Pharmacoinformatics-based investigation of bioactive compounds of Rasam (South Indian recipe) against human cancer.

Spice-rich recipes are referred to as "functional foods" because they include a variety of bioactive chemicals that have health-promoting properties, in addition to their nutritional value. Using pharmacoinformatics-based analysis, we explored the relevance of bioactive chemicals found in Rasam (a South Indian cuisine) against oxidative stress-induced human malignancies. The Rasam is composed of twelve main ingredients, each of which contains a variety of bioactive chemicals. Sixty-six bioactive compounds were found from these ingredients, and their structures were downloaded from Pubchem. To find the right target via graph theoretical analysis (mitogen-activated protein kinase 6 (MAPK6)) and decipher their signaling route, a network was built. Sixty-six bioactive compounds were used for in silico molecular docking study against MAPK6 and compared with known MAPK6 inhibitor drug (PD-173955). The top four compounds were chosen for further study based on their docking scores and binding energies. In silico analysis predicted ADMET and physicochemical properties of the selected compounds and were used to assess their drug-likeness. Molecular dynamics (MD) simulation modelling methodology was also used to analyse the effectiveness and safety profile of selected bioactive chemicals based on the docking score, as well as to assess the stability of the MAPK6-ligand complex. Surprisingly, the discovered docking scores against MAPK6 revealed that the selected bioactive chemicals exhibit varying binding ability ranges between - 3.5 and - 10.6 kcal mol-1. MD simulation validated the stability of four chemicals at the MAPK6 binding pockets, including Assafoetidinol A (ASA), Naringin (NAR), Rutin (RUT), and Tomatine (TOM). According to the results obtained, fifty of the sixty-six compounds showed higher binding energy (- 6.1 to - 10.6 kcal mol-1), and four of these compounds may be used as lead compounds to protect cells against oxidative stress-induced human malignancies.

Crystal Structure and Inhibitor Identifications Reveal Targeting Opportunity for the Atypical MAPK Kinase ERK3.

Extracellular signal-regulated kinase 3 (ERK3), known also as mitogen-activated protein kinase 6 (MAPK6), is an atypical member of MAPK kinase family, which has been poorly studied. Little is known regarding its function in biological processes, yet this atypical kinase has been suggested to play important roles in the migration and invasiveness of certain cancers. The lack of tools, such as a selective inhibitor, hampers the study of ERK3 biology. Here, we report the crystal structure of the kinase domain of this atypical MAPK kinase, providing molecular insights into its distinct ATP binding pocket compared to the classical MAPK ERK2, explaining differences in their inhibitor binding properties. Medium-scale small molecule screening identified a number of inhibitors, several of which unexpectedly exhibited remarkably high inhibitory potencies. The crystal structure of CLK1 in complex with CAF052, one of the most potent inhibitors identified for ERK3, revealed typical type-I binding mode of the inhibitor, which by structural comparison could likely be maintained in ERK3. Together with the presented structural insights, these diverse chemical scaffolds displaying both reversible and irreversible modes of action, will serve as a starting point for the development of selective inhibitors for ERK3, which will be beneficial for elucidating the important functions of this understudied kinase.

Biochemical, cellular and structural characterization of novel and selective ERK3 inhibitors.

Triazolo[4,5-d]pyrimidin-5-amines were identified from kinase selectivity screening as novel ERK3 inhibitors with sub-100 nanomolar potencies in a biochemical assay using MK5 as substrate and with an attractive kinase selectivity profile. ERK3 crystal structures clarified the inhibitor binding mode in the ATP pocket with impact on A-loop, GC-loop and αC-helix conformations suggesting a potential structural link towards MK5 interaction via the FHIEDE motif. The inhibitors also showed sub-100 nM potencies in a cellular ERK3 NanoBRET assay and with excellent correlation to the biochemical IC50s. This novel series provides valuable tool compounds to further investigate the biological function and activation mechanism of ERK3.

MicroRNA-138 inhibits proliferation and induces apoptosis of laryngeal carcinoma via targeting MAPK6.

OBJECTIVE: To explore whether microRNA-138 could regulate the incidence and progression of laryngeal carcinoma through modulating proliferation and apoptosis of laryngeal carcinoma cells via MAPK6. PATIENTS AND METHODS: MicroRNA-138 expression in laryngeal carcinoma tissues and paracancerous tissues were detected by qRT-PCR (Quantitative Real-Time Polymerase Chain Reaction). The regulatory effects of microRNA-138 on proliferation and apoptosis of laryngeal carcinoma cells were detected by colony formation assay and flow cytometry, respectively. Target gene of microRNA-138 was predicted by online software and verified by luciferase reporter gene assay. Corresponding plasmids of microRNA-138 and the target gene were constructed. Rescue experiments were conducted to explore the regulatory effect of microRNA-138 on the target gene. RESULTS: MicroRNA-138 was downregulated in laryngeal carcinoma tissues than that of paracancerous tissues. MicroRNA-138 knockdown resulted in increased proliferation and decreased apoptosis of laryngeal carcinoma cells. MAPK6 was predicted as the target gene of microRNA-138. Luciferase reporter gene assay further verified that MAPK6 could directly bind to microRNA-138. Both mRNA and protein levels of MAPK6 were downregulated after microRNA-13 overexpression in laryngeal carcinoma cells. Rescue experiment results indicated that increased proliferation and decreased apoptosis of laryngeal carcinoma cells resulted from microRNA-13 knockdown were partially reversed by MAPK6 overexpression. CONCLUSIONS: MicroRNA-138 is downregulated in laryngeal carcinoma patients. MicroRNA-138 knockdown promotes proliferation and inhibits apoptosis of laryngeal carcinoma cells via inhibiting MAPK6 expression.

The atypical mitogen-activated protein kinase ERK3 is essential for establishment of epithelial architecture.

Epithelia contribute to physical barriers that protect internal tissues from the external environment and also support organ structure. Accordingly, establishment and maintenance of epithelial architecture are essential for both embryonic development and adult physiology. Here, using gene knockout and knockdown techniques along with gene profiling, we show that extracellular signal-regulated kinase 3 (ERK3), a poorly characterized atypical mitogen-activated protein kinase (MAPK), regulates the epithelial architecture in vertebrates. We found that in Xenopus embryonic epidermal epithelia, ERK3 knockdown impairs adherens and tight-junction protein distribution, as well as tight-junction barrier function, resulting in epidermal breakdown. Moreover, in human epithelial breast cancer cells, inhibition of ERK3 expression induced thickened epithelia with aberrant adherens and tight junctions. Results from microarray analyses suggested that transcription factor AP-2α (TFAP2A), a transcriptional regulator important for epithelial gene expression, is involved in ERK3-dependent changes in gene expression. Of note, TFAP2A knockdown phenocopied ERK3 knockdown in both Xenopus embryos and human cells, and ERK3 was required for full activation of TFAP2A-dependent transcription. Our findings reveal that ERK3 regulates epithelial architecture, possibly together with TFAP2A.

Substrate-Trapped Interactors of PHD3 and FIH Cluster in Distinct Signaling Pathways.

Amino acid hydroxylation is a post-translational modification that regulates intra- and inter-molecular protein-protein interactions. The modifications are regulated by a family of 2-oxoglutarate- (2OG) dependent enzymes and, although the biochemistry is well understood, until now only a few substrates have been described for these enzymes. Using quantitative interaction proteomics, we screened for substrates of the proline hydroxylase PHD3 and the asparagine hydroxylase FIH, which regulate the HIF-mediated hypoxic response. We were able to identify hundreds of potential substrates. Enrichment analysis revealed that the potential substrates of both hydroxylases cluster in the same pathways but frequently modify different nodes of signaling networks. We confirm that two proteins identified in our screen, MAPK6 (Erk3) and RIPK4, are indeed hydroxylated in a FIH- or PHD3-dependent mechanism. We further determined that FIH-dependent hydroxylation regulates RIPK4-dependent Wnt signaling, and that PHD3-dependent hydroxylation of MAPK6 protects the protein from proteasomal degradation.

MAP Kinase 6-mediated activation of vacuolar processing enzyme modulates heat shock-induced programmed cell death in Arabidopsis.

• Vacuolar processing enzyme (VPE), a cysteine protease, has been intensively studied in plant hypersensitive cell death, but the role and molecular mechanism of VPEs in response to abiotic stresses remain unclear. This work investigated the involvement of VPEs in Arabidopsis response to heat stress. • Under heat shock (HS), Arabidopsis VPE activity and the transcript level of γVPE were both upregulated, and γVPE deficiency suppressed vacuolar disruption and delayed caspase-3-like activation in HS-induced programmed cell death (PCD). Moreover, the change of VPE activity generally paralleled the alteration of caspase-1-like activity under HS treatment, indicating that HS-induced VPE activity might exhibit the caspase-1-like activity. • Further studies showed that MAP Kinase 6 (MPK6) activity was increased after HS treatment, and experiments with inhibitors and mutants suggested that MPK6 was responsible for the γVPE activation after HS treatment. In response to HS stress, reactive oxygen species (ROS) production, increase of cytoplasmic calcium concentration ([Ca(2+) ](cyt)) and the upregulation of calmodulin 3 (CaM3) transcript level occurred upstream of MPK6 activation. • Our results suggested that activation of Arabidopsis γVPE was mediated by MPK6 and played an important role in HS-induced Arabidopsis PCD, providing new insight into the mechanistic study of plant VPEs.

Effects of epigallocatechin-3-gallate on rat retinal ganglion cells after optic nerve axotomy.

The purpose of this study was to investigate the effects of epigallocatechin-3-gallate (EGCG) in axotomized eyes and the pathways related to its action. Wistar rats received intracranial optic nerve (ON) axotomy 2 mm behind the globe in left eyes, whereas right eyes received sham operations. EGCG was administrated via intraperitoneal injection 30 min before and 4 days after axotomy. The density of retinal ganglion cell (RGC) was examined by a retrograde labeling technique. Western blot analysis was used to assess the expression of neuronal nitric oxide synthase (nNOS), Bax, Bcl-2, ERK and Akt. Optic nerve axotomy caused 54% RGC loss 7 days following surgery, and EGCG treatment reduced RGC loss by 12% (P = 0.017). The expression of the nNOS and pro-apoptotic Bax proteins were increased 5 days after axotomy, while EGCG treatment significantly blunted the up-regulation of the above two proteins (P = 0.04 and 0.02, respectively). Axotomy-induced p-ERK 1/2 and p-Akt proteins expression 5 days and 3 days following injury, respectively. Treatment with EGCG further enhanced p-ERK 1/2 and p-Akt expressions after axotomy. Inhibition of ERK and Akt pathways attenuated the protection of EGCG on RGC against axotomy damage. Thus, we demonstrated that administration of EGCG prior to axotomy promotes RGC survival. The neuroprotective capacity of EGCG appears to act through mediating nitric oxide, anti-apoptotic, and cell survival signaling pathways.

Identification of extracellular signal-regulated kinase 1/2 and p38 MAPK as regulators of human sperm motility and acrosome reaction and as predictors of poor spermatozoan quality.

Mature spermatozoa acquire progressive motility only after ejaculation. Their journey in the female reproductive tract also includes suppression of progressive motility, reactivation, capacitation, and hyperactivation of motility (whiplash), the mechanisms of which are obscure. MAPKs are key regulatory enzymes in cell signaling, participating in diverse cellular functions such as growth, differentiation, stress, and apoptosis. Here we report that ERK1/2 and p38 MAPK are primarily localized to the tail of mature human spermatozoa. Surprisingly, c-Jun N-terminal kinase 1/2, which is thought to be ubiquitously expressed, could not be detected in mature human spermatozoa. ERK1/2 stimulation is downstream to protein kinase C (PKC) activation, which is also present in the human sperm tail (PKCbetaI and PKCepsilon). ERK1/2 stimulates and p38 inhibits forward and hyperactivated motility, respectively. Both ERK1/2 and p38 MAPK are involved in the acrosome reaction. Using a proteomic approach, we identified ARHGAP6, a RhoGAP, as an ERK substrate in PMA-stimulated human spermatozoa. Inverse correlation was obtained between the relative expression level of ERK1 or the relative activation level of p38 and sperm motility, forward progression motility, sperm morphology, and viability. Therefore, increased expression of ERK1 and activated p38 can predict poor human sperm quality.