Inhibition of MARK4 by serotonin as an attractive therapeutic approach to combat Alzheimer's disease and neuroinflammation

Abstract

Mitogen-activated protein kinases (MAPKs) govern various cellular programs and crucial intermediate pathways in signaling. Microtubule affinity-regulating kinase 4 (MARK4) is a part of the kinase family recognized for actively phosphorylating neural microtubule-associated proteins (MAPs) like MAP2, MAP4 and most importantly, tau. The Ser/Thr kinase MARK4 overexpression is associated with various life-threatening conditions such as neurodegenerative disorders, diabetic neuropathy, and cancer. Functionally, MARK4 is correlated with many important signaling cascades and transcription factors contributing to neurodegeneration and cancer onset and progression. Serotonin is a key molecule associated with regulating mood, stress, and various behavioral aspects. Low serotonin levels promote the progression of neurological and psychotic disorders, which is also a consequence of tau accumulation. MARK4 being a major contributor to phosphorylating tau, leading to its accumulation, and contributing to tauopathy, is targeted for inhibition by serotonin. The study deals with the inhibition of MARK4 by serotonin using combined computational and experimental studies. The results presented in this paper provide strong evidence for the direct physical binding of serotonin to recombinant MARK4 and subsequent inhibition of its kinase activity. In addition, we have performed molecular docking, followed by 100 ns MD simulations of MARK4 in the presence of serotonin, to estimate the stability of the protein-ligand complex. Since MARK4 is a potential drug target and can be exploited for drug design and discovery for cancer and neurodegenerative disorders, the results presented here are of interest and may be further exploited for Alzheimer's disease (AD) and other neurodegenerative diseases.

Fig. 1. (A) Serotonin in the binding pocket of MARK4. (B) Magnified view of the MARK4–serotonin interaction drawn in PyMoL using the top docked pose obtained through InstaDock. (C) Charged surface view of serotonin occupying the binding pocket of MARK4. (D) 2D diagram of serotonin binding to MARK4 with critical interacting residues labeled.

Fig. 2. (A) RMSD plots of backbone atoms (red is serotonin bound protein; black is apo protein). (B) RMSD plot of the ligand during the 100 ns run.

Fig. 3. (A) Radius of gyration plotted during the 100 ns MD run of apo (black) and serotonin bound MARK4 (red). (B) SASA plots of the protein backbone atoms as a function of the number of frames in the production runs.

Fig. 4. (A) Backbone hydrogen bonds (both apo and bound Mark4) plotted as a function of the number of snapshots. (B) Intermolecular hydrogen bonds monitored between the protein and serotonin.

Fig. 5. LIE energies are plotted as a function of the number of snapshots.

Fig. 6. (A) Fluorescence emission spectra of MARK4 in the absence and presence of varying serotonin concentrations (0–1.5 μM). (B) Modified Stern–Volmer plot of the MARK4–serotonin complex.

Fig. 7. (A) ITC measurements for the titration of serotonin with MARK4 at 25 °C. (B) Kinase assay of MARK4 with varying serotonin concentrations (0–15 μM).