Discovery of the 3-Amino-1,2,4-triazine-Based Library as Selective PDK1 Inhibitors with Therapeutic Potential in Highly Aggressive Pancreatic Ductal Adenocarcinoma

Pyruvate dehydrogenase kinases (PDKs) are serine/threonine kinases, that are directly involved in altered cancer cell metabolism, resulting in cancer aggressiveness and resistance. Dichloroacetic acid (DCA) is the first PDK inhibitor that has entered phase II clinical; however, several side effects associated with weak anticancer activity and excessive drug dose (100 mg/kg) have led to its limitation in clinical application. Building upon a molecular hybridization approach, a small library of 3-amino-1,2,4-triazine derivatives has been designed, synthesized, and characterized for their PDK inhibitory activity using in silico, in vitro, and in vivo assays. Biochemical screenings showed that all synthesized compounds are potent and subtype-selective inhibitors of PDK. Accordingly, molecular modeling studies revealed that a lot of ligands can be properly placed inside the ATP-binding site of PDK1. Interestingly, 2D and 3D cell studies revealed their ability to induce cancer cell death at low micromolar doses, being extremely effective against human pancreatic KRAS mutated cancer cells. Cellular mechanistic studies confirm their ability to hamper the PDK/PDH axis, thus leading to metabolic/redox cellular impairment, and to ultimately trigger apoptotic cancer cell death. Remarkably, preliminary in vivo studies performed on a highly aggressive and metastatic Kras-mutant solid tumor model confirm the ability of the most representative compound 5i to target the PDH/PDK axis in vivo and highlighted its equal efficacy and better tolerability profile with respect to those elicited by the reference FDA approved drugs, cisplatin and gemcitabine. Collectively, the data highlights the promising anticancer potential of these novel PDK-targeting derivatives toward obtaining clinical candidates for combatting highly aggressive KRAS-mutant pancreatic ductal adenocarcinomas

A multinuclear magnetic resonance study of vanadium (V) complexes and equilibria

The use of high-field n.m.r. spectroscopy to study the aqueous chemistry of vanadium(V) has yielded information not only about the species present but, also, a qualitative understanding of the ¹⁷O n.m.r. chemical shifts in polyanions. The peroxo and sulphido complexes of vanadium(V) are reported as well as the interaction of vanadate with ADP and ATP, and oxygen exchange in the polyanion tetradecavanadophosphate (9-) ([PV₁₄O₄₂]⁹ˉ). In general, peroxovanadate complexes tend to contain two peroxide ligands per vanadium, although some mono-, tri- and tetraperoxovanadates are found. The mono- and diperoxovanadates undergo a change in co-ordination from octahedral to tetrahedral on removal of their final proton (˜pH 14). Four new dimeric species have been observed, three of which are unsymmetrical. The pH-dependent differential rates of oxygen exchange of tetradecavanadophosphate (9-) can be explained in terms of the stabilisation of the anion by the two pentaco-ordinate VO caps with respect to the "equator" of the Keggin type structure. The four half-hemisphere units resist exchange. Bulk distortions of this anion and decavanadate (6-) can be deduced from the observation of their ¹⁷O chemical shifts with pH. The three known sulphido and oxosulphido- vanadium(V) species [VS₄]ᶟˉ, [VOS₃]ᶟˉ and [VO₂S₂]ᶟˉ have been identified by high-field vanadium-51 n.m.r., together with the previously unobserved ions VO₃S]ᶟˉ, [V₂S₇]⁴ˉ, [O₃VSVO₃]⁴ˉ, [SO₂VSVO₂S]⁴ˉ and the protonated monomers. No equilibria between monomeric species are observed other than for protonation. Vanadate complexes with the polyphosphate chain in both ATP and ADP, probably as [VO₂]⁺ bridging two adjacent phosphates. Exchange with species containing [VO₃]ˉ bound to the terminal phosphate and, in ATP only, "[VO]ᶟ⁺ bound to all the phosphates may occur

Structural basis for VIPP1 oligomerization and maintenance of thylakoid membrane integrity

Vesicle-inducing protein in plastids 1 (VIPP1) is essential for the biogenesis and maintenance of thylakoid membranes, which transform light into life. However, it is unknown how VIPP1 performs its vital membrane-remodeling functions. Here, we use cryo-electron microscopy to determine structures of cyanobacterial VIPP1 rings, revealing how VIPP1 monomers flex and interweave to form basket-like assemblies of different symmetries. Three VIPP1 monomers together coordinate a non-canonical nucleotide binding pocket on one end of the ring. Inside the ring's lumen, amphipathic helices from each monomer align to form large hydrophobic columns, enabling VIPP1 to bind and curve membranes. In vivo mutations in these hydrophobic surfaces cause extreme thylakoid swelling under high light, indicating an essential role of VIPP1 lipid binding in resisting stress-induced damage. Using cryo-correlative light and electron microscopy (cryo-CLEM), we observe oligomeric VIPP1 coats encapsulating membrane tubules within the Chlamydomonas chloroplast. Our work provides a structural foundation for understanding how VIPP1 directs thylakoid biogenesis and maintenance

Introgression of an adult-plant powdery mildew resistance gene Pm4VL from Dasypyrum villosum chromosome 4V into bread wheat

Powdery mildew caused by Blumeria graminis f. sp. tritici (Bgt) seriously threatens wheat production worldwide. It is imperative to identify novel resistance genes from wheat and its wild relatives to control this disease by host resistance. Dasypyrum villosum (2n = 2x = 14, VV) is a relative of wheat and harbors novel genes for resistance against multi-fungal diseases. In the present study, we developed a complete set of new wheat-D. villosum disomic introgression lines through genomic in situ hybridization (GISH), fluorescence in situ hybridization (FISH) and molecular markers analysis, including four disomic substitution lines (2n=42) containing respectively chromosomes 1V#6, 2V#6, 3V#6, and 6V#6, and four disomic addition lines (2n=44) containing respectively chromosomes 4V#6, 5V#6, 6V#6 and 7V#6. These lines were subsequently evaluated for their responses to a mixture Bgt isolates at both seedling and adult-plant stages. Results showed that introgression lines containing chromosomes 3V#6, 5V#6, and 6V#6 exhibited resistance at both seedling and adult-plant stages, whereas the chromosome 4V#6 disomic addition line NAU4V#6-1 exhibited a high level of adult plant resistance to powdery mildew. Moreover, two translocation lines were further developed from the progenies of NAU4V#6-1 and the Ph1b mutation line NAU0686-ph1b. They were T4DL·4V#6S whole-arm translocation line NAU4V#6-2 and T7DL·7DS-4V#6L small-fragment translocation line NAU4V#6-3. Powdery mildew tests of the two lines confirmed the presence of an adult-plant powdery mildew resistance gene, Pm4VL, located on the terminal segment of chromosome arm 4V#6L (FL 0.6-1.00). In comparison with the recurrent parent NAU0686 plants, the T7DL·7DS-4V#6L translocation line NAU4V#6-3 showed no obvious negative effect on yield-related traits, providing a new germplasm in breeding for resistance

SPOP Promotes Ubiquitination and Degradation of the ERG Oncoprotein to Suppress Prostate Cancer Progression

The ERG gene is fused to TMPRSS2 in approximately 50% of prostate cancers (PrCa), resulting in its overexpression. However, whether this is the sole mechanism underlying ERG elevation in PrCa is currently unclear. Here we report that ERG ubiquitination and degradation are governed by the Cullin 3-based ubiquitin ligase SPOP and that deficiency in this pathway leads to aberrant elevation of the ERG oncoprotein. Specifically, we find that truncated ERG (ΔERG), encoded by the ERG fusion gene, is stabilized by evading SPOP-mediated destruction, whereas prostate cancer-associated SPOP mutants are also deficient in promoting ERG ubiquitination. Furthermore, we show that the SPOP/ERG interaction is modulated by CKI-mediated phosphorylation. Importantly, we demonstrate that DNA damage drugs, topoisomerase inhibitors, can trigger CKI activation to restore the SPOP/ΔERG interaction and its consequent degradation. Therefore, SPOP functions as a tumor suppressor to negatively regulate the stability of the ERG oncoprotein in prostate cancer

The effects of early visual experience on cerebral RNA metabolism in the young chick.

1. A method for preparing nuclei in high yield from brain tissue of one-day old chicks was developed. These nuclei appeared to be essentially free from contamination by cytoplasm and endothelial cells. A further fractionation of nuclei according to size (i.e. of presumed neuronal arid glial origins) proved impractical. 2. The activity and some characteristics of the enzyme RNA polymerase in the nuclei were determined and compared to the data available on the same enzyme from other sources. The activity of the chick enzyme was higher than that from mammals but was basically similar in its response to ions in the assay medium. 3.The activity of RNA polymerase was measured in three brain regions of chicks either exposed for 30 minutes to an imprinting stimulus or kept in darkness. There was a 34% higher activity in the forebrain roof of the exposed than the dark-maintained birds. 4. A new set of equipment for exposing chicks to the imprinting stimulus or diffuse light was built and tested, both by the birds' behaviour after exposure and by their relative degrees of incorporation of 14c-uracil into brain RNA. These results were compared to those obtained previously on other equipment and found to be in general agreement. 5. Using the new equipment, the effects of different periods of exposure to the stimulus on the birds’ cerebral RNA polymerase activity were determined. These results were compared with those from the uracil incorporation experiments. 6. Possible extensions of the work are discussed in conjunction with other suggested directions in which such research could be pursued