Apoptosis signal-regulating kinase 1 (ASK1) is the ubiquitously expressed mitogen-activated protein kinase kinase kinase 5 that has a broad range of biological functions depending on the cell type and cellular context. ASK1 is associated with several diseases. For example, ASK1 was identified as an essential component in the neuronal death signaling pathway induced by expanded polyglutamine (polyQ) repeats. ASK1 activation by amyloid beta protein is an important step in Alzheimer's disease pathogenesis. Recently, it has been revealed that ASK1 is also involved in motor neuron cell death during familial amyotrophic lateral sclerosis. The inhibition of ASK1 by the recombinant adeno-associated virus expressing a dominant-negative ASK1?N-KR mutant was capable of suppressing heart failure progression by preventing cardiomyocyte apoptosis. In vivo study supports the role of ASK1 in the immune response, since ASK1-deficient mice were resistant to lipopolysaccharide (LPS)-induced septic shock. It is ground for the search of potential ASK1 inhibitors. The main aims of our research are to identify the small molecule inhibitors of ASK1 and to predict binding modes of these ligands with ASK1 ATP binding site. In order to discover novel ASK1 inhibitors we have performed screening program, using both in silico and in vitro approaches. AutoDock and DOCK software were used to conduct receptor-ligand flexible docking. The virtual screening experiments were carried out targeting the ATP binding site of ASK1 by browsing the commercially available OTAVA chemical library of 164,840 compounds. The high-ranking 300 compounds have been taken for the kinase assay study. In vitro experiments revealed that a number of 2-thioxo-thiazolidin-4-one derivatives showed inhibitory activity towards ASK1. The most active compound inhibited ASK1 with IC50 = 2 microM. Then, we performed in-depth study of this chemical class using the pre-selected library of 8,425 2-thioxo-thiazolidin-4-one derivatives. Ten compounds were selected and taken for the kinase assay. Compound 2-{5-[5-(3,4-dichloro-phenyl)-furan-2-ylmethylene]-4-oxo-2-thioxo-thiazolidin-3-yl}-propanoic acid (PFTA-1) possesses submicromolar activity (IC50 = 0.65 microM). The inhibitory profiles of PFTA-1 were investigated in vitro using protein kinases CK2, JNK3, Aurora А, FGFR1, HGFR1 and Tie2. Our preliminary selectivity studies have demonstrated that compound seems to be potent inhibitor of ASK1. To inspect the binding mode of studied compounds we analyzed ASK1 complexes with 2-thioxo-thiazolidin-4-one derivatives generated by molecular docking software. It turned out that peculiarity of the PFTA-1 in the comparison to other inactive nine compounds is its ability to bind simultaneously to the part of kinase domain known as "hinge region" and the phosphate-binding region of the ATP-binding cleft. Our further goal was to use structural core of PFTA-1 to develop more potent and selective inhibitors of ASK1. 32 derivatives of 5-(5-phenyl-furan-2-ylmethylene)-2-thioxo-thiazolidin-4-one were synthesized and tested. Comparing SAR and modeling data, it can be assumed that the presence of phenyl ring in the compound structure, which takes part in the hydrophobic interaction with hinge region, is very important for the inhibitory activity against ASK1. However, the substituents on the phenyl ring only slightly affect the potency of investigated compounds. It was also turned out that the carboxylic acid in the structure of 5-(5-phenyl-furan-2-ylmethylene)-2-thioxo-thiazolidin-4-one derivatives is necessary for inhibitory activity toward ASK1. Virtual screening also allowed us to identify small-molecule inhibitor of ASK1 among 3H-naphtho[1,2,3-de]quinoline-2,7-diones. In vitro experiments revealed that ethyl 2,7-dioxo-2,7-dihydro-3H-naphtho[1,2,3-de]quinoline-1-carboxylate (NQDI-1) inhibited ASK1 with IC50=3 microM. Accordingly to the in silico modeling results, the mechanism of ASK1 inhibition involves the hydrogen bond formation between carbonyl of NQDI-1 and ASK1 hinge region. Our preliminary selectivity studies have demonstrated that this compound is a selective inhibitor of ASK1. At the next step, compounds PFTA-1 and NQDI-1 were investigated in HEK293 and HT22 cells during apoptosis induced by CHI3L2 and glutamate, respectively, to determine whether they are also active in cellular systems. In these experiments inhibitors demonstrated cytoprotective effect. However, high concentrations of the inhibitors were toxic. These results strongly suggest that the core structure of studied compounds can be used for further optimization to improve biological properties.
