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[Image: see text] Glycogen synthase kinase-3β (GSK3β) is involved in many pathological conditions and represents an attractive drug target. We previously reported dual GSK3β/p38α mitogen-activated protein kinase inhibitors and identified N-(4-(4-(4-fluorophenyl)-2-methyl-1H-imidazol-5-yl)pyridin-2-yl)cyclopropanecarboxamide (1) as a potent dual inhibitor of both target kinases. In this study, we aimed to design selective GSK3β inhibitors based on our pyridinylimidazole scaffold. Our efforts resulted in several novel and potent GSK3β inhibitors with IC(50) values in the low nanomolar range. 5-(2-(Cyclopropanecarboxamido)pyridin-4-yl)-4-cyclopropyl-1H-imidazole-2-carboxamide (6g) displayed very good kinase selectivity as well as metabolical stability and inhibited GSK3β activity in neuronal SH-SY5Y cells. Interestingly, we observed the importance of the 2-methylimidazole’s tautomeric state for the compound activity. Finally, we reveal how this crucial tautomerism effect is surmounted by imidazole-2-carboxamides, which are able to stabilize the binding via enhanced water network interactions, regardless of their tautomeric state.

In small molecule binding, water is not a passive bystander but rather takes an active role in the binding site, which may be decisive for the potency of the inhibitor. Here, by addressing a high-energy water, we improved the IC50 value of our co-crystallized glycogen synthase kinase-3β (GSK-3β) inhibitor by nearly two orders of magnitude. Surprisingly, our results demonstrate that this high-energy water was not displaced by our potent inhibitor (S)-3-(3-((7-ethynyl-9H-pyrimido[4,5-b]indol-4-yl)(methyl)amino)piperidin-1-yl)propanenitrile ((S)-15, IC50 value of 6 nM). Instead, only a subtle shift in the location of this water molecule resulted in a dramatic decrease in the energy of this high-energy hydration site, as shown by the WaterMap analysis combined with microsecond timescale molecular dynamics simulations. (S)-15 demonstrated both a favorable kinome selectivity profile and target engagement in a cellular environment and reduced GSK-3 autophosphorylation in neuronal SH-SY5Y cells. Overall, our findings highlight that even a slight adjustment in the location of a high-energy water can be decisive for ligand binding.

Glycogen synthase kinase-3&beta, (GSK-3&beta, ) is a potential target in the field of Alzheimer&rsquo, s disease drug discovery. We recently reported a new class of 9H-pyrimido[4,5-b]indole-based GSK-3&beta, inhibitors, of which 3-(3-((7-chloro-9H-pyrimido[4,5-b]indol-4-yl)(methyl)amino)piperidin-1-yl)propanenitrile (1) demonstrated promising inhibitory potency. However, this compound underwent rapid degradation by human liver microsomes. Starting from 1, we prepared a series of amide-based derivatives and studied their structure&ndash, activity relationships against GSK-3&beta, supported by 1 &micro, s molecular dynamics simulations. The biological potency of this series was substantially enhanced by identifying the eutomer configuration at the stereocenter. Moreover, the introduction of an amide bond proved to be an effective strategy to eliminate the metabolic hotspot. The most potent compounds, (R)-3-(3-((7-chloro-9H-pyrimido[4,5-b]indol-4-yl)(methyl)amino)piperidin-1-yl)-3-oxopropanenitrile ((R)-2) and (R)-1-(3-((7-bromo-9Hpyrimido[4,5-b]indol-4-yl)(methyl)amino)piperidin-1-yl)propan-1-one ((R)-28), exhibited IC50 values of 480 nM and 360 nM, respectively, and displayed improved metabolic stability. Their favorable biological profile is complemented by minimal cytotoxicity and neuroprotective properties.