Choline Analogues Research Articles

New insight into the mechanism of accumulation and intraerythrocytic compartmentation of albitiazolium, a new type of antimalarial.

Bis-thiazolium salts constitute a new class of antihematozoan drugs that inhibit parasite phosphatidylcholine biosynthesis. They specifically accumulate in Plasmodium- and Babesia-infected red blood cells (IRBC). Here, we provide new insight into the choline analogue albitiazolium, which is currently being clinically tested against severe malaria. Concentration-dependent accumulation in P. falciparum-infected erythrocytes reached steady state after 90 to 120 min and was massive throughout the blood cycle, with cellular accumulation ratios of up to 1,000. This could not occur through a lysosomotropic effect, and the extent did not depend on the food vacuole pH, which was the case for the weak base chloroquine. Analysis of albitiazolium accumulation in P. falciparum IRBC revealed a high-affinity component that was restricted to mature stages and suppressed by pepstatin A treatment, and thus likely related to drug accumulation in the parasite food vacuole. Albitiazolium also accumulated in a second high-capacity component present throughout the blood cycle that was likely not related to the food vacuole and also observed with Babesia divergens-infected erythrocytes. Accumulation was strictly glucose dependent, drastically inhibited by H+/K+ and Na+ ionophores upon collapse of ionic gradients, and appeared to be energized by the proton-motive force across the erythrocyte plasma membrane, indicating the importance of transport steps for this permanently charged new type of antimalarial agent. This specific, massive, and irreversible accumulation allows albitiazolium to restrict its toxicity to hematozoa-infected erythrocytes. The intraparasitic compartmentation of albitiazolium corroborates a dual mechanism of action, which could make this new type of antimalarial agent resistant to parasite resistance.

The role of positron emission tomography/computed tomography imaging with radiolabeled choline analogs in prostate cancer

IntroductionProstate cancer is the most frequent solid malignant tumor in Western countries. Positron emission tomography/X-ray computed tomography imaging with radiolabeled choline analogs is a useful tool for restaging prostate cancer in patients with rising prostate-specific antigen after radical treatment (in whom conventional imaging techniques have important limitations) as well as in the initial assessment of a selected group of prostate cancer patients. For this reason a literature review is necessary in order to evaluate the usefulness of this imaging test for the diagnosis and treatment of prostate cancer. Evidence acquisitionA MEDLINE (PubMed way) literature search was performed using the search parameters: «Prostate cancer» and «Choline-PET/CT». Other search terms were «Biochemical failure» and/or «Staging» and/or «PSA kinetics». English and Spanish papers were selected; original articles, reviews, systematic reviews and clinical guidelines were included. ConclusionsAccording to available data, radiolabeled choline analogs plays an important role in the management of prostate cancer, especially in biochemical relapse because technique accuracy is properly correlated with prostate-specific antigen values and kinetics. Although is an emerging diagnostic technique useful in treatment planning of prostate cancer, final recommendations have not been submitted.

False-Positive Result in 18F-Fluorocholine PET/CT Due to Incidental and Ectopic Parathyroid Hyperplasia

PET/CT with F-fluorocholine (a positron-labeled choline analog) is currently used as a diagnostic tool for restaging prostate cancer patients with increasing prostate-specific antigen. We present an unusual case of a false-positive result using F-fluorocholine PET/CT because of incidental and ectopic parathyroid hyperplasia.

Hyperpolarized choline as an MR imaging molecular probe: feasibility of in vivo imaging in a rat model.

To assess the feasibility of choline MRI using a new choline molecular probe for dynamic nuclear polarization (DNP) hyperpolarized MRI. Male Sprague-Dawley rats with an average weight of 400 ± 20 g (n = 5), were anesthetized and injection tubing was placed in the tail vein. [1,1,2,2-D4 , 1-(13) C]choline chloride (CMP1) was hyperpolarized by DNP and injected into rats at doses ranging from 12.6 to 50.0 mg/kg. Coronal projection (13) C imaging was performed on a 3 Tesla clinical MRI scanner (bore size 60 cm) using a variable flip angle gradient echo sequence. Images were acquired 15 to 45 s after the start of bolus injection. Signal intensities in regions of interest were determined at each time point and compared. (13) C MRI images of hyperpolarized CMP1 at a 50 mg/kg dose showed time-dependent organ distribution patterns. At 15 s, high intensities were observed in the inferior vena cava, heart, aorta, and kidneys. At 30 s, most of the signal intensity was localized to the kidneys. These distribution patterns were reproduced using 12.6 and 25 mg/kg doses. At 45 s, only signal in the kidneys was detected. Hyperpolarized choline imaging with MRI is feasible using a stable-isotope labeled choline analog (CMP1). Nonradioactive imaging of choline accumulation may provide a new investigatory dimension for kidney physiology. J. Magn. Reson. Imaging 2015;41:917-923. © 2014 Wiley Periodicals, Inc.

Comparative uptake of 18F-FEN-DPAZn2, 18F-FECH, 18F-fluoride, and 18F-FDG in fibrosarcoma and aseptic inflammation

The aim of this study is to evaluate uptake of 2-18F-fluoroethyl-bis(zinc(II)-dipicolylamine) (18F-FEN-DPAZn2) as a promising cell death imaging agent, a choline analog 18F-fluoroethylcholine (18F-FECH), 18F-fluoride as a bone imaging agent, and a glucose analog 2−18F-fluoro-2-deoxy-d-glucose (18F-FDG) in the combined S180 fibrosarcoma and turpentine-induced inflammation mice models. The results showed that 18F-FDG had the highest tumor-to-blood uptake ratio and tumor-to-muscle ratio, and high inflammation-to-blood ratio and inflammation-to-muscle ratio. 18F –FECH showed moderate tumor-to-blood ratio and tumor-to-muscle ratio, and low inflammation-to-blood ratio and inflammation-to-muscle ratio. However, accumulation of 18F FEN-DPAZn2 in tumor was similar to that in normal muscle. Also, 18F-FEN-DPAZn2 and 18F-fluoride exhibited the best selectivity to inflammation. 18F-FECH positron emission tomography (PET) imaging demonstrates some advantages over 18F-FDG PET for the differentiation of tumor from inflammation. 18F FEN-DPAZn2 and 18F-fluoride can be used for PET imaging of aseptic inflammation.

Radiopharmaceuticals in the evaluation and treatment of liver lesions

Radiopharmaceuticals have long been used to study the liver, giving interesting results. Nonetheless, for most indications, radionuclide imaging has not tended to be a first-choice approach, due to the better diagnostic performance of other imaging methods, namely, contrastenhanced computed tomography (ceCT) and ultrasound (US). However, the emergence, in recent years of radiolabelled tracers for use with positron emission tomography (PET) has led to renewed interest in radionuclide imaging of the liver. Applications (both new and more established ones) include the evaluation of hepatocellular carcinoma using choline PET, the use of FDG PET/CT for identifying secondary liver lesions and for localising unknown primary tumours in patients with liver metastases, as well as innovative approaches, such as the use of hybrid PET/ magnetic resonance imaging (PET/MRI) and PET-guided liver biopsies. Furthermore, radioembolisation as a treatment for liver lesions has recently been successfully introduced. This issue of Clinical and Translational Imaging is devoted to these topics. It has been compiled by a group of experts in this field, some of whom are pioneers and leaders at European and international level. In addition to setting out the rationale and background behind these approaches, they have endeavoured to provide an overview of the current state of the art, comparing their experience with that of their contemporaries in an effort to reach to a consensus. An increasingly important area is that of PET/CT imaging of hepatocellular carcinoma (HCC) using different radiopharmaceuticals. The group of Jean-Noel Talbot in Paris pioneered the use of F-fluorocholine (FCH), the fluorinated analogue of choline, for imaging HCC with PET [1], obtaining promising results. It is, in fact, well known that F-fluorodeoxyglucose (FDG), the reference PET tracer in oncology, has limitations in the detection of intra-hepatic well-differentiated HCC, and therefore, does not allow accurate study of affected patients. At present, dual-tracer PET with FCH and FDG could constitute a better approach, as the aggressiveness of lesions can vary and some lesions may take up only one tracer. Dual-tracer PET can also be used for disease staging and for predicting recurrence [2] and can therefore be useful in the selection of patients for liver transplantation or HCC tumour resection. Metastases from non-HCC liver malignancies, on the other hand, are not usually FCH avid, while cholangiocarcinoma (CC) can be visualised as a hot focus. As regards benign liver tumours, it is important from a prognostic point of view to differentiate hepatocellular adenoma, which can degenerate in up to 4 % of lesions from FNH, which is composed of normal hepatocytes occurring in a normal liver: promising results have recently been published which suggest that FCH PET/CT could play a role in differentiating between these two benign lesion types. Despite the aforementioned limitations of FDG as a radiopharmaceutical for HCC imaging, there are some indications for the use of this tracer in liver lesion imaging, and this issue is covered by Ilaria Grassi et al. from the Bologna group. The identification of secondary lesions using FDG is undoubtedly the most frequent such S. Fanti (&) Department of Nuclear Medicine, S. Orsola University Hospital, PAD 30, Via Massarenti 9, 40135 Bologna, Italy e-mail: stefano.fanti@aosp.bo.it

Papel de la tomografía por emisión de positrones/tomografía computarizada con análogos radiomarcados de colina en el cáncer de próstata

Introductionprostate cancer is the most frequent solid malignant tumor in Western Countries. Positron emission tomography/x-ray computed tomography imaging with radiolabeled choline analogues is a useful tool for restaging prostate cancer in patients with rising prostate-specific antigen after radical treatment (in whom conventional imaging techniques have important limitations) as well as in the initial assessment of a selected group of prostate cancer patients. For this reason a literature review is necessary in order to evaluate the usefulness of this imaging test for the diagnosis and treatment of prostate cancer. Evidence acquisitiona MEDLINE (PubMed way) literature search was performed using the search parameters: «Prostate cancer» and «Choline-PET/CT». Other search terms were «Biochemical failure» and/or «Staging» and/or «PSA kinetics». English and Spanish papers were selected; original articles, reviews, systematic reviews and clinical guidelines were included. Conclusionsaccording to available data, radiolabeled choline analogues plays an important role in the management of prostate cancer, especially in biochemical relapse because technique accuracy is properly correlated with prostate-specific antigen values and kinetics. Although is an emerging diagnostic technique useful in treatment planning of prostate cancer, final recommendations have not been submitted.

Specific and Reversible Immobilization of Proteins Tagged to the Affinity Polypeptide C-LytA on Functionalized Graphite Electrodes

We have developed a general method for the specific and reversible immobilization of proteins fused to the choline-binding module C-LytA on functionalized graphite electrodes. Graphite electrode surfaces were modified by diazonium chemistry to introduce carboxylic groups that were subsequently used to anchor mixed self-assembled monolayers consisting of N,N-diethylethylenediamine groups, acting as choline analogs, and ethanolamine groups as spacers. The ability of the prepared electrodes to specifically bind C-LytA-tagged recombinant proteins was tested with a C-LytA-β-galactosidase fusion protein. The binding, activity and stability of the immobilized protein was evaluated by electrochemically monitoring the formation of an electroactive product in the enzymatic hydrolysis of the synthetic substrate 4-aminophenyl β-D-galactopyranoside. The hybrid protein was immobilized in an specific and reversible way, while retaining the catalytic activity. Moreover, these functionalized electrodes were shown to be highly stable and reusable. The method developed here can be envisaged as a general, immobilization procedure on the protein biosensor field.

Superparamagnetic iron oxide/oleic acid nanoparticles with immobilized organosilicon derivatives of N‐(2‐hydroxyethyl)tetrahydroisoquinoline: synthesis, morphology and interaction with normal and tumour cells

Superparamagnetic iron oxide/oleic acid nanoparticles bearing lipid‐like organosilicon N‐(2‐hydroxyethyl)‐1,2,3,4‐tetrahydroisoquinoline derivatives have been synthesized with the aim of their potential biomedical application. X‐ray diffraction analysis, Dynamic light‐scattering measurements, method of magnetogranulometry and some others have been employed to investigate the morphology and properties of the nanoparticles synthesized. The magnetic core diameter of mixed covered nanoparticles ranged between 4.8 and 9.6 nm. The magnetization analyses showed that the particles are superparamagnetic at room temperature. In vitro cell cytotoxicity and intracellular NO generation caused by the water magnetic solution of nanoparticles possessing cytotoxic organosilicon heterocyclic choline analogue, namely N‐(2‐dimethyl‐n‐hexadecylsiloxyethyl)‐N‐methyl‐1,2,3,4‐tetrahydroisoquinolinium iodide, was examined in relation to monolayer human fibrosarcoma (HT‐1080) and mouse hepatoma (MG‐22A) tumour cell lines and normal mouse fibroblasts (NIH 3T3). The biological studies have revealed its selective cytotoxicity in tumour cells and strong effect on MG‐22A cell morphology. Incorporation of the synthesized nanoparticles into cells was observed. Copyright © 2013 John Wiley & Sons, Ltd.

Crystal structures of CbpF complexed with atropine and ipratropium reveal clues for the design of novel antimicrobials against Streptococcus pneumoniae

BackgroundStreptococcus pneumoniae is a major pathogen responsible of important diseases worldwide such as pneumonia and meningitis. An increasing resistance level hampers the use of currently available antibiotics to treat pneumococcal diseases. Consequently, it is desirable to find new targets for the development of novel antimicrobial drugs to treat pneumococcal infections. Surface choline-binding proteins (CBPs) are essential in bacterial physiology and infectivity. In this sense, esters of bicyclic amines (EBAs) such as atropine and ipratropium have been previously described to act as choline analogs and effectively compete with teichoic acids on binding to CBPs, consequently preventing in vitro pneumococcal growth, altering cell morphology and reducing cell viability. MethodsWith the aim of gaining a deeper insight into the structural determinants of the strong interaction between CBPs and EBAs, the three-dimensional structures of choline-binding protein F (CbpF), one of the most abundant proteins in the pneumococcal cell wall, complexed with atropine and ipratropium, have been obtained. ResultsThe choline analogs bound both to the carboxy-terminal module, involved in cell wall binding, and, unexpectedly, also to the amino-terminal module, that possesses a regulatory role in pneumococcal autolysis. ConclusionsAnalysis of the complexes confirmed the importance of the tropic acid moiety of the EBAs on the strength of the binding, through π–π interactions with aromatic residues in the binding site. General significanceThese results represent the first example describing the molecular basis of the inhibition of CBPs by EBA molecules and pave the way for the development of new generations of antipneumococcal drugs.

Functional expression of choline transporter-like protein 1 (CTL1) in small cell lung carcinoma cells: A target molecule for lung cancer therapy

Choline is essential for the synthesis of the major membrane phospholipid phosphatidylcholine and the neurotransmitter acetylcholine (ACh). Elevated levels of choline and up-regulated choline kinase activity have been detected in cancer cells. Thus, the intracellular accumulation of choline through choline transporters is the rate-limiting step in phospholipid metabolism and a prerequisite for cancer cell proliferation. However, the uptake system for choline and the functional expression of choline transporters in lung cancer cells are poorly understood. We examined the molecular and functional characterization of choline uptake in the small cell lung carcinoma cell line NCI-H69. Choline uptake was saturable and mediated by a single transport system. Interestingly, removal of Na+ from the uptake buffer strongly enhanced choline uptake. This increase in choline uptake under the Na+-free conditions was inhibited by dimethylamiloride (DMA), a Na+/H+ exchanger (NHE) inhibitor. Various organic cations and the choline analog hemicholinium-3 (HC-3) inhibited the choline uptake and cell viability. A correlation analysis of the potencies of organic cations for the inhibition of choline uptake and cell viability showed a strong correlation (R=0.8077). RT-PCR revealed that choline transporter-like protein 1 (CTL1) mRNA and NHE1 are mainly expressed. HC-3 and CTL1 siRNA inhibited choline uptake and cell viability, and increased caspase-3/7 activity. The conversion of choline to ACh was confirmed, and this conversion was enhanced under Na+-free conditions, which in turn was sensitive to HC-3. These results indicate that choline uptake through CTL1 is used for ACh synthesis. Both an acetylcholinesterase inhibitor (eserine) and a butyrylcholinesterase inhibitor (ethopropazine) increased cell proliferation, and these effects were inhibited by 4-DAMP, a mAChR3 antagonist. We conclude that NCI-H69 cells express the choline transporter CTL1 which uses a directed H+ gradient as a driving force, and its transport functions in co-operation with NHE1. This system primarily supplies choline for the synthesis of ACh and secretes ACh to act as an autocrine/paracrine growth factor, and the functional inhibition of CTL1 could promote apoptotic cell death. Identification of this new CTL1-mediated choline transport system provides a potential new target for therapeutic intervention.

Characterization of choline uptake in Trypanosoma brucei procyclic and bloodstream forms

Choline is an essential nutrient for eukaryotic cells, where it is used as precursor for the synthesis of choline-containing phospholipids, such as phosphatidylcholine (PC). According to published data, Trypanosoma brucei parasites are unable to take up choline from the environment but instead use lyso-phosphatidylcholine as precursor for choline lipid synthesis. We now show that T. brucei procyclic forms in culture readily incorporate [3H]-labeled choline into PC, indicating that trypanosomes express a transporter for choline at the plasma membrane. Characterization of the transport system in T. brucei procyclic and bloodstream forms shows that uptake of choline is independent of sodium and potassium ions and occurs with a Km in the low micromolar range. In addition, we demonstrate that choline uptake can be blocked by the known choline transport inhibitor, hemicholinium-3, and by synthetic choline analogs that have been established as anti-malarials. Together, our results show that T. brucei parasites express an uptake system for choline and that exogenous choline is used for PC synthesis.

Heart dysfunction induced by choline-deficiency in adult rats: The protective role of l-carnitine

Choline is a B vitamin co-factor and its deficiency seems to impair heart function. Carnitine, a chemical analog of choline, has been used as adjunct in the management of cardiac diseases. The study investigates the effects of choline deficiency on myocardial performance in adult rats and the possible modifications after carnitine administration. Wistar Albino rats (n=24), about 3 months old, were randomized into four groups fed with: (a) standard diet (control-CA), (b) choline deficient diet (CDD), (c) standard diet and carnitine in drinking water 0.15% w/v (CARN) and (d) choline deficient diet and carnitine (CDD+CARN). After four weeks of treatment, we assessed cardiac function under isometric conditions using the Langendorff preparations [Left Ventricular Developed Pressure (LVDP-mmHg), positive and negative first derivative of LVDP were evaluated], measured serum homocysteine and brain natriuretic peptide (BNP) levels and performed histopathology analyses. In the CDD group a compromised myocardium contractility compared to control (P=0.01), as assessed by LVDP, was noted along with a significantly impaired diastolic left ventricular function, as assessed by (−) dp/dt (P=0.02) that were prevented by carnitine. Systolic force, assessed by (+) dp/dt, showed no statistical difference between groups. A significant increase in serum BNP concentration was found in the CDD group (P<0.004) which was attenuated by carnitine (P<0.05), whereas homocysteine presented contradictory results (higher in the CDD+CARN group). Heart histopathology revealed a lymphocytic infiltration of myocardium and valves in the CDD group that was reduced by carnitine. In conclusion, choline deficiency in adult rats impairs heart performance; carnitine acts against these changes.

High-Resolution Optical Molecular Imaging of Changes in Choline Metabolism in Oral Neoplasia

This study was aimed at developing an optical molecular imaging approach to measure differences in uptake and intracellular retention of choline in clinically isolated tissue biopsies from head and neck cancer patients. An optically detectable analogue of choline (propargyl choline) was synthesized and evaluated in 2D and 3D models and clinically isolated paired biopsies (n = 22 biopsies). Fluorescence contrast between clinically abnormal and normal tissues based on uptake and intracellular retention of propargyl choline was measured and correlated with pathologic diagnosis. Results in 2D and 3D models demonstrated a rapid uptake of propargyl choline in cancer cells, uniform permeation in tissue models, and specific detection of intracellular entrapped propargyl choline using the click chemistry reaction with an azide-modified Alexa 488 dye. Fluorescence imaging measurements following topical delivery of propargyl choline in clinically isolated biopsies showed that the mean fluorescence intensity (MFI) of neoplastic tissues was four-fold to five-fold higher than the MFI of clinically and pathologically normal samples. This difference in fluorescence contrast was measured on the basis of comparison of paired biopsy sets isolated from individual patients as well as comparison of clinically abnormal and normal biopsies independent of anatomic locations in the head and neck cavity and across diverse patients. In conclusion, a novel imaging approach based on monoalkyne-modified choline was developed and validated using cell and tissue models. Results in clinically isolated tissue biopsies demonstrate a significant fluorescent contrast between neoplastic and normal tissues and illustrate high specificity of the optical imaging approach.

In vitro visualization of betaine aldehyde synthesis and oxidation using hyperpolarized magnetic resonance spectroscopy

Real-time monitoring of betaine aldehyde metabolism at high temporal resolution was accomplished using a hyperpolarized choline analog and (13)C-NMR. This represents the first observation of an aldehyde intermediate on hyperpolarized MR and opens the way for kinetic studies of oxidase/dehydrogenase enzymes in vitro and in vivo.

Purification and Characterization of an Alkaliphilic Choline Oxidase ofFusarium oxysporum

A novel choline oxidase found in a fungus, Fusarium oxysporum strain V2, was purified to homogeneity as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme has a molecular mass of 128 kDa and consists of two identical subunits. The purified enzyme showed adsorption peaks at 340 nm and 450 nm. It showed alkaliphilic pH characteristics: its optimum pH was 9.0-10.0, and it was stable at pH 8.0-10.2. The Michaelis constant (Km) values for choline and betaine aldehyde were 0.28 mM and 0.39 mM respectively. Trimethylamino-alcohols, dimethylamino-alcohols, and diethylaminoethanol were substrates for the enzyme, but the Km values for them increased with decreasing numbers of methyl groups on the ammonium headgroup. A marked decrease in the maximum velocity (Vmax) and Vmax/Km values was observed when N-replaced choline analogs were used as substrate instead of choline. The enzyme had a remarkably higher affinity for choline and betaine aldehyde than do previously reported enzymes. The enzyme oxidized these two substrates more quickly than a choline oxidase from Arthrobacter globiformis, and oxidation by the V2 enzyme was accompanied by an increase in the stoichometric amount of hydrogen peroxide.

Transport and pharmacodynamics of albitiazolium, an antimalarial drug candidate

BACKGROUND AND PURPOSECholine analogues, a new type of antimalarials, exert potent in vitro and in vivo antimalarial activity. This has given rise to albitiazolium, which is currently in phase II clinical trials to cure severe malaria. Here we dissected its mechanism of action step by step from choline entry into the infected erythrocyte to its effect on phosphatidylcholine (PC) biosynthesis.EXPERIMENTAL APPROACHWe biochemically unravelled the transport and enzymatic steps that mediate de novo synthesis of PC and elucidated how albitiazolium enters the intracellular parasites and affects the PC biosynthesis.KEY RESULTSCholine entry into Plasmodium falciparum-infected erythrocytes is achieved both by the remnant erythrocyte choline carrier and by parasite-induced new permeability pathways (NPP), while parasite entry involves a poly-specific cation transporter. Albitiazolium specifically prevented choline incorporation into its end-product PC, and its antimalarial activity was strongly antagonized by choline. Albitiazolium entered the infected erythrocyte mainly via a furosemide-sensitive NPP and was transported into the parasite by a poly-specific cation carrier. Albitiazolium competitively inhibited choline entry via the parasite-derived cation transporter and also, at a much higher concentration, affected each of the three enzymes conducting de novo synthesis of PC.CONCLUSIONS AND IMPLICATIONSInhibition of choline entry into the parasite appears to be the primary mechanism by which albitiazolium exerts its potent antimalarial effect. However, the pharmacological response to albitiazolium involves molecular interactions with different steps of the de novo PC biosynthesis pathway, which would help to delay the development of resistance to this drug.

Disulfide Prodrugs of Albitiazolium (T3/SAR97276): Synthesis and Biological Activities

We report herein the design, synthesis, and biological screening of a series of 15 disulfide prodrugs as precursors of albitiazolium bromide (T3/SAR97276, compound 1), a choline analogue which is currently being evaluated in clinical trials (phase II) for severe malaria. The corresponding prodrugs are expected to revert back to the active bis-thiazolium salt through an enzymatic reduction of the disulfide bond. To enhance aqueous solubility of these prodrugs, an amino acid residue (valine or lysine) or a phosphate group was introduced on the thiazolium side chain. Most of the novel derivatives exhibited potent in vitro antimalarial activity against P. falciparum. After oral administration, the cyclic disulfide prodrug 8 showed the best improvement of oral efficacy in comparison to the parent drug.

Conditional Mutagenesis of a Novel Choline Kinase Demonstrates Plasticity of Phosphatidylcholine Biogenesis and Gene Expression in Toxoplasma gondii

The obligate intracellular and promiscuous protozoan parasite Toxoplasma gondii needs an extensive membrane biogenesis that must be satisfied irrespective of its host-cell milieu. We show that the synthesis of the major lipid in T. gondii, phosphatidylcholine (PtdCho), is initiated by a novel choline kinase (TgCK). Full-length (∼70-kDa) TgCK displayed a low affinity for choline (K(m) ∼0.77 mM) and harbors a unique N-terminal hydrophobic peptide that is required for the formation of enzyme oligomers in the parasite cytosol but not for activity. Conditional mutagenesis of the TgCK gene in T. gondii attenuated the protein level by ∼60%, which was abolished in the off state of the mutant (Δtgck(i)). Unexpectedly, the mutant was not impaired in its growth and exhibited a normal PtdCho biogenesis. The parasite compensated for the loss of full-length TgCK by two potential 53- and 44-kDa isoforms expressed through a cryptic promoter identified within exon 1. TgCK-Exon1 alone was sufficient in driving the expression of GFP in E. coli. The presence of a cryptic promoter correlated with the persistent enzyme activity, PtdCho synthesis, and susceptibility of T. gondii to a choline analog, dimethylethanolamine. Quite notably, the mutant displayed a regular growth in the off state despite a 35% decline in PtdCho content and lipid synthesis, suggesting a compositional flexibility in the membranes of the parasite. The observed plasticity of gene expression and membrane biogenesis can ensure a faithful replication and adaptation of T. gondii in disparate host or nutrient environments.

Evaluation of Deuterated 18F- and 11C-Labeled Choline Analogs for Cancer Detection by Positron Emission Tomography

(11)C-Choline-positron emission tomography (PET) has been exploited to detect the aberrant choline metabolism in tumors. Radiolabeled choline uptake within the imaging time is primarily a function of transport, phosphorylation, and oxidation. Rapid choline oxidation, however, complicates interpretation of PET data. In this study, we investigated the biologic basis of the oxidation of deuterated choline analogs and assessed their specificity in human tumor xenografts. (11)C-Choline, (11)C-methyl-[1,2-(2)H(4)]-choline ((11)C-D4-choline), and (18)F-D4-choline were synthesized to permit comparison. Biodistribution, metabolism, small-animal PET studies, and kinetic analysis of tracer uptake were carried out in human colon HCT116 xenograft-bearing mice. Oxidation of choline analogs to betaine was highest with (11)C-choline, with reduced oxidation observed with (11)C-D4-choline and substantially reduced with (18)F-D4-choline, suggesting that both fluorination and deuteration were important for tracer metabolism. Although all tracers were converted intracellularly to labeled phosphocholine (specific signal), the higher rate constants for intracellular retention (K(i) and k(3)) of (11)C-choline and (11)C-D4-choline, compared with (18)F-D4-choline, were explained by the rapid conversion of the nonfluorinated tracers to betaine within HCT116 tumors. Imaging studies showed that the uptake of (18)F-D4-choline in three tumors with similar radiotracer delivery (K(1)) and choline kinase α expression-HCT116, A375, and PC3-M-were the same, suggesting that (18)F-D4-choline has utility for cancer detection irrespective of histologic type. We have shown here that both deuteration and fluorination combine to provide protection against choline oxidation in vivo. (18)F-D4-choline showed the highest selectivity for phosphorylation and warrants clinical evaluation.