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Glycosylated antitumor ether lipids (GAELs) have superior anticancer properties relative to the alkyllysophospholipid class, but there have been no studies of the mechanisms of these compounds. The prototype GAEL, 1-O-hexadecyl-2-O-methyl-3-O-(2'-amino-2'-deoxy-beta-D-glucopyranosyl)-sn-glycerol (Gln), effectively killed mouse embryonic fibroblasts (MEFs) lacking key molecules involved in caspase-dependent apoptosis, and cell death was not prevented by caspase inhibitors. Gln did not cause a loss of mitochondrial membrane potential, even in rounded-up dying cells. Gln stimulated the appearance and accumulation of LC3-II, a protein marker for autophagy, in a variety of cells, including wild-type MEFs, but not in MEFs lacking ATG5, a key protein required for autophagy. Gln induced LC3 puncta formation in Chinese hamster ovary cells stably expressing a LC3-green fluorescent protein fusion protein. Thus, Gln appears to induce autophagy. Autophagy was mTOR-independent and was not inhibited by 3-methyladenine or wortmannin. Although Gln is toxic, cellular ability to undergo autophagy was not essential for its toxicity. Furthermore, the GAEL analog 2-deoxy-C-Glc induced LC3 puncta formation but did not kill the cells. Gln, but not 2-deoxy-C-Glc, caused the accumulation of cytoplasmic acidic vacuoles in the cells. Our data suggest that GAELs may activate autophagy; however, GAELs do not kill cells by apoptosis or autophagy but rather by a paraptosis-like cell death mechanism.

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We describe metabolically inert l-glucosamine-based glycosylated antitumor ether lipids (L-GAELs) that retain the cytotoxic effects of the D-GAELs including the ability to kill BT-474 breast cancer stem cells (CSCs). When compared to adriamycin, cisplatin, and the anti-CSC agent salinomycin, L-GAELs display superior activity to kill cancer stem cells (CSCs). Mode of action studies indicate that L-GAELs like the D-GAELs kill cells via an apoptosis-independent mechanism that was not due to membranolytic effects.

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Two ether glucosyl diglyceride analogs were synthesized, and their antiproliferative activity against four epithelial cancer cell lines was evaluated. 1-O-Hexadecyl-2-O-methyl-3-O-(2'-acetamido-2'-deoxy-beta-D- glucopyranosyl)-sn-glycerol (4) was synthesized by reaction of 2-acetamido-2-deoxy-3,4,6-tri-O-acetyl-alpha-D-glucopyranosyl chloride with 1-O-hexadecyl-2-O-methyl-sn-glycerol followed by deacetylation by methanolic hydrolysis. The N-acetyl group of 4 was removed by hydrolysis with ethanolic potassium hydroxide to form 1-O-hexadecyl-2-O-methyl-3-O-(2'-amino-2'-deoxy-beta-D-glucopyranosyl)- sn-glycerol (5). Compounds 4 and 5 inhibited the proliferation of MCF-7, A549, A427, and T84 cancer cell lines. The IC(50) values for 5 ranged from 6.5 to 12.2 microM, whereas 4 was more effective against A549 cells (IC(50) 9 microM) than against MCF-7 (IC(50) 17 microM) and A427 (IC(50) 25 microM) cells and was inactive against T84 cells. Under identical incubation conditions, compounds 4 and 5 were potent inhibitors of the proliferation of OVCAR-3 cells with IC(50) values of 12 and 4 microM, respectively, whereas ET-18-OCH(3), hexadecylphosphocholine, and erucylphosphocholine had IC(50) values of 24, >30, and >30 microM, respectively. The cell-inhibitory profile of these ether-linked glucosyl diglycerides strengthens the hypothesis that such glycolipids represent a distinct group of antitumor ether lipids, having antineoplastic activities that differ from the well-known alkylphosphocholines and alkyllysophospholipids.

Objectives To develop a multifunctional adjuvant molecule that can rescue β-lactam antibiotics and β-lactam/β-lactamase inhibitor combinations from resistance in carbapenem-resistant Pseudomonas aeruginosa clinical isolates. Methods Preparation of adjuvant was guided by structure–activity relationships, following standard protocols. Susceptibility and chequerboard studies were assessed using serial 2-fold dilution assays. Toxicity was evaluated against porcine erythrocytes, human embryonic kidney (HEK293) cells and liver carcinoma (HepG2) cells via MTS assay. Preliminary in vivo efficacy was evaluated using a Galleria mellonella infection model. Results Conjugation of tobramycin and cyclam abrogates the ribosomal effects of tobramycin but confers a potent adjuvant property that restores full antibiotic activity of meropenem and aztreonam against carbapenem-resistant P. aeruginosa. Therapeutic levels of susceptibility, as determined by CLSI susceptibility breakpoints, were attained in several MDR clinical isolates, and time–kill assays revealed a synergistic dose-dependent pharmacodynamic relationship. A triple combination of the adjuvant with ceftazidime/avibactam (approved), aztreonam/avibactam (Phase III) and meropenem/avibactam enhances the efficacies of β-lactam/β-lactamase inhibitors against recalcitrant strains, suggesting rapid access of the combination to their periplasmic targets. The newly developed adjuvants, and their combinations, were non-haemolytic and non-cytotoxic, and preliminary in vivo evaluation in G. mellonella suggests therapeutic potential for the double and triple combinations. Conclusions Non-ribosomal tobramycin–cyclam conjugate mitigates the effect of OprD/OprF porin loss in P. aeruginosa and potentiates β-lactam/β-lactamase inhibitors against carbapenem-resistant clinical isolates, highlighting the complexity of resistance to β-lactam antibiotics. Our strategy presents an avenue to further preserve the therapeutic utility of β-lactam antibiotics.

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