title: Evaluation of human microtubule affinity-regulating kinase 4 inhibitors: fluorescence binding studies, enzyme, and cell assays.
abstract: 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. IC50values 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.
authors.affiliation: a Center for Interdisciplinary Research in Basic Sciences , Jamia Millia Islamia , Jamia Nagar, New Delhi 110025 , India.
authors.forename: Farha
authors.initials: F
authors.lastname: Naz
authors.affiliation: a Center for Interdisciplinary Research in Basic Sciences , Jamia Millia Islamia , Jamia Nagar, New Delhi 110025 , India.
authors.forename: Neha
authors.initials: N
authors.lastname: Sami
authors.affiliation: a Center for Interdisciplinary Research in Basic Sciences , Jamia Millia Islamia , Jamia Nagar, New Delhi 110025 , India.
authors.forename: Abu Turab
authors.initials: AT
authors.lastname: Naqvi
authors.affiliation: a Center for Interdisciplinary Research in Basic Sciences , Jamia Millia Islamia , Jamia Nagar, New Delhi 110025 , India.
authors.forename: Asimul
authors.initials: A
authors.lastname: Islam
authors.affiliation: a Center for Interdisciplinary Research in Basic Sciences , Jamia Millia Islamia , Jamia Nagar, New Delhi 110025 , India.
authors.forename: Faizan
authors.initials: F
authors.lastname: Ahmad
authors.affiliation: a Center for Interdisciplinary Research in Basic Sciences , Jamia Millia Islamia , Jamia Nagar, New Delhi 110025 , India.
authors.forename: Md
authors.identifier: http://orcid.org/0000-0002-3663-4940
authors.initials: M
authors.lastname: Imtaiyaz Hassan
mesh_terms: D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000737:chemistry; D001665:Binding Sites; D002460:Cell Line; D055808:Drug Discovery; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D008024:Ligands; D062105:Molecular Docking Simulation; D056004:Molecular Dynamics Simulation; D011485:Protein Binding; D047428:Protein Kinase Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry*
chemical_list: D005456:Fluorescent Dyes; D008024:Ligands; D047428:Protein Kinase Inhibitors; C483047:MARK4 protein, human; D017346:Protein Serine-Threonine Kinases; D000251:Adenosine Triphosphatases
keywords: MAP/microtubule affinity-regulating kinase 4; MTT assay; drug target; high affinity ligand; ligand binding