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Choline kinase alpha (ChoKα) is a therapeutic target being developed for a variety of diseases, from cancer to rheumatoid arthritis and from parasites to bacterial infections. Nevertheless, the therapeutic potential of this drug target seems not exhausted and may end up as a possible solution for a larger variety of conditions. Here we present our working model for how ChoKα could play a role in obesity and for how drugs being developed as therapeutics for other diseases using ChoKα as a target, could be repurposed as prophylactic treatments for obesity. We also present preliminary observations in support of our model. [ABSTRACT FROM AUTHOR]

Choline kinase alpha (ChoKα) is a therapeutic target being developed for a variety of diseases, from cancer to rheumatoid arthritis and from parasites to bacterial infections. Nevertheless, the therapeutic potential of this drug target seems not exhausted and may end up as a possible solution for a larger variety of conditions. Here we present our working model for how ChoKα could play a role in obesity and for how drugs being developed as therapeutics for other diseases using ChoKα as a target, could be repurposed as prophylactic treatments for obesity. We also present preliminary observations in support of our model.

Abstract Background Angioimmunoblastic T-cell lymphoma (AITL) is a malignancy with very poor survival outcome, in urgent need of more specific therapeutic strategies. The drivers of malignancy in this disease are CD4+ follicular helper T cells (Tfh). The metabolism of these malignant Tfh cells was not yet elucidated. Therefore, we decided to identify their metabolic requirements with the objective to propose a novel therapeutic option. Methods To reveal the prominent metabolic pathways used by the AITL lymphoma cells, we relied on metabolomic and proteomic analysis of murine AITL (mAITL) T cells isolated from our established mAITL model. We confirmed these results using AITL patient and healthy T cell expression data. Results Strikingly, the mAITL Tfh cells were highly dependent on the second branch of the Kennedy pathway, the choline lipid pathway, responsible for the production of the major membrane constituent phosphatidylcholine. Moreover, gene expression data from Tfh cells isolated from AITL patient tumors, confirmed the upregulation of the choline lipid pathway. Several enzymes involved in this pathway such as choline kinase, catalyzing the first step in the phosphatidylcholine pathway, are upregulated in multiple tumors other than AITL. Here we showed that treatment of our mAITL preclinical mouse model with a fatty acid oxydation inhibitor, significantly increased their survival and even reverted the exhausted CD8 T cells in the tumor into potent cytotoxic anti-tumor cells. Specific inhibition of Chokα confirmed the importance of the phosphatidylcholine production pathway in neoplastic CD4 + T cells, nearly eradicating mAITL Tfh cells from the tumors. Finally, the same inhibitor induced in human AITL lymphoma biopsies cell death of the majority of the hAITL PD-1high neoplastic cells. Conclusion Our results suggest that interfering with choline metabolism in AITL reveals a specific metabolic vulnerability and might represent a new therapeutic strategy for these patients.

Mitochondrial fatty acid oxidation (FAO) contributes to the proton motive force that drives ATP synthesis in many mammalian tissues. In eutherian (placental) mammals, brown adipose tissue (BAT) can also dissipate this proton gradient through uncoupling protein 1 (UCP1) to generate heat, but the evolutionary events underlying the emergence of BAT are unknown. An essential step in FAO is the transport of cytoplasmic long chain acyl-coenzyme A (acyl-CoA) into the mitochondrial matrix, which requires the action of carnitine palmitoyltransferase 1B (CPT1B) in striated muscle and BAT. In eutherians, the CPT1B gene is closely linked to the choline kinase beta (CHKB) gene, which is transcribed from the same DNA strand and terminates just upstream of CPT1B. CHKB is a rate-limiting enzyme in the synthesis of phosphatidylcholine (PC), a predominant mitochondrial membrane phospholipid, suggesting that the coordinated expression of CHKB and CPT1B may cooperatively enhance mitochondrial FAO. The present findings show that transcription of the eutherian CHKB and CPT1B genes is linked within a unitary epigenetic domain targeted to the CHKB gene, and that that this regulatory linkage appears to have resulted from an intergenic deletion in eutherians that significantly altered the distribution of CHKB and CPT1B expression. Informed by the timing of this event relative to the emergence of BAT, the phylogeny of CHKB-CPT1B synteny, and the insufficiency of UCP1 to account for eutherian BAT, these data support a mechanism for the emergence of BAT based on the acquisition of a novel capacity for adipocyte FAO in a background of extant UCP1.

Choline is a vitamin-like nutrient that is taken up via specific transporters and metabolized by choline kinase, which converts it to phosphocholine needed for de novo synthesis of phosphatidylcholine (PC), the main phospholipid of cellular membranes. We found that Toll-like receptor (TLR) activation enhances choline uptake by macrophages and microglia through induction of the choline transporter CTL1. Inhibition of CTL1 expression or choline phosphorylation attenuated NLRP3 inflammasome activation and IL-1β and IL-18 production in stimulated macrophages. Mechanistically, reduced choline uptake altered mitochondrial lipid profile, attenuated mitochondrial ATP synthesis, and activated the energy sensor AMP-activated protein kinase (AMPK). By potentiating mitochondrial recruitment of DRP1, AMPK stimulates mitophagy, which contributes to termination of NLRP3 inflammasome activation. Correspondingly, choline kinase inhibitors ameliorated acute and chronic models of IL-1β-dependent inflammation.

[Display omitted] • Overexpression of ChoKα1 in cancer cells makes it an excellent antitumor target. • Fa22 and PL48 inhibit ChoKα1 but have low specificity. • Coupling ChoKα1 inhibitors to BMNPs enhances specificity. • ChoKα1 inhibitors-BMNPs enable combined chemo and hyperthermia therapies. Targeting phospholipid biosynthesis, specifically phosphatidylcholine (PC), which is enhanced in tumor cells, has been proven a suitable antitumor strategy. In fact, the overexpression of the choline kinase α1 (ChoKα1) isoform has been found in malignant cells and tumors, thus becoming an excellent antitumor target. ChoKα1 inhibitors are being synthesized at the present that show a large inhibitory activity. Two of them have been chosen in this study as representatives of different structural families: a biscationic biphenyl derivative of thieno[3,2- d ]pyrimidinium substituted with a cyclic amine (here referred to as Fa22) and a biscationic biphenyl thioethano derivative of 7-chloro-quinolinium substituted with a pyrrolidinic moiety (here referred to as PL48). However, the potential use of these types of compounds in systemic treatments is hampered because of their low specificity. In fact, to enter the cell and reach their target, these inhibitors use choline transporters and inhibit choline uptake, being that one of the causes of their toxicity. One way to solve this problem could be allowing their entrance into the cells by alternative ways. With this goal, MamC-mediated magnetic nanoparticles (BMNPs), already proven effective drug nanocarriers, have been used to immobilize Fa22 and PL48. The idea is to let BMNPs enter the cell (they enter the cell by endocytosis) carrying these molecules, and, therefore, offering another way in for these compounds. In the present study, we demonstrate that the coupling of Fa22 and PL48 to BMNPs allows these molecules to enter the tumoral cell without completely inhibiting choline uptake, so, therefore, the use of Fa22 and PL48 in these nanoformulations reduces the toxicity compared to that of the soluble drugs. Moreover, the nanoassemblies Fa22-BMNPs and PL48-BMNPs allow the combination of chemotherapy and local hyperthermia therapies for a enhanced cytotoxic effect on the tumoral HepG2 cell line. The consistency of the results, independently of the drug structure, may indicate that this behavior could be extended to other ChoKα1 inhibitors, opening up a possibility for their potential use in clinics. [ABSTRACT FROM AUTHOR]

BackgroundMagnetic resonance spectroscopy (MRS) studies have identified elevated levels of the phospholipid precursor phosphocholine (PC) and phosphoethanolamine (PE) as metabolic hallmarks of cancer. Unusually, however, PC and PE levels are reduced in mutant isocitrate dehydrogenase 1 (IDHmut) gliomas that produce the oncometabolite 2-hydroxyglutarate (2-HG) relative to wild-type IDH1 (IDHwt) gliomas. The goal of this study was to determine the molecular mechanism underlying this unusual metabolic reprogramming in IDHmut gliomas.MethodsSteady-state PC and PE were quantified using 31P-MRS. To quantify de novo PC and PE synthesis, we used 13C-MRS and measured flux to 13C-PC and 13C-PE in cells incubated with [1,2-13C]-choline and [1,2-13C]-ethanolamine. The activities of choline kinase (CK) and ethanolamine kinase (EK), the enzymes responsible for PC and PE synthesis, were quantified using 31P-MR-based assays. To interrogate the role of 2-HG, we examined IDHwt cells incubated with 2-HG and, conversely, IDHmut cells treated with the IDHmut inhibitor AGI-5198. To examine the role of hypoxia-inducible factor 1-α (HIF-1α), we silenced HIF-1α using RNA interference. To confirm our findings in vivo and in the clinic, we studied IDHwt and IDHmut orthotopic tumor xenografts and glioma patient biopsies.ResultsDe novo synthesis of PC and PE was reduced in IDHmut cells relative to IDHwt. Concomitantly, CK activity and EK activity were reduced in IDHmut cells. Pharmacological manipulation of 2-HG levels established that 2-HG was responsible for reduced CK activity, EK activity, PC and PE. 2-HG has previously been reported to stabilize levels of HIF-1α, a known regulator of CK activity. Silencing HIF-1α in IDHmut cells restored CK activity, EK activity, PC and PE to IDHwt levels. Our findings were recapitulated in IDHmut orthotopic tumor xenografts and, most importantly, in IDHmut patient biopsies, validating our findings in vivo and in the clinic.ConclusionsThis study identifies, to our knowledge for the first time, a direct role for 2-HG in the downregulation of CK and EK activity, and thereby, PC and PE synthesis in IDHmut gliomas. These results highlight the unusual reprogramming of phospholipid metabolism in IDHmut gliomas and have implications for the identification of MRS-detectable metabolic biomarkers associated with 2-HG status.

The availability of nanoparticles (NPs) to deliver small interfering RNA (siRNA) has significantly expanded the specificity and range of 'druggable' targets for precision medicine in cancer. This is especially important for cancers such as triple negative breast cancer (TNBC) for which there are no targeted treatments. Our purpose here was to understand the role of tumor vasculature and vascular endothelial growth factor (VEGF) overexpression in a TNBC xenograft in improving the delivery and function of siRNA NPs using in vivo as well as ex vivo imaging. We used triple negative MDA-MB-231 human breast cancer xenografts derived from cells engineered to overexpress VEGF to understand the role of VEGF and vascularization in NP delivery and function. We used polyethylene glycol (PEG) conjugated polyethylenimine (PEI) NPs to deliver siRNA that downregulates choline kinase alpha (Chkα), an enzyme that is associated with malignant transformation and tumor progression. Because Chkα converts choline to phosphocholine, effective delivery of Chkα siRNA NPs resulted in functional changes of a significant decrease in phosphocholine and total choline that was detected with 1H magnetic resonance spectroscopy (MRS). We observed a significant increase in NP delivery and a significant decrease in Chkα and phosphocholine in VEGF overexpressing xenografts. Our results demonstrated the importance of tumor vascularization in achieving effective siRNA delivery and downregulation of the target gene Chkα and its function. [ABSTRACT FROM AUTHOR]

Lung cancer is one of the main causes of death in developed countries, and non-small cell lung cancer (NSCLC) is the most frequent type (80% of patients). In advanced NSCLC, platinum-based chemotherapy is the frontline palliative treatment, but less than 5% of patients achieve prolonged survival. Immunotherapy has recently been proposed as the standard of care (SoC) as either monotherapy or in combination with chemotherapy for advanced NSCLC. The levels of expression of PD-L1 are the only predictive biomarkers for patient assessment. Although around 30% of patients receiving immunotherapy achieve 5-year survival, a significant number does not benefit from this novel therapeutic approach. Therefore, there is a need for novel strategies to improve clinical outcomes. The expression level of choline kinase α (ChoKα) is increased in a large number of human tumors, including NSCLC tumors, and constitutes an independent prognostic factor for early-stage NSCLC patients. Thus, ChoKα has been postulated as a new target drug in cancer therapy. The combination of cisplatin with novel targeted drugs such as choline kinase inhibitors may improve both the survival rates and the quality of life of NSCLC patients and may serve as the basis for the development of new therapeutic approaches. To that aim, we developed several in vitro and in vivo approaches to assess the antitumor activity of a novel combination regimen using cisplatin and ChoKα inhibitors. Our results suggest that a proper combination of specific inhibitors of the NSCLC prognostic factor ChoKα and platinum-based conventional chemotherapy might constitute a new, efficient treatment approach for NSCLC patients. This novel approach may help reduce the toxicity profile associated with cisplatin since, despite the advances in NSCLC management in recent years, the overall 5-year survival rate is still poor. [ABSTRACT FROM AUTHOR]

Background: Rapid tumor growth inevitably results in energy stress, including deficiency of glutamine, a critical amino acid for tumor cell proliferation. However, whether glutamine deficiency allows tumor cells to use lipid droplets as an energy resource and the mechanism underlying this potential regulation remain unclear. Methods: We purified lipid droplets from H322 and H358 human non-small-cell lung cancer (NSCLC) cells under glutamine deprivation conditions and performed immunoblotting to determine the binding of choline kinase (CHK) α2 to lipid droplets. Immunofluorescence was used to quantify lipid droplet numbers and sizes. Immunoprecipitation and immunoblotting were performed to examine AMPK activation and CHKα2 phosphorylation. Cellular fatty acid levels, mitochondrial acetyl coenzyme A and ATP production, and cell apoptosis and proliferation were measured. Immunohistochemical analyses were performed to determine the expression levels of ACC pS79 and CHKα2 pS279 in tumor specimens from NSCLC patients. The prognostic value of ACC pS79 and CHKα2 pS279 was assessed using the Kaplan-Meier method and Cox regression models. Results: Glutamine deficiency induces AMPK-mediated CHKα2 S279 phosphorylation, which promotes the binding of CHKα2 to lipid droplets, resulting in recruitment of cytosolic lipase ATGL and autophagosomes and subsequent lipolysis of lipid droplets to sustain tumor cell survival and proliferation. In addition, the levels of ACC pS79 and CHKα S279 were much higher in human NSCLC specimens than in their adjacent normal tissues and positively correlated with each other. Notably, ACC pS79 and CHKα pS279 expression levels alone were associated with poor prognosis of NSCLC patients, and combined values of both phosphorylation levels were correlated with worse prognosis of the patients. Conclusion: CHKα2 plays a critical role in lipolysis of lipid droplets in NSCLC. ACC pS79 and CHKα2 pS279 alone or in combination can be used as prognostic markers in NSCLC. [ABSTRACT FROM AUTHOR]

Abstract Background Choline kinase-α (ChoKα/CHKA) overexpression and hyper-activation sustain altered choline metabolism conferring the cholinic phenotype to epithelial ovarian cancer (OC), the most lethal gynecological tumor. We previously proved that CHKA down-modulation reduced OC cell aggressiveness and increased sensitivity to in vitro chemotherapeutics’ treatment also affecting intracellular content of one-carbon metabolites. In tumor types other than ovary, methionine decrease was shown to increase sensitivity to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-receptor 2 triggering. These effects were suggestive of a potential role for ChoKα in regulating susceptibility to TRAIL cytokine. Methods The relationship between ChoKα/CHKA and TRAIL-receptor 2 (TRAIL-R2) expression was investigated in silico in OC patients’ GEO datasets and in vitro in a panel of OC cell lines upon transient CHKA silencing (siCHKA). The effect of siCHKA on metabolites content was assessed by LC-MS. The triggered apoptotic signalling was studied following soluble-TRAIL or anti-TRAIL-R2 agonist antibody treatment. Lipid rafts were isolated by Triton X-100 fractionation. Preclinical ex vivo studies were performed in OC cells derived from patients’ ascites using autologous PBLs as effectors and a bispecific anti-TRAIL-R2/anti-CD3 antibody as triggering agent. Results Here we demonstrate that siCHKA specifically overcomes resistance to TRAIL-mediated apoptosis in OC cells. Upon siCHKA we detected: a significant sensitization to caspase-dependent apoptosis triggered by both soluble TRAIL and anti-TRAIL-R2 agonist antibody, a specific increase of TRAIL-R2 expression and TRAIL-R2 relocation into lipid rafts. In siCHKA-OC cells the acquired TRAIL sensitivity was completely reverted upon recovery of ChoKα expression but, at variance of other tumor cell types, TRAIL sensitivity was not efficiently phenocopied by methionine deprivation. Of note, we were also able to show that siCHKA sensitized tumor cells derived ex vivo from OC patients’ ascites to the cytotoxic activity of autologous lymphocytes redirected by a bispecific anti-TRAIL-R2/anti-CD3 antibody. Conclusions Our findings suggest that ChoKα/CHKA impairment, by restoring drug-induced or receptor-mediated cell death, could be a suitable therapeutic strategy to be used in combination with chemotherapeutics or immunomodulators to improve OC patients’ outcome.

BackgroundRapid tumor growth inevitably results in energy stress, including deficiency of glutamine, a critical amino acid for tumor cell proliferation. However, whether glutamine deficiency allows tumor cells to use lipid droplets as an energy resource and the mechanism underlying this potential regulation remain unclear.MethodsWe purified lipid droplets from H322 and H358 human non-small-cell lung cancer (NSCLC) cells under glutamine deprivation conditions and performed immunoblotting to determine the binding of choline kinase (CHK) α2 to lipid droplets. Immunofluorescence was used to quantify lipid droplet numbers and sizes. Immunoprecipitation and immunoblotting were performed to examine AMPK activation and CHKα2 phosphorylation. Cellular fatty acid levels, mitochondrial acetyl coenzyme A and ATP production, and cell apoptosis and proliferation were measured. Immunohistochemical analyses were performed to determine the expression levels of ACC pS79 and CHKα2 pS279 in tumor specimens from NSCLC patients. The prognostic value of ACC pS79 and CHKα2 pS279 was assessed using the Kaplan-Meier method and Cox regression models.ResultsGlutamine deficiency induces AMPK-mediated CHKα2 S279 phosphorylation, which promotes the binding of CHKα2 to lipid droplets, resulting in recruitment of cytosolic lipase ATGL and autophagosomes and subsequent lipolysis of lipid droplets to sustain tumor cell survival and proliferation. In addition, the levels of ACC pS79 and CHKα S279 were much higher in human NSCLC specimens than in their adjacent normal tissues and positively correlated with each other. Notably, ACC pS79 and CHKα pS279 expression levels alone were associated with poor prognosis of NSCLC patients, and combined values of both phosphorylation levels were correlated with worse prognosis of the patients.ConclusionCHKα2 plays a critical role in lipolysis of lipid droplets in NSCLC. ACC pS79 and CHKα2 pS279 alone or in combination can be used as prognostic markers in NSCLC.

In the past 30 years there has been a significant increase in the number of publications on phospholipid (PL) metabolism, both for the medical purposes of detection and diagnosis of cancer and for the monitoring of the treatment of human cancers. Most of the work has focused on the pathway that produces phosphatidylcholine, the major component of human cell membranes. The trigger for this research was the advent of applications of NMR spectroscopy in vitro and in vivo in the 1980’s and observations that most cancer cells and tumors had significant increases in the water-soluble PL precursors and breakdown products. Increased phosphocholine (PC) has been focused on as a marker for cancer using Magnetic Resonance Spectroscopy (MRS) and Positron Emission Tomography (PET). MRS is now used clinically to aid in the diagnosis and severity of some brain tumors; and choline PET is used for the diagnosis and staging of recurrent prostate cancer, paid for by medical insurance companies. Another major area of research starting in the 1990’s was the development of specific choline kinase (CK) inhibitors aimed at the isoenzyme CK-a. This isoenzyme is markedly upregulated in cancer cells and unexpectedly was found to have a role in oncogenic transformation independent of its enzyme function.

ObjectivesLittle is known about targeting the metabolome in non-cancer conditions. Choline kinase (ChoKα), an essential enzyme for phosphatidylcholine biosynthesis, is required for cell proliferation and has been implicated in cancer invasiveness. Aggressive behaviour of fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA) led us to evaluate whether this metabolic pathway could play a role in RA FLS function and joint damage.MethodsCholine metabolic profile of FLS cells was determined by (1)H magnetic resonance spectroscopy ((1)HMRS) under conditions of ChoKα inhibition. FLS function was evaluated using the ChoKα inhibitor MN58b (IC₅₀=4.2 μM). For arthritis experiments, mice were injected with K/BxN sera. MN58b (3 mg/kg) was injected daily intraperitoneal beginning on day 0 or day 4 after serum administration.ResultsThe enzyme is expressed in synovial tissue and in cultured RA FLS. Tumour necrosis factor (TNF) and platelet-derived growth factor (PDGF) stimulation increased ChoKα expression and levels of phosphocholine in FLS measured by Western Blot (WB) and metabolomic studies of choline-containing compounds in cultured RA FLS extracts respectively, suggesting activation of this pathway in RA synovial environment. A ChoKα inhibitor also suppressed the behaviour of cultured FLS, including cell migration and resistance to apoptosis, which might contribute to cartilage destruction in RA. In a passive K/BxN arthritis model, pharmacologic ChoKα inhibition significantly decreased arthritis in pretreatment protocols as well as in established disease.ConclusionsThese data suggest that ChoKα inhibition could be an effective strategy in inflammatory arthritis. It also suggests that targeting the metabolome can be a new treatment strategy in non-cancer conditions.

Liver regeneration is a key compensatory process in response to liver injury serving to contain damages and to rescue liver functions. Hepatocytes, having temporarily exited the cell cycle after embryogenesis, resume proliferation to regenerate the injured liver parenchyma. In the present study we investigated the transcriptional regulation of choline kinase alpha (Chka) in hepatocytes in the context of liver regeneration. We report that Chka expression was significantly up-regulated in the regenerating livers in the partial hepatectomy (PHx) model and the acetaminophen (APAP) injection model. In addition, treatment with hepatocyte growth factor (HGF), a strong pro-proliferative cue, stimulated Chka expression in primary hepatocytes. Chka depletion attenuated HGF-induced proliferation of hepatocytes as evidenced by quantitative PCR and Western blotting measurements of pro-proliferative genes as well as EdU incorporation into replicating DNA. Of interest, deletion of Brahma-related gene 1 (Brg1), a chromatin remodeling protein, attenuated Chka induction in the regenerating livers in mice and in cultured hepatocytes. Further analysis revealed that Brg1 interacted with hypoxia-inducible factor 1 alpha (HIF-1α) to directly bind to the Chka promoter and activate Chka transcription. Finally, examination of human acute liver failure (ALF) specimens identified a positive correlation between Chka expression and Brg1 expression. In conclusion, our data suggest that Brg1-dependent trans-activation of Chka expression may contribute to liver regeneration.

Abstract Background Group I Paks are serine/threonine kinases that function as major effectors of the small GTPases Rac1 and Cdc42, and they regulate cytoskeletal dynamics, cell polarity, and transcription. We previously demonstrated that Pak1 and Pak2 function redundantly to promote skeletal myoblast differentiation during postnatal development and regeneration in mice. However, the roles of Pak1 and Pak2 in adult muscle homeostasis are unknown. Choline kinase β (Chk β) is important for adult muscle homeostasis, as autosomal recessive mutations in CHKβ are associated with two human muscle diseases, megaconial congenital muscular dystrophy and proximal myopathy with focal depletion of mitochondria. Methods We analyzed mice conditionally lacking Pak1 and Pak2 in the skeletal muscle lineage (double knockout (dKO) mice) over 1 year of age. Muscle integrity in dKO mice was assessed with histological stains, immunofluorescence, electron microscopy, and western blotting. Assays for mitochondrial respiratory complex function were performed, as was mass spectrometric quantification of products of choline kinase. Mice and cultured myoblasts deficient for choline kinase β (Chk β) were analyzed for Pak1/2 phosphorylation. Results dKO mice developed an age-related myopathy. By 10 months of age, dKO mouse muscles displayed centrally-nucleated myofibers, fibrosis, and signs of degeneration. Disease severity occurred in a rostrocaudal gradient, hindlimbs more strongly affected than forelimbs. A distinctive feature of this myopathy was elongated and branched intermyofibrillar (megaconial) mitochondria, accompanied by focal mitochondrial depletion in the central region of the fiber. dKO muscles showed reduced mitochondrial respiratory complex I and II activity. These phenotypes resemble those of rmd mice, which lack Chkβ and are a model for human diseases associated with CHKβ deficiency. Pak1/2 and Chkβ activities were not interdependent in mouse skeletal muscle, suggesting a more complex relationship in regulation of mitochondria and muscle homeostasis. Conclusions Conditional loss of Pak1 and Pak2 in mice resulted in an age-dependent myopathy with similarity to mice and humans with CHKβ deficiency. Protein kinases are major regulators of most biological processes but few have been implicated in muscle maintenance or disease. Pak1/Pak2 dKO mice offer new insights into these processes.

The availability of nanoparticles (NPs) to deliver small interfering RNA (siRNA) has significantly expanded the specificity and range of ‘druggable’ targets for precision medicine in cancer. This is especially important for cancers such as triple negative breast cancer (TNBC) for which there are no targeted treatments. Our purpose here was to understand the role of tumor vasculature and vascular endothelial growth factor (VEGF) overexpression in a TNBC xenograft in improving the delivery and function of siRNA NPs using in vivo as well as ex vivo imaging. We used triple negative MDA-MB-231 human breast cancer xenografts derived from cells engineered to overexpress VEGF to understand the role of VEGF and vascularization in NP delivery and function. We used polyethylene glycol (PEG) conjugated polyethylenimine (PEI) NPs to deliver siRNA that downregulates choline kinase alpha (Chkα), an enzyme that is associated with malignant transformation and tumor progression. Because Chkα converts choline to phosphocholine, effective delivery of Chkα siRNA NPs resulted in functional changes of a significant decrease in phosphocholine and total choline that was detected with 1H magnetic resonance spectroscopy (MRS). We observed a significant increase in NP delivery and a significant decrease in Chkα and phosphocholine in VEGF overexpressing xenografts. Our results demonstrated the importance of tumor vascularization in achieving effective siRNA delivery and downregulation of the target gene Chkα and its function.

Lung cancer is one of the main causes of death in developed countries, and non-small cell lung cancer (NSCLC) is the most frequent type (80% of patients). In advanced NSCLC, platinum-based chemotherapy is the frontline palliative treatment, but less than 5% of patients achieve prolonged survival. Immunotherapy has recently been proposed as the standard of care (SoC) as either monotherapy or in combination with chemotherapy for advanced NSCLC. The levels of expression of PD-L1 are the only predictive biomarkers for patient assessment. Although around 30% of patients receiving immunotherapy achieve 5-year survival, a significant number does not benefit from this novel therapeutic approach. Therefore, there is a need for novel strategies to improve clinical outcomes. The expression level of choline kinase α (ChoKα) is increased in a large number of human tumors, including NSCLC tumors, and constitutes an independent prognostic factor for early-stage NSCLC patients. Thus, ChoKα has been postulated as a new target drug in cancer therapy. The combination of cisplatin with novel targeted drugs such as choline kinase inhibitors may improve both the survival rates and the quality of life of NSCLC patients and may serve as the basis for the development of new therapeutic approaches. To that aim, we developed several in vitro and in vivo approaches to assess the antitumor activity of a novel combination regimen using cisplatin and ChoKα inhibitors. Our results suggest that a proper combination of specific inhibitors of the NSCLC prognostic factor ChoKα and platinum-based conventional chemotherapy might constitute a new, efficient treatment approach for NSCLC patients. This novel approach may help reduce the toxicity profile associated with cisplatin since, despite the advances in NSCLC management in recent years, the overall 5-year survival rate is still poor.

Endoplasmic reticulum (ER) is a central organelle in eukaryotic cells that regulates protein synthesis and maturation. Perturbation of ER functions leads to ER stress, which has been previously associated with a broad variety of diseases. ER stress is generally regarded as compensatory, but prolonged ER stress has been involved in apoptosis induced by several cytotoxic agents. Choline kinase α (ChoKα), the first enzyme in the Kennedy pathway, is responsible for the generation of phosphorylcholine (PCho) that ultimately renders phosphatidylcholine. ChoKα overexpression and high PCho levels have been detected in several cancer types. Inhibition of ChoKα has demonstrated antiproliferative and antitumor properties; however, the mechanisms underlying these activities remain poorly understood. Here, we demonstrate that ChoKα inhibitors (ChoKIs), MN58b and RSM932A, induce cell death in cancer cells (T47D, MCF7, MDA-MB231, SW620 and H460), through the prolonged activation of ER stress response. Evidence of ChoKIs-induced ER stress includes enhanced production of glucose-regulated protein, 78 kDa (GRP78), protein disulfide isomerase, IRE1α, CHOP, CCAAT/enhancer-binding protein beta (C/EBPβ) and TRB3. Although partial reduction of ChoKα levels by small interfering RNA was not sufficient to increase the production of ER stress proteins, silencing of ChoKα levels also show a decrease in CHOP overproduction induced by ChoKIs, which suggests that ER stress induction is due to a change in ChoKα protein folding after binding to ChoKIs. Silencing of CHOP expression leads to a reduction in C/EBPβ, ATF3 and GRP78 protein levels and abrogates apoptosis in tumor cells after treatment with ChoKIs, suggesting that CHOP maintains ER stress responses and triggers the pro-apoptotic signal. Consistent with the differential effect of ChoKIs in cancer and primary cells previously described, ChoKIs only promoted a transient and moderated ER stress response in the non-tumorogenic cells MCF10A. In conclusion, pharmacological inhibition of ChoKα induces cancer cell death through a mechanism that involves the activation of exaggerated and persistent ER stress supported by CHOP overproduction.

Background: Choline kinase-α (ChoKα/CHKA) overexpression and hyper-activation sustain altered choline metabolism conferring the cholinic phenotype to epithelial ovarian cancer (OC), the most lethal gynecological tumor. We previously proved that CHKA down-modulation reduced OC cell aggressiveness and increased sensitivity to in vitro chemotherapeutics' treatment also affecting intracellular content of one-carbon metabolites. In tumor types other than ovary, methionine decrease was shown to increase sensitivity to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-receptor 2 triggering. These effects were suggestive of a potential role for ChoKα in regulating susceptibility to TRAIL cytokine. Methods: The relationship between ChoKα/CHKA and TRAIL-receptor 2 (TRAIL-R2) expression was investigated in silico in OC patients' GEO datasets and in vitro in a panel of OC cell lines upon transient CHKA silencing (siCHKA). The effect of siCHKA on metabolites content was assessed by LC-MS. The triggered apoptotic signalling was studied following soluble-TRAIL or anti-TRAIL-R2 agonist antibody treatment. Lipid rafts were isolated by Triton X-100 fractionation. Preclinical ex vivo studies were performed in OC cells derived from patients' ascites using autologous PBLs as effectors and a bispecific anti-TRAIL-R2/anti-CD3 antibody as triggering agent. Results: Here we demonstrate that siCHKA specifically overcomes resistance to TRAIL-mediated apoptosis in OC cells. Upon siCHKA we detected: a significant sensitization to caspase-dependent apoptosis triggered by both soluble TRAIL and anti-TRAIL-R2 agonist antibody, a specific increase of TRAIL-R2 expression and TRAIL-R2 relocation into lipid rafts. In siCHKA-OC cells the acquired TRAIL sensitivity was completely reverted upon recovery of ChoKα expression but, at variance of other tumor cell types, TRAIL sensitivity was not efficiently phenocopied by methionine deprivation. Of note, we were also able to show that siCHKA sensitized tumor cells derived ex vivo from OC patients' ascites to the cytotoxic activity of autologous lymphocytes redirected by a bispecific anti-TRAIL-R2/anti-CD3 antibody. Conclusions: Our findings suggest that ChoKα/CHKA impairment, by restoring drug-induced or receptor-mediated cell death, could be a suitable therapeutic strategy to be used in combination with chemotherapeutics or immunomodulators to improve OC patients' outcome. [ABSTRACT FROM AUTHOR]

Hypoxia is a complex microenvironmental condition known to regulate choline kinase α (CHKA) activity and choline transport through transcription factor hypoxia-inducible factor-1α (HIF-1α) and, therefore, may confound the uptake of choline radiotracer [18F]fluoromethyl-[1,2-2H4]-choline ([18F]-D4-FCH). The aim of this study was to investigate how hypoxia affects the choline radiotracer dynamics. Three underlying mechanisms by which hypoxia could potentially alter the uptake of the choline radiotracer, [18F]-D4-FCH, were investigated: 18F-D4-FCH import, CHKA phosphorylation activity, and the efflux of [18F]-D4-FCH and its phosphorylated product [18F]-D4-FCHP. The effects of hypoxia on [18F]-D4-FCH uptake were studied in CHKA-overexpressing cell lines of prostate cancer, PC-3, and breast cancer MDA-MB-231 cells. The mechanisms of radiotracer efflux were assessed by the cell uptake and immunofluorescence in vitro and examined in vivo (n = 24). The mathematical modelling methodology was further developed to verify the efflux hypothesis using [18F]-D4-FCH dynamic PET scans from non-small cell lung cancer (NSCLC) patients (n = 17). We report a novel finding involving the export of phosphorylated [18F]-D4-FCH and [18F]-D4-FCHP via HIF-1α-responsive efflux transporters, including ABCB4, when the HIF-1α level is augmented. This is supported by a graphical analysis of human data with a compartmental model (M2T6k + k5) that accounts for the efflux. Hypoxia/HIF-1α increases the efflux of phosphorylated radiolabelled choline species, thus supporting the consideration of efflux in the modelling of radiotracer dynamics.

Both nosocomial pathogens, such as Streptococcus pneumoniae and Haemophilus influenzae and food-borne pathogens, such as Bacillus cereus and Clostridium perfringens are known to be detrimental to human and animal health. The effectiveness of currently used treatments for these pathogens becomes limited as resistant strains emerge. Therefore, new methods for eliminating bacterial pathogens must be developed continuously. This includes establishing novel targets to which drug discovery efforts could be focused. A promising method for discovering new drug targets in prokaryotes is to take advantage of the information available regarding the enzymatic pathways that have been established as drug targets in eukaryotic systems and explore the analogous pathways found in bacterial systems. This is an efficient strategy because the same inhibitors developed at considerable expense to block these pathways in eukaryotic systems could also be employed in prokaryotes. Drugs that are used to prevent diseases involving eukaryotic cells could be repurposed as antibiotics and antimicrobials for the control of bacteria pathogens. This strategy could be pursued whenever the primary and tertiary structures of a target are are conserved between eukaryotic and prokaryotes. A possible novel target fitting these parameters is choline kinase (ChoK), whose active site sequences are conserved (Figure 1) and whose tertiary structure (Figure 2) is maintained. Here, we describe why ChoK is a putative drug target by describing its role in the growth and pathogenesis of Gram-positive bacteria S. pneumoniae and the Gram-negative bacteria H. influenzae. Using S. pneumoniae as a model, we also present promising preliminary information that repurposing of drugs known to inhibit the human isoform of ChoK (hChoK), is a promising strategy for blocking the growth of S. pneumoniae cells and inhibiting the activity of the S. pneumoniae isoform of ChoK (sChok), with downstream physiological effects on the cell wall.

Background: Angioimmunoblastic T-cell lymphoma (AITL) is a malignancy with very poor survival outcome, in urgent need of more specific therapeutic strategies. The drivers of malignancy in this disease are CD4 + follicular helper T cells (Tfh). The metabolism of these malignant Tfh cells was not yet elucidated. Therefore, we decided to identify their metabolic requirements with the objective to propose a novel therapeutic option., Methods: To reveal the prominent metabolic pathways used by the AITL lymphoma cells, we relied on metabolomic and proteomic analysis of murine AITL (mAITL) T cells isolated from our established mAITL model. We confirmed these results using AITL patient and healthy T cell expression data., Results: Strikingly, the mAITL Tfh cells were highly dependent on the second branch of the Kennedy pathway, the choline lipid pathway, responsible for the production of the major membrane constituent phosphatidylcholine. Moreover, gene expression data from Tfh cells isolated from AITL patient tumors, confirmed the upregulation of the choline lipid pathway. Several enzymes involved in this pathway such as choline kinase, catalyzing the first step in the phosphatidylcholine pathway, are upregulated in multiple tumors other than AITL. Here we showed that treatment of our mAITL preclinical mouse model with a fatty acid oxydation inhibitor, significantly increased their survival and even reverted the exhausted CD8 T cells in the tumor into potent cytotoxic anti-tumor cells. Specific inhibition of Chokα confirmed the importance of the phosphatidylcholine production pathway in neoplastic CD4 + T cells, nearly eradicating mAITL Tfh cells from the tumors. Finally, the same inhibitor induced in human AITL lymphoma biopsies cell death of the majority of the hAITL PD-1 high neoplastic cells., Conclusion: Our results suggest that interfering with choline metabolism in AITL reveals a specific metabolic vulnerability and might represent a new therapeutic strategy for these patients., (© 2024. The Author(s).)

Aberrant choline metabolism, characterized by an increase in total choline-containing compounds, phosphocholine and phosphatidylcholine (PC), is a metabolic hallmark of carcinogenesis and tumor progression. This aberration arises from alterations in metabolic enzymes that control PC biosynthesis and catabolism. Among these enzymes, choline kinase α (CHKα) exhibits the most frequent alterations and is commonly overexpressed in human cancers. CHKα catalyzes the phosphorylation of choline to generate phosphocholine, the first step in de novo PC biosynthesis. CHKα overexpression is associated with the malignant phenotype, metastatic capability and drug resistance in human cancers, and thus has been recognized as a robust biomarker and therapeutic target of cancer. Of clinical importance, increased choline metabolism and CHKα activity can be detected by non-invasive magnetic resonance spectroscopy (MRS) or positron emission tomography/computed tomography (PET/CT) imaging with radiolabeled choline analogs for diagnosis and treatment monitoring of cancer patients. Both choline-based MRS and PET/CT imaging have also been clinically applied for lymphoid malignancies, including non-Hodgkin lymphoma, multiple myeloma and central nervous system lymphoma. However, information on how choline kinase is dysregulated in lymphoid malignancies is very limited and has just begun to be unraveled. In this review, we provide an overview of the current understanding of choline kinase in B cell and T cell malignancies with the goal of promoting future investigation in this area.

Choline kinase (ChoK) is a cytosolic enzyme that catalyzes the phosphorylation of choline to form phosphorylcholine (PCho) in the presence of ATP and magnesium. ChoK is required for the synthesis of key membrane phospholipids and is involved in malignant transformation in a large variety of human tumours. Active compounds against ChoK have been identified and proposed as antitumor agents. The ChoK inhibitory and antiproliferative activities of symmetrical bispyridinium and bisquinolinium compounds have been defined using quantitative structure–activity relationships (QSARs) and structural parameters. The design strategy followed in the development of the most active molecules is presented. The selective anticancer activity of these structures is also described. One promising anticancer compound has even entered clinical trials. Recently, ChoKα inhibitors have also been proposed as a novel therapeutic approach against parasites, rheumatoid arthritis, inflammatory processes, and pathogenic bacteria. The evidence for ChoKα as a novel drug target for approaches in precision medicine is discussed.

Both nosocomial pathogens, such as Streptococcus pneumoniae and Haemophilus influenzae and food-borne pathogens, such as Bacillus cereus and Clostridium perfringens are known to be detrimental to human and animal health. The effectiveness of currently used treatments for these pathogens becomes limited as resistant strains emerge. Therefore, new methods for eliminating bacterial pathogens must be developed continuously. This includes establishing novel targets to which drug discovery efforts could be focused. A promising method for discovering new drug targets in prokaryotes is to take advantage of the information available regarding the enzymatic pathways that have been established as drug targets in eukaryotic systems and explore the analogous pathways found in bacterial systems. This is an efficient strategy because the same inhibitors developed at considerable expense to block these pathways in eukaryotic systems could also be employed in prokaryotes. Drugs that are used to prevent diseases involving eukaryotic cells could be repurposed as antibiotics and antimicrobials for the control of bacteria pathogens. This strategy could be pursued whenever the primary and tertiary structures of a target are are conserved between eukaryotic and prokaryotes. A possible novel target fitting these parameters is choline kinase (ChoK), whose active site sequences are conserved (Figure 1) and whose tertiary structure (Figure 2) is maintained. Here, we describe why ChoK is a putative drug target by describing its role in the growth and pathogenesis of Gram-positive bacteria S. pneumoniae and the Gram-negative bacteria H. influenzae. Using S. pneumoniae as a model, we also present promising preliminary information that repurposing of drugs known to inhibit the human isoform of ChoK (hChoK), is a promising strategy for blocking the growth of S. pneumoniae cells and inhibiting the activity of the S. pneumoniae isoform of ChoK (sChok), with downstream physiological effects on the cell wall. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Obtaining a halotolerant CKI by computational design without affecting its activity. • Increase of acidic residues is assumed to contribute to halotolerance. • CKI mutants show high tolerance in over 1200 mM acetate solutions. • CDP-choline producing using a halophilic CKI shows increased titer and productivity. Abstract Biocatalysis has become the main approach to produce cytidine diphosphate choline (CDP-choline), which has been applied for treatment of acute craniocerebral injury and consciousness after brain surgery. However, salt accumulates with the production and inhibits enzyme activity, and eventually reduces yield and product accumulation rate. Our work provided a possible solution to this problem by applying a computational designed halophilic choline kinase. The halotolerant CKI (choline kinase) was designed following a unique strategy considering the most variable residue positions on the protein surface among target enzymes from different sources. The basic and neutral surface residues were replaced with acidic ones. This approach was enlightened by features of natural halophilic enzymes. Mutants in the work represented higher catalytic activities and IC50 (inhibit activity by 50%) at high salt concentrations (over 1200 mM). Furthermore, when the mutant was used in fed-batch production, the CDP-choline accumulation rate doubled comparing with process using wild-type CKI at acetate concentration of over 700 mM. The maximum titer was 151 ± 3.2 mM, the productivity was 5.8 ± 0.1 mM·L−1 h−1, and molar yield to CMP and utilization efficiency of energy were 85.3 and 63.5%. The idea of computational design in our work can also be applied to modify other enzymes in industry, and sheds light on alleviating effect of salt accumulation during industrial manufacturing process. [ABSTRACT FROM AUTHOR]

The process of neuronal differentiation is associated with neurite elongation and membrane biogenesis, and phosphatidylcholine (PtdCho) is the major membrane phospholipid in mammalian cells. During neuroblast differentiation, the transcription of two genes involved in PtdCho biosynthesis are stimulated: Chka gene for choline kinase (CK) alpha isoform and Pcyt1a gene for CTP:phosphocholine cytidylyltransferase (CCT) alpha isoform. Here we show that CKα is essential for neuronal differentiation. In addition, we demonstrated that KDM2B regulates CKα expression and, as a consequence, neuronal differentiation. This factor is up-regulated in the course of the neuroblasts proliferative and undifferentiated state and down-regulated during differentiation induced by retinoic acid (RA). During proliferation, KDM2B binds to the Box2 located in the Chka promoter repressing its transcription. Interestingly, KDM2B knockdown enhances the levels of CKα expression in neuroblast cells and induces neuronal differentiation even in the absence of RA. These results suggest that KDM2B is required for the appropriate regulation of CKα during neuronal differentiation and to the maintaining of the undifferentiated stage of neuroblast cells. [ABSTRACT FROM AUTHOR]

Among the malaria-causing parasites, the deadliest is Plasmodium falciparum, which accounts for the majority of the fatalities. As the infection progresses inside erythrocytes, major cellular and metabolic changes take place. For its own growth, the parasite relies on the accumulation of phospholipids, which are essential for membrane synthesis. Within the Kennedy pathway, the P. falciparum choline kinase (PfChoK) has a central role in the biosynthesis of phosphatidylcholine and its selective inhibition leads to the parasite arrest and eradication. Here, we report the crystal structure of the apo and the ADP-bound form of choline kinase from Plasmodium falciparum at 2.0 and 2.2 Å resolution, respectively. These new structural data will facilitate the implementation of effective structure-based drug development strategies against PfChoK in the fight against malaria.

Although gemcitabine plus cisplatin is the gold standard chemotherapy regimen for advanced cholangiocarcinoma, the response rate has been disappointing. This study aims to investigate a novel therapeutic regimen [gemcitabine plus everolimus (rad001), an mTOR inhibitor] for cholangiocarcinoma. Gemcitabine, oxaliplatin, cetuximab and rad001 in various combinations were first evaluated in vitro using six cholangiocarcinoma cell lines. In vivo therapeutic efficacies of gemcitabine and rad001 alone and their combination were further evaluated using a xenograft mouse model and a chemically induced orthotopic cholangiocarcinoma rat model. In the in vitro study, gemcitabine plus rad001 exerted a synergistic therapeutic effect on the cholangiocarcinoma cells, irrespective of the KRAS mutation status. In the xenograft study, gemcitabine plus rad001 showed the best therapeutic effect on tumor volume change, and was associated with increased caspase-3 expression, decreased eIF4E expression, as well as overexpression of both death receptor- and mitochondrial apoptotic pathway-related genes. In a chemically induced cholangiocarcinoma-afflicted rat model, the gemcitabine plus rad001 treatment suppressed tumor glycolysis as measured by 18F-fludeoxyglucose micro-positron emission tomography. Also, increased intratumoral free choline, decreased glycerophosphocholine and nearly undetectable phosphocholine levels were demonstrated by proton nuclear magnetic resonance, supported by results of decreased choline kinase expression in western blotting. We concluded that gemcitabine plus rad001 has a synergistic antiproliferative effect on cholangiocarcinoma, irrespective of the KRAS mutation status. The antitumor effect is associated with activation of both death receptor and mitochondrial pathways, as well as the downregulation of choline kinase activity, resulting in a characteristic change in choline metabolism.

The major phospholipid present in most eukaryotic membranes is phosphatidylcholine (PC), comprising ~ 50% of phospholipid content. PC metabolic pathways are highly conserved from yeast to humans. The main pathway for the synthesis of PC is the Kennedy (CDP‐choline) pathway. In this pathway, choline is converted to phosphocholine by choline kinase, phosphocholine is metabolized to CDP‐choline by the rate‐determining enzyme for this pathway, CTP:phosphocholine cytidylyltransferase, and cholinephosphotransferase condenses CDP‐choline with diacylglycerol to produce PC. This Review discusses how PC synthesis via the Kennedy pathway is regulated, its role in cellular and biological processes, as well as diseases known to be associated with defects in PC synthesis. Finally, we present the first model for the making of a membrane via PC synthesis. [ABSTRACT FROM AUTHOR]

A tri-enzyme system consisting of choline kinase/choline oxidase/horseradish peroxidase was used in the rapid and specific determination of the biomarker for bacterial sepsis infection, secretory phospholipase Group 2-IIA (sPLA2-IIA). These enzymes were individually immobilized onto the acrylic microspheres via succinimide groups for the preparation of an electrochemical biosensor. The reaction of sPLA2-IIA with its substrate initiated a cascading enzymatic reaction in the tri-enzyme system that led to the final production of hydrogen peroxide, which presence was indicated by the redox characteristics of potassium ferricyanide, K3Fe(CN)6 . An amperometric biosensor based on enzyme conjugated acrylic microspheres and gold nanoparticles composite coated onto a carbon-paste screen printed electrode (SPE) was fabricated and the current measurement was performed at a low potential of 0.20 V. This enzymatic biosensor gave a linear range 0.01-100 ng/mL (R2 = 0.98304) with a detection limit recorded at 5 × 10-3 ng/mL towards sPLA2-IIA. Moreover, the biosensor showed good reproducibility (relative standard deviation (RSD) of 3.04% (n = 5). The biosensor response was reliable up to 25 days of storage at 4°C. Analysis of human serum samples for sPLA2-IIA indicated that the biosensor has potential for rapid bacterial sepsis diagnosis in hospital emergency department. [ABSTRACT FROM AUTHOR]

Choline kinase (Chok) is an enzyme found in eukaryotes and Gram-positive bacteria. Chok catalyzes the production of phosphocholine from choline and ATP. This enzyme has been validated as a drug target in Streptococcus pneumonia, but the role Chok enzymatic activity plays in bacterial cell growth and division is not well understood. Phosphocholine production by Chok and its attenuation by inhibitors in the context of complex samples such as cell extracts can currently be quantified by several methods. These include choline depletion measurements, radioactive methods, mass-spectrometry, and nuclear magnetic resonance. The first does not measure phosphocholine directly, the second requires elaborate safety procedures, and the third and fourth require significant capital investments and technical expertise. For these reasons, a less expensive, higher throughput, more easily accessible assay is needed to facilitate further study in Gram-positive Choks. Here, we present the development of a triiodide/activated charcoal/molybdenum blue system for detecting and quantifying choline and phosphocholine in parallel. We demonstrate that this system can reliably quantify changes in choline and phosphocholine concentrations over time in Chok enzymatic assays using cell extracts as the source of the enzyme. This is an easily accessible, convenient, robust, and economical method for studying Chok activity in complex samples. The triiodide/activated charcoal/molybdenum blue system opens new doors into the study choline kinase in Gram-positive pathogens. [ABSTRACT FROM AUTHOR]

Gram-positive pathogens, such as Streptococcus pneumoniae, can have deleterious effects on both human and animal health. Antibiotics and antimicrobials have been developed to treat infections caused by such pathogens and to prevent food contamination. However, these strategies have been increasingly thwarted by the emergence of resistant bacteria strains. Thus, new methods for controlling Gram-positive pathogen growth need to be continuously developed. Choline analogs, such as Hemicholinium-3 (HC-3), have been shown to be useful in blocking cell division in eukaryotic cells through the inhibition of choline kinase, an enzyme which catalyzes the production of phosphocholine from choline and ATP. In some Gram-positive pathogens, choline kinase is an important enzyme in the production of the cell wall element, lipoteichoic acid. However, it is not known if inhibiting this enzyme has any effect on cell division in Gram-positive bacteria. Using the R6 strain as a model, we tested the ability of HC-3 to block the activity of choline kinase in S. pneumoniae and inhibit cell growth. Mass-spectrometry measurements of crude extracts revealed that HC-3 blocked choline kinase activity. Turbidity measurements and population counts showed that HC-3 inhibited cell growth. Competition assays with choline suggested that HC-3 also blocked choline transporters. Western blots showed that lipoteichoic acid production was blocked in the presence of HC-3, and autolytic assays showed that this decrease in lipoteichoic acids caused cells to be more resistant to autolysis. Scanning electron microscopy revealed that HC-3 distorted the cell wall. This study thus establishes choline kinase as a novel drug target for S. pneumoniae. [ABSTRACT FROM AUTHOR]

In this paper, five novel cholinium-based organic magnetic ionic liquids (MILs) were synthesized. The carbon chain links to the nitrogen atom from cationic nucleus. The MILs were comprehensively identified by FT-IR, HPLC, UV–Vis, 1 H NMR and ESI-MS. The MILs have interesting performance to form aqueous two-phase system and can respond to the external magnetic field after the formation of aqueous two-phase system, which can assists magnetically phase separation. The magnetic susceptibility of the MILs was measured at 298 K. Other physical properties such as conductivities, acidity and density were measured in range of 278.15 K to 323.15 K for further application investigation. Satisfactory results were obtained after the data of physical properties were correlated. Based on the measurements and correlations above, some discussions were carried out, including the ion association and ion diffusion, the influences of MILs carbon chain and temperature on the molar conductivity, the influences of the cation size on the molar volume, acidity variation of the MILs. [ABSTRACT FROM AUTHOR]

In this study, we elucidated the mechanism by which human choline kinase-α (hCKα) interacts with nonstructural protein 5A (NS5A) and phosphatidylinositol-4-kinase IIIα (PI4KIIIα), the lipid kinase crucial for maintaining the integrity of virus-induced membranous webs and modulates hepatitis C virus (HCV) replication. hCKα activity positively modulated phosphatidylinositol-4-phosphate (PI4P) levels in HCV-expressing cells, and hCKα-mediated PI4P accumulation was abolished by AL-9, a PI4KIIIα- specific inhibitor. hCKα colocalized with NS5A and PI4KIIIα or PI4P; NS5A expression increased hCKα and PI4KIIIα colocalization; and hCKα formed a ternary complex with PI4KIIIα and NS5A, supporting the functional interplay of hCKα with PI4KIIIα and NS5A. PI4KIIIα inactivation by AL-9 or hCKα inactivation by CK37, a specific hCKα inhibitor, impaired the endoplasmic reticulum (ER) localization and colocalization of these three molecules. Interestingly, hCKα knockdown or inactivation inhibited PI4KIIIα- NS5A binding. In an in vitro PI4KIIIα activity assay, hCKα activity slightly increased PI4KIIIα basal activity but greatly augmented NS5A-induced PI4KIIIα activity, supporting the essential role of ternary complex formation in robust PI4KIIIα activation. Concurring with the upregulation of PI4P production and viral replication, overexpression of active hCKα-R (but not the D288A mutant) restored PI4KIIIα and NS5A translocation to the ER in hCKα stable knockdown cells. Furthermore, active PI4KIIIα overexpression restored PI4P production, PI4KIIIα and NS5A translocation to the ER, and viral replication in CK37-treated cells. Based on our results, hCKα functions as an indispensable regulator that bridges PI4KIIIα and NS5A and potentiates NS5Astimulated PI4KIIIα activity, which then facilitates the targeting of the ternary complex to the ER for viral replication.α IMPORTANCE The mechanisms by which hCKα activity modulates the transport of the hCKα-NS5A complex to the ER are not understood. In the present study, we investigated how hCKα interacts with PI4KIIIα (a key element that maintains the integrity of the "membranous web" structure) and NS5A to regulate viral replication. We demonstrated that HCV hijacks hCKα to bridge PI4KIIIα and NS5A, forming a ternary complex, which then stimulates PI4KIIIα activity to produce PI4P. Pronounced PI4P synthesis then redirects the translocation of the ternary complex to the ER-derived, PI4P-enriched membrane for assembly of the viral replication complex and viral replication. Our study provides novel insights into the indispensable modulatory role of hCKα in the recruitment of PI4KIIIα to NS5A and in NS5A-stimulated PI4P production and reveals a new perspective for understanding the impact of profound PI4KIIIα activation on the targeting of PI4KIIIα and NS5A to the PI4P-enriched membrane for viral replication complex formation. [ABSTRACT FROM AUTHOR]

Metabolites linked to changes in choline kinase-α (CK-α) expression and drug resistance, which contribute to survival and autophagy mechanisms, are attractive targets for breast cancer therapies. We previously reported that autophagy played a causative role in driving tamoxifen (TAM) resistance of breast cancer cells (BCCs) and was also promoted by CK-α knockdown, resulting in the survival of TAM-resistant BCCs. There is no comparative study yet about the metabolites resulting from BCCs with TAM-resistance and CK-α knockdown. Therefore, the aim of this study was to explore the discriminant metabolic biomarkers responsible for TAM resistance as well as CK-α expression, which might be linked with autophagy through a protective role. A total of 33 intracellular metabolites, including a range of amino acids, energy metabolism-related molecules and others from cell extracts of the parental cells (MCF-7), TAM-resistant cells (MCF-7/TAM) and CK-α knockdown cells (MCF-7/shCK-α, MCF-7/TAM/shCK-α) were analyzed by proton nuclear magnetic resonance spectroscopy (1H-NMRS). Principal component analysis (PCA) and partial least square discriminant analysis (PLS-DA) revealed the existence of differences in the intracellular metabolites to separate the 4 groups: MCF-7 cells, MCF-7/TAM cells, MCF-7-shCK-α cells, and MCF-7/TAM/shCK-α cells. The metabolites with VIP>1 contributed most to the differentiation of the cell groups, and they included fumarate, UA (unknown A), lactate, myo-inositol, glycine, phosphocholine, UE (unknown E), glutamine, formate, and AXP (AMP/ADP/ATP). Our results suggest that these altered metabolites would be promising metabolic biomarkers for a targeted therapeutic strategy in BCCs that exhibit TAM-resistance and aberrant CK-α expression, which triggers a survival and drug resistance mechanism. [ABSTRACT FROM AUTHOR]

Understanding protein interaction networks and their dynamic changes is a major challenge in modern biology. Currently, several experimental and in silico approaches allow the screening of protein interactors in a large-scale manner. Therefore, the bulk of information on protein interactions deposited in databases and peer-reviewed published literature is constantly growing. Multiple databases interfaced from user-friendly web tools recently emerged to facilitate the task of protein interaction data retrieval and data integration. Nevertheless, as we evidence in this report, despite the current efforts towards data integration, the quality of the information on protein interactions retrieved by in silico approaches is frequently incomplete and may even list false interactions. Here we point to some obstacles precluding confident data integration, with special emphasis on protein interactions, which include gene acronym redundancies and protein synonyms. Three human proteins (choline kinase, PPIase and uromodulin) and three different web-based data search engines focused on protein interaction data retrieval (PSICQUIC, DASMI and BIPS) were used to explain the potential occurrence of undesired errors that should be considered by researchers in the field. We demonstrate that, despite the recent initiatives towards data standardization, manual curation of protein interaction networks based on literature searches are still required to remove potential false positives. A three-step workflow consisting of: (i) data retrieval from multiple databases, (ii) peer-reviewed literature searches, and (iii) data curation and integration, is proposed as the best strategy to gather updated information on protein interactions. Finally, this strategy was applied to compile bona fide information on human DREAM protein interactome, which constitutes liable training datasets that can be used to improve computational predictions.

Triple negative breast cancer (TNBC), defined by lack of estrogen and progesterone receptors in the absence of protein overexpression/gene amplification of human epidermal growth factor receptor 2 (HER2), is still a clinical challenge despite progress in breast cancer (BC) care. 1H magnetic resonance spectroscopy (MRS) allows identification and non-invasive monitoring of TNBC metabolic aberrations and elucidation of some key mechanisms underlying tumor progression. Thus, it has the potential to improve in vivo diagnosis and follow-up, and identify new targets for treatment. Several studies have shown an altered phosphatidylcholine (PtdCho) metabolism in TNBCs, both in patients and in experimental models. Up-regulation of choline kinase-alpha, an enzyme of the Kennedy pathway that phosphorylates free choline (Cho) to phosphocholine (PCho), is a major contributor to the increased PCho content detected in TNBCs. Phospholipase-mediated PtdCho headgroup hydrolysis also contributes to the build-up of a PCho pool in TNBC cells. The oncogene-driven PtdCho cycle appears to be fine tuned in TNBC cells in at least three ways: by modulating the choline import, by regulating the activity or expression of specific metabolic enzymes, and by contributing to the rewiring of the entire metabolic network. Thus, only by thoroughly dissecting these mechanisms, it will be possible to effectively translate this basic knowledge into further development and implementation of Cho-based imaging techniques and novel classes of therapeutics.

Elucidation of molecular mechanisms underlying the aberrant phosphatidylcholine-cycle in cancer cells plays in favor of the use of metabolic imaging in oncology and opens the way for designing new targeted therapies. The anomalous choline metabolic profile detected in cancer by magnetic resonance spectroscopy (MRS) and spectroscopic imaging (MRSI) provides molecular signatures of tumor progression and response to therapy.The increased level of intracellular phosphocholine (PCho) typically detected in cancer cells is mainly attributed to upregulation of choline kinase, responsible for choline phosphorylation in the biosynthetic Kennedy pathway, but can also be partly produced by activation of phosphatidylcholine-specific phospholipase C (PC-PLC). This hydrolytic enzyme, known for implications in bacterial infection and in plant survival to hostile environmental conditions, is reported to be activated in mitogen- and oncogene-induced phosphatidylcholine cycles in mammalian cells, with effects on cell signaling, cell cycle regulation and cell proliferation.Recent investigations showed that PC-PLC activation could account for 20-to-50% of the intracellular PCho production in ovarian and breast cancer cells of different subtypes. Enzyme activation was associated with PC-PLC protein overexpression and subcellular redistribution in these cancer cells compared with non-tumoral counterparts. Moreover, PC-PLC co-immunoprecipitated with the human epidermal growth factor receptor-2 (HER2) and EGFR in HER2-overexpressing breast and ovarian cancer cells, while pharmacological PC-PLC inhibition resulted into long-lasting HER2 downregulation, retarded receptor re-expression on plasma membrane and anti-proliferative effects.This body of evidence points to PC-PLC as a potential target for newly designed therapies, whose effects can be pre-clinically and clinically monitored by metabolic imaging methods.