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Crystallographic analysis depicting the interaction of the kinase inhibitor SCH772984 with ERK1/2 reveals a unique binding pocket distinct from off-targets such as haspin and is associated with slow binding kinetics and prolonged inhibitory activity. Activation of the ERK pathway is a hallmark of cancer, and targeting of upstream signaling partners led to the development of approved drugs. Recently, SCH772984 has been shown to be a selective and potent ERK1/2 inhibitor. Here we report the structural mechanism for its remarkable selectivity. In ERK1/2, SCH772984 induces a so-far-unknown binding pocket that accommodates the piperazine-phenyl-pyrimidine decoration. This new binding pocket was created by an inactive conformation of the phosphate-binding loop and an outward tilt of helix αC. In contrast, structure determination of SCH772984 with the off-target haspin and JNK1 revealed two canonical but distinct type I binding modes. Notably, the new binding mode with ERK1/2 was associated with slow binding kinetics in vitro as well as in cell-based assay systems. The described binding mode of SCH772984 with ERK1/2 enables the design of a new type of specific kinase inhibitors with prolonged on-target activity.

Non-steroidal anti-inflammatory drugs (NSAIDs) showed promising clinical efficacy toward COVID-19 (Coronavirus disease 2019) patients as potent painkillers and anti-inflammatory agents. However, the prospective anti-COVID-19 mechanisms of NSAIDs are not evidently exposed. Therefore, we intended to decipher the most influential NSAIDs candidate(s) and its novel mechanism(s) against COVID-19 by network pharmacology. FDA (U.S. Food & Drug Administration) approved NSAIDs (19 active drugs and one prodrug) were used for this study. Target proteins related to selected NSAIDs and COVID-19 related target proteins were identified by the Similarity Ensemble Approach, Swiss Target Prediction, and PubChem databases, respectively. Venn diagram identified overlapping target proteins between NSAIDs and COVID-19 related target proteins. The interactive networking between NSAIDs and overlapping target proteins was analyzed by STRING. RStudio plotted the bubble chart of the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis of overlapping target proteins. Finally, the binding affinity of NSAIDs against target proteins was determined through molecular docking test (MDT). Geneset enrichment analysis exhibited 26 signaling pathways against COVID-19. Inhibition of proinflammatory stimuli of tissues and/or cells by inactivating the RAS signaling pathway was identified as the key anti-COVID-19 mechanism of NSAIDs. Besides, MAPK8, MAPK10, and BAD target proteins were explored as the associated target proteins of the RAS. Among twenty NSAIDs, 6MNA, Rofecoxib, and Indomethacin revealed promising binding affinity with the highest docking score against three identified target proteins, respectively. Overall, our proposed three NSAIDs (6MNA, Rofecoxib, and Indomethacin) might block the RAS by inactivating its associated target proteins, thus may alleviate excessive inflammation induced by SARS-CoV-2.

Mitogen-activated protein kinases (MAPKs), important in a large array of signalling pathways, are tightly controlled by a cascade of protein kinases and by MAPK phosphatases (MKPs). MAPK signalling efficiency and specificity is modulated by protein–protein interactions between individual MAPKs and the docking motifs in cognate binding partners. Two types of docking interactions have been identified: D-motif-mediated interaction and FXF-docking interaction. Here we report the crystal structure of JNK1 bound to the catalytic domain of MKP7 at 2.4-Å resolution, providing high-resolution structural insight into the FXF-docking interaction. The 285 FNFL 288 segment in MKP7 directly binds to a hydrophobic site on JNK1 that is near the MAPK insertion and helix αG. Biochemical studies further reveal that this highly conserved structural motif is present in all members of the MKP family, and the interaction mode is universal and critical for the MKP-MAPK recognition and biological function. The important MAPK family of signalling proteins is controlled by MAPK phosphatases (MKPs). Here, the authors report the structure of MKP7 bound to JNK1 and characterise the conserved MKP-MAPK interaction.

The c‐jun N‐terminal kinase (JNK) signaling pathway is regulated by JNK‐interacting protein‐1 (JIP1), which is a scaffolding protein assembling the components of the JNK cascade. Overexpression of JIP1 deactivates the JNK pathway selectively by cytoplasmic retention of JNK and thereby inhibits gene expression mediated by JNK, which occurs in the nucleus. Here, we report the crystal structure of human JNK1 complexed with pepJIP1, the peptide fragment of JIP1, revealing its selectivity for JNK1 over other MAPKs and the allosteric inhibition mechanism. The van der Waals contacts by the three residues (Pro157, Leu160, and Leu162) of pepJIP1 and the hydrogen bonding between Glu329 of JNK1 and Arg156 of pepJIP1 are critical for the selective binding. Binding of the peptide also induces a hinge motion between the N‐ and C‐terminal domains of JNK1 and distorts the ATP‐binding cleft, reducing the affinity of the kinase for ATP. In addition, we also determined the ternary complex structure of pepJIP1‐bound JNK1 complexed with SP600125, an ATP‐competitive inhibitor of JNK, providing the basis for the JNK specificity of the compound.

c-Jun N-terminal kinase (JNK) is activated by dual phosphorylation of both threonine and tyrosine residues in the phosphorylation loop of the protein in response to several stress factors. However, the precise molecular mechanisms for activation after phosphorylation remain elusive. Here we show that Pin1, a peptidyl-prolyl isomerase, has a key role in the JNK1 activation process by modulating a phospho-Thr-Pro motif in the phosphorylation loop. Pin1 overexpression in human breast cancer cell lines correlates with increased JNK activity. In addition, small interfering RNA (siRNA) analyses showed that knockdown of Pin1 in a human breast cancer cell line decreased JNK1 activity. Pin1 associates with JNK1, and then catalyzes prolyl isomerization of the phospho-Thr-Pro motif in JNK1 from trans- to cis -conformation. Furthermore, Pin1 enhances the association of JNK1 with its substrates. As a result, Pin1 −/− cells are defective in JNK activation and resistant to oxidative stress. These results provide novel insights that, following stress-induced phosphorylation of Thr in the Thr-Pro motif of JNK1, JNK1 associates with Pin1 and undergoes conformational changes to promote the binding of JNK1 to its substrates, resulting in cellular responses from extracellular signals.

Biosensor-based fragment screening is a valuable tool in the drug discovery process. This method is advantageous over many biochemical methods because primary hits can be distinguished from non-specific or non-ideal interactions by examining binding profiles and responses, resulting in reduced false-positive rates. Biolayer interferometry (BLI), a technique that measures changes in an interference pattern generated from visible light reflected from an optical layer and a biolayer containing proteins of interest, is a relatively new method for monitoring small molecule interactions. The BLI format is based on a disposable sensor that is immersed in 96-well or 384-well plates. BLI has been validated for small molecule detection and fragment screening with model systems and well-characterized targets where affinity constants and binding profiles are generally similar to those obtained with surface plasmon resonsance (SPR). Screens with challenging targets involved in protein–protein interactions including BCL-2, JNK1, and eIF4E were performed with a fragment library of 6,500 compounds, and hit rates were compared for these targets. For eIF4E, a protein containing a PPI site and a nucleotide binding site, results from a BLI fragment screen were compared to results obtained in biochemical HTS screens. Overlapping hits were observed for the PPI site, and hits unique to the BLI screen were identified. Hit assessments with SPR and BLI are described.

Grateloupia lanceolata is a red alga native to coastal areas of East Asia. In this study, extract from G. lanceolata (EGL) was investigated for suppressive effects on lipopolysaccharide (LPS)-induced inflammatory responses in RAW 264.7 macrophages. EGL was found to have anti-inflammatory properties with the inhibition of nitric oxide (NO), pro-inflammatory cytokine production, and MAPK signaling in LPS-induced RAW 264.7 macrophages. Moreover, treatment of RAW 264.7 macrophage with EGL inhibited LPS-induced IL-1β production in a dose-dependent manner. These inhibitory effects were found with the blockage of p38 mitogen-activated protein kinases (MAPK), extracellular signal regulated kinases 1 and 2 (ERK1/2), and also c-Jun N-terminal kinases 1 and 2 (JNK1/2). These results indicated that anti-inflammatory actions of EGL in RAW 264.7 macrophages involved in the inhibition of LPS-induced p38MAPK/ERK/JNK signaling pathways. In addition, our findings suggest that EGL holds great promise for use in the treatment of various inflammatory diseases.

The posttranslational regulation of mammalian clock proteins has been assigned a time‐keeping function, but seems to have more essential roles. Here we show that c‐Jun N‐terminal kinase (JNK), identified by inhibitor screening of BMAL1 phosphorylation at Ser 520/Thr 527/Ser 592, confers dynamic regulation on the clock. Knockdown of JNK1 and JNK2 abrogates BMAL1 phosphorylation and lengthens circadian period in fibroblasts. Mice deficient for neuron‐specific isoform JNK3 have altered behavioural rhythms, with longer free‐running period and compromised phase shifts to light. The locomotor rhythms are insensitive to intensity variance of constant light, deviating from Aschoff's rule. Thus, JNK regulates a core characteristic of the circadian clock by controlling the oscillation speed and the phase in response to light. Deletion of neuron‐specific JNK in mice alters behavioural rhythms and phase shifts to light. JNK deletion also inhibits BMAL1 phosphorylation and lengthens circadian period, indicating that this kinase regulates a core aspect of the mammalian clock.

Dihydroartemisinin (DHA) and its analogs are reported to possess selective anticancer activity. Here, we reported a novel DHA derivative DHA-37 that exhibited more potent anticancer activity on the cells tested. Distinct from DHA-induced apoptosis, DHA-37 triggered excessive autophagic cell death, and became the main contributor to DHA-37-induced A549 cell death. Incubation of the cells with DHA-37 but not DHA produced increased dots distribution of GFP-LC3 and expression ratio of LC3-II/LC3-I, and enhanced the formation of autophagic vacuoles as revealed by TEM. Treatment with the autophagy inhibitor 3-MA, LY294002, or chloroquine could reverse DHA-37-induced cell death. In addition, DHA-37-induced cell death was associated significantly with the increased expression of HMGB1, and knockdown of HMGB1 could reverse DHA-37-induced cell death. More importantly, the elevated HMGB1 expression induced autophagy through the activation of the MAPK signal but not PI3K-AKT–mTOR pathway. In addition, DHA-37 also showed a wonderful performance in A549 xenograft mice model. These findings suggest that HMGB1 as a target candidate for apoptosis-resistant cancer treatment and artemisinin-based drugs could be used in inducing autophagic cell death.

The retinal pigment epithelium (RPE) is constantly exposed to external injuries which lead to degeneration, dysfunction or loss of RPE cells. The balance between RPE cells death and proliferation may be responsible for several diseases of the underlying retina, including age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR). Signaling pathways able to control cells proliferation or death usually involve the MAPK (mitogen-activated protein kinases) pathways, which modulate the activity of transcription factors by phosphorylation. UV exposure induces DNA breakdown and causes cellular damage through the production of reactive oxygen species (ROS) leading to programmed cell death. In this study, human retinal pigment epithelial cells ARPE19 were exposed to 100 J/m² of UV-C and MAPK pathways were studied. We first showed the expression of the three major MAPK pathways. Then we showed that activator protein-1 (AP-1) was activated through phosphorylation of cJun and cFos, induced by JNK and p38, respectively. Specific inhibitors of both kinases decreased their respective activities and phosphorylation of their nuclear targets (cJun and cFos) and reduced UV-induced cell death. The use of specific kinases inhibitors may provide excellent tools to prevent RPE apoptosis specifically in RPE diseases involving ROS and other stress-related compounds such as in AMD.