Multifunctional matrix protein (M) of rabies virus (RABV) plays essential roles in the pathogenesis of rabies infection. Identification of M protein interacting partners in target hosts could help to elucidate the biological pathways and molecular mechanisms involved in the pathogenesis of this virus. In this study, two-dimensional Far-western blotting (2D-Far-WB) technique was applied to find possible matrix protein partners in the rat brainstem. Recombinant RABV M was expressed in Pichia pastoris and was partially purified. Subsequently, 2D-Far-WB-determined six rat brainstem proteins interacted with recombinant M proteins that were identified by mass spectrometry. Functional annotation by gene ontology analysis determined these proteins were involved in the regulation of synaptic transmission processes, metabolic process and cell morphogenesis–cytoskeleton organization. The interaction of viral M protein with selected host proteins in mouse Neuro-2a cells infected with RABV was verified by super-resolution confocal microscopy. Molecular docking simulations also demonstrated the formation of RABV M complexes. However, further confirmation with co-immunoprecipitation was only successful for M-actin cytoplasmic 1 interaction. Our study revealed actin cytoplasmic 1 as a binding partner of M protein, which might have important role(s) in rabies pathogenesis.

The efficiency of interferon beta (IFNβ)-based drugs is considerably limited due to their undesirable properties, especially high immunogenicity. In this study, for the first time we investigated the impact of a computationally designed peptide mimetic of IFNβ, called MSPEP27, in the animal model of MS. A peptide library was constructed using the Rosetta program based on the predominant IFNAR1-binding site of IFNβ. Molecular docking studies were carried out using ClusPro and HADDOCK tools. The GROMACS package was subsequently used for molecular dynamics (MD) simulations. Validation of peptide-receptor interaction was carried out using intrinsic fluorescence measurements. To explore in silico findings further, experimental autoimmune encephalomyelitis (EAE) was induced by subcutaneous immunization of myelin oligodendrocyte glycoprotein (MOG35-55). Mice were then separated into distinct groups and intravenously received 10 or 20mgkg of MSPEP27 or IFNβ. The inflammatory mediators were monitored by immunohistochemistry (IL17, CD11b, CD45), quantitative real-time PCR (MMP2, MMP9, TIMP-1) and enzyme-linked immunosorbent assay (IL1β, TNFα) methods. Among the library of tolerated peptides, MSPEP27, a peptide with favorable physicochemical properties, was chosen for further experiments. This peptide was shown to significantly interact with IFNAR1 in a dose-dependent manner. Like IFNβ, MSPEP27 could efficiently bind to IFNAR1 and form a stable peptide-receptor complex during 30ns MD simulations. In vivo analyses revealed that MSPEP27 could lessen inflammation by modulating the levels of inflammatory mediators. According to our results, MSPEP27 peptide could efficiently bind to IFNAR1 and suppress neuroinflammation in vivo. We conclude that MSPEP27 has protective effects against MOG-induced EAE via reduction of immune dysfunction and inflammation.

Benzodiazepines (BZD) are widely used in neurological disorders. The use of classical benzodiazepines is limited due to side effects. In this study, based on the structure-activity relationship (SAR) of Benzodiazepine receptors, new derivatives of 4-amino-3,5-diphenyl-1,2,4-triazole as benzodiazepine agonists with selective effects were designed and synthesized. Docking studies showed that pharmacophore groups of the designed structures and zolpidem, a benzodiazepine receptor agonist, are properly matched and are located well in the GABA receptor. The triazole group of the compound 4j, N N-(3,5-diphenyl-4H-1,2,4-triazol-4-yl)-2-((4-fluorobenzyl)amino)acetamide, was near the nitrogen moiety of the imidazole ring of zolpidem providing the hydrogen bond acceptor in the suitable direction in the BDZ-binding site of GABAA receptor model (α1β2ϒ2). The compounds were synthesized with acceptable yield and in-vitro affinity for the BZD receptor was determined. Compound 4j had the best affinity for the BZD site of action on GABAA receptor complex (Ki = 2.56 nM and IC50 = 6.10 nM). In addition, the sedative-hypnotic effect, the locomotor activity, and evaluated memory of the novel compounds were assessed by pentobarbital-induced sleeping, open field, and passive avoidance tests respectively. Most of the novel compounds showed significant hypnotic activity with no impairment on learning and memory performance in the mouse. The pharmacological effects of the compounds were antagonized by flumazenil, a BZD antagonist, which confirms the involvement of BZD receptors in the biological effects.