The immune response to RNA suppresses nucleic acid synthesis by limiting ribose 5-phosphate.

During infection viruses hijack host cell metabolism to promote their replication. Here, analysis of metabolite alterations in macrophages exposed to poly I:C recognises that the antiviral effector Protein Kinase RNA-activated (PKR) suppresses glucose breakdown within the pentose phosphate pathway (PPP). This pathway runs parallel to central glycolysis and is critical to producing NADPH and pentose precursors for nucleotides. Changes in metabolite levels between wild-type and PKR-ablated macrophages show that PKR controls the generation of ribose 5-phosphate, in a manner distinct from its established function in gene expression but dependent on its kinase activity. PKR phosphorylates and inhibits the Ribose 5-Phosphate Isomerase A (RPIA), thereby preventing interconversion of ribulose- to ribose 5-phosphate. This activity preserves redox control but decreases production of ribose 5-phosphate for nucleotide biosynthesis. Accordingly, the PKR-mediated immune response to RNA suppresses nucleic acid production. In line, pharmacological targeting of the PPP during infection decreases the replication of the Herpes simplex virus. These results identify an immune response-mediated control of host cell metabolism and suggest targeting the RPIA as a potential innovative antiviral treatment.

Bio-Mimicking Brain Vasculature to Investigate the Role of Heterogeneous Shear Stress in Regulating Barrier Integrity.

A continuous, sealed endothelial membrane is essential for the blood-brain barrier (BBB) to protect neurons from toxins present in systemic circulation. Endothelial cells are critical sensors of the capillary environment, where factors like fluid shear stress (FSS) and systemic signaling molecules activate intracellular pathways that either promote or disrupt the BBB. The brain vasculature exhibits complex heterogeneity across the bed, which is challenging to recapitulate in BBB microfluidic models with fixed dimensions and rectangular cross-section microchannels. Here, a Cayley-tree pattern, fabricated using lithography-less, fluid shaping technique in a modified Hele-Shaw cell is used to emulate the brain vasculature in a microfluidic chip. This geometry generates an inherent distribution of heterogeneous FSS, due to smooth variations in branch height and width. hCMEC/D3 endothelial cells cultured in the Cayley-tree designed chip generate a 3D monolayer of brain endothelium with branching hierarchy, enabling the study of the effect of heterogeneous FSS on the brain endothelium. The model is employed to study neuroinflammatory conditions by stimulating the brain endothelium with tumor necrosis factor-α under heterogeneous FSS conditions. The model has immense potential for studies involving drug transport across the BBB, which can be misrepresented in fixed dimension models.

Chemoresistant Cancer Cell Lines Are Characterized by Migratory, Amino Acid Metabolism, Protein Catabolism and IFN1 Signalling Perturbations.

Chemoresistance remains the major barrier to effective ovarian cancer treatment. The molecular features and associated biological functions of this phenotype remain poorly understood. We developed carboplatin-resistant cell line models using OVCAR5 and CaOV3 cell lines with the aim of identifying chemoresistance-specific molecular features. Chemotaxis and CAM invasion assays revealed enhanced migratory and invasive potential in OVCAR5-resistant, compared to parental cell lines. Mass spectrometry analysis was used to analyse the metabolome and proteome of these cell lines, and was able to separate these populations based on their molecular features. It revealed signalling and metabolic perturbations in the chemoresistant cell lines. A comparison with the proteome of patient-derived primary ovarian cancer cells grown in culture showed a shared dysregulation of cytokine and type 1 interferon signalling, potentially revealing a common molecular feature of chemoresistance. A comprehensive analysis of a larger patient cohort, including advanced in vitro and in vivo models, promises to assist with better understanding the molecular mechanisms of chemoresistance and the associated enhancement of migration and invasion.

Reaction hijacking of tyrosine tRNA synthetase as a new whole-of-life-cycle antimalarial strategy.

Aminoacyl transfer RNA (tRNA) synthetases (aaRSs) are attractive drug targets, and we present class I and II aaRSs as previously unrecognized targets for adenosine 5'-monophosphate-mimicking nucleoside sulfamates. The target enzyme catalyzes the formation of an inhibitory amino acid-sulfamate conjugate through a reaction-hijacking mechanism. We identified adenosine 5'-sulfamate as a broad-specificity compound that hijacks a range of aaRSs and ML901 as a specific reagent a specific reagent that hijacks a single aaRS in the malaria parasite Plasmodium falciparum, namely tyrosine RS (PfYRS). ML901 exerts whole-life-cycle-killing activity with low nanomolar potency and single-dose efficacy in a mouse model of malaria. X-ray crystallographic studies of plasmodium and human YRSs reveal differential flexibility of a loop over the catalytic site that underpins differential susceptibility to reaction hijacking by ML901.

Cystathionine-ß-synthase is essential for AKT-induced senescence and suppresses the development of gastric cancers with PI3K/AKT activation.

Hyperactivation of oncogenic pathways downstream of RAS and PI3K/AKT in normal cells induces a senescence-like phenotype that acts as a tumor-suppressive mechanism that must be overcome during transformation. We previously demonstrated that AKT-induced senescence (AIS) is associated with profound transcriptional and metabolic changes. Here, we demonstrate that human fibroblasts undergoing AIS display upregulated cystathionine-ß-synthase (CBS) expression and enhanced uptake of exogenous cysteine, which lead to increased hydrogen sulfide (H2S) and glutathione (GSH) production, consequently protecting senescent cells from oxidative stress-induced cell death. CBS depletion allows AIS cells to escape senescence and re-enter the cell cycle, indicating the importance of CBS activity in maintaining AIS. Mechanistically, we show this restoration of proliferation is mediated through suppressing mitochondrial respiration and reactive oxygen species (ROS) production by reducing mitochondrial localized CBS while retaining antioxidant capacity of transsulfuration pathway. These findings implicate a potential tumor-suppressive role for CBS in cells with aberrant PI3K/AKT pathway activation. Consistent with this concept, in human gastric cancer cells with activated PI3K/AKT signaling, we demonstrate that CBS expression is suppressed due to promoter hypermethylation. CBS loss cooperates with activated PI3K/AKT signaling in promoting anchorage-independent growth of gastric epithelial cells, while CBS restoration suppresses the growth of gastric tumors in vivo. Taken together, we find that CBS is a novel regulator of AIS and a potential tumor suppressor in PI3K/AKT-driven gastric cancers, providing a new exploitable metabolic vulnerability in these cancers.

Glutaminase inhibition impairs CD8 T cell activation in STK11-/Lkb1-deficient lung cancer.

The tumor microenvironment (TME) contains a rich source of nutrients that sustains cell growth and facilitate tumor development. Glucose and glutamine in the TME are essential for the development and activation of effector T cells that exert antitumor function. Immunotherapy unleashes T cell antitumor function, and although many solid tumors respond well, a significant proportion of patients do not benefit. In patients with KRAS-mutant lung adenocarcinoma, KEAP1 and STK11/Lkb1 co-mutations are associated with impaired response to immunotherapy. To investigate the metabolic and immune microenvironment of KRAS-mutant lung adenocarcinoma, we generated murine models that reflect the KEAP1 and STK11/Lkb1 mutational landscape in these patients. Here, we show increased glutamate abundance in the Lkb1-deficient TME associated with CD8 T cell activation in response to anti-PD1. Combination treatment with the glutaminase inhibitor CB-839 inhibited clonal expansion and activation of CD8 T cells. Thus, glutaminase inhibition negatively impacts CD8 T cells activated by anti-PD1 immunotherapy.

Red Blood Cell BCL-xL Is Required for Plasmodium falciparum Survival: Insights into Host-Directed Malaria Therapies.

The development of antimalarial drug resistance is an ongoing problem threatening progress towards the elimination of malaria, and antimalarial treatments are urgently needed for drug-resistant malaria infections. Host-directed therapies (HDT) represent an attractive strategy for the development of new antimalarials with untapped targets and low propensity for resistance. In addition, drug repurposing in the context of HDT can lead to a substantial decrease in the time and resources required to develop novel antimalarials. Host BCL-xL is a target in anti-cancer therapy and is essential for the development of numerous intracellular pathogens. We hypothesised that red blood cell (RBC) BCL-xL is essential for Plasmodium development and tested this hypothesis using six BCL-xL inhibitors, including one FDA-approved compound. All BCL-xL inhibitors tested impaired proliferation of Plasmodium falciparum 3D7 parasites in vitro at low micromolar or sub-micromolar concentrations. Western blot analysis of infected cell fractions and immunofluorescence microscopy assays revealed that host BCL-xL is relocated from the RBC cytoplasm to the vicinity of the parasite upon infection. Further, immunoprecipitation of BCL-xL coupled with mass spectrometry analysis identified that BCL-xL forms unique molecular complexes with human µ-calpain in uninfected RBCs, and with human SHOC2 in infected RBCs. These results provide interesting perspectives for the development of host-directed antimalarial therapies and drug repurposing efforts.

Ceramide-induced integrated stress response overcomes Bcl-2 inhibitor resistance in acute myeloid leukemia.

Inducing cell death by the sphingolipid ceramide is a potential anticancer strategy, but the underlying mechanisms remain poorly defined. In this study, triggering an accumulation of ceramide in acute myeloid leukemia (AML) cells by inhibition of sphingosine kinase induced an apoptotic integrated stress response (ISR) through protein kinase R-mediated activation of the master transcription factor ATF4. This effect led to transcription of the BH3-only protein Noxa and degradation of the prosurvival Mcl-1 protein on which AML cells are highly dependent 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 anticancer effects of ceramide and preclinical evidence for new approaches to augment Bcl-2 inhibition in the therapy of AML and other cancers with high Mcl-1 dependency.

The sphingosine 1-phosphate receptor 2/4 antagonist JTE-013 elicits off-target effects on sphingolipid metabolism.

Sphingosine 1-phosphate (S1P) is a signaling lipid that has broad roles, working either intracellularly through various protein targets, or extracellularly via a family of five G-protein coupled receptors. Agents that selectively and specifically target each of the S1P receptors have been sought as both biological tools and potential therapeutics. JTE-013, a small molecule antagonist of S1P receptors 2 and 4 (S1P2 and S1P4) has been widely used in defining the roles of these receptors in various biological processes. Indeed, our previous studies showed that JTE-013 had anti-acute myeloid leukaemia (AML) activity, supporting a role for S1P2 in the biology and therapeutic targeting of AML. Here we examined this further and describe lipidomic analysis of AML cells that revealed JTE-013 caused alterations in sphingolipid metabolism, increasing cellular ceramides, dihydroceramides, sphingosine and dihydrosphingosine. Further examination of the mechanisms behind these observations showed that JTE-013, at concentrations frequently used in the literature to target S1P2/4, inhibits several sphingolipid metabolic enzymes, including dihydroceramide desaturase 1 and both sphingosine kinases. Collectively, these findings demonstrate that JTE-013 can have broad off-target effects on sphingolipid metabolism and highlight that caution must be employed in interpreting the use of this reagent in defining the roles of S1P2/4.

Resensitising proteasome inhibitor-resistant myeloma with sphingosine kinase 2 inhibition.

The introduction of the proteasome inhibitor bortezomib into treatment regimens for myeloma has led to substantial improvement in patient survival. However, whilst bortezomib elicits initial responses in many myeloma patients, this haematological malignancy remains incurable due to the development of acquired bortezomib resistance. With other patients presenting with disease that is intrinsically bortezomib resistant, it is clear that new therapeutic approaches are desperately required to target bortezomib-resistant myeloma. We have previously shown that targeting sphingolipid metabolism with the sphingosine kinase 2 (SK2) inhibitor K145 in combination with bortezomib induces synergistic death of bortezomib-naïve myeloma. In the current study, we have demonstrated that targeting sphingolipid metabolism with K145 synergises with bortezomib and effectively resensitises bortezomib-resistant myeloma to this proteasome inhibitor. Notably, these effects were dependent on enhanced activation of the unfolded protein response, and were observed in numerous separate myeloma models that appear to have different mechanisms of bortezomib resistance, including a new bortezomib-resistant myeloma model we describe which possesses a clinically relevant proteasome mutation. Furthermore, K145 also displayed synergy with the next-generation proteasome inhibitor carfilzomib in bortezomib-resistant and carfilzomib-resistant myeloma cells. Together, these findings indicate that targeting sphingolipid metabolism via SK2 inhibition may be effective in combination with a broad spectrum of proteasome inhibitors in the proteasome inhibitor resistant setting, and is an approach worth clinical exploration.

Polymyxin-Induced Metabolic Perturbations in Human Lung Epithelial Cells.

Inhaled polymyxins are associated with toxicity in human lung epithelial cells that involves multiple apoptotic pathways. However, the mechanism of polymyxin-induced pulmonary toxicity remains unclear. This study aims to investigate polymyxin-induced metabolomic perturbations in human lung epithelial A549 cells. A549 cells were treated with 0.5 or 1.0 mM polymyxin B or colistin for 1, 4, and 24 h. Cellular metabolites were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS), and significantly perturbed metabolites (log2 fold change [log2FC] ≥ 1; false-discovery rate [FDR] ≤ 0.2) and key pathways were identified relative to untreated control samples. At 1 and 4 h, very few significant changes in metabolites were observed relative to the untreated control cells. At 24 h, taurine (log2FC = -1.34 ± 0.64) and hypotaurine (log2FC = -1.20 ± 0.27) were significantly decreased by 1.0 mM polymyxin B. The reduced form of glutathione (GSH) was significantly depleted by 1.0 mM polymyxin B at 24 h (log2FC = -1.80 ± 0.42). Conversely, oxidized glutathione (GSSG) was significantly increased by 1.0 mM both polymyxin B (log2FC = 1.38 ± 0.13 at 4 h and 2.09 ± 0.20 at 24 h) and colistin (log2FC = 1.33 ± 0.24 at 24 h). l-Carnitine was significantly decreased by 1.0 mM of both polymyxins at 24 h, as were several key metabolites involved in biosynthesis and degradation of choline and ethanolamine (log2FC ≤ -1); several phosphatidylserines were also increased (log2FC ≥ 1). Polymyxins perturbed key metabolic pathways that maintain cellular redox balance, mitochondrial ß-oxidation, and membrane lipid biogenesis. These mechanistic findings may assist in developing new pharmacokinetic/pharmacodynamic strategies to attenuate the pulmonary toxicities of inhaled polymyxins and in the discovery of new-generation polymyxins.

Genetic and pharmacological evidence for kinetic competition between alternative poly(A) sites in yeast.

Most eukaryotic mRNAs accommodate alternative sites of poly(A) addition in the 3' untranslated region in order to regulate mRNA function. Here, we present a systematic analysis of 3' end formation factors, which revealed 3'UTR lengthening in response to a loss of the core machinery, whereas a loss of the Sen1 helicase resulted in shorter 3'UTRs. We show that the anti-cancer drug cordycepin, 3' deoxyadenosine, caused nucleotide accumulation and the usage of distal poly(A) sites. Mycophenolic acid, a drug which reduces GTP levels and impairs RNA polymerase II (RNAP II) transcription elongation, promoted the usage of proximal sites and reversed the effects of cordycepin on alternative polyadenylation. Moreover, cordycepin-mediated usage of distal sites was associated with a permissive chromatin template and was suppressed in the presence of an rpb1 mutation, which slows RNAP II elongation rate. We propose that alternative polyadenylation is governed by temporal coordination of RNAP II transcription and 3' end processing and controlled by the availability of 3' end factors, nucleotide levels and chromatin landscape.

Sphingolipid imbalance and inflammatory effects induced by uremic toxins in heart and kidney cells are reversed by dihydroceramide desaturase 1 inhibition.

Non-dialysable protein-bound uremic toxins (PBUTs) contribute to the development of cardiovascular disease (CVD) in chronic kidney disease (CKD) and vice versa. PBUTs have been shown to alter sphingolipid imbalance. Dihydroceramide desaturase 1 (Des1) is an important gatekeeper enzyme which controls the non-reversible conversion of sphingolipids, dihydroceramide, into ceramide. The present study assessed the effect of Des1 inhibition on PBUT-induced cardiac and renal effects in vitro, using a selective Des1 inhibitor (CIN038). Des1 inhibition attenuated hypertrophy in neonatal rat cardiac myocytes and collagen synthesis in neonatal rat cardiac fibroblasts and renal mesangial cells induced by the PBUTs, indoxyl sulfate and p-cresol sulfate. This is at least attributable to modulation of NF-κB signalling and reductions in ß-MHC, Collagen I and TNF-α gene expression. Lipidomic analyses revealed Des1 inhibition restored C16-dihydroceramide levels reduced by indoxyl sulfate. In conclusion, PBUTs play a critical role in mediating sphingolipid imbalance and inflammatory responses in heart and kidney cells, and these effects were attenuated by Des1 inhibition. Therefore, sphingolipid modifying agents may have therapeutic potential for the treatment of CVD and CKD and warrant further investigation.

ß-Adrenoceptor regulation of metabolism in U937 derived macrophages.

Macrophages have important roles in the immune system including clearing pathogens and wound healing. Metabolic phenotypes in macrophages have been associated with functional phenotypes, where pro-inflammatory macrophages have an increased rate of glycolysis and anti-inflammatory macrophages primarily use oxidative phosphorylation. ß-adrenoceptor (ßAR) signalling in macrophages has been implicated in disease states such as cancer, atherosclerosis and rheumatoid arthritis. The impact of ßAR signalling on macrophage metabolism has not been defined. Using metabolomics and proteomics, we describe the impact of ßAR signalling on macrophages treated with isoprenaline. We found that ßAR signalling alters proteins involved in cytoskeletal rearrangement and redox homeostasis of the cell. We showed that ßAR signalling in macrophages shifts glucose metabolism from glycolysis towards the tricarboxylic acid cycle and pentose phosphate pathways. We also show that ßAR signalling perturbs purine metabolism by accumulating adenylate and guanylate pools. Taken together, these results indicate that ßAR signalling shifts metabolism to support redox processes and upregulates proteins involved in cytoskeletal changes, which may contribute to ßAR effects on macrophage function.

Microbial metabolism of L-tyrosine protects against allergic airway inflammation.

The constituents of the gut microbiome are determined by the local habitat, which itself is shaped by immunological pressures, such as mucosal IgA. Using a mouse model of restricted antibody repertoire, we identified a role for antibody-microbe interactions in shaping a community of bacteria with an enhanced capacity to metabolize L-tyrosine. This model led to increased concentrations of p-cresol sulfate (PCS), which protected the host against allergic airway inflammation. PCS selectively reduced CCL20 production by airway epithelial cells due to an uncoupling of epidermal growth factor receptor (EGFR) and Toll-like receptor 4 (TLR4) signaling. Together, these data reveal a gut microbe-derived metabolite pathway that acts distally on the airway epithelium to reduce allergic airway responses, such as those underpinning asthma.

Measuring Sulforaphane and Its Metabolites in Human Plasma: A High Throughput Method.

(1) Background: There is increasing understanding of the potential health benefits of cruciferous vegetables. In particular sulforaphane (SFN), found in broccoli, and its metabolites sulforaphane-glutathione (SFN-GSH), sulforaphane-cysteine (SFN-Cys), sulforaphane cysteine-glycine (SFN-CG) and sulforaphane-N-acetyl-cysteine (SFN-NAC) have potent antioxidant effects that may offer therapeutic value. Clinical investigation of sulforaphane as a therapeutic antioxidant requires a sensitive and high throughput process for quantification of sulforaphane and metabolites; (2) Methods: We collected plasma samples from healthy human volunteers before and for eight hours after consumption of a commercial broccoli extract supplement rich in sulforaphane. A rapid and sensitive method for quantification of sulforaphane and its metabolites in human plasma using Liquid Chromatography-Mass Spectrometry (LC-MS) has been developed; (3) Results: The LC-MS analytical method was validated at concentrations ranging between 3.9 nM and 1000 nM for SFN-GSH, SFN-CG, SFN-Cys and SFN-NAC and between 7.8 nM and 1000 nM in human plasma for SFN. The method displayed good accuracy (1.85%-14.8% bias) and reproducibility (below 9.53 %RSD) including low concentrations 3.9 nM and 7.8 nM. Four SFN metabolites quantitation was achieved using external standard calibration and in SFN quantitation, SFN-d8 internal standardization was used. The reported method can accurately quantify sulforaphane and its metabolites at low concentrations in plasma; (4) Conclusions: We have established a time- and cost-efficient method of measuring sulforaphane and its metabolites in human plasma suitable for high throughput application to clinical trials.

Multi-omic Characterization of the Mode of Action of a Potent New Antimalarial Compound, JPC-3210, Against Plasmodium falciparum.

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 ß-hematin polymerization assays and showed JPC-3210 to be an intermediate inhibitor of ß-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.

Mechanistic Insights From Global Metabolomics Studies into Synergistic Bactericidal Effect of a Polymyxin B Combination With Tamoxifen Against Cystic Fibrosis MDR Pseudomonas aeruginosa.

Polymyxins are amongst the most important antibiotics in modern medicine, in recent times their clinical utility has been overshadowed by nosocomial outbreaks of polymyxin resistant MDR Gram-negative 'superbugs'. An effective strategy to surmount polymyxin resistance is combination therapy with FDA-approved non-antibiotic drugs. Herein we used untargeted metabolomics to investigate the mechanism(s) of synergy between polymyxin B and the selective estrogen receptor modulator (SERM) tamoxifen against a polymyxin-resistant MDR cystic fibrosis (CF) Pseudomonas aeruginosa FADDI-PA006 isolate (polymyxin B MIC=8 mg/L , it is an MDR polymyxin resistant P. aeruginosa isolated from the lungs of a CF patient). The metabolome of FADDI-PA006 was profiled at 15 min, 1 and 4 h following treatment with polymyxin B (2 mg/L), tamoxifen (8 mg/L) either as monotherapy or in combination. At 15 min, the combination treatment induced a marked decrease in lipids, primarily fatty acid and glycerophospholipid metabolites that are involved in the biosynthesis of bacterial membranes. In line with the polymyxin-resistant status of this strain, at 1 h, both polymyxin B and tamoxifen monotherapies produced little effect on bacterial metabolism. In contrast to the combination which induced extensive reduction (≥ 1.0-log2-fold, p ≤ 0.05; FDR ≤ 0.05) in the levels of essential intermediates involved in cell envelope biosynthesis. Overall, these novel findings demonstrate that the primary mechanisms underlying the synergistic bactericidal effect of the combination against the polymyxin-resistant P. aeruginosa CF isolate FADDI-PA006 involves a disruption of the cell envelope biogenesis and an inhibition of aminoarabinose LPS modifications that confer polymyxin resistance.

Benzoxaborole treatment perturbs S-adenosyl-L-methionine metabolism in Trypanosoma brucei.

The parasitic protozoan Trypanosoma brucei causes Human African Trypanosomiasis and Nagana in other mammals. These diseases present a major socio-economic burden to large areas of sub-Saharan Africa. Current therapies involve complex and toxic regimens, which can lead to fatal side-effects. In addition, there is emerging evidence for drug resistance. AN5568 (SCYX-7158) is a novel benzoxaborole class compound that has been selected as a lead compound for the treatment of HAT, and has demonstrated effective clearance of both early and late stage trypanosomiasis in vivo. The compound is currently awaiting phase III clinical trials and could lead to a novel oral therapeutic for the treatment of HAT. However, the mode of action of AN5568 in T. brucei is unknown. This study aimed to investigate the mode of action of AN5568 against T. brucei, using a combination of molecular and metabolomics-based approaches.Treatment of blood-stage trypanosomes with AN5568 led to significant perturbations in parasite metabolism. In particular, elevated levels of metabolites involved in the metabolism of S-adenosyl-L-methionine, an essential methyl group donor, were found. Further comparative metabolomic analyses using an S-adenosyl-L-methionine-dependent methyltransferase inhibitor, sinefungin, showed the presence of several striking metabolic phenotypes common to both treatments. Furthermore, several metabolic changes in AN5568 treated parasites resemble those invoked in cells treated with a strong reducing agent, dithiothreitol, suggesting redox imbalances could be involved in the killing mechanism.

Synergistic Killing of Polymyxin B in Combination With the Antineoplastic Drug Mitotane Against Polymyxin-Susceptible and -Resistant Acinetobacter baumannii: A Metabolomic Study.

Polymyxins are currently used as the last-resort antibiotics against multidrug-resistant Acinetobacter baumannii. As resistance to polymyxins emerges in A. baumannii with monotherapy, combination therapy is often the only remaining treatment option. A novel approach is to employ the combination of polymyxin B with non-antibiotic drugs. In the present study, we employed metabolomics to investigate the synergistic mechanism of polymyxin B in combination with the antineoplastic drug mitotane against polymyxin-susceptible and -resistant A. baumannii. The metabolomes of four A. baumannii strains were analyzed following treatment with polymyxin B, mitotane and the combination. Polymyxin B monotherapy induced significant perturbation in glycerophospholipid (GPL) metabolism and histidine degradation pathways in polymyxin-susceptible strains, and minimal perturbation in polymyxin-resistant strains. Mitotane monotherapy induced minimal perturbation in the polymyxin-susceptible strains, but caused significant perturbation in GPL metabolism, pentose phosphate pathway and histidine degradation in the LPS-deficient polymyxin-resistant strain (FADDI-AB065). The polymyxin B - mitotane combination induced significant perturbation in all strains except the lipid A modified polymyxin-resistant FADDI-AB225 strain. For the polymyxin-susceptible strains, the combination therapy significantly perturbed GPL metabolism, pentose phosphate pathway, citric acid cycle, pyrimidine ribonucleotide biogenesis, guanine ribonucleotide biogenesis, and histidine degradation. Against FADDI-AB065, the combination significantly perturbed GPL metabolism, pentose phosphate pathway, citric acid cycle, and pyrimidine ribonucleotide biogenesis. Overall, these novel findings demonstrate that the disruption of the citric acid cycle and inhibition of nucleotide biogenesis are the key metabolic features associated with synergistic bacterial killing by the combination against polymyxin-susceptible and -resistant A. baumannii.