A sulfoximine-based inhibitor of human asparagine synthetase kills l-asparaginase-resistant leukemia cells.

An adenylated sulfoximine transition-state analogue 1, which inhibits human asparagine synthetase (hASNS) with nanomolar potency, has been reported to suppress the proliferation of an l-asparagine amidohydrolase (ASNase)-resistant MOLT-4 leukemia cell line (MOLT-4R) when l-asparagine is depleted in the medium. We now report the synthesis and biological activity of two new sulfoximine analogues of 1 that have been studied as part of systematic efforts to identify compounds with improved cell permeability and/or metabolic stability. One of these new analogues, an amino sulfoximine 5 having no net charge at cellular pH, is a better hASNS inhibitor (K(I)(∗)=8nM) than 1 and suppresses proliferation of MOLT-4R cells at 10-fold lower concentration (IC(50)=0.1mM). More importantly, and in contrast to the lead compound 1, the presence of sulfoximine 5 at concentrations above 0.25mM causes the death of MOLT-4R cells even when ASNase is absent in the culture medium. The amino sulfoximine 5 exhibits different dose-response behavior when incubated with an ASNase-sensitive MOLT-4 cell line (MOLT-4S), supporting the hypothesis that sulfoximine 5 exerts its effect by inhibiting hASNS in the cell. Our work provides further evidence for the idea that hASNS represents a chemotherapeutic target for the treatment of leukemia, and perhaps other cancers, including those of the prostate

The biosynthetic pathway of potato solanidanes diverged from that of spirosolanes due to evolution of a dioxygenase

ジャガイモの毒α-ソラニンはトマトの苦味成分から分岐進化したことを解明. 京都大学プレスリリース. 2021-03-03.Potato (Solanum tuberosum), a worldwide major food crop, produces the toxic, bitter tasting solanidane glycoalkaloids α-solanine and α-chaconine. Controlling levels of glycoalkaloids is an important focus on potato breeding. Tomato (Solanum lycopersicum) contains a bitter spirosolane glycoalkaloid, α-tomatine. These glycoalkaloids are biosynthesized from cholesterol via a partly common pathway, although the mechanisms giving rise to the structural differences between solanidane and spirosolane remained elusive. Here we identify a 2-oxoglutarate dependent dioxygenase, designated as DPS (Dioxygenase for Potato Solanidane synthesis), that is a key enzyme for solanidane glycoalkaloid biosynthesis in potato. DPS catalyzes the ring-rearrangement from spirosolane to solanidane via C-16 hydroxylation. Evolutionary divergence of spirosolane-metabolizing dioxygenases contributes to the emergence of toxic solanidane glycoalkaloids in potato and the chemical diversity in Solanaceae

Identification of a tomato UDP-arabinosyltransferase for airborne volatile reception

植物間コミュニケーションの仕組みを解明 --受容した香りを防御物質に変える遺伝子発見--. 京都大学プレスリリース. 2023-02-28.Volatiles from herbivore-infested plants function as a chemical warning of future herbivory for neighboring plants. (Z)-3-Hexenol emitted from tomato plants infested by common cutworms is taken up by uninfested plants and converted to (Z)-3-hexenyl β-vicianoside (HexVic). Here we show that a wild tomato species (Solanum pennellii) shows limited HexVic accumulation compared to a domesticated tomato species (Solanum lycopersicum) after (Z)-3-hexenol exposure. Common cutworms grow better on an introgression line containing an S. pennellii chromosome 11 segment that impairs HexVic accumulation, suggesting that (Z)-3-hexenol diglycosylation is involved in the defense of tomato against herbivory. We finally reveal that HexVic accumulation is genetically associated with a uridine diphosphate-glycosyltransferase (UGT) gene cluster that harbors UGT91R1 on chromosome 11. Biochemical and transgenic analyses of UGT91R1 show that it preferentially catalyzes (Z)-3-hexenyl β-D-glucopyranoside arabinosylation to produce HexVic in planta

Occurrence of a bacterial membrane microdomain at the cell division site enriched in phospholipids with polyunsaturated hydrocarbon chains.

In this study, we found that phospholipids containing an eicosapentaenyl group form a novel membrane microdomain at the cell division site of a Gram-negative bacterium, Shewanella livingstonensis Ac10, using chemically synthesized fluorescent probes. The occurrence of membrane microdomains in eukaryotes and prokaryotes has been demonstrated with various imaging tools for phospholipids with different polar headgroups. However, few studies have focused on the hydrocarbon chain-dependent localization of membrane-resident phospholipids in vivo. We previously found that lack of eicosapentaenoic acid (EPA), a polyunsaturated fatty acid found at the sn-2 position of glycerophospholipids, causes a defect in cell division after DNA replication of S. livingstonensis Ac10. Here, we synthesized phospholipid probes labeled with a fluorescent 7-nitro-2,1,3-benzoxadiazol-4-yl (NBD) group to study the localization of EPA-containing phospholipids by fluorescence microscopy. A fluorescent probe in which EPA was bound to the glycerol backbone via an ester bond was found to be unsuitable for imaging because EPA was released from the probe by in vivo hydrolysis. To overcome this problem, we synthesized hydrolysis-resistant ether-type phospholipid probes. Using these probes, we found that the fluorescence localized between two nucleoids at the cell center during cell division when the cells were grown in the presence of the eicosapentaenyl group-containing probe (N-NBD-1-oleoyl-2-eicosapentaenyl-sn-glycero-3-phosphoethanolamine), whereas this localization was not observed with the oleyl group-containing control probe (N-NBD-1-oleoyl-2-oleyl-sn-glycero-3-phosphoethanolamine). Thus, phospholipids containing an eicosapentaenyl group are specifically enriched at the cell division site. Formation of a membrane microdomain enriched in EPA-containing phospholipids at the nucleoid occlusion site probably facilitates cell division

Glutathione-analogous peptidyl phosphorus esters as mechanism-based inhibitors of γ-glutamyl transpeptidase for probing cysteinyl-glycine binding site.

γ-Glutamyl transpeptidase (GGT) catalyzing the cleavage of γ-glutamyl bond of glutathione and its S-conjugates is involved in a number of physiological and pathological processes through glutathione homeostasis. Defining its Cys-Gly binding site is extremely important not only in defining the physiological function of GGT, but also in designing specific and effective inhibitors for pharmaceutical purposes. Here we report the synthesis and evaluation of a series of glutathione-analogous peptidyl phosphorus esters as mechanism-based inhibitors of human and Escherichia coli GGTs to probe the structural and stereochemical preferences in the Cys-Gly binding site. Both enzymes were inhibited strongly and irreversibly by the peptidyl phosphorus esters with a good leaving group (phenoxide). Human GGT was highly selective for l-aliphatic amino acid such as l-2-aminobutyrate (l-Cys mimic) at the Cys binding site, whereas E. coli GGT significantly preferred l-Phe mimic at this site. The C-terminal Gly and a l-amino acid analogue at the Cys binding site were necessary for inhibition, suggesting that human GGT was highly selective for glutathione (γ-Glu-l-Cys-Gly), whereas E. coli GGT are not selective for glutathione, but still retained the dipeptide (l-AA-Gly) binding site. The diastereoisomers with respect to the chiral phosphorus were separated. Both GGTs were inactivated by only one of the stereoisomers with the same stereochemistry at phosphorus. The strict recognition of phosphorus stereochemistry gave insights into the stereochemical course of the catalyzed reaction. Ion-spray mass analysis of the inhibited E. coli GGT confirmed the formation of a 1:1 covalent adduct with the catalytic subunit (small subunit) with concomitant loss of phenoxide, leaving the peptidyl moiety that presumably occupies the Cys-Gly binding site. The peptidyl phosphonate inhibitors are highly useful as a ligand for X-ray structural analysis of GGT for defining hitherto unidentified Cys-Gly binding site to design specific inhibitors

Structure-activity relationship of volatile compounds that induce defense-related genes in maize seedlings

Volatile organic compounds mediate plant-to-plant communication, and plants receiving volatile cues can acquire greater defenses against attackers. It has been expected that volatiles are received by factors that eventually lead to the induction of defense-related gene expression; however, the nature of these factors remain unclear. Structure-activity relationship analysis of gene expression induction by volatiles should provide insights into the nature of these factors. We conducted a structure-activity relationship study using maize seedlings and (Z)-3-hexen-1-yl acetate (Z3HAC) as the lead compound. The acid portion of Z3HAC was not essential, and (Z)-3-hexen-1-ol (Z3HOL), which is formed after the hydrolysis of Z3HAC, is likely the structure essential for the upregulation of the genes. The double bond of Z3HOL is essential; however, its geometry is indistinguishable. Strict specificity was detected regarding the length of the methylene chain on the α- and ω-sides of the double bond, and therefore, the 3-hexen-1-ol structure was found to be the ultimate structure. This finding provides insight into the nature of the factors that interact with a volatile compound and subsequently activate signaling pathways, leading to the upregulation of a subset of defense genes

Characterization of melanin and optimal conditions for pigment production by an endophytic fungus, Spissiomyces endophytica SDBR-CMU319.

Melanin is a natural pigment that is produced by filamentous fungi. In this study, the endophytic species, Spissiomyces endophytica (strain SDBR-CMU319), produced a brown-black pigment in the mycelia. Consequently, the pigment was extracted from the dried fungal biomass. This was followed by pigment purification, characterization and identification. Physical and chemical characteristics of the pigment showed acid precipitation, alkali solubilization, decolorization with oxidizing agents, and insolubility in most organic solvents and water. The pigment was confirmed as melanin based on ultraviolet-visible spectroscopy, Fourier-transform infrared, and electron paramagnetic resonance spectra analyses. The analyses of the elemental composition indicated that the pigment possessed a low percentage of nitrogen, and therefore, was not 3,4-dihydroxyphenylalanine melanin. Inhibition studies involving specific inhibitors, both tricyclazole and phthalide, and suggest that fungal melanin could be synthesized through the 1,8-dihydroxynaphthalene pathway. The optimum conditions for fungal pigment production from this species were investigated. The highest fungal pigment yield was observed in glucose yeast extract peptone medium at an initial pH value of 6.0 and at 25°C over three weeks of cultivation. This is the first report on the production and characterization of melanin obtained from the genus Spissiomyces

Enhancement of production of eugenol and its glycosides in transgenic aspen plants via genetic engineering.

Eugenol, a volatile phenylpropene found in many plant species, exhibits antibacterial and acaricidal activities. This study attempted to modify the production of eugenol and its glycosides by introducing petunia coniferyl alcohol acetyltransferase (PhCFAT) and eugenol synthase (PhEGS) into hybrid aspen. Gas chromatography analyses revealed that wild-type hybrid aspen produced small amount of eugenol in leaves. The heterologous overexpression of PhCFAT alone resulted in up to 7-fold higher eugenol levels and up to 22-fold eugenol glycoside levels in leaves of transgenic aspen plants. The overexpression of PhEGS alone resulted in a subtle increase in either eugenol or eugenol glycosides, and the overexpression of both PhCFAT and PhEGS resulted in significant increases in the levels of both eugenol and eugenol glycosides which were nonetheless lower than the increases seen with overexpression of PhCFAT alone. On the other hand, overexpression of PhCFAT in transgenic Arabidopsis and tobacco did not cause any synthesis of eugenol. These results indicate that aspen leaves, but not Arabidopsis and tobacco leaves, have a partially active pathway to eugenol that is limited by the level of CFAT activity and thus the flux of this pathway can be increased by the introduction of a single heterologous gene