Cytotoxic and Anti-Plasmodial Activities of Stephania dielsiana Y.C. Wu Extracts and the Isolated Compounds.

Natural products remain a viable source of novel therapeutics, and as detection and extraction techniques improve, we can identify more molecules from a broader set of plant tissues. The aim of this study was an investigation of the cytotoxic and anti-plasmodial activities of the methanol extract from Stephania dielsiana Y.C. Wu leaves and its isolated compounds. Our study led to the isolation of seven alkaloids, among which oxostephanine (1) is the most active against several cancer cell lines including HeLa, MDA-MB231, MDA-MB-468, MCF-7, and non-cancer cell lines, such as 184B5 and MCF10A, with IC50 values ranging from 1.66 to 4.35 µM. Morever, oxostephanine (1) is on average two-fold more active against cancer cells than stephanine (3), having a similar chemical structure. Cells treated with oxostephanine (1) are arrested at G2/M cell cycle, followed by the formation of aneuploidy and apoptotic cell death. The G2/M arrest appears to be due, at least in part, to the inactivation of Aurora kinases, which is implicated in the onset and progression of many forms of human cancer. An in-silico molecular modeling study suggests that oxostephanine (1) binds to the ATP binding pocket of Aurora kinases to inactivate their activities. Unlike oxostephanine (1), thailandine (2) is highly effective against only the triple-negative MDA-MB-468 breast cancer cells. However, it showed excellent selectivity against the cancer cell line when compared to its effects on non-cancer cells. Furthermore, thailandine (2) showed excellent anti-plasmodial activity against both chloroquine-susceptible 3D7 and chloroquine-resistant W2 Plasmodium falciparum strains. The structure-activity relationship of isolated compound was also discussed in this study. The results of this study support the traditional use of Stephania dielsiana Y.C. Wu and the lead molecules identified can be further optimized for the development of highly effective and safe anti-cancer and anti-plasmodial drugs.

Metagenomics of Wastewater Influent from Wastewater Treatment Facilities across Ontario in the Era of Emerging SARS-CoV-2 Variants of Concern.

We report metagenomic sequencing analyses of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in composite wastewater influent from 10 regions in Ontario, Canada, during the transition between Delta and Omicron variants of concern. The Delta and Omicron BA.1/BA.1.1 and BA.2-defining mutations occurring in various frequencies were reported in the consensus and subconsensus sequences of the composite samples.

Lead optimization of novel quinolone chalcone compounds by a structure-activity relationship (SAR) study to increase efficacy and metabolic stability.

Many agents targeting the colchicine binding site in tubulin have been developed as potential anticancer agents. However, none has successfully made it to the clinic, due mainly to dose limiting toxicities and the emergence of multi-drug resistance. Chalcones targeting tubulin have been proposed as a safe and effective alternative. We have shown previously that quinolone chalcones target tubulin and maintain potent anti-proliferative activity vis-à-vis colchicine, while also having high tolerability and low toxicity in mouse models of cancer and refractivity to multi-drug resistance mechanisms. To identify the most effective anticancer chalcone compound, we synthesized 17 quinolone-chalcone derivatives based on our previously published CTR-17 and CTR-20, and then carried out a structure-activity relationship study. We identified two compounds, CTR-21 [((E)-8-Methoxy-3-(3-(2-methoxyphenyl)-3-oxoprop-1-enyl) quinolin-2(1H)-one)] and CTR-32 [((E)-3-(3-(2-ethoxyphenyl)-3-oxoprop-1-enyl) quinolin-2(1H)-one)] as potential leads, which contain independent moieties that play a significant role in their enhanced activities. At the nM range, CTR-21 and CTR-32 effectively kill a panel of different cancer cells originated from a variety of different tissues including breast and skin. Both compounds also effectively kill multi-drug resistant cancer cells. Most importantly, CTR-21 and CTR-32 show a high degree of selectivity against cancer cells. In silico, both of them dock near the colchicine-binding site with similar energies. Whereas both CTR-21 and CTR-32 effectively prevents tubulin polymerization, leading to the cell cycle arrest at G2/M, CTR-21 has more favorable metabolic properties. Perhaps not surprisingly, the combination of CTR-21 and ABT-737, a Bcl-2 inhibitor, showed synergistic effect in killing cancer cells, since we previously found the "parental" CTR-20 also exhibited synergism. Taken together, CTR-21 can potentially be a highly effective and relatively safe anticancer drug.

Genetic interaction network of the Saccharomyces cerevisiae type 1 phosphatase Glc7.

BACKGROUND: Protein kinases and phosphatases regulate protein phosphorylation, a critical means of modulating protein function, stability and localization. The identification of functional networks for protein phosphatases has been slow due to their redundant nature and the lack of large-scale analyses. We hypothesized that a genome-scale analysis of genetic interactions using the Synthetic Genetic Array could reveal protein phosphatase functional networks. We apply this approach to the conserved type 1 protein phosphatase Glc7, which regulates numerous cellular processes in budding yeast. RESULTS: We created a novel glc7 catalytic mutant (glc7-E101Q). Phenotypic analysis indicates that this novel allele exhibits slow growth and defects in glucose metabolism but normal cell cycle progression and chromosome segregation. This suggests that glc7-E101Q is a hypomorphic glc7 mutant. Synthetic Genetic Array analysis of glc7-E101Q revealed a broad network of 245 synthetic sick/lethal interactions reflecting that many processes are required when Glc7 function is compromised such as histone modification, chromosome segregation and cytokinesis, nutrient sensing and DNA damage. In addition, mitochondrial activity and inheritance and lipid metabolism were identified as new processes involved in buffering Glc7 function. An interaction network among 95 genes genetically interacting with GLC7 was constructed by integration of genetic and physical interaction data. The obtained network has a modular architecture, and the interconnection among the modules reflects the cooperation of the processes buffering Glc7 function. CONCLUSION: We found 245 genes required for the normal growth of the glc7-E101Q mutant. Functional grouping of these genes and analysis of their physical and genetic interaction patterns bring new information on Glc7-regulated processes.

Novel quinolone chalcones targeting colchicine-binding pocket kill multidrug-resistant cancer cells by inhibiting tubulin activity and MRP1 function.

Agents targeting colchicine-binding pocket usually show a minimal drug-resistance issue, albeit often associated with high toxicity. Chalcone-based compounds, which may bind to colchicine-binding site, are found in many edible fruits, suggesting that they can be effective drugs with less toxicity. Therefore, we synthesized and examined 24 quinolone chalcone compounds, from which we identified ((E)-3-(3-(2-Methoxyphenyl)-3-oxoprop-1-enyl) quinolin-2(1H)-one) (CTR-17) and ((E)-6-Methoxy-3-(3-(2-methoxyphenyl)-3-oxoprop-1-enyl) quinolin-2(1H)-one) (CTR-20) as promising leads. In particular, CTR-20 was effective against 65 different cancer cell lines originated from 12 different tissues, largely in a cancer cell-specific manner. We found that both CTR-17 and CTR-20 reversibly bind to the colchicine-binding pocket on ß-tubulin. Interestingly however, both the CTRs were highly effective against multidrug-resistant cancer cells while colchicine, paclitaxel and vinblastine were not. Our study with CTR-20 showed that it overcomes multidrug-resistance through its ability to impede MRP1 function while maintaining strong inhibition against microtubule activity. Data from mice engrafted with the MDA-MB-231 triple-negative breast cancer cells showed that both CTR-17 and CTR-20 possess strong anticancer activity, alone or in combination with paclitaxel, without causing any notable side effects. Together, our data demonstrates that both the CTRs can be effective and safe drugs against many different cancers, especially against multidrug-resistant tumors.

Cdk1-mediated phosphorylation of Cdc7 suppresses DNA re-replication.

To maintain genetic stability, the entire mammalian genome must replicate only once per cell cycle. This is largely achieved by strictly regulating the stepwise formation of the pre-replication complex (pre-RC), followed by the activation of individual origins of DNA replication by Cdc7/Dbf4 kinase. However, the mechanism how Cdc7 itself is regulated in the context of cell cycle progression is poorly understood. Here we report that Cdc7 is phosphorylated by a Cdk1-dependent manner during prometaphase on multiple sites, resulting in its dissociation from origins. In contrast, Dbf4 is not removed from origins in prometaphase, nor is it degraded as cells exit mitosis. Our data thus demonstrates that constitutive phosphorylation of Cdc7 at Cdk1 recognition sites, but not the regulation of Dbf4, prevents the initiation of DNA replication in normally cycling cells and under conditions that promote re-replication in G2/M. As cells exit mitosis, PP1α associates with and dephosphorylates Cdc7. Together, our data support a model where Cdc7 (de)phosphorylation is the molecular switch for the activation and inactivation of DNA replication in mitosis, directly connecting Cdc7 and PP1α/Cdk1 to the regulation of once-per-cell cycle DNA replication in mammalian cells.

Dynamic regulation of histone H3K9 is linked to the switch between replication and transcription at the Dbf4 origin-promoter locus.

The co-regulation of DNA replication and gene transcription is still poorly understood. To gain a better understanding of this important control mechanism, we examined the DNA replication and transcription using the Dbf4 origin-promoter and Dbf4 pseudogene models. We found that origin firing and Dbf4 transcription activity were inversely regulated in a cell cycle-dependent manner. We also found that proteins critical for the regulation of replication (ORC, MCM), transcription (SP1, TFIIB), and cohesin (Smc1, Smc3) and Mediator functions (Med1, Med12) interact with specific sites within and the surrounding regions of the Dbf4 locus in a cell cycle-dependent manner. As expected, replication initiation occurred within a nucleosome-depleted region, and nucleosomes flanked the 2 replication initiation zones. Further, the histone H3 in this region was distinctly acetylated or trimethylated on lysine 9 in a cell cycle-dependent fluctuation pattern: H3K9ac was most prevalent when the Dbf4 transcription level was highest whereas the H3K9me3 level was greatest during and just after replication. The KDM4A histone demethylase, which is responsible for the H3K9me3 modification, was enriched at the Dbf4 origin in a manner coinciding with H3K9me3. Finally, HP1γ, a protein known to interact with H3K9me3 in the heterochromatin was also found enriched at the origin during DNA replication, indicating that H3K9me3 may be required for the regulation of replication at both heterochromatin and euchromatin regions. Taken together, our data show that mammalian cells employ an extremely sophisticated and multilayered co-regulation mechanism for replication and transcription in a highly coordinated manner.

Same partners, different dance: involvement of DNA replication proteins in centrosome regulation.

Genome replication is the most fundamental element of the continuity of life. In eukaryotes, DNA replication is regulated by an elegant network of many different protein factors to ensure the timely and accurate copying of their entire genome once per cell cycle. The replication factors include the maintenance (MCM) proteins, Cdt1, Cdc6, Cdc7, Cdc45, and geminin. All of these proteins are involved in the regulation of DNA replication at the initiation step. Interestingly, recent studies have shown that some of these replication proteins also localize to the centrosome, often throughout the entire cell cycle. These centrosomally localized replication proteins appear to play essential roles in the regulation of centrosome biogenesis, suggesting that genome replication and segregation are regulated interdependently. In this review, we summarize and discuss the inter-dependent regulation played by some of the replication proteins.

The COMA complex is required for Sli15/INCENP-mediated correction of defective kinetochore attachments.

Before anaphase, kinetochores of sister chromatid (SC) pairs must be attached to microtubules emanating from opposing spindle poles. The conserved Aurora B kinase Ipl1 and its adaptor INCENP/Sli15 correct defective attachments and promote SC bi-orientation. Ame1 and Okp1 are essential components of the COMA sub-complex of the budding yeast central kinetochore, and Ame1 has been demonstrated to physically interact with Sli15. Here, we examine the significance of the Ame1-Sli15 interaction in vivo. We find Sli15 localization at kinetochores is reduced in the absence of Ame1. We demonstrate a role for Ame1 in the correction of defective attachments. While overexpression of OKP1 restores kinetochore localization of the mutant Ame1-4 protein, Sli15 localization is not restored and defective attachments are not corrected. Our findings reveal a new role for the central kinetochore in promoting Sli15 function, and suggest functional Ame1 is required for the correction of defective attachments by promoting the localization of Sli15/INCENP at kinetochores.

Spindle checkpoint maintenance requires Ame1 and Okp1.

Kinetochore proteins are required for high fidelity chromosome segregation and as a platform for checkpoint signaling. Ame1 is an essential component of the COMA (Ctf19, Okp1, Mcm21, Ame1) sub-complex of the central kinetochore of budding yeast. In this study, we describe the isolation and characterization of an Ame1 conditional mutant, ame1-4. ame1-4 cells exhibit chromosome segregation defects and Mad2-dependent cell cycle delay similar to okp1-5 cells. However, the viability of ame1-4 cells is markedly reduced relative to wild type and okp1-5 cells after three hours at restrictive temperature. To determine if ame1-4 cells enter anaphase with mis-segregated chromosomes, we monitored the localization of Bub3:VFP as a marker for anaphase onset. ame1-4 cells containing mis-segregated sister chromatids initially accumulate Bub3:VFP at kinetochores, indicating checkpoint activation and a metaphase arrest. Subsequently, Bub3:VFP de-localizes and cells reinitiate DNA duplication and budding without cytokinesis in the presence of un-segregated chromosomes. Overexpression of OKP1 in ame1-4 cells restores ame1-4 protein localization and a stable arrest. Based on our results, we propose that Ame1 and Okp1 are required for a sustained checkpoint arrest in the presence of mis-segregated chromosomes. Our results suggest that checkpoint response might be controlled not only at the level of activation but also via signals that ensure maintenance of the response.