shown that Rho kinase (ROCK) activation is involved in many neurodegenerative diseases, and some ROCK inhibitors might promote the degradation of abnormal protein aggregates. However, it is not known if fasudil, the only ROCK inhibitor available in clinical setting, could promote the degradation of α-syn, and ameliorate the α-syn induced neurotoxicity. In this regard, we investigated the effect of fasudil on neurite injury caused by A53T α-syn overexpression and the implicated pathway it might mediate. In the current study, we found that under the condition of A53T α-syn overexpression, the neurite outgrowth decreased significantly with the increasing expression of ROCK2. Fasudil, the ROCK inhibitor, ameliorated such neurotoxicity and promoted the clearance of A53T α-syn. Its underlying mechanism was supported by that fasudil could increase the macroautophagy activation via JNK 1 and Bcl-2 phosphorylation and beclin 1/Vps34 complex formation. Taken together, fasudil might be able to provide a novel and promising strategy for PD treatment by enhancing α-syn clearance and activating the JNK 1/Bcl-2/beclin 1 pathway.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; alpha-Synuclein; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Cell Line, Tumor; Humans; Membrane Proteins; Mitogen-Activated Protein Kinase 8; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-bcl-2; rho-Associated Kinases; Signal Transduction

Parkinson's disease (PD) is the most common neurodegenerative movement disorder, yet disease-modifying treatments do not currently exist. Rho-associated protein kinase (ROCK) was recently described as a novel neuroprotective target in PD. Since alpha-synuclein (α-Syn) aggregation is a major hallmark in the pathogenesis of PD, we aimed to evaluate the anti-aggregative potential of pharmacological ROCK inhibition using the isoquinoline derivative Fasudil, a small molecule inhibitor already approved for clinical use in humans. Fasudil treatment significantly reduced α-Syn aggregation in vitro in a H4 cell culture model as well as in a cell-free assay. Nuclear magnetic resonance spectroscopy analysis revealed a direct binding of Fasudil to tyrosine residues Y133 and Y136 in the C-terminal region of α-Syn. Importantly, this binding was shown to be biologically relevant using site-directed mutagenesis of these residues in the cell culture model. Furthermore, we evaluated the impact of long-term Fasudil treatment on α-Syn pathology in vivo in a transgenic mouse model overexpressing human α-Syn bearing the A53T mutation (α-Syn(A53T) mice). Fasudil treatment improved motor and cognitive functions in α-Syn(A53T) mice as determined by Catwalk(TM) gait analysis and novel object recognition (NOR), without apparent side effects. Finally, immunohistochemical analysis revealed a significant reduction of α-Syn pathology in the midbrain of α-Syn(A53T) mice after Fasudil treatment. Our results demonstrate that Fasudil, next to its effects mediated by ROCK-inhibition, directly interacts with α-Syn and attenuates α-Syn pathology. This underscores the translational potential of Fasudil as a disease-modifying drug for the treatment of PD and other synucleinopathies.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; alpha-Synuclein; Amides; Animals; Brain; Carrier Proteins; Cell Line, Tumor; Disease Models, Animal; Enzyme Inhibitors; Gene Expression Regulation; Humans; Mice; Mice, Transgenic; Mutation; Nerve Tissue Proteins; Parkinson Disease; Protein Aggregates; Protein Kinase Inhibitors; Psychomotor Performance; Pyridines; Recognition, Psychology; Time Factors; Tyrosine 3-Monooxygenase