Role of MARK4 in methamphetamine-induced acute kidney injury. / MARK4在甲基苯丙胺诱导急性肾损伤中的作用.

OBJECTIVES: Methamphetamine (METH) is an illicit psychoactive substance that can damage various organs, with the urinary system being one of its significant targets. This study aims to explore the role of microtubule affinity-regulating kinase 4 (MARK4) in METH-induced acute kidney injury (AKI). METHODS: A total of 10 healthy adult male C57BL/6 mice were randomly divided into a control group and a METH group, 5 mice in each group. The METH group was administered METH (20 mg/kg, intraperitoneally, once daily for 3 consecutive days), while the control group received an equal volume of physiological saline. The mice were executed 24 hours after the final injection, and the success of the AKI model was detected by blood serum creatinine, blood urea nitrogen, and renal HE staining. Proteins differentially expressed between kidney tissues with METH-induced AKI and normal kidney tissues were screened by proteomics techniques and subjected to gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and bioinformatics analysis. The accuracy of proteomic data was validated using Western blotting, and the expression levels of MARK4 and cleaved caspase-3 in mouse kidneys were measured. We further explored the role of MARK4 in METH-induced AKI. Firstly, a METH toxicity model was established in BUMPT cells to screen the appropriate concentration and time of METH treatment; the viability of BUMPT cells after METH treatment and the expression of cleaved caspase-3 were detected by interfering with MARK4 expression through inhibitors. RESULTS: The proteomic analysis of kidney tissues from METH and control groups screened for a total of 17 differentially expressed proteins, of which 11 were up-regulated and 6 were down-regulated (all P<0.05). The expression levels of MARK4 and cleaved caspase-3 were elevated in the kidneys of METH-treated mice (both P<0.05). The activity of BUMPT cells gradually decreased with increasing METH treatment concentration (all P<0.05), where the viability of BUMPT cells decreased to about 60% after METH treatment at 4 mmol/L. Compared with the control group, expression levels of MARK4 and cleaved caspase-3 were increased with higher METH concentrations and longer exposure times in a concentration- and time-dependent manner (all P<0.05). Inhibition of MARK4 expression improved METH-induced decrease in BUMPT cell activity, down-regulated the expression of cleaved caspase-3, and decreased the apoptosis of BUMPT cells induced by METH. CONCLUSIONS: MARK4 is highly expressed in a mouse model of METH-induced AKI, and MARK4 mediates METH-induced AKI by regulating cell apoptosis.

Ropinirole reverses the effects of neuroinflammation, and cellular demise by downregulating the MARK4-NFκß signaling system in Alzheimer's disease.

Ropinirole (ROP) is a dopamine agonist that can cross the blood-brain barrier (BBB), which is crucial for drugs targeting neurological conditions like Alzheimer's disease (AD). The rationale for the current research is to investigate the potential of ROP as an inhibitor of Microtubule affinity regulating kinase 4 (MARK4)-NFκß in neurodegenerative diseases, specifically AD. The interaction between ROP and MARK4-NFκß holds significant promise in the realm of drug discovery and therapeutic interventions for diseases like AD. Molecular docking and biophysical characterization demonstrate how ROP effectively hinders MARK4 activity, offering detailed insights into their molecular interactions. The present research also investigates the biological aspect of MARK4 shows promise in treating AD, with neuroinflammation playing a crucial role in the disease's progression. Aß42 and ROP were co-administered directly into the cells for the establishment of the AD model. We confirmed that ROP can inhibit the path of MARK4 activity, as evidenced by biophysical characterization, and can enhance the cell viability, lowers the expression of MARK4, decrease the rate of oxidative stress, and attenuate the expression of NFκß, leading to reduced neuronal apoptosis in an in vitro-induced Aß model. Overall, this research provides valuable mechanistic insights into the neuroprotective potential of ROP and its ability to target the MARK4-NFκß pathway.

A cell-penetrating peptide derived from SARS-CoV-2 protein Orf9b allosterically inhibits MARK4 activity and mitigates tau toxicity.

Abnormal activation of microtubule affinity-regulating kinase 4 (MARK4) and its phosphorylation of the microtubule-associated protein tau are believed to play a role in the pathogenesis of Alzheimer's disease, and MARK4 inhibition can be a strategy to develop disease-modifying therapy. Here we report the development of a membrane-permeable peptide that inhibits MARK4 activity in an allosteric manner. The SARS-CoV-2-derived protein Orf9b inhibited MARK4-mediated tau phosphorylation in primary neurons and Drosophila. Orf9b inhibited MARK4 activity in an allosteric manner and did not inhibit the activity of MARK2, which is another MARK family member and is closely related to MARK4. Co-expression of Orf9b in the fly retina expressing human tau and MARK4 suppressed phosphorylation of tau at the microtubule-binding repeats and tau-induced neurodegeneration. We identified the minimal sequence of Orf9b required to suppress MARK4 activity and fused it to a cell-permeable sequence (TAT-Orf9b10-18_78-95). Extracellular supplementation of TAT-Orf9b10-18_78-95 inhibited MARK4 activity in primary neurons, and feeding TAT-Orf9b10-18_78-95 to a fly model of tauopathy lowered phospho-tau levels and suppressed neurodegeneration. These results suggest that TAT-Orf9b10-18_78-95 is a unique class of MARK4 inhibitor and can be used to modify tau toxicity.

Therapeutic targeting of microtubule affinity-regulating kinase 4 in cancer and neurodegenerative diseases.

Microtubule affinity-regulating kinase 4 (MARK4) is a member of the Ser/Thr protein kinase family, phosphorylates the microtubule-connected proteins and plays a vital role in causing cancers and neurodegenerative diseases. This kinase modulates multiple signaling pathways, including mammalian target of rapamycin, nuclear factor-κB, and Hippo-signaling, presumably responsible for cancer and Alzheimer's. MARK4 acts as a negative controller of the Hippo-kinase cassette for promoting YAP/TAZ action, and the loss of MARK4 detains the tumorigenic properties of cancer cells. MARK4 is involved in tau hyperphosphorylation that consequently affects neurodegeneration. MARK4 is a promising drug target for cancer, diabetes, and Alzheimer's. Developing the potent and selective inhibitors of MAKR4 are promising in the therapeutic management of associated diseases. Despite its great significance, a few reviews are available to discuss its structure, function and clinical significance. In the current review, we aimed to provide detailed information on the structural features of MARK4 targeted in drug development and its role in various signaling pathways related to cancer and neurodegenerative diseases. We further described the therapeutic potential of MARK4 inhibitors in preventing numerous diseases. Finally, the updated information on MARK4 will be helpful in the further development of effective therapeutic molecules.

Targeting inhibition of microtubule affinity regulating kinase 4 by Harmaline: Strategy to combat Alzheimer's disease.

Microtubule-affinity regulating kinase 4 (MARK4) is linked with the development of cancer, diabetes and neurodegenerative diseases. Due to its direct role in the hyperphosphorylation of tau protein, MARK4 is considered as an attractive target to fight Alzheimer's disease (AD) and neuroinflammation. In the present study, we have selected Harmaline (HAR), an alkaloid of Paganum harmala, to investigate its MARK4 inhibitory potential and its binding mechanism. Molecular docking and fluorescence binding studies were carried out to estimate the binding affinity of the HAR with the MARK4. We observed an excellent binding affinity of HAR to the MARK4 (K = 107 M-1), further complemented by isothermal titration calorimetric measurements. In addition, HAR significantly inhibits the kinase activity of MARK4 (IC50 value of 4.46 µM). Structural investigations suggested that HAR binds to the active site pocket and forms several non-covalent interactions with biologically important residues of MARK4. All-atom molecular dynamics simulation studies further advocated that the MARK4-HAR complex is stabilized throughout the trajectory of 200 ns and causes a little conformational change. All these findings suggest that HAR is a potential MARK4 inhibitor that can be implicated in managing MARK4-associated diseases, including AD.

Microtubule-affinity regulating kinase 4: A potential drug target for cancer therapy.

The human genome encodes more than 500 protein kinases that work by transferring the γ-phosphate group from ATP to serine, threonine, or tyrosine (Ser/Thr/Tyr) residues. Various kinases are associated with the onset of cancer and its further progression. The recent advancements in developing small-molecule kinase inhibitors to treat different cancer types have shown noticeable results in clinical therapies. Microtubule-affinity regulating kinase 4 (MARK-4) is a Ser/Thr protein kinase that relates structurally to AMPK/Snf1 subfamily of the CaMK kinases. The protein kinase modulates major signalling pathways such as NF-κB, mTOR and the Hippo-signalling pathway. MARK4 is associated with various cancer types due to its important role in regulating microtubule dynamics and subsequent cell division. Aberrant expression of MARK4 is linked with several pathologies such as cancer, Alzheimer's disease, obesity, etc. This review provides detailed information on structural aspects of MARK4 and its role in various signalling pathways related to cancer. Several therapeutic molecules were designed to inhibit the MARK4 activity from controlling associated diseases. The review further highlights kinase-targeted drug discovery and development in oncology and cancer therapies. Finally, we summarize the latest findings regarding the role of MARK4 in cancer, diabetes, and neurodegenerative disease path to provide a solid rationale for future investigation and therapeutic intervention.

SET8 mitigates hepatic ischemia/reperfusion injury in mice by suppressing MARK4/NLRP3 inflammasome pathway.

AIMS: Hepatic ischemia/reperfusion (I/R) injury is a critical factor affecting the prognosis of liver surgery. The aim of this study is to explore the effects of SET8 on hepatic I/R injury and the putative mechanisms. MAIN METHODS: The expression of SET8 and MARK4 in I/R group and sham group were detected both in vivo and in vitro. In addition, mouse and RAW 264.7 cells were transfected with MARK4 siRNA and SET8 siRNA knockdown of MARK4 and SET8, respectively. The expression of SET8, MARK4 and NLRP3-associated proteins were detected after different treatments. The pathology of liver and the serologic detection were detected after different treatments. KEY FINDINGS: Our present study identified SET domain-containing protein 8 (SET8) as an efficient protein, which can negatively regulate hepatic I/R-mediated inflammatory response and ameliorate hepatic I/R injury by suppressing microtubule affinity-regulating kinase 4 (MARK4)/ NLR family pyrin domain containing 3 (NLRP3) inflammasome pathway. The data showed that MARK4 deficiency inhibited hypoxia/reoxygenation (H/R)-induced NLRP3 inflammasome activation, while SET8 deficiency showed the opposite effect. We further demonstrated that SET8 restrained NLRP3 inflammasome activation by inhibiting MARK4. Moreover, we verified SET8 made protective effect on hepatic I/R injury. SIGNIFICANCE: SET8 plays an essential role in hepatic ischemia/reperfusion injury in mice by suppressing MARK4/NLRP3 inflammasome pathway. Our results may offer a new strategy to mitigate hepatic I/R injury.

Impact of glioblastoma multiforme associated mutations on the structure and function of MAP/microtubule affinity regulating kinase 4.

MAP/Microtubule affinity regulating kinase 4 (MARK4) plays an important role in the regulation of microtubule dynamics by phosphorylation of tau protein. A higher expression of MARK4 is observed in the glioblastoma multiforme (GBM) cell lines. We identified eight synonymous and non-synonymous mutations in the MARK4 gene related to GBM in The Cancer Genome Atlas (TCGA) consortium. Out of these, three non-synonymous mutations were found in the catalytic domain of the protein (Lys231Asn, Tyr247His and Arg265Gln), were selected to see the possible deleterious effects on the structure and function using the cutting-edge in-silico tools. In addition, molecular dynamics simulation, principal component analysis, dynamic cross correlation matrix analysis and correlation network analysis were performed to gain insights into the conformation of the MARK4 and its mutants. We found that, Tyr247His shows a maximum deleterious impact, reflected from structural deviation in comparison to Lys231Asn and Arg265Gln. In conclusion, Tyr247His mutant of MARK4 has relatively higher chances of affecting the structure and function of the protein thus leading to abnormal MARK4 activity which is associated to GBM.Communicated by Ramaswamy H. Sarma.

Microtubule affinity-regulating kinase 4 with an Alzheimer's disease-related mutation promotes tau accumulation and exacerbates neurodegeneration.

Accumulation of the microtubule-associated protein tau is associated with Alzheimer's disease (AD). In AD brain, tau is abnormally phosphorylated at many sites, and phosphorylation at Ser-262 and Ser-356 plays critical roles in tau accumulation and toxicity. Microtubule affinity-regulating kinase 4 (MARK4) phosphorylates tau at those sites, and a double de novo mutation in the linker region of MARK4, ΔG316E317D, is associated with an elevated risk of AD. However, it remains unclear how this mutation affects phosphorylation, aggregation, and accumulation of tau and tau-induced neurodegeneration. Here, we report that MARK4ΔG316E317D increases the abundance of highly phosphorylated, insoluble tau species and exacerbates neurodegeneration via Ser-262/356-dependent and -independent mechanisms. Using transgenic Drosophila expressing human MARK4 (MARK4wt) or a mutant version of MARK4 (MARK4ΔG316E317D), we found that coexpression of MARK4wt and MARK4ΔG316E317D increased total tau levels and enhanced tau-induced neurodegeneration and that MARK4ΔG316E317D had more potent effects than MARK4wt Interestingly, the in vitro kinase activities of MARK4wt and MARK4ΔG316E317D were similar. When tau phosphorylation at Ser-262 and Ser-356 was blocked by alanine substitutions, MARK4wt did not promote tau accumulation or exacerbate neurodegeneration, whereas coexpression of MARK4ΔG316E317D did. Both MARK4wt and MARK4ΔG316E317D increased the levels of oligomeric forms of tau; however, only MARK4ΔG316E317D further increased the detergent insolubility of tau in vivo Together, these findings suggest that MARK4ΔG316E317D increases tau levels and exacerbates tau toxicity via a novel gain-of-function mechanism and that modification in this region of MARK4 may affect disease pathogenesis.

Suberoylanilide Hydroxamic Acid Attenuates Interleukin-1ß-Induced Interleukin-6 Upregulation by Inhibiting the Microtubule Affinity-Regulating Kinase 4/Nuclear Factor-κB Pathway in Synovium-Derived Mesenchymal Stem Cells from the Temporomandibular Joint.

Synovium-derived mesenchymal stem cells (SMSCs) can migrate to the site of destroyed condylar cartilage and differentiate into chondrocytes to repair temporomandibular joint (TMJ) damage. Interleukin (IL)-1ß-induced IL-6 secretion has been shown to inhibit the chondrogenic potential of SMSCs. The histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) has recently been shown to be closely related to the inflammation induced by IL-1ß. However, the relationship between SAHA and IL-6 secretion induced by IL-1ß in SMSCs remains unclear. In this study, we evaluated the relationships between IL-1ß and IL-6 in synovial specimens from patients with TMD and in model rats with osteoarthritis (OA). We found that IL-1ß and IL-6 were positively correlated and that IL-6 expression in SMSCs increased with IL-1ß stimulation in vitro. Moreover, microtubule affinity-regulating kinase 4 (MARK4) was significantly upregulated in IL-1ß-stimulated SMSCs and in the synovium of rats with OA. MARK4 knockdown inhibited IL-6 secretion and nuclear factor (NF)-κB pathway activation in IL-1ß-stimulated SMSCs. SAHA attenuated IL-6 secretion in IL-1ß-induced SMSCs through NF-κB pathway inhibition, and MARK4 was also downregulated in SAHA-treated SMSCs. However, inhibition of the NF-κB pathway did not suppress MARK4 expression. Thus, these results showed that SAHA attenuated IL-6 secretion in IL-1ß-induced SMSCs through inhibition of the MARK4/NF-κB pathway.

Evaluation of pyrazolopyrimidine derivatives as microtubule affinity regulating kinase 4 inhibitors: Towards therapeutic management of Alzheimer's disease.

Microtubule affinity regulating kinase 4 (MARK4) plays essential role in the tau-assisted regulation of microtubule dynamics. Over expression of MARK4 causes early phosphorylation of Ser262 of tau protein which is essential for microtubule binding. Hyperphosphorylation of tau protein causes the formation of paired helical fragments and neurofibrillary tangles, the hallmarks of Alzheimer's disease. Targeting the modulation of MARK4 activity is an effective strategy for therapeutic intervention of Alzheimer's and other MARK4 associated neurodegenerative diseases. Having role of pyrazolopyrimidine derivatives in the therapeutic management of neurodegenerative diseases, we have tried to estimate their binding affinity with the MARK4. We performed in silico screening of 59 pyrazolopyrimidine derivatives against MARK4 and obtained a few best possible inhibitors. Molecular docking-based interaction analysis suggested five potential leads that were further analyzed using molecular dynamics simulations, MM/PBSA, principal component analysis and graph theory based dynamic network analysis to observe structural changes caused due to ligand binding. All these computational analyses suggested that compounds with PubChem IDs: 91895678, 91895679, 91895692, 91145515 and 90794095 may be further exploited to address Alzheimer's and other neurodegenerative diseases.Communicated by Ramaswamy H. Sarma.

Identification and evaluation of bioactive natural products as potential inhibitors of human microtubule affinity-regulating kinase 4 (MARK4).

Microtubule affinity-regulating kinase 4 (MARK4) has recently been identified as a potential drug target for several complex diseases including cancer, diabetes and neurodegenerative disorders. Inhibition of MARK4 activity is an appealing therapeutic option to treat such diseases. Here, we have performed structure-based virtual high-throughput screening of 100,000 naturally occurring compounds from ZINC database against MARK4 to find its potential inhibitors. The resulted hits were selected, based on the binding affinities, docking scores and selectivity. Further, binding energy calculation, Lipinski filtration and ADMET prediction were carried out to find safe and better hits against MARK4. Best 10 compounds bearing high specificity and binding efficiency were selected, and their binding pattern to MARK4 was analyzed in detail. Finally, 100 ns molecular dynamics simulation was performed to evaluate; the dynamics stability of MARK4-compound complex. In conclusion, these selected natural compounds from ZINC database might be potential leads against MARK4, and can further be exploited in drug design and development for associated diseases.

Design and development of Isatin-triazole hydrazones as potential inhibitors of microtubule affinity-regulating kinase 4 for the therapeutic management of cell proliferation and metastasis.

Microtubule affinity-regulating kinase 4 (MARK4) is a potential drug target as the same is found to be over expressed in several types of cancers. In search of effective MARK4 inhibitors, we have synthesized and characterized Isatin-triazole hydrazones (9a-i) and evaluated their inhibitory potential. Of all the compounds, 9g showed better binding affinity and enzyme inhibition potential in sub micromolar range. Human serum albumin (HSA) binding assay suggested an easy transportation of 9g in blood stream due to its binding affinity. In vitro anticancer studies performed on MCF-7, MDA-MB-435s and HepG2 cells using 9g showed inhibition of cell proliferation and cell migration. Further, 9g induces apoptosis in these cancerous cells, with IC50 values of 6.22, 9.94 and 8.14 µM, respectively. Putatively, 9g seems to cause oxidative stress resulting in apoptosis. Functional assay of 9g with a panel of 26 kinases showed MARK4 specific profile. In conclusion, 9g seems to possess an effective inhibitory potential towards MARK4 adding an additional repertoire to anticancer therapeutics.

Evaluation of human microtubule affinity-regulating kinase 4 inhibitors: fluorescence binding studies, enzyme, and cell assays.

Human microtubule affinity-regulating kinase 4 (MARK4) is considered as an encouraging drug target for the design and development of inhibitors to cure several life-threatening diseases such as Alzheimer disease, cancer, obesity, and type-II diabetes. Recently, we have reported four ligands namely, BX-912, BX-795, PKR-inhibitor, and OTSSP167 (hydrochloride) which bind preferentially to the two different constructs of human MARK4 containing kinase domain. To ensure the role of ubiquitin-associated (UBA) domain in the ligand binding, we made a newer construct of MARK4 which contains both kinase and UBA domains, named as MARK4-F3. We observed that OTSSP167 (hydrochloride) binds to the MARK4-F3 with a binding constant (K) of 3.16 × 106, M-1 (±.21). However, UBA-domain of MARK4-F3 doesn't show any interaction with ligands directly as predicted by the molecular docking. To validate further, ATPase inhibition assays of all three constructs of MARK4 in the presence of mentioned ligands were carried out. An appreciable correlation between the binding experiments and ATPase inhibition assays of MARK4 was observed. In addition, cell-proliferation inhibition activity for all four ligands on the Human embryonic kidney (HEK-293) and breast cancer cell lines (MCF-7) was performed using MTT assay. IC50 values of OTSSP167 for HEK-293 and MCF-7 were found to be 58.88 (±1.5), and 48.2 (±1.6), respectively. OTSSP167 among all four inhibitors, showed very good enzyme inhibition activity against three constructs of MARK4. Moreover, all four inhibitors showed anti-neuroblastoma activity and anticancer properties. In conclusion, OTSSP167 may be considered as a promising scaffold to discover novel inhibitors of MARK4.

Ubiquitin-associated domain of MARK4 provides stability at physiological pH.

Microtubule affinity regulating Kinase 4 (MARK4) belongs to the family of AMP-activated protein kinase. It phosphorylates microtubule associated proteins at specific sites (Serine in KXGS motifs) in the microtubule-binding repeats. In our previous studies, two constructs, namely, kinase domain with 59 N-terminal residues (residues 1-310) and only kinase domain (residues 59-310) of MARK4 show aggregation at physiological pH. However, these two constructs were stable at extremes of pH conditions. Now the question arises: how is MARK4 stable at physiological pH in-vivo? To answer this question, we have successfully cloned, expressed, and purified UBA-domain along with the kinase domain of MARK4 and performed spectroscopic measurements and activity assays. We observed a pronounced secondary and tertiary structure and ATPase activity in the MARK4 at physiological pH. In conclusion, UBA domain may be important to maintain the structure, stability and activity of MARK4 under physiological conditions.

miR-515-5p controls cancer cell migration through MARK4 regulation.

Here, we show that miR-515-5p inhibits cancer cell migration and metastasis. RNA-seq analyses of both oestrogen receptor receptor-positive and receptor-negative breast cancer cells overexpressing miR-515-5p reveal down-regulation of NRAS, FZD4, CDC42BPA, PIK3C2B and MARK4 mRNAs. We demonstrate that miR-515-5p inhibits MARK4 directly 3' UTR interaction and that MARK4 knock-down mimics the effect of miR-515-5p on breast and lung cancer cell migration. MARK4 overexpression rescues the inhibitory effects of miR-515-5p, suggesting miR-515-5p mediates this process through MARK4 down-regulation. Furthermore, miR-515-5p expression is reduced in metastases compared to primary tumours derived from both in vivo xenografts and samples from patients with breast cancer. Conversely, miR-515-5p overexpression prevents tumour cell dissemination in a mouse metastatic model. Moreover, high miR-515-5p and low MARK4 expression correlate with increased breast and lung cancer patients' survival, respectively. Taken together, these data demonstrate the importance of miR-515-5p/MARK4 regulation in cell migration and metastasis across two common cancers.

Human microtubule affinity-regulating kinase 4 is stable at extremes of pH.

MAP/microtubule affinity-regulating kinase 4 (MARK4) is a member of adenosine monophosphate-activated protein kinases, directly associated with cancer and neurodegenerative diseases. Here, we have cloned, expressed, and purified two variants of MARK4 [the kinase domain (MARK4-F2), and kinase domain along with 59 N-terminal residues (MARK4-F1)] and compared their stability at varying pH range. Structural and functional changes were observed by incubating both forms of MARK4 in buffers of different pH. We measured the secondary structure of MARK4 using circular dichroism and tertiary structure by measuring intrinsic fluorescence and absorbance properties along with the size of proteins by dynamic light scattering. We observed that at extremes of pH (below pH 3.5 and above pH 9.0), MARK4 is quite stable. However, a remarkable aggregate formation was observed at intermediate pH (between pH 3.5 and 9.0). To further validate this result, we have modeled both forms of MARK4 and performed molecular dynamics simulation for 15 ns. The spectroscopic observations are in excellent agreement with the findings of molecular dynamics simulation. We also performed ATPase activity at varying pH and found a significant correlation of structure of MARK4 with its enzyme activity. It is interesting to note that both forms of MARK4 are showing a similar pattern of structure changes with reference to pH.

PKR-inhibitor binds efficiently with human microtubule affinity-regulating kinase 4.

MAP/microtubule affinity-regulating kinase 4 (MARK4) plays a central role in the cellular physiology, and it is inseparably linked with many human diseases including cancer, diet induced obesity, type2 diabetes and neurodegenerative disorders. Here, we studied the interaction of PKR-inhibitor with two variants of human MARK4. One variant is named as MARK4-F1 which has 59 N-terminal residues along with kinase domain while another variant is MARK4-F2 which has kinase domain only. Molecular-docking, molecular dynamics (MD) simulation and fluorescence-binding studies were undertaken to understand the role of N-terminal 59-residues in the binding of substrate/inhibitors. Molecular docking studies revealed that the PKR-inhibitor binds in the large hydrophobic cavity of the kinase domain of MARK4 through several hydrophobic and hydrogen-bonded interactions. Furthermore, MD simulation showed a stable parameters for the complexes of both MARK4-F1 and MARK4-F2 to PKR-inhibitor with marginal difference in their binding affinities. A significant decrease in the fluorescence intensity of MARK4 was observed on successive addition of the PKR-inhibitor. Using fluorescence data we have calculated the binding-affinity and the number of binding site of PKR-inhibitor to the MARK4. A significantly high binding affinity was observed for the PKR-inhibitor to the MARK4 variants. However, there is no any significant difference in the binding affinity of PKR-inhibitor to the MARK4 variants was observed, indicating that 59 N-terminal residues of MARK4-F1 are not playing a crucial role in the ligand binding. The present study will provide an insights into designing of new PKR-inhibitor derivative as potent and selective therapeutic agent against many life threatening diseases which are associated with MARK4.