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  • Herein we disclose novel dihydropyrano c pyrazoles that inhi


    Herein, we disclose novel 2,4-dihydropyrano[2,3-c]pyrazoles that inhibit human ATX (hATX). Our results demonstrate and further support previous findings showing that a desired biological action can be achieved without targeting the catalytic site of the enzyme. Moreover, this study validated hen egg white ATX (ewATX) activity assay as a convenient and truly affordable method for initial discovery of ATX inhibitors. Noteworthy, molecular modeling was able to correctly predict the biologically active enantiomer of the 2,4-dihydropyrano[2,3-c]pyrazoles, as verified by MRS 2365 crystallization and biological testing.
    Material and Methods
    Results and Discussion
    Conclusions Using mATX crystal structure in virtual screening and validated ewATX preparation in the preliminary phase of inhibitor screening, we have identified a novel ATX inhibitor (S)-25 that also inhibits hATX with an IC50-value of 134nM. We further validated (S)-25 in melanoma cell migration assays, where the compound blocked ATX-mediated cell migration with no observed effect on LPA receptors or downstream signaling mediating cell migration. The 2,4-dihydropyrano[2,3-c]pyrazoles represent a totally different type of inhibitor as compared to the traditional ATX inhibitors, as they most likely bind to the lipophilic pocket instead of the catalytic site. Interestingly, the molecular modeling was able to clearly distinguish the enantiomers of 2,4-dihydropyrano[2,3-c]pyrazoles, predicting correctly the biologically active enantiomer. Worth noting, a compound with the similar 2,4-dihydropyrano[2,3-c]pyrazole scaffold has been previously disclosed, although not further exploited, in HTS of ATX inhibitors (Albers et al., 2010). We noticed that the reference inhibitor S32826 was not effectively inhibiting LPC hydrolysis although it performed well in assays with the artificial substrate pNP-TMP. This can be taken as a cautionary example of using an unnatural substrate in the screening assay, generating the possibility for false positive results. In addition, the use of natural substrate enabled us to identify inhibitors which leave the active site unhindered or do not compete with the substrate or both. On the other hand, with the artificial substrate, we were able to provide additional evidence that 2,4-dihydropyrano[2,3-c]pyrazoles leave the enzyme's active site unhindered, as inhibition of pNP-TMP hydrolysis was minor with these compounds. Our data suggested that the novel 2,4-dihydropyrano[2,3-c]pyrazoles likely inhibit ATX by binding to the lipophilic pocket instead of the enzyme's active-site. Unfortunately, as the pocket of ATX is highly-lipophilic, it drives the inhibitors which target this site to have high logP values. To overcome this phenomenon, new strategies need to be considered when aiming at targeting this pocket. One potential approach could be to find all plausible hydrogen bond contacts within the pocket to decrease the logP values of the inhibitors. Especially S170, which lies deep in the pocket, could be an attractive target to establish hydrogen bond in order to obtain inhibitors with lower logP values. Another possibility would be utilizing the open solvent interface of the binding pocket. For instance, attaching a hydrophilic moiety towards the solvent phase from the inhibitor, could solve the high logP issue.
    Conflict of interest
    Acknowledgements We would like to thank Taina Vihavainen and Tiina Koivunen for technical assistance, and the Finland's CSC - IT Center for Science Ltd. for the computational resources. This study was supported by the Academy of Finland (grant 276509 to AP; grant 278212 to JTL) and Biocenter Finland/DDCB (TL).
    Autotaxin (ATX) is a secreted glycoprotein phosphodiesterase, which is a member of the ectonucleotide pyrophosphatase and phosphodiesterase family (ENPP). ATX hydrolyzes extracellular lysophosphatidylcholine (LPC) into the lipid mediator lysophosphatidic acid (LPA) and choline., LPA acts on a series of G-protein coupled receptors (GPCRs) which are known as LPA receptors. This leads to the activation of multiple signaling cascades and various cellular responses. Six LPA receptors (LPA 1–6) have been known. They are expressed differently and possess distinct signaling properties. Cellular responses include differentiation, migration, proliferation and survival. Besides, inhibitory responses have also been reported., ATX-LPA signaling pathway is also associated with wound healing, inflammation, platelet aggregation as well as vascular and neuronal development. Deregulation of this pathway is implicated in a variety of pathological processes and diseases including cancer,, , , , , vascularization diseases, autoimmune diseases, fibrotic diseases, cardiovascular diseases and neurodegenerative diseases.,