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Microtubules play crucial role in process of mitosis and cell proliferation, which have been considered as attractive drug targets for anticancer therapy. The aim of this study was to discover novel and chemically diverse tubulin inhibitors for treatment of cancer. In this investigation, the multilayer virtual screening methods, including common feature pharmacophore model, structure-based pharmacophore model and molecular docking, were developed to screen BioDiversity database with 30,000 compounds. A total of 102 compounds were obtained by the virtual screening, and further filtered by diverse chemical clusters with desired properties and PAINS analysis. Finally, 50 compounds were selected and submitted to the biological evaluation. Among these hits, hits 8 and 30 with novel scaffolds displayed stronger antiproliferative activity on four human tumor cells including Hela, A549, MCF-7, and HepG2. Moreover, the two hits were subsequently submitted to molecular dynamic simulations of 90 ns with the aim of exploring the stability of ligand-protein interactions into the binding pocket, and further probing the mechanism of the interaction between tubulin and hits. The molecular dynamic simulation results revealed there had stronger interactions between tubulin and hits in equilibrium state. Therefore, the hits 8 and 30 have been well characterized as lead compounds for developing new tubulin inhibitors with potential anticancer activity.

In land plants, chloroplast transcripts undergo post-transcriptional modifications, including splicing, editing, trimming, etc., before translation, and a set of nuclear-encoded proteins regulate this essential step. In this study, we characterized a rice (Oryza sativa) seedling-lethal albino mutant sla4 from the progeny of tissue culture plants of the japonica cultivar Zhonghua 11. The sla4 mutant exhibited an albino phenotype from germination through the third-leaf stage, and then gradually died. The sla4 mutants lacked photosynthetic pigments and had severe defects in photosynthesis and early chloroplast development. Map-based cloning showed that a 13-bp deletion in the coding region of OsSLA4 on chromosome 7 resulted in the albino phenotype and albino mutants were also generated by knocking-out OsSLA4 in wild type with the CRISPR/Cas9 system. OsSLA4 encodes a chloroplast-localized pentatricopeptide repeat (PPR) protein with 15 PPR motifs and an atypical DYW-like motif. Loss-of-function of OsSLA4 resulted in severe defects in the intron splicing of atpF, ndhA, petB, rpl2, rpl16, rps12-2, and trnG, as well as a significant reduction in the transcript levels of chloroplast ribosomal RNAs and some chloroplast development- and photosynthesis-related genes. These results indicate that OsSLA4 is indispensable for early chloroplast development and seedling growth in rice, most likely acting by influencing the intron splicing of multiple chloroplast group II introns.

Background The large family of pentatricopeptide repeat (PPR) proteins is widely distributed among land plants. Such proteins play vital roles in intron splicing, RNA editing, RNA processing, RNA stability and RNA translation. However, only a small number of PPR genes have been identified in rice. Results In this study, we raised a mutant from tissue-culture-derived plants of Oryza sativa subsp. japonica ‘Zhonghua 11’, which exhibited a lethal chlorosis phenotype from germination to the third-leaf stage. The mutant was designated seedling-lethal chlorosis 1 (slc1). The slc1 mutant leaves showed extremely low contents of photosynthetic pigments and abnormal chloroplast development, and were severely defective in photosynthesis. Map-based cloning of OsSLC1 revealed that a single base (G) deletion was detected in the first exon of Os06g0710800 in the slc1 mutant, which caused a premature stop codon. Knockout and complementation experiments further confirmed that OsSLC1 is responsible for the seedling-lethal chlorosis phenotype in the slc1 mutant. OsSLC1 was preferentially expressed in green leaves, and encoded a chloroplast-localized PPR protein harboring 12 PPR motifs. Loss-of-function of OsSLC1 affected the intron splicing of multiple group II introns, and especially precluded the intron splicing of rps16, and resulted in significant increase in the transcript levels of 3 chloroplast ribosomal RNAs and 16 chloroplast development-related and photosynthesis-related genes, and in significant reduction in the transcript levels of 1 chloroplast ribosomal RNAs and 2 chloroplast development-related and photosynthesis-related genes. Conclusion We characterized a novel chloroplast-localized PPR protein, OsSLC1, which plays a vital role in the intron splicing of multiple group II introns, especially the rps16 intron, and is essential for early chloroplast development and seedling survival in rice.

Protein kinase casein kinase 2 (CK2), a member of the serine/threonine kinase family, has been established as one of the most attractive targets for molecularly targeted cancer therapy. The discovery of CK2 inhibitors has thus attracted much attention in recent years. In this investigation, a hybrid virtual screening approach based on Bayesian classification model, pharmacophore hypothesis and molecular docking was proposed and employed to identify CK2 inhibitors. We first established a naïve Bayes classification model of CK2 inhibitors/non-inhibitors and pharmacophore hypotheses of CK2 inhibitors. The docking parameters and scoring functions were also optimized in advance. The three virtual screening methods were sequentially used to screen two large chemical libraries, Specs and Enamine, for retrieving new CK2 inhibitors. Finally 30 compounds were selected from the final hits for in vitro CK2 kinase inhibitory assays. Five compounds with completely novel scaffolds showed a good inhibitory potency against CK2, which have good potentials for a future hit-to-lead optimization.

SKLB1028, a novel oral multikinase inhibitor of EGFR, FLT3 and Abl, displays exceptional activity in models of FLT3-driven AML and considerable potency in models of CML harboring Abl mutants

IKK2 (IκB kinase 2) inhibitors have been identified as potential drug candidates in the treatment of various immune/inflammatory disorders as well as cancer. So far more than one hundred small molecule inhibitors against IKK2 have been reported publicly. In this investigation, pharmacophore modeling was carried out to clarify the essential structure-activity relationship for the known IKK2 inhibitors. One of the established pharmacophore hypotheses, namely Hypo8, which has the best prediction ability to an external test data set, was suggested as a template for virtual screening. Evaluation of the performances of Hypo8 and a hybrid method (Hypo81docking) in virtual screening indicated that the use of the hybrid virtual screening considerably increased the hit rate and enrichment factor. The hybrid method was therefore adopted for screening several commercially available chemical databases, including Specs, NCI, Maybridge and Chinese Nature Product Database (CNPD), for novel potent IKK2 inhibitors. The hit compounds were subsequently subjected to filtering by Lipinski's rule of five. Finally some of the final hit compounds were selected and suggested for further experimental investigations.

Aldehydes are genotoxic and cytotoxic molecules and have received considerable attention for their associations with the pathogenesis of various human diseases. In addition, exposure to anthropogenic aldehydes increases human health risks. The general mechanism of aldehyde toxicity involves adduct formation with biomolecules such as DNA and proteins. Although the genotoxic effects of aldehydes such as mutations and chromosomal aberrations are directly related to DNA damage, the role of DNA damage in the cytotoxic effects of aldehydes is poorly understood because concurrent protein damage by aldehydes has similar effects. In this study, we have analysed how saturated and α,β-unsaturated aldehydes exert cytotoxic effects through DNA and protein damage. Interestingly, DNA repair is essential for alleviating the cytotoxic effect of weakly toxic aldehydes such as saturated aldehydes but not highly toxic aldehydes such as long α,β-unsaturated aldehydes. Thus, highly toxic aldehydes inactivate cells exclusively by protein damage. Our data suggest that DNA interstrand crosslinks, but not DNA-protein crosslinks and DNA double-strand breaks, are the critical cytotoxic DNA damage induced by aldehydes. Further, we show that the depletion of intracellular glutathione and the oxidation of thioredoxin 1 partially account for the DNA damage-independent cytotoxicity of aldehydes. On the basis of these findings, we have proposed a mechanistic model of aldehyde cytotoxicity mediated by DNA and protein damage.

Background: Many anticancer agents kill cancer cells by inducing lethal damage in DNA, but the capacity of DNA repair of cells reduce the therapeutic efficacy of anticancer agents. RuvB-like (RUVBL) 2 is part of large protein complexes such as TIP60 and INO80 that are involved in chromatin remodeling and DNA damage responses and repair. Relatively few studies have investigated the role of RUVBL2 in the survival of cells after exposure to anticancer agents. Methods: We depleted RUVBL2 in human MRC5-SV cells by small interfering (si) RNA and assessed the sensitivity of the cells to chemotherapeutic anticancer agents including cisplatin (cisPt), 2’-deoxy-5-azacytidine (azadC), and mitomycin C (MMC), and to physical DNA-damaging agents including X-rays. Results: The knockdown efficiency with 10 nM siRUVBL2 was 80% on day 3 post-transfection, and knockdown (>65%) persisted on day 6. The cell growth slowed significantly due to depletion of RUVBL2 when compared to mock- and control siRNA-treated cells, indicating that RUVBL2 is essential for the proliferation of cells. The RUVBL2-depleted cells were moderately sensitive to cisPt, azadC, and X-rays. The increase in the sensitivity to MMC was marginal. Conclusion: Depletion of RUVBL2 in cells confers moderate sensitivity to anticancer agents and X-rays, presumably through partial impairment of the homologous recombination repair of DNA double-strand break intermediates formed directly or indirectly by anticancer agents or X-rays. Further studies are necessary to clarify the exact role of RUVBL2 in this process.

The kinetics method of the substrate reaction during modification of enzyme activity previously described by Tsou (Adv. Enzymol. Related. Areas Mol. Biol., 1988, 61, 381-436) has been applied to study the kinetics of the course of inactivation of green crab alkaline phosphatase by phenylglyoxal. The obtained results shows that the inactivation of the enzyme by phenylglyoxal is a slow reversible reaction. The microscopic rate constants for the reaction of the inhibitor with the enzyme were determined. The presence of substrate offers marked protection of this enzyme against inactivation by phenylglyoxal. The above results suggest that the arginine residue is essential for activity and is situated at the active site of the enzyme.

Green crab (Scylla serrata) alkaline phosphatase (EC 3.1.3.1) is a metalloenzyme, which catalyzes the nonspecific hydrolysis of phosphate monoesters. The present paper deals with the study of the effect of some kinds of metal ions on the enzyme. The positive monovalent alkali metal ions (Li(+), Na(+) and K(+)) have no effect on the enzyme; positive bivalent alkaline-earth metal ions (Mg(2+), Ca(2+) and Ba(2+)) and transition metal ions (Mn(2+), Co(2+), Ni(2+) and Cd(2+)) activate the enzyme; heavy metal ions (Hg(2+), Ag(+), Bi(2+), Cu(2+) and Zn(2+)) inhibit the enzyme. The activation of magnesium ion on the enzyme appears to be a partial noncompetitive type. The kinetic model has been set up and a new plot to determine the activation constant of Mg(2+) was put forward. From the plot, we can easily determine the activation constant (K(a)) value and the activation ratio of Mg(2+) on the enzyme. The inhibition effects of Cu(2+) and Hg(2+) on the enzyme are of noncompetitive type. The inhibition constants have been determined. The inhibition effect of Hg(2+) is stronger than that of Cu(2+).

The toxicity of NO3- and NO2- to mammals has been widely publicized. However, the kinetic mechanism of inhibition of human muscle creatine kinase by NO3- and NO2- has not been explored. The kinetic theory of the substrate reaction during the modification of enzyme activity previously described by Tsou (Adv. Enzymol. Related Areas Mol. Biol. 1988, 61, 381-436) has been applied to a study of the kinetics of slow reversible inhibition of human muscle creatine kinase by planar anions (NO3- and NO2-). The kinetic equation of the substrate reaction was derived from theoretical analysis and experimental data, then simplified. The microscopic rate constants for the reaction of the inhibitors with the enzyme were obtained from the simplified equation for the substrate reaction in the presence of the inhibitors. The results show that the apparent forward rate constant A is dependent on ATP concentration, indicating competition between the inhibitor (NO3- or NO2-) and ATP. The results also suggest that binding of creatine-MgADP and the anion with the enzyme is very tight, since their binding constants are much higher than those for normal substrates.