Landomycins as glutathione-depleting agents and natural fluorescent probes for cellular Michael adduct-dependent quinone metabolism

Abstract

Landomycins are angucyclines with promising antineoplastic activity produced by Streptomyces bacteria. The aglycone landomycinone is the distinctive core, while the oligosaccharide chain differs within derivatives. Herein, we report that landomycins spontaneously form Michael adducts with biothiols, including reduced cysteine and glutathione, both cell-free or intracellularly involving the benz[a]anthraquinone moiety of landomycinone. While landomycins generally do not display emissive properties, the respective Michael adducts exerted intense blue fluorescence in a glycosidic chain-dependent manner. This allowed label-free tracking of the short-lived nature of the mono-SH-adduct followed by oxygen-dependent evolution with addition of another SH-group. Accordingly, hypoxia distinctly stabilized the fluorescent mono-adduct. While extracellular adduct formation completely blocked the cytotoxic activity of landomycins, intracellularly it led to massively decreased reduced glutathione levels. Accordingly, landomycin E strongly synergized with glutathione-depleting agents like menadione but exerted reduced activity under hypoxia. Summarizing, landomycins represent natural glutathione-depleting agents and fluorescence probes for intracellular anthraquinone-based angucycline metabolism.

Targeted Isolation of N-Acetylcysteine-Containing Angucycline Derivatives from Streptomyces sp. MC16 and Their Antiproliferative Effects

“…Rohr observed the nonenzymatic formation of methanethiol on angucycline urdamycin E through a reaction with methionine . More recently, Terenzi et al demonstrated that Michael adducts of landomycin derivatives with biothiols, such as reduced glutathione and cysteine, can form spontaneously both inside and outside cells …”

“…19 More recently, Terenzi et al demonstrated that Michael adducts of landomycin derivatives with biothiols, such as reduced glutathione and cysteine, can form spontaneously both inside and outside cells. 20 The inhibitory effects of all of the isolated compounds on the proliferation of A431, A549, and HeLa cell lines were evaluated. Antibiotics 100-1 (3) and vineomycinone B 2 (4) exhibited moderate inhibitory effects on all three cell lines, with IC 50 values ranging from 18.5 to 59.0 μM.…”

Synthesis and Photochemical Properties of Fluorescent Metabolites Generated from Fluorinated Benzoylmenadiones in Living Cells

“…66 On the other hand, (benz[a]anthraquinone derivatives, Figure 11) from the angucycline family (natural antibiotics produced by Streptomyces bacteria) were shown, either in cell-free conditions or intracellularly, to react through the spontaneous formation of Michael adducts with biothiols (e.g., reduced cysteine or glutathione). 67 With landomycin E (LE) and N-acetylcycteine (NAC), the authors evidenced the formation of an oxidized LE-NAC (LE-NAC ox ) that can be reduced to a short-lived (several minutes) fluorescent species (LE-NAC red , λ em = 428 nm) that is reoxidized to form a L-NAC ox species (dissociation of the substituted fluorescence was also observed live-cell fluorescence microscopy in the cytoplasm and nucleus of human LN229 glioblastoma and MG63 osteosarcoma cells. These two studies thus suggest that F-PDO could also lead, under photoactivation, to the formation of an anthrone-like tetracyclic compound capable of associating like LE with a biothiol and allowing, according to the same mechanism, an explanation of the fluorescence observed in both the cytoplasm and the nucleus of BY-2 tobacco cells.…”

Double-acting anticancer drugs to overcome the acquired resistance of malignant cells to chemotherapy

“…LA 1 μM +Q 5 μM +Q 10 μM + M 5 μM + M 10 μM + DC 2 μM + DC 5 μM + DC 10 μM + DC 25 μM 1 5,18±0,13 3,95±0,19 1 4,75±0,48 2,40±0,12 2 2,19±0,06 2 4,33±0,11 3,96±0,2 1 3,75±0,11 1 4,41±0,22 3 3,40±0,08 2,42±0,05 3 3,34±0,17 1,81±0,07 4 1,69±0,08 4 2,42±0,18 3 1,86±0,28 4 1,67±0,25 4 2,38±0,24 [22,23]. Такі аддукти мають більшу гідрофільність, ніж молекули ландоміцинів, що й перешкоджає їх проникненню шляхом дифузії у клітини-мішені.…”

“…Отже, зниження продукції АФК ландоміцинами під дією NAC пояснюється тим, що аддукти ландоміцинів з NAC фізично не можуть проникнути у клітини і, відповідно, не зв'язуються з NQO1 і не генерують АФК. Однак, як показали фізико-хімічні дослідження, ці аддукти не є стабільними, швидко розпадаються, а вивільнена молекула ландоміцину знову приєднує до себе наступну молекулу NAC [22]. Цим і пояснюється втрата АФК-протекторної активності NAC на 12 і 24 год дії ландоміцинів, що було показано нами раніше [26].…”