MD pharmacophore-based search for novel MARK4 inhibitors: in silico and in vitro studies Alina Kutlushina, Olena Mokshyna, Lenka Hruba, Sona Gurska, Marian Hajduch, Petr Dzubak, Pavel Polishchuk Institute of Molecular and Translational Medicine, Palacký University and University Hospital in Olomouc, Olomouc, Czech Republic alina.kutlushina@upol.cz This work was supported by the INTER-EXCELLENCE LTARF18013 project (MEYS), the European Regional Development Fund - Project ENOCH (No. CZ.02.1.01/0.0/0.0/16_019/0000868) and ELIXIR CZ research infrastructure project (MEYS Grant No: LM2018131). MD snapshot timeline MD pharmacophores 3D pharmacophore hashes MD representative pharmacophores Materials and Methods Hit1 MARK1 MARK2 MARK3 MARK4 IC50, μM >50 14.72 0.0088 0.032 Hit2 MARK1 MARK2 MARK3 MARK4 IC50, μM >50 19.79 >50 12.01 Results Summary We developed and validated a workflow for virtual screening based on our previous implementation of MD pharmacophores and molecular docking. We were able to find a novel MARK inhibitor which was selective over 1 and 2 subtypes of MARK family and can be promising for further research. github.com/ci-lab-cz/pharmd A B D F G 1 2 3 4 5 compound conformers 4 5 = 0.8 CCA score RTB <= 5 MW <= 500 Da MD snapshot timeline 150 ns 5965 5221 4015 2532 MD representative pharmacophores 3938 2331 4989 5876 5+ features MD representative pharmacophores MD pharmacophore virtual screening 68337 molecules 3.8 % up to 50 conformers Conformers coverage Approach (CCA) 618 molecules 0.035 % Molecular Docking 235 molecules 0.013 % Visual inspection of the docked poses 24 hits 1783794 molecules 100 % The complexes of MARK4 with the 4 initial inhibitors MARK1, μM MARk2, μM MARK3, μM MARK4, μM >50 >50 >50 0.95 >50 >50 38.8 0.59 >50 >50 >50 0.47 >50 >50 >50 1.29 CCA score is the number of conformers which were mapped by at least one pharmacophore from the representative set divided by the total number of conformers. A B C D E F G b36..87 53b..ef 53b..ef e4f..ce 53b..ef 4af..f3 79f..31 MARK4 protein is a Serine/Threonine protein kinase, structurally related too the AMPK/Snf1 subfamily of the CaMK group of kinases. MARK4 proteins’ abnormal function has been associated with several pathological conditions including Alzheimer’s disease, which makes it a promising target for drug development. In the current study we searched for selective MARK4 inhibitors of new chemotypes too extend structural diversity of compounds for further selection and optimization . For this purpose, we used the workflow based on molecular dynamics (MD) pharmacophores. This approach allows us too take intoo account the conformational flexibility of the protein, while a pharmacophore is an abstract representation of the molecular interactions of the ligand with the protein, which is not related too the specific compound structure. Introduction 5RC MARK1 MARK2 MARK3 MARK4 IC50, μM 0.0041 0.0025 0.0039 0.0046 The MARK4 crystal structure exists in complex with a pyrazolopyrimidine (PDB ID: 5es1). The inhibitor (5RC) is highly active too MARK4 but not specific. Correlation of CCA and docking scores Distribution of the docking scores up to 50 conformers ChEMBL + proprietary 90 inactive and 29 active Virtual screening on validation set Quality assessment of MD representative pharmacophores PharMD MD pharmacophore virtual screening 68337 molecules 3.8 % up to 50 conformers Conformers coverage Approach (CCA) 618 molecules 0.035 % Molecular Docking 235 molecules 0.013 % Visual inspection of the docked poses 24 hits 1783794 molecules 100 % AutoDock Vina ChEMBL Proprietary Found hit Database Molecular space visualization of known MARK4 inhibitors (t-SNE) Tanimotoo 0.202 The nearest structure 0.205 White structures are poses determined by pharmacophores Pink structures are docked poses by AutoDock Vina -8.6 -8.1 -7.6 -8.8 -9.4 Docking score Proprietary reference compounds 0.03 The most distantMol 2Mol 3