Synthesis and Molecular Docking Studies of N,N-Dimethyl Arylpyranopyrimidinedione Derivatives

The synthesis of N,N-dimethyl arylpyranopyrimidinedione derivatives from aromatic aldehydes, N-methyl-1-(methylthio)-2-nitroethamine (NMSM) and 1,3-dimethyl barbituric acid, in the presence of piperidine as a catalyst, is reported. The reaction mechanism involves a Knoevenagel condensation, followed by Michael addition and intramolecular O-cyclization reaction sequence. The synthesized compounds were docked with human kinesin Eg5 protein to calculate binding energy, inhibition constant and H-bond interaction. All the compounds show good binding affinity towards the protein, with significant docking score.

Exploring the N1 Position of Biginelli Compounds: New Insights and Trends for Chemical Diversity Generation of Bioactive Derivatives

: Dihydropyrimidinones (DHPMs) are heterocycles obtained by the multicomponent Biginelli reaction. Recently, new synthetic protocols have allowed us to explore functionalisation at less explored positions of DHPMs, such as the N1 position. In this context, we have performed a full literature survey of N1-substituted DHPMs. We analysed 27 papers and identified 379 compounds with substituents at the N1 position, most of them with alkyl groups, and of 28% with aromatic substituents attached at the N1 position. N1-substituted DHPMs are explored mainly due to their effects on cancer cell proliferation via numerous targets, such as kinesin Eg5, heat shock protein 70, heat shock protein 90 and the epidermal growth factor receptor. Similarity analyses were performed using the data of 379 DHPMs from different cheminformatic approaches, i.e. chemical property correlations, principal component analysis, similarity networks and compound clustering.

Eg5 as a Prognostic Biomarker and Potential Therapeutic Target for Hepatocellular Carcinoma

Background: The kinesin Eg5, a mitosis-associated protein, is overexpressed in many cancers. Here we explored the clinical significance of Eg5 in hepatocellular carcinoma (HCC). Methods: HCC tissues from surgical resection were collected. Total RNA was prepared from tumorous and nontumorous parts. Eg5 expression levels were correlated with overall survival (OS) and disease-free survival (DFS). In vitro efficacy of LGI-147, a specific Eg5 inhibitor, was tested in HCC cell lines. In vivo efficacy of Eg5 inhibition was investigated in a xenograft model. Results: A total of 108 HCC samples were included. The patients were divided into three tertile groups with high, medium, and low Eg5 expression levels. OS of patients with low Eg5 expression was better than that of patients with medium and high Eg5 expression (median, 155.6 vs. 75.3 vs. 57.7 months, p = 0.002). DFS of patients with low Eg5 expression was also better than that of patients with medium and high Eg5 expression (median, 126.3 vs. 46.2 vs. 39.4 months, p = 0.001). In multivariate analyses, the associations between Eg5 expression and OS (p < 0.001) or DFS remained (p < 0.001). LGI-147 reduced cell growth via cell cycle arrest and apoptosis and induced accumulation of abnormal mitotic cells. In the xenograft model, the tumor growth rate under LGI-147 treatment was significantly slower than under the control. Conclusion: High Eg5 expression was associated with poor HCC prognosis. In vitro and in vivo evidence suggests that Eg5 may be a reasonable therapeutic target for HCC.

Novel photochromic inhibitor for mitotic kinesin Eg5 which forms multiple isomerization states

The mitotic kinesin Eg5 is a plus-end directed homotetrameric molecular motor essential for the formation of bipolar spindles during cell division. Kinesin Eg5 is overexpressed in cancer cells and hence considered as a target for cancer therapy; the inhibitors specific for Eg5 have been developed as anticancer drugs. In this study, we synthesized a novel functional photoresponsive inhibitor composed of spiropyran and azobenzene derivatives to control Eg5 function with multistage inhibitory activity accompanied by the formation of different isomerization states. The photochromic inhibitor spiropyran-sulfo-azobenzene (SPSAB) exhibited three isomerization states: spiro (SP)-trans, merocyanine (MC)-cis and MC-trans, upon exposure to visible light, ultraviolet and in the dark, respectively. SPSAB-induced reversible changes in the inhibitory activity of ATPase and motor activities correlating with photoisomerization among the three states. Among the three isomerization states of SPSAB, the SP-trans isomer showed potent inhibitory activity at an IC50 value of 30 µM in the basal ATPase assay. MC-trans and MC-cis exhibited less inhibitory activity at IC50 values of 38 and 86 µM, respectively. The results demonstrated that the novel photochromic inhibitor enabled precise control of Eg5 function at three different levels using light irradiation.

Evaluating Phenyl Propanoids Isolated from Citrus medica as Potential Inhibitors for Mitotic kinesin Eg5

Background: Human mitotic kinesins play an essential role in mitotic cell division. Targeting the spindle separation phase of mitosis has gained much attention in cancer chemotherapy. Spindle segregation is carried out mainly by the kinesin, Eg5. Many Eg5 inhibitors are in different phases of clinical trials as cancer drugs. This enzyme has two allosteric binding sites to which the inhibitors can bind. The first site is formed by loop L5, helix α2 and helix α3 and all the current drug candidates bind un-competitively to this site with ATP/ADP. The second site, formed by helix α4 and helix α6, which has gained attention recently, has not been explored well. Some inhibitors that bind to this site are competitive, while others are uncompetitive to ATP/ADP. Phenylpropanoids are pharmacologically active secondary metabolites. Methods: In this study, we have evaluated fourteen phenyl propanoids extracted from Citrus medica for inhibitory activity against human mitotic kinesin Eg5 in vitro steady-state ATPase assay. Ther interactions and stability using molecular docking and molecular dynamics simulations. Results and Discussions: Of the fourteen compounds tested, naringin and quercetin showed good activity with IC50 values in the micromolar range. Molecular docking studies of these complexes showed that both the molecules interact with the key residues of the active site predominantly thorough hydrophobic & aromatic π–π interactions consistent with the known inhibitors. Besides, these molecules also form hydrogen bonding interactions stabilizing the complexes. Molecular dynamics simulations of these complexes confirm the stability of these interactions. Conclusion: These results can be used as a strong basis for further modification of these compounds to design new inhibitors with higher potency using structure-based drug design.

Role of Eg5 in Prognosis Prediction and Treatment of Hepatocellular Carcinoma

Background: The kinesin Eg5, a mitosis-associated protein, is overexpressed in many cancers. Here we explored the clinical significance of Eg5 in HCC.Methods: HCC tissues from surgical resection were collected. Total RNA was prepared from tumorous and nontumorous parts. Eg5 expression levels were correlated with overall survival (OS) and disease-free survival (DFS). In vitro efficacy of LGI-147, a specific Eg5 inhibitor, was tested in HCC cell lines. In vivo efficacy of Eg5 inhibition was investigated in a xenograft model.Results: A total of 108 HCC samples were included. The patients were divided into three tertile groups with high, medium, and low Eg5 expression levels. OS of patients with low Eg5 expression was better than that of patients with medium and high Eg5 expression (median, 155.6 vs. 75.3 vs. 57.7 months, p = 0.002). DFS of patients with low Eg5 expression was also better than that of patients with medium and high Eg5 expression (median, 126.3 vs. 46.2 vs. 39.4 months, p = 0.001). In multivariate analyses, the associations between Eg5 expression and OS (p < 0.001) or DFS remained (p < 0.001). LGI-147 reduced cell growth via cell cycle arrest and apoptosis and induced accumulation of abnormal mitotic cells. In the xenograft model, the tumor growth rate under LGI-147 treatment was significantly slower than the control.Conclusion: High Eg5 expression was associated with poor HCC prognosis. In vitro and in vivo evidence suggests that Eg5 may be a reasonable therapeutic target for HCC.

Lead Generation for Human Mitotic Kinesin Eg5 using Structure-based Virtual Screening and Validation by in vitro and Cell-based Assays

Background: Human mitotic kinesins play a crucial role in mitotic cell division. Targeting the spindle separation phase of mitosis has gained much attention pharmaceutically in cancer chemotherapy. Spindle segregation is carried out mainly by Eg5 kinesin, and currently, it has many inhibitors in different phases of clinical trials. All the current drug candidates bind un-competitively with ATP/ADP at allosteric site 1 (formed by loop L5, helix α2 and helix α3). Recent experiments show that inhibitors that bind to the site 2 (formed by helix α4 and helix α6) are either competitive or uncompetitive to ATP/ADP. Objectives: To identify suitable lead compounds that target the mitotic kinesin Eg5, using in silico screening and their validation using in vitro and cell-based assays. Methodology: We have screened for potential inhibitors for human Eg5 (kinesin-5) through structure-based virtual screening and validated the top-scoring compounds using steady-state ATPase assay, differential scanning fluorimetry and microscale thermophoresis. The anticancer activity of the compounds was evaluated in the epithelial (A549) and chronic myelogenous leukaemia (K562) cancer cell lines. A known strong binding inhibitor S-trityl-L-cystine is used as a reference compound. Results & Conclusion: Of the many compounds tested, MM01 and MM03 showed good cell-based activity against the cancer cell lines A549 and K562 and can be further studied in animal models.

New pharmacological findings linked to biphenyl DHPMs, kinesin Eg5 ligands: anticancer and antioxidant effects

Background: Dihydropyrimidin-2-thiones (DHPMs) are a class of heterocyclic compound which have been intensively investigated mainly due to their anticancer activity as kinesin Eg5 inhibitors. Materials & methods: A library of N1 aryl substituted DHPMs were tested against glioma and bladder cancer cell lines. Quantitative structure–activity relationship (QSAR) investigation was performed in order to identify key elements of DHPMs linked with their antiproliferative effect. The toxicity of most active compounds was investigated using Caenorhabditis elegans as the model. Results & conclusion: DHPMs 9, 13 and 17 have been identified as having improved activity against glioma and bladder cell lines as compared with monastrol. Flow cytometry investigations showed that the new compounds induce cell cycle arrest in phase G2/M and cell death by apoptosis. In addition, compound 13 was able to modulate the reactive oxygen species production in vivo in C. elegans. The biphenyl dihydropyrimidinthiones provided a safety profile in C. elegans.