Metabolic networks are interconnected and influence diverse cellular processes. The protein-metabolite interactions that mediate these networks are frequently low affinity and challenging to systematically discover. We developed mass spectrometry integrated with equilibrium dialysis for the discovery of allostery systematically (MIDAS) to identify such interactions. Analysis of 33 enzymes from human carbohydrate metabolism identified 830 protein-metabolite interactions, including known regulators, substrates, and products as well as previously unreported interactions. We functionally validated a subset of interactions, including the isoform-specific inhibition of lactate dehydrogenase by long-chain acyl–coenzyme A. Cell treatment with fatty acids caused a loss of pyruvate-lactate interconversion dependent on lactate dehydrogenase isoform expression. These protein-metabolite interactions may contribute to the dynamic, tissue-specific metabolic flexibility that enables growth and survival in an ever-changing nutrient environment.

The activation of c-Jun N-terminal kinases (JNKs) plays an important role in physiological processes including neuronal function, immune activity, and development, and thus, JNKs have been a therapeutic target for various diseases such as neurodegenerative diseases, inflammation, and cancer. Efforts to develop JNK-specific inhibitors have been ongoing for several decades. In this process, the structures of JNK in complex with various inhibitors have contributed greatly to the design of novel compounds and to the elucidation of structure-activity relationships. Almost 100 JNK structures with various compounds have been determined. Here we summarize the information gained from these structures and classify the inhibitors into several groups based on the binding mode. These groups include inhibitors in the open conformation and closed conformation of the gatekeeper residue, non-ATP site binders, peptides, covalent inhibitors, and type II kinase inhibitors. Through this work, deep insight into the interaction of inhibitors with JNKs can be gained and this will be helpful for developing novel, potent, and selective inhibitors.

The c-Jun N-terminal kinases (JNKs) are implicated in many neuropathological conditions, including neurodegenerative diseases. To explore potential JNK3 inhibitors from the U.S. Food and Drug Administration-approved drug library, we performed structure-based virtual screening and identified azelastine (Aze) as one of the candidates. NMR spectroscopy indicated its direct binding to the ATP-binding site of JNK3, validating our observations. Although the antihistamine effect of Aze is well documented, the involvement of the JNK pathway in its action remains to be elucidated. This study investigated the effects of Aze on lipopolysaccharide (LPS)-induced JNK phosphorylation, pro-inflammatory mediators, and cell migration in BV2 microglial cells. Aze was found to inhibit the LPS-induced phosphorylation of JNK and c-Jun. It also inhibited the LPS-induced production of pro-inflammatory mediators, including interleukin-6, tumor necrosis factor-α, and nitric oxide. Wound healing and transwell migration assays indicated that Aze attenuated LPS-induced BV2 cell migration. Furthermore, Aze inhibited LPS-induced IκB phosphorylation, thereby suppressing nuclear translocation of NF-κB. Collectively, our data demonstrate that Aze exerts anti-inflammatory and anti-migratory effects through inhibition of the JNK/NF-κB pathway in BV2 cells. Based on our findings, Aze may be a potential candidate for drug repurposing to mitigate neuroinflammation in various neurodegenerative disorders, including Alzheimer’s and Parkinson’s diseases.

Fisetin (3,3′,4′,7-tetrahydroxyflavone) is a widely distributed natural flavonol. It interacts with albumin, and thereby generates a fluorescence signal quantitatively. Based on such optical characteristics, we postulated that fisetin was applicable to the quantitation of albumin as an indicator. To establish the fisetin-based albumin assay, we examined the optical properties of fisetin and fisetin–albumin complex. The assay conditions were fine-tuned to fit for the actual concentration of serum albumin and to generate an optimal signal with a high signal-to-background ratio. The reaction between fisetin and albumin was linear in a wide range of concentrations. Non-protein serum components did not interfere with the reaction. The reactivity of fisetin was apparently specific for albumin among serum proteins. Both plasma and serum were compatible with the assay. The samples could be stored in a refrigerator or a freezer without the loss of reactivity toward fisetin. The generation and decay rates of the signal were acceptable for manual handling. The recovery of fortified albumin in serum was confirmed and the assay was validated with human sera. Fisetin-based albumin assay is suitable for clinical laboratory testing, considering the simple and short procedure, high specificity and sensitivity, linearity over a wide range of albumin concentrations, and, presumably, potential automatability.