New Inhibitors of Bcr-Abl Based on 2,6,9-Trisubstituted Purine Scaffold Elicit Cytotoxicity in Chronic Myeloid Leukemia-Derived Cell Lines Sensitive and Resistant to TKIs.

Bcr-Abl is an oncoprotein with aberrant tyrosine kinase activity involved in the progression of chronic myeloid leukemia (CML) and has been targeted by inhibitors such as imatinib and nilotinib. However, despite their efficacy in the treatment of CML, a mechanism of resistance to these drugs associated with mutations in the kinase region has emerged. Therefore, in this work, we report the synthesis of 14 new 2,6,9-trisubstituted purines designed from our previous Bcr-Abl inhibitors. Here, we highlight 11b, which showed higher potency against Bcr-Abl (IC50 = 0.015 µM) than imatinib and nilotinib and exerted the most potent antiproliferative properties on three CML cells harboring the Bcr-Abl rearrangement (GI50 = 0.7-1.3 µM). In addition, these purines were able to inhibit the growth of KCL22 cell lines expressing Bcr-AblT315I, Bcr-AblE255K, and Bcr-AblY253H point mutants in micromolar concentrations. Imatinib and nilotinib were ineffective in inhibiting the growth of KCL22 cells expressing Bcr-AblT315I (GI50 > 20 µM) compared to 11b-f (GI50 = 6.4-11.5 µM). Molecular docking studies explained the structure-activity relationship of these purines in Bcr-AblWT and Bcr-AblT315I. Finally, cell cycle cytometry assays and immunodetection showed that 11b arrested the cells in G1 phase, and that 11b downregulated the protein levels downstream of Bcr-Abl in these cells.

BBX21 integrates brassinosteroid biosynthesis and signalling in the inhibition of hypocotyl growth under shade.

B-Box-containing zinc finger transcription factors (BBX) are involved in light-mediated growth, affecting processes such as hypocotyl elongation in Arabidopsis thaliana. However, the molecular and hormonal framework that regulates plant growth through BBX proteins is incomplete. Here, we demonstrate that BBX21 inhibits the hypocotyl elongation through the brassinosteroid (BR) pathway. BBX21 reduces the sensitivity to 24-epiBL, a synthetic active BR, principally at very-low concentrations in simulated shade. The biosynthesis profile of BRs showed that two active BR -brassinolide (BL) and 28-homobrassinolide (28-homoBL)- and 8 of 11 intermediates can be repressed by BBX21 under white light (WL) or simulated shade. Furthermore, BBX21 represses the expression of CYTOCHROME P450 90B1 (DWF4/CYP90B1), BRASSINOSTEROID-6-OXIDASE 1 (BR6OX1, CYP85A1) and BR6OX2 (CYP85A2) genes involved in the BR biosynthesis in WL while specifically promoting DWF4 and PHYB ACTIVATION TAGGED SUPPRESSOR 1 (CYP2B1/BAS1) expression in WL supplemented with far-red (WL+FR), a treatment that simulates shade. In addition, BBX21 represses BR signalling genes such as PACLOBUTRAZOL RESISTANCE1 (PRE1), PRE3 and ARABIDOPSIS MYB-LIKE 2 (MYBL2), and auxin-related and expansin genes, such as INDOLE-3-ACETIC ACID INDUCIBLE 1 (IAA1), IAA4 and EXPANSIN 11 (EXP11) in short-term shade. By a genetic approach we found that BBX21 acts genetically upstream of BRASSINAZOLE-RESISTANT 1 (BZR1) for the promotion of DWF4 and BAS1 gene expression in shade. We propose that BBX21 integrates the BR homeostasis and shade-light signalling allowing the fine-tuning of hypocotyl elongation in Arabidopsis.

Endemic parkinsonism: clusters, biology and clinical features.

The term 'endemic parkinsonism' refers to diseases that manifest with a dominant parkinsonian syndrome, which can be typical or atypical, and are present only in a particular geographically defined location or population. Ten phenotypes of endemic parkinsonism are currently known: three in the Western Pacific region; two in the Asian-Oceanic region; one in the Caribbean islands of Guadeloupe and Martinique; and four in Europe. Some of these disease entities seem to be disappearing over time and therefore are probably triggered by unique environmental factors. By contrast, other types persist because they are exclusively genetically determined. Given the geographical clustering and potential overlap in biological and clinical features of these exceptionally interesting diseases, this Review provides a historical reference text and offers current perspectives on each of the 10 phenotypes of endemic parkinsonism. Knowledge obtained from the study of these disease entities supports the hypothesis that both genetic and environmental factors contribute to the development of neurodegenerative diseases, not only in endemic parkinsonism but also in general. At the same time, this understanding suggests useful directions for further research in this area.

AZG1 is a cytokinin transporter that interacts with auxin transporter PIN1 and regulates the root stress response.

An environmentally responsive root system is crucial for plant growth and crop yield, especially in suboptimal soil conditions. This responsiveness enables the plant to exploit regions of high nutrient density while simultaneously minimizing abiotic stress. Despite the vital importance of root systems in regulating plant growth, significant gaps of knowledge exist in the mechanisms that regulate their architecture. Auxin defines both the frequency of lateral root (LR) initiation and the rate of LR outgrowth. Here, we describe a search for proteins that regulate root system architecture (RSA) by interacting directly with a key auxin transporter, PIN1. The native separation of Arabidopsis plasma membrane protein complexes identified several PIN1 co-purifying proteins. Among them, AZG1 was subsequently confirmed as a PIN1 interactor. Here, we show that, in Arabidopsis, AZG1 is a cytokinin (CK) import protein that co-localizes with and stabilizes PIN1, linking auxin and CK transport streams. AZG1 expression in LR primordia is sensitive to NaCl, and the frequency of LRs is AZG1-dependent under salt stress. This report therefore identifies a potential point for auxin:cytokinin crosstalk, which shapes RSA in response to NaCl.

Organ-specific COP1 control of BES1 stability adjusts plant growth patterns under shade or warmth.

Under adverse conditions such as shade or elevated temperatures, cotyledon expansion is reduced and hypocotyl growth is promoted to optimize plant architecture. The mechanisms underlying the repression of cotyledon cell expansion remain unknown. Here, we report that the nuclear abundance of the BES1 transcription factor decreased in the cotyledons and increased in the hypocotyl in Arabidopsis thaliana under shade or warmth. Brassinosteroid levels did not follow the same trend. PIF4 and COP1 increased their nuclear abundance in both organs under shade or warmth. PIF4 directly bound the BES1 promoter to enhance its activity but indirectly reduced BES1 expression. COP1 physically interacted with the BES1 protein, promoting its proteasome degradation in the cotyledons. COP1 had the opposite effect in the hypocotyl, demonstrating organ-specific regulatory networks. Our work indicates that shade or warmth reduces BES1 activity by transcriptional and post-translational regulation to inhibit cotyledon cell expansion.

Mal de Río Cuarto virus infection causes hormone imbalance and sugar accumulation in wheat leaves.

BACKGROUND: Mal de Río Cuarto virus (MRCV) infects several monocotyledonous species including maize and wheat. Infected plants show shortened internodes, partial sterility, increased tillering and reduced root length. To better understand the molecular basis of the plant-virus interactions leading to these symptoms, we combined RNA sequencing with metabolite and hormone measurements. RESULTS: More than 3000 differentially accumulated transcripts (DATs) were detected in MRCV-infected wheat plants at 21 days post inoculation compared to mock-inoculated plants. Infected plants exhibited decreased levels of TaSWEET13 transcripts, which are involved in sucrose phloem loading. Soluble sugars, starch, trehalose 6-phosphate (Tre6P), and organic and amino acids were all higher in MRCV-infected plants. In addition, several transcripts related to plant hormone metabolism, transport and signalling were increased upon MRCV infection. Transcripts coding for GA20ox, D14, MAX2 and SMAX1-like proteins involved in gibberellin biosynthesis and strigolactone signalling, were reduced. Transcripts involved in jasmonic acid, ethylene and brassinosteroid biosynthesis, perception and signalling and in auxin transport were also altered. Hormone measurements showed that jasmonic acid, brassinosteroids, abscisic acid and indole-3-acetic acid were significantly higher in infected leaves. CONCLUSIONS: Our results indicate that MRCV causes a profound hormonal imbalance that, together with alterations in sugar partitioning, could account for the symptoms observed in MRCV-infected plants.

New insights into auxin metabolism in Bradyrhizobium japonicum.

Bacterial metabolism of phytohormones includes several processes such as biosynthesis, catabolism, conjugation, hydrolysis and homeostatic regulation. However, only biosynthesis and occasionally catabolism are studied in depth in microorganisms. In this work, we evaluated and reconsidered IAA metabolism in Bradyrhizobiumjaponicum E109, one of the most widely used strains for soybean inoculation around the world. The genomic analysis of the strain showed the presence of several genes responsible for IAA biosynthesis, mainly via indole-3-acetonitrile (IAN), indole-3-acetamide (IAM) and tryptamine (TAM) pathways. However; in vitro experiments showed that IAA is not accumulated in the culture medium in significant amounts. On the contrary, a strong degradation activity was observed after exogenous addition of 0.1 mM of IAA, IBA or NAA to the medium. B. japonicum E109 was not able to grow in culture medium containing IAA as a sole carbon source. In YEM medium, the bacteria degraded IAA and hydrolyzed amino acid auxin conjugates with alanine (IAAla), phenylalanine (IAPhe), and leucine (IAPhe), releasing IAA which was quickly degraded. Finally, the presence of exogenous IAA induced physiological changes in the bacteria such as increased biomass and exopolysaccharide production, as well as infection effectiveness and symbiotic behavior in soybean plants.

Specialized Plant Metabolism Characteristics and Impact on Target Molecule Biotechnological Production.

Plant secondary metabolism evolved in the context of highly organized and differentiated cells and tissues, featuring massive chemical complexity operating under tight environmental, developmental and genetic control. Biotechnological demand for natural products has been continuously increasing because of their significant value and new applications, mainly as pharmaceuticals. Aseptic production systems of plant secondary metabolites have improved considerably, constituting an attractive tool for increased, stable and large-scale supply of valuable molecules. Surprisingly, to date, only a few examples including taxol, shikonin, berberine and artemisinin have emerged as success cases of commercial production using this strategy. The present review focuses on the main characteristics of plant specialized metabolism and their implications for current strategies used to produce secondary compounds in axenic cultivation systems. The search for consonance between plant secondary metabolism unique features and various in vitro culture systems, including cell, tissue, organ, and engineered cultures, as well as heterologous expression in microbial platforms, is discussed. Data to date strongly suggest that attaining full potential of these biotechnology production strategies requires being able to take advantage of plant specialized metabolism singularities for improved target molecule yields and for bypassing inherent difficulties in its rational manipulation.

Synthesis and in vitro anticancer activity of 23(23')E-benzylidenespirostanols derived from steroid sapogenins.

Benzylidenespirostanols were prepared by two-step synthesis including BF3·Et2O-catalyzed aldol condensation of several acetylated steroid sapogenins with benzaldehyde followed by saponification. The obtained compounds showed moderate cytotoxicity against three cancer cell lines (T-lymphoblastic leukemia cell line CEM, breast carcinoma cell line MCF7 and cervical carcinoma cell line HeLa) and normal human fibroblasts (BJ). The most active of the five tested substances was 3c (lowest IC50 for MCF7 cells 19.9±0.1µM) without any selectivity towards human cancer and normal cells, respectively.

Synthesis and antiproliferative activity of novel steroidal dendrimer conjugates.

We describe the synthesis of steroidal dendrimer conjugates of first and second generation with tetramethylene core and 5-hydroxy-isophtalic acid dimethyl ester as branching unit modified to incorporate ethynylestradiol or 17α-estradiol as terminal units. The steroidal dendrimer conjugates, the free drug (steroids) and dendrimer were tested against a panel of cancer cell lines (CEM, MCF7, HeLa) and normal human fibroblast (BJ). The steroidal dendrimer conjugates of first generation exhibited cytotoxic activity and induced apoptosis in chronic leukemia (CEM) as resultant activation of caspase cascade which is mainly provoked in G2/M arrested cells.

Synthesis and biological activity of 23-ethylidene-26-hydroxy-22-oxocholestane derivatives from spirostanic sapogenins.

The synthesis and biological evaluation of three new cholestane frameworks of the type: (25R)-3ß,16ß-Diacetoxy-23-ethylidene-26-hydroxy-22-oxocholestane, starting from spirostanic sapogenins of the 25R series, is described. The compounds were obtained by the reductive cleavage of the F ring of 22-oxo-23(1),26-epoxycholestane derivatives using 9-BBN. These modified derivatives exhibit cytotoxic activity against CEM and MCF7 cells and are able to induce apoptosis in them. Its effect on the cell cycle was determined.