To investigate the role of endothelin-1 (ET-1) in the pathogenesis of hypoxic pulmonary hypertension, we studied the effects of a recently described endothelin-receptor antagonist (ETA), BQ123, on the development of this process. Intraperitoneal osmotic pumps were placed into 8-wk-old Sprague-Dawley rats that received either saline or BQ123 (0.15 mg/h). The rats were maintained in room air normoxia or placed in a hypobaric chamber (380 Torr) for 2 wk to induce hypoxic pulmonary hypertension. There were no hemodynamic differences between normoxic rats treated with either saline or BQ123. However, treatment with BQ123 attenuated the hypoxia-induced increase in pulmonary arterial mean pressure and total pulmonary resistance index by 60 and 87% respectively. There was also a reduction in hypoxia-induced right ventricular hypertrophy in the BQ123 group. Histological studies performed using a barium-gelatin fixation technique in hypoxic BQ123-treated animals demonstrated a decrease in medial wall thickness in arteries corresponding to the respiratory and terminal bronchioles, respectively. Similarly, there was a significant reduction in the degree of muscularization of more distal vessels at the level of alveolar ducts in BQ123-treated hypoxic rats. We conclude that the ETA-receptor antagonist BQ123 attenuates the development of hypoxic pulmonary hypertension in rats in vivo, thereby suggesting a possible contributing role for ET-1 and the ETA receptor in the pathogenesis of this process.

Multidrug-resistant presents a global medical challenge, and polymyxins are a key last-resort therapeutic option. Unfortunately, polymyxin resistance in has been increasingly reported. The present study was designed to define metabolic differences between paired polymyxin-susceptible and -resistant strains using untargeted metabolomics and lipidomics analyses. The metabolomes of wild-type strain K ([PAK] polymyxin B MIC, 1 mg/liter) and its paired mutant strains, PAK and PAK (polymyxin B MICs of 16 mg/liter and 64 mg/liter, respectively) were characterized using liquid chromatography-mass spectrometry, and metabolic differences were identified through multivariate and univariate statistics. PAK and PAK, which displayed lipid A modifications with 4-amino-4-deoxy-l-arabinose, showed significant perturbations in amino acid and carbohydrate metabolism, particularly the intermediate metabolites from 4-amino-4-deoxy-l-arabinose synthesis and the methionine salvage cycle pathways. The genomics result showed a premature termination (Y275stop) in (encoding spermidine synthase) in PAK, and metabolomics data revealed a decreased intracellular level of spermidine in PAK compared to that in PAK Our results indicate that spermidine may play an important role in high-level polymyxin resistance in Interestingly, both mutants had decreased levels of phospholipids, fatty acids, and acyl-coenzyme A compared to those in the wild-type PAK. Moreover, the more resistant PAK mutant exhibited much lower levels of phospholipids than the PAK mutant, suggesting that the decreased phospholipid level was associated with polymyxin resistance. In summary, this study provides novel mechanistic information on polymyxin resistance in and highlights its impacts on bacterial metabolism.

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The increasing incidence of antimalarial drug resistance to the first-line artemisinin combination therapies underpins an urgent need for new antimalarial drugs, ideally with a novel mode of action. The recently developed 2-aminomethylphenol, JPC-3210, (MMV 892646) is an erythrocytic schizonticide with potent in vitro antimalarial activity against multidrug-resistant Plasmodium falciparum lines, low cytotoxicity, potent in vivo efficacy against murine malaria, and favorable preclinical pharmacokinetics including a lengthy plasma elimination half-life. To investigate the impact of JPC-3210 on biochemical pathways within P. falciparum-infected red blood cells, we have applied a “multi-omics” workflow based on high resolution orbitrap mass spectrometry combined with biochemical approaches. Metabolomics, peptidomics and hemoglobin fractionation analyses revealed a perturbation in hemoglobin metabolism following JPC-3210 exposure. The metabolomics data demonstrated a specific depletion of short hemoglobin-derived peptides, peptidomics analysis revealed a depletion of longer hemoglobin-derived peptides, and the hemoglobin fractionation assay demonstrated decreases in hemoglobin, heme and hemozoin levels. To further elucidate the mechanism responsible for inhibition of hemoglobin metabolism, we used in vitro β-hematin polymerization assays and showed JPC-3210 to be an intermediate inhibitor of β-hematin polymerization, about 10-fold less potent then the quinoline antimalarials, such as chloroquine and mefloquine. Further, quantitative proteomics analysis showed that JPC-3210 treatment results in a distinct proteomic signature compared with other known antimalarials. While JPC-3210 clustered closely with mefloquine in the metabolomics and proteomics analyses, a key differentiating signature for JPC-3210 was the significant enrichment of parasite proteins involved in regulation of translation. These studies revealed that the mode of action for JPC-3210 involves inhibition of the hemoglobin digestion pathway and elevation of regulators of protein translation. Importantly, JPC-3210 demonstrated rapid parasite killing kinetics compared with other quinolones, suggesting that JPC-3210 warrants further investigation as a potentially long acting partner drug for malaria treatment.

Inducing cell death by the sphingolipid ceramide is a potential anti-cancer strategy, but the underlying mechanisms remain poorly defined. Here, we show that triggering accumulation of ceramide in acute myeloid leukaemia (AML) cells by inhibition of sphingosine kinase induces an apoptotic integrated stress response (ISR) through protein kinase R-mediated activation of the master transcription factor ATF4. This leads to transcription of the BH3-only protein, Noxa, and degradation of the pro-survival Mcl-1 protein on which AML cells are highly dependent on for survival. Targeting this novel ISR pathway in combination with the Bcl-2 inhibitor venetoclax synergistically killed primary AML blasts, including those with venetoclax-resistant mutations, as well as immunophenotypic leukemic stem cells, and reduced leukemic engraftment in patient-derived AML xenografts. Collectively, these findings provide mechanistic insight into the anti-cancer effects of ceramide and pre-clinical evidence for new approaches to augment Bcl-2 inhibition in the therapy of AML and other cancers with high Mcl-1 dependency.