ABSTRACT
The Mnk-eIF4E axis plays a crucial role in tumor development, and inhibiting Mnk kinases is a promising approach for cancer therapy. Starting with fragment WS23, a series of 4-(indolin-1-yl)-6-substituted-pyrido[3,2-d]pyrimidine derivatives were designed and synthesized. Among these derivatives, compound 15b showed the highest potency with IC50 values of 0.8 and 1.5 nM against Mnk1 and Mnk2, respectively. Additionally, it demonstrated good selectivity among 30 selected kinases. 15b significantly suppressed MOLM-13 and K562 cell lines growth and caused cell cycle arrest. Furthermore, the Western blot assay revealed that 15b effectively downregulated the downstream proteins p-eIF4E, Mcl-1, and c-myc. Additionally, 15b exhibited remarkable stability in rat plasma and rat and human microsomes. In vivo anti-tumor activity study suggested that treatment with 15b suppressed tumor growth in LL/2 syngeneic models. These findings highlight the potential of 15b as a novel and potent Mnks inhibitor, which deserves further investigation.
Subject(s)
Antineoplastic Agents , Cell Proliferation , Drug Design , Intracellular Signaling Peptides and Proteins , Protein Kinase Inhibitors , Protein Serine-Threonine Kinases , Pyrimidines , Humans , Pyrimidines/pharmacology , Pyrimidines/chemistry , Pyrimidines/chemical synthesis , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Cell Proliferation/drug effects , Rats , Structure-Activity Relationship , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/metabolism , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Intracellular Signaling Peptides and Proteins/metabolism , Molecular Structure , Drug Screening Assays, Antitumor , Dose-Response Relationship, Drug , Cell Line, Tumor , Mice , Neoplasms, Experimental/drug therapy , Neoplasms, Experimental/pathology , Neoplasms, Experimental/metabolismABSTRACT
Macleaya cordata is a dominant plant of mine tailings and a zinc (Zn) accumulator with high Zn tolerance. In this study, M. cordata seedlings cultured in Hoagland solution were treated with 200 µmol·L-1 of Zn for 1 day or 7 days, and then, their leaves were taken for a comparative analysis of the transcriptomes and proteomes between the leaves of the control and Zn treatments. Differentially expressed genes included those that were iron (Fe)-deficiency-induced, such as vacuolar iron transporter VIT, ABC transporter ABCI17 and ferric reduction oxidase FRO. Those genes were significantly upregulated by Zn and could be responsible for Zn transport in the leaves of M. cordata. Differentially expressed proteins, such as chlorophyll a/b-binding proteins, ATP-dependent protease, and vacuolar-type ATPase located on the tonoplast, were significantly upregulated by Zn and, thus, could be important in chlorophyll biosynthesis and cytoplasm pH stabilization. Moreover, the changes in Zn accumulation, the production of hydrogen peroxide, and the numbers of mesophyll cells in the leaves of M. cordata were consistent with the expression of the genes and proteins. Thus, the proteins involved in the homeostasis of Zn and Fe are hypothesized to be the keys to the tolerance and accumulation of Zn in M. cordata. Such mechanisms in M. cordata can suggest novel approaches to genetically engineering and biofortifying crops.
ABSTRACT
BACKGROUND: Macleaya cordata is a traditional medicinal herb, and it has high tolerance and accumulation ability to heavy metals, which make it a good candidate species for studying phytoremediation. The objectives of this study were to investigate response and tolerance of M. cordata to lead (Pb) toxicity based on comparative analysis of transcriptome and proteome. RESULTS: In this study, the seedlings of M. cordata cultured in Hoagland solution were treated with 100 µmol·L- 1 Pb for 1 day (Pb 1d) or 7 days (Pb 7d), subsequently leaves of M. cordata were taken for the determination of Pb accumulation and hydrogen peroxide production (H2O2), meanwhile a total number of 223 significantly differentially expressed genes (DEGs) and 296 differentially expressed proteins (DEPs) were screened between control and Pb treatments. The results showed leaves of M. cordata had a special mechanism to maintain Pb at an appropriate level. Firstly, some DEGs were iron (Fe) deficiency-induced transporters, for example, genes of vacuolar iron transporter and three ABC transporter I family numbers were upregulated by Pb, which can maintain Fe homeostasis in cytoplasm or chloroplast. In addition, five genes of calcium (Ca2+) binding proteins were downregulated in Pb 1d, which may regulate cytoplasmic Ca2+ concentration and H2O2 signaling pathway. On the other hand, the cysteine synthase upregulated, glutathione S-transferase downregulated and glutathione reductase downregulated in Pb 7d can cause reduced glutathione accumulation and decrease Pb detoxification in leaves. Furthermore, DEPs of eight chlorophyll a/b binding proteins, five ATPases and eight ribosomal proteins can play a pivotal role on chloroplast turnover and ATP metabolism. CONCLUSIONS: Our results suggest that the proteins involved in Fe homeostasis and chloroplast turnover in mesophyll cells may play key roles in tolerance of M. cordata to Pb. This study offers some novel insights into Pb tolerance mechanism of plants, and the potential valuable for environmental remediation of this important medicinal plant.
Subject(s)
Hydrogen Peroxide , Lead , Lead/toxicity , Chlorophyll A , ATP-Binding Cassette Transporters , Adenosine TriphosphatasesABSTRACT
CRISPR-mediated genome editing has become a powerful tool for the genetic modification of biological traits. However, developing an efficient, site-specific, gene knock-in system based on homology-directed DNA repair (HDR) remains a significant challenge in plants, especially in woody species like poplar. Here, we show that simultaneous inhibition of non-homologous end joining (NHEJ) recombination cofactor XRCC4 and overexpression of HDR enhancer factors CtIP and MRE11 can improve HDR efficiency for gene knock-in. Using this approach, the BleoR gene was integrated onto the 3' end of the MKK2 MAP kinase gene to generate a BleoR-MKK2 fusion protein. Based on fully edited nucleotides evaluated by TaqMan real-time PCR, the HDR-mediated knock-in efficiency was up to 48% when using XRCC4 silencing incorporated with a combination of CtIP and MRE11 overexpression compared with no HDR enhancement or NHEJ silencing. Furthermore, this combination of HDR enhancer overexpression and NHEJ repression also increased genome targeting efficiency and gave 7-fold fewer CRISPR-induced insertions and deletions (InDels), resulting in no functional effects on MKK2-based salt stress responses in poplar. Therefore, this approach may be useful not only in poplar and plants or crops but also in mammals for improving CRISPR-mediated gene knock-in efficiency.
