Protein kinases regulate protein phosphorylation, which are involved in fundamental cellular processes such as inflammatory response. In this study, we discovered a novel multi-protein kinase inhibitor, KMU-1170, a derivative of indolin-2-one, and investigated the mechanisms of its inflammation-inhibiting signaling in both THP-1 cells and human osteoarthritic fibroblast-like synoviocytes (FLS). We demonstrated that in THP-1 cells, KMU-1170 inhibited lipopolysaccharide (LPS)-induced upregulation of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), and, furthermore, suppressed LPS-induced phosphorylation of transforming growth factor-β-activated kinase 1, JNK, ERK, inhibitor of NF-κB kinase α/β (IKKα/β), and NF-κB p65 as well as nuclear translocation of NF-κB p65. Moreover, KMU-1170 suppressed LPS-induced upregulation of proinflammatory cytokines such as IL-1β, TNF-α, and IL-6, and, notably, inhibited LPS-induced upregulation of the NLRP3 inflammasome in THP-1 cells. Importantly, KMU-1170 attenuated LPS-mediated inflammatory responses in human osteoarthritic FLS, such as the upregulation of IL-1β, TNF-α, IL-6, iNOS, and COX-2 and the phosphorylation of IKKα/β and NF-κB p65. Collectively, these results suggest that KMU-1170 inhibits inflammatory signal transduction and could be developed as a potential anti-inflammatory agent.

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Pim proteins consisting of three isoforms (Pim-1, Pim-2, and Pim-3) are a family of serine/threonine kinases that regulate fundamental cellular responses such as cell growth, differentiation, and apoptosis. Overexpression of the Pim kinases has been linked to a wide variety of hematological and solid tumors. Thus, all three Pim kinases have been studied as promising targets for anticancer therapy. Here, we report on the development and optimization of an immobilized metal ion affinity partitioning (IMAP) fluorescence polarization (FP) method for Pim kinases. In this homogeneous 384-well assay method, fluorescein-labeled phosphopeptides are captured on cationic nanoparticles through interactions with immobilized trivalent metals, resulting in high polarization values. The apparent K_m values for adenosine triphosphate (ATP) were determined to be 45 ± 7, 6.4 ± 2, and 29 ± 5 μM for Pim-1, Pim-2, and Pim-3, respectively. The assay yielded robustness with Z′-factors of >0.75 and low day-to-day variability (CV <5%) for all three Pim kinases. The IMAP FP assay was further validated by determining IC₅₀ values for staurosporine and a known Pim inhibitor. We have also used an IMAP FP assay to examine whether compound 1, an ATP mimetic inhibitor designed through structure-based drug design, is indeed an ATP-competitive inhibitor of Pim kinases. Kinetic analysis based on Lineweaver–Burk plots showed that the inhibition mechanism of compound 1 is ATP competitive against all three Pim isoforms. The optimized IMAP assay for Pim kinases not only allows for high-throughput screening but also facilitates the characterization of novel Pim inhibitors for drug development.

Head and neck squamous cell carcinoma (HNSCC) is the 11th leading cancer by incidence worldwide. Surgery and radiotherapy have been the major treatment for patients with HNSCC while chemotherapy has become an important treatment option for locally advanced HNSCC. Understanding of the molecular mechanisms underlying HNSCC impelled the development of targeted therapeutic agents. The development and combinations of targeted therapies in different cellular pathways may be needed to fulfill the unmet needs of current HNSCC chemotherapy.