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The two branches of the Kennedy pathways (CDP-choline and CDP-ethanolamine) are the predominant pathways responsible for the synthesis of the most abundant phospholipids, phosphatidylcholine and phosphatidylethanolamine, respectively, in mammalian membranes. Recently, hereditary diseases associated with single gene mutations in the Kennedy pathways have been identified. Interestingly, genetic diseases within the same pathway vary greatly, ranging from muscular dystrophy to spastic paraplegia to a childhood blinding disorder to bone deformations. Indeed, different point mutations in the same gene (PCYT1; CCTα) result in at least three distinct diseases. In this review, we will summarize and review the genetic diseases associated with mutations in genes of the Kennedy pathway for phospholipid synthesis. These single-gene disorders provide insight, indeed direct genotype-phenotype relationships, into the biological functions of specific enzymes of the Kennedy pathway. We discuss potential mechanisms of how mutations within the same pathway can cause disparate disease., (Copyright © 2020 © 2020 Tavasoli et al. Published by Elsevier Inc. All rights reserved.) more...

Seeking for new anticancer drugs with strong antiproliferative activity and simple molecular structure, we designed a novel series of compounds based on our previous reported pharmacophore model composed of five moieties. Antiproliferative assays on four tumoral cell lines and evaluation of Human Choline Kinase CKα1 enzymatic activity was performed for these compounds. Among tested molecules, those ones with biphenyl spacer showed betters enzymatic and antiproliferative activities (n-v). Docking and crystallization studies validate the hypothesis and confirm the results. The most active compound (t) induces a significant arrest of the cell cycle in G0/G1 phase that ultimately lead to apoptosis, following the mitochondrial pathway, as demonstrated for other choline kinase inhibitors. However additional assays reveal that the inhibition of choline uptake could also be involved in the antiproliferative outcome of this class of compounds., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.) more...

Novel antimicrobial agents are crucial to combat antibiotic resistance in pathogenic bacteria. Choline kinase (ChoK) in bacteria catalyzes the synthesis of phosphorylcholine, which is subsequently incorporated into the cell wall or outer membrane. In certain species of bacteria, phosphorylcholine is also used to synthesize membrane phosphatidylcholine. Numerous human ChoK inhibitors (ChoKIs) have been synthesized and tested for anticancer properties. Inhibition of S . pneumoniae ChoK by human ChoKIs showed a promising effect by distorting the cell wall and retarded the growth of this pathogen. Comparison of amino acid sequences at the catalytic sites of putative choline kinases from pathogenic bacteria and human enzymes revealed striking sequence conservation that supports the potential application of currently available ChoKIs for inhibiting bacterial enzymes. We also propose the combined use of ChoKIs and nanoparticles for targeted delivery to the pathogen while shielding the human host from any possible side effects of the inhibitors. More research should focus on the verification of putative bacterial ChoK activities and the characterization of ChoKIs with active enzymes. In conclusion, the presence of ChoK in a wide range of pathogenic bacteria and the distinct function of this enzyme has made it an attractive drug target. This review highlighted the possibility of "choking" bacterial ChoKs by using human ChoKIs., Competing Interests: The authors declare no conflict of interest., (Copyright © 2020 Moad Khalifa et al.) more...

Choline kinase alpha is a 457-residue protein that catalyzes the reaction between ATP and choline to yield ADP and phosphocholine. This metabolic action has been well studied because of choline kinase's link to cancer malignancy and poor patient prognosis. As the myriad of x-ray crystal structures available for this enzyme show, chemotherapeutic drug design has centered on stopping the catalytic activity of choline kinase and reducing the downstream metabolites it produces. Furthermore, these crystal structures only reveal the catalytic domain of the protein, residues 80-457. However, recent studies provide evidence for a non-catalytic protein-binding role for choline kinase alpha. Here, we show that choline kinase alpha interacts with the SH3 domain of c-Src. Co-precipitation assays, surface plasmon resonance, and crystallographic analysis of a 1.5 Å structure demonstrate that this interaction is specific and is mediated by the poly-proline region found N-terminal to the catalytic domain of choline kinase. Taken together, these data offer strong evidence that choline kinase alpha has a heretofore underappreciated role in protein-protein interactions, which offers an exciting new way to approach drug development against this cancer-enhancing protein. more...

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., (Copyright © 2018. Published by Elsevier B.V.) more...

Choline kinase α (ChoKα) is an enzyme that is upregulated in many types of cancer and has been shown to be tumorigenic. As such, it makes a promising target for inhibiting tumor growth. Though there have been several inhibitors synthesized for ChoKα, not all of them demonstrate the same efficacy in vivo, though the reasons behind this difference in potency are not clear. One particular inhibitor, designated TCD-717, has recently completed phase I clinical trials. Cell culture and in vitro studies support the powerful inhibitory effect TCD-717 has on ChoKα, but an examination of the inhibitor's interaction with the ChoKα enzyme has been missing prior to this work. Here we detail the 2.35 Å structure of ChoKα in complex with TCD-717. Examination of this structure in conjunction with kinetic assays reveals that TCD-717 does not bind directly in the choline pocket as do previously characterized ChoKα inhibitors, but rather in a proximal but novel location near the surface of the enzyme. The unique binding site identified for TCD-717 lends insight for the future design of more potent in vivo inhibitors for ChoKα. more...

Choline, a hydrophilic cation, has versatile physiological roles throughout the body, including cholinergic neurotransmission, memory consolidation and membrane biosynthesis and metabolism. Choline kinases possess enzyme activity that catalyses the conversion of choline to phosphocholine, which is further converted to cytidine diphosphate-coline (CDP-choline) in the biosynthesis of phosphatidylcholine (PC). PC is a major constituent of the phospholipid bilayer which constitutes the eukaryotic cell membrane, and regulates cell signal transduction. Choline Kinase consists of three isoforms, CHKα1, CHKα2 and CHKβ, encoded by two separate genes (CHKA(Human)/Chka(Mouse) and CHKB(Human)/Chkb(Mouse)). Both isoforms have similar structures and enzyme activity, but display some distinct molecular structural domains and differential tissue expression patterns. Whilst Choline Kinase was discovered in early 1950, its pivotal role in the development of muscular dystrophy, bone deformities, and cancer has only recently been identified. CHKα has been proposed as a cancer biomarker and its inhibition as an anti-cancer therapy. In contrast, restoration of CHKβ deficiency through CDP-choline supplements like citicoline may be beneficial for the treatment of muscular dystrophy, bone metabolic diseases, and cognitive conditions. The molecular structure and expression pattern of Choline Kinase, the differential roles of Choline Kinase isoforms and their potential as novel therapeutic targets for muscular dystrophy, bone deformities, cognitive conditions and cancer are discussed., (Copyright © 2016. Published by Elsevier Ltd.) more...

Malaria is a life-threatening disease caused by different species of the protozoan parasite Plasmodium, with P. falciparum being the deadliest. Increasing parasitic resistance to existing antimalarials makes the necessity of novel avenues to treat this disease an urgent priority. The enzymes responsible for the synthesis of phosphatidylcholine and phosphatidylethanolamine are attractive drug targets to treat malaria as their selective inhibition leads to an arrest of the parasite's growth and cures malaria in a mouse model. We present here a detailed study that reveals a mode of action for two P. falciparum choline kinase inhibitors both in vitro and in vivo. The compounds present distinct binding modes to the choline/ethanolamine-binding site of P. falciparum choline kinase, reflecting different types of inhibition. Strikingly, these compounds primarily inhibit the ethanolamine kinase activity of the P. falciparum choline kinase, leading to a severe decrease in the phosphatidylethanolamine levels within P. falciparum, which explains the resulting growth phenotype and the parasites death. These studies provide an understanding of the mode of action, and act as a springboard for continued antimalarial development efforts selectively targeting P. falciparum choline kinase. more...

A novel family of compounds derivative of 1,1'-(((ethane-1,2-diylbis(oxy))bis(4,1-phenylene))bis(methylene))-bispyridinium or -bisquinolinium bromide (10a-l) containing a pair of oxygen atoms in the spacer of the linker between the biscationic moieties, were synthesized and evaluated as inhibitors of choline kinase against a panel of cancer-cell lines. The most promising compounds in this series were 1,1'-(((ethane-1,2-diylbis(oxy))bis(4,1-phenylene))bis(methylene))bis(4-(dimethylamino)pyridinium) bromide (10a) and 1,1'-(((ethane-1,2-diylbis(oxy))bis(4,1-phenylene))bis(methylene))-bis(7-chloro-4-(pyrrolidin-1-yl)quinolinium) bromide (10l), which inhibit human choline kinase (ChoKα1) with IC50 of 1.0 and 0.92 μM, respectively, in a range similar to that of the previously reported biscationic compounds MN58b and RSM932A. Our compounds show greater antiproliferative activities than do the reference compounds, with unprecedented values of GI50 in the nanomolar range for several of the cancer-cell lines assayed, and more importantly they present low toxicity in non-tumoral cell lines, suggesting a cancer-cell-selective antiproliferative activity. Docking studies predict that the compounds interact with the choline-binding site in agreement with the binding mode of most previously reported biscationic compounds. Moreover, the crystal structure of ChoKα1 with compound 10a reveals that this compound binds to the choline-binding site and mimics HC-3 binding mode as never before. more...

The reversible phosphorylation of proteins catalyzed by protein kinases in eukaryotes supports an important role for eukaryotic protein kinases (ePKs) in the emergence of nucleated cells in the third superkingdom of life. Choline kinases (ChKs) could also be critical in the early evolution of eukaryotes, because of their function in the biosynthesis of phosphatidylcholine, which is unique to eukaryotic membranes. However, the genomic origins of ePKs and ChKs are unclear. The high degeneracy of protein sequences and broad expansion of ePK families have made this fundamental question difficult to answer. In this study, we identified two class-I aminoacyl-tRNA synthetases with high similarities to consensus amino acid sequences of human protein-serine/threonine kinases. Comparisons of primary and tertiary structures supported that ePKs and ChKs evolved from a common ancestor related to glutaminyl aminoacyl-tRNA synthetases, which may have been one of the key factors in the successful of emergence of ancient eukaryotic cells from bacterial colonies., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.) more...

A strategy is described to identify new antimalarial agents to overcome the drug resistance and/or failure issues through in silico screening of multiple biological targets. As a part of this, three enzymes namely CTPS, CK, and GST were selected, from among 56 drug targets of P. falciparum, and used them in virtual screening of ZINC database entries which led to the design and synthesis of arylsulfonyloxy acetimidamides as their consensus inhibitors. From these, two compounds showed good activity against sensitive (3D7; IC50, 1.10 and 1.45 μM) and resistant (K1; IC50, 2.10 and 2.13 μM) strains of the parasite, and they were further investigated through docking and molecular dynamics simulations. The findings of this study collectively paved the way for arylsulfonyloxy acetimidamides as a new class of antimalarial agents. more...

Choline kinase (CK) catalyses the transfer of the ATP γ-phosphate to choline to generate phosphocholine and ADP in the presence of magnesium leading to the synthesis of phosphatidylcholine. Of the three isoforms of CK described in humans, only the α isoforms (HsCKα) are strongly associated with cancer and have been validated as drug targets to treat this disease. Over the years, a large number of Hemicholinium-3 (HC-3)-based HsCKα biscationic inhibitors have been developed though the relevant common features important for the biological function have not been defined. Here, selecting a large number of previous HC-3-based inhibitors, we discover through computational studies a pharmacophore model formed by five moieties that are included in the 1-benzyl-4-(N-methylaniline)pyridinium fragment. Using a pharmacophore-guided virtual screening, we then identified 6 molecules that showed binding affinities in the low μM range to HsCKα1. Finally, protein crystallization studies suggested that one of these molecules is bound to the choline and ATP-binding sites. In conclusion, we have developed a pharmacophore model that not only allowed us to dissect the structural important features of the previous HC-3 derivatives, but also enabled the identification of novel chemical tools with good ligand efficiencies to investigate the biological functions of HsCKα1., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.) more...

LicA plays a key role in the cell-wall phosphorylcholine biosynthesis of Streptococcus pneumonia. Here we determined the crystal structures of apo-form LicA at 1.94 Å and two complex forms LicA-choline and LicA-AMP-MES, at 2.01 and 1.45 Å resolution, respectively. The overall structure adopts a canonical protein kinase-like fold, with the active site located in the crevice of the N- and C-terminal domains. The three structures present distinct poses of the active site, which undergoes an open-closed-open conformational change upon substrate binding and product release. The structure analyses combined with mutageneses and enzymatic assays enabled us to figure out the key residues for the choline kinase activity of LicA. In addition, structural comparison revealed the loop between helices α7 and α8 might modulate the substrate specificity and catalytic activity. These findings shed light on the structure and mechanism of the prokaryotic choline kinase LicA, and might direct the rational design of novel anti-pneumococcal drugs. more...

Aim: Research of the antitumor properties of biscationic compounds has received significant attention over the last few years., Results: A novel family of 1,1'-([2,2'-bipyridine]-5,5'-diylbis(methylene))bis-substituted bromide (9a-k), containing two nitrogen atoms in the linker, considered as hypothetical hydrogen bond acceptors, were synthesized and evaluated as ChoK inhibitors and their antiproliferative activity against six cancer cell lines., Conclusion: The most promising compounds in this series are 1,1'-([2,2'-bipyridine]-5,5'-diylbis(methylene))bis(4-(methyl(phenyl)amino)-quinolinium bromide derivatives 9g-i (analogs to RSM932A), that significantly inhibit cancer cell growth at even submicromolar concentrations, especially against leukemia cells. Compounds 9g-i also inhibit the ChoKα1 with good or moderate values, as predicted by initial docking studies. In addition, the most active compound 9h remarkably induces apoptosis in two cell lines following the mitochondrial pathway. more...

Cation-π interactions to cognate ligands in enzymes have key roles in ligand binding and enzymatic catalysis. We have deciphered the key functional role of both charged and aromatic residues within the choline binding subsite of CTP:phosphocholine cytidylyltransferase and choline kinase from Plasmodium falciparum. Comparison of quaternary ammonium binding site structures revealed a general composite aromatic box pattern of enzyme recognition sites, well distinguished from the aromatic box recognition site of receptors., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.) more...

Human choline kinase α (CKα) is a validated drug target for the treatment of cancer. In recent years, a large number of CK inhibitors have been synthesized, and one of them is currently being evaluated in Phase I clinical trials as a treatment for solid tumors. Here we have evaluated a new series of asymmetrical biscationic CK inhibitors by means of enzymatic, crystallographic, and antitumor studies. We demonstrate that one of these structures adopts a completely new binding mode not observed before inducing the aperture of an adjacent binding site. This compound shows antiproliferative and apoptotic effects on cancer cells through activation of caspase-3. Therefore, this study not only provides fruitful insights into the design of more efficient compounds that may target different regions in CKα1 but also explains how these compounds induce apoptosis in cancer cells. more...

Choline kinase (CK) is a homodimeric enzyme that catalyses the transfer of the ATP γ-phosphate to choline, generating phosphocholine and ADP in the presence of magnesium. Several isoforms of CK are present in humans but only the HsCKα has been associated with cancer and validated as a drug target to treat this disease. As a consequence a large number of compounds based on Hemicholinium (HC-3) have been described. Two compounds, previously reported to inhibit the human enzyme, have recently been shown to inhibit P. falciparum CK (PfCK) and therefore their potential applications might be anticipated to other pathogens. Herein, using molecular dynamic simulations, we have firstly observed that the ATP and the choline binding site of different CK in pathogens and human are conserved, suggesting that previous compounds inhibiting the human enzyme may also interact with CKs from different pathogens. We have substantiated such observation with experimental assays showing that HsCKα1, PfCK and CpCK bind to two compounds with distinct structural features in the low μM range. Collectively, these results uncover similarities among the choline kinase binding site from different pathogenic species and the human enzyme, highlighting the feasibility of designing novel inhibitors based on the choline binding pocket. more...

We have investigated the mechanism of action of inhibition of the choline kinase of P. falciparum (p.f.-ChoK) by two inhibitors of the human ChoKα, MN58b and RSM-932A, which have previously been shown to be potent antitumoral agents. The efficacy of these inhibitors against p.f.-ChoK is investigated using enzymatic and in vitro assays. While MN58b may enter the choline/phosphocholine binding site, RSM-932A appears to have an altogether novel mechanism of inhibition and is synergistic with respect to both choline and ATP. A model of inhibition for RSM-932A in which this inhibitor traps p.f.-ChoK in a phosphorylated intermediate state blocking phosphate transfer to choline is presented. Importantly, MN58b and RSM-932A have in vitro inhibitory activity in the low nanomolar range and are equally effective against chloroquine-sensitive and chloroquine-resistant strains. RSM-932A and MN58b significantly reduced parasitemia and induced the accumulation of trophozoites and schizonts, blocking intraerythrocytic development and interfering with parasite egress or invasion, suggesting a delay of the parasite maturation stage. The present data provide two new potent structures for the development of antimalarial compounds and validate p.f.-ChoK as an accessible drug target against the parasite. more...

Dual binding modes: Combined empirical and computational studies of a series of compounds showed adenine and 1-benzyl-4-(dimethylamino)pyridinium fragments to function most efficiently in binding CHOKα1, and also determined how the latter fragment interacts with the choline binding site through two different binding modes. These data provide a basis for the future design of better and more selective inhibitors., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.) more...

Choline Kinase is a key component of the Kennedy pathway that converts choline into a number of structural and signalling lipids that are essential for cell growth and survival. One member of the family, Choline Kinase-α (ChoKα) is frequently up-regulated in human cancers, and expression of ChoKα is sufficient to transform cells. Consequently ChoKα has been studied as a potential target for therapeutic agents in cancer research. Despite great interest in the enzyme, mechanistic studies have not been reported. In this study, a combination of initial velocity and product inhibition studies, together with the kinetic and structural characterisation of a novel ChoKα inhibitor is used to support a mechanism of action for human ChoKα. Substrate and inhibition kinetics are consistent with an iso double displacement mechanism, in which the γ-phosphate from ATP is transferred to choline in two distinct steps via a phospho-enzyme intermediate. Co-crystal structures, and existing site-specific mutation studies, support an important role for Asp306, in stabilising the phospho-enzyme intermediate. The kinetics also indicate a distinct kinetic (isomerisation) step associated with product release, which may be attributed to a conformational change in the protein to disrupt an interaction between Asp306 and the phosphocholine product, facilitating product release. This study describes a mechanism for ChoKα that is unusual amongst kinases, and highlights the availability of different enzyme states that can be exploited for drug discovery., (Copyright © 2013 Elsevier B.V. All rights reserved.) more...

Applying a CHOK hold: Combined experimental and computational studies of the binding mode of a rationally designed inhibitor of the dimeric choline kinase α1 (CHOKα1) explain the molecular mechanism of negative cooperativity (see scheme) and how the monomers are connected. The results give insight into how the symmetry of the dimer can be partially conserved despite a lack of conservation in the static crystal structures., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.) more...

Choline kinase-α expression and activity are increased in multiple human neoplasms as a result of growth factor stimulation and activation of cancer-related signaling pathways. The product of choline kinase-α, phosphocholine, serves as an essential metabolic reservoir for the production of phosphatidylcholine, the major phospholipid constituent of membranes and substrate for the production of lipid second messengers. Using in silico screening for small molecules that may interact with the choline kinase-α substrate binding domain, we identified a novel competitive inhibitor, N-(3,5-dimethylphenyl)-2-[[5-(4-ethylphenyl)-1H-1,2,4-triazol-3-yl]sulfanyl] acetamide (termed CK37) that inhibited purified recombinant human choline kinase-α activity, reduced the steady-state concentration of phosphocholine in transformed cells, and selectively suppressed the growth of neoplastic cells relative to normal epithelial cells. Choline kinase-α activity is required for the downstream production of phosphatidic acid, a promoter of several Ras signaling pathways. CK37 suppressed mitogen-activated protein kinase and phosphatidylinositol 3-kinase/AKT signaling, disrupted actin cytoskeletal organization, and reduced plasma membrane ruffling. Finally, administration of CK37 significantly decreased tumor growth in a lung tumor xenograft mouse model, suppressed tumor phosphocholine, and diminished activating phosphorylations of extracellular signal-regulated kinase and AKT in vivo. Together, these results further validate choline kinase-α as a molecular target for the development of agents that interrupt Ras signaling pathways, and indicate that receptor-based computational screening should facilitate the identification of new classes of choline kinase-α inhibitors. more...

We have summarized here the importance of ChoKα1 in human carcinogenesis. ChoKα1 displays its oncogenic activity through activation of specific signaling pathways that influence on cell proliferation and survival. It is overexpressed in a large number of human tumors with an incidence of 40-60% of all tumors investigated. Currently, there is an active effort in the development of strategies to knockdown the activity of ChoKα through specific siRNA or small molecules inhibitors. Results from genetic silencing or from treatment with MN58b, a well characterized ChoKα inhibitor showing antiproliferative and antitumoral effect in mice xenografts, provide strong support to this concept, indicating that the design of new antitumoral drugs must be selective against this isoform. However, affecting the other two known isoforms of ChoK may have also therapeutic consequences since the physiologically active form of ChoK may be constituted by homo or heterodimers. Furthermore, alteration of the ChoKβ activity might lead to a change in the lipid content of the cells of particular tissues such as skeletal muscle as described in the ChoKβ null mice (Sher et al., 2006). Finally, the identification of the ChoKα1 isoform as an excellent novel tool for the diagnosis and prognosis of cancer patients may have clinical consequences of immediate usefulness. On one hand, the use of specific monoclonal antibodies against ChoKα1 as a tool for diagnosis in paraffin embedded samples from patient biopsies, through standard immunohistochemistry techniques, can now be achieved (Gallego-Ortega et al., 2006). On the other hand, it has been recently described the prognostic value of determination of ChoKα1 expression levels in non-small cell lung cancer using real time quantitative PCR technology (Ramírez de Molina et al., 2007). Therefore, further research should be supported on the utility of ChoK isoforms as a promising area to improve cancer diagnosis and treatment. more...

Human choline kinase (ChoK) catalyzes the first reaction in phosphatidylcholine biosynthesis and exists as ChoKalpha (alpha1 and alpha2) and ChoKbeta isoforms. Recent studies suggest that ChoK is implicated in tumorigenesis and emerging as an attractive target for anticancer chemotherapy. To extend our understanding of the molecular mechanism of ChoK inhibition, we have determined the high resolution x-ray structures of the ChoKalpha1 and ChoKbeta isoforms in complex with hemicholinium-3 (HC-3), a known inhibitor of ChoK. In both structures, HC-3 bound at the conserved hydrophobic groove on the C-terminal lobe. One of the HC-3 oxazinium rings complexed with ChoKalpha1 occupied the choline-binding pocket, providing a structural explanation for its inhibitory action. Interestingly, the HC-3 molecule co-crystallized with ChoKbeta was phosphorylated in the choline binding site. This phosphorylation, albeit occurring at a very slow rate, was confirmed experimentally by mass spectroscopy and radioactive assays. Detailed kinetic studies revealed that HC-3 is a much more potent inhibitor for ChoKalpha isoforms (alpha1 and alpha2) compared with ChoKbeta. Mutational studies based on the structures of both inhibitor-bound ChoK complexes demonstrated that Leu-401 of ChoKalpha2 (equivalent to Leu-419 of ChoKalpha1), or the corresponding residue Phe-352 of ChoKbeta, which is one of the hydrophobic residues neighboring the active site, influences the plasticity of the HC-3-binding groove, thereby playing a key role in HC-3 sensitivity and phosphorylation. more...

The proliferation of the malaria-causing parasite Plasmodium falciparum within the erythrocyte is concomitant with massive phosphatidylcholine and phosphatidylethanolamine biosynthesis. Based on pharmacological and genetic data, de novo biosynthesis pathways of both phospholipids appear to be essential for parasite survival. The present study characterizes PfCK (P. falciparum choline kinase) and PfEK (P. falciparum ethanolamine kinase), which catalyse the first enzymatic steps of these essential metabolic pathways. Recombinant PfCK and PfEK were expressed as His6-tagged fusion proteins from overexpressing Escherichia coli strains, then purified to homogeneity and characterized. Using murine polyclonal antibodies against recombinant kinases, PfCK and PfEK were shown to be localized within the parasite cytoplasm. Protein expression levels increased during erythrocytic development. PfCK and PfEK appeared to be specific to their respective substrates and followed Michaelis-Menten kinetics. The Km value of PfCK for choline was 135.3+/-15.5 microM. PfCK was also able to phosphorylate ethanolamine with a very low affinity. PfEK was found to be an ethanolamine-specific kinase (Km=475.7+/-80.2 microM for ethanolamine). The quaternary ammonium compound hemicholinium-3 and an ethanolamine analogue, 2-amino-1-butanol, selectively inhibited PfCK or PfEK. In contrast, the bis-thiazolium compound T3, which was designed as a choline analogue and is currently in clinical trials for antimalarial treatment, affected PfCK and PfEK activities similarly. Inhibition exerted by T3 was competitive for both PfCK and PfEK and correlated with the impairment of cellular phosphatidylcholine biosynthesis. Comparative analyses of sequences and structures for both kinase types gave insights into their specific inhibition profiles and into the dual capacity of T3 to inhibit both PfCK and PfEK. more...

Choline kinase (ChoK) is reported to involve in cell signaling pathways and cell growth by regulating the intermediate, phosphocholine (PCho), which is the first step to biosynthesis a membrane phospholipid, phosphatidylcholine. The PCho levels are overexpressed due to elevated activation of the protein under carcinogenesis conditions. ChoK has thus evolved as a novel target for various cancers and a range of compounds has been reported in this course as potent ChoK inhibitors. However, not much information is known about the binding site of the inhibitors. Therefore, we ventured to unravel the possible binding site of 39 bis-quinolinium inhibitors from which the structural requirement for better protein-ligand complex was delved. Molecular docking and 3D-QSAR studies namely comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) were performed on the series. The knowledge of the active site was obtained from the site id search and molcad surface calculations of Sybyl, which was further considered for docking studies. In 3D-QSAR, the best predictions were obtained from the model where 29 compounds were considered in the training set and remaining 10 in the test set. The best CoMFA statistics were obtained with r(2) of 0.99 and q(2) of 0.81 while, CoMSIA was resulted with r(2) of 0.98 and q(2) of 0.77. A comparative analysis was done with the resulted 3D-QSAR maps and the docked poses by overlaying the maps on the active site residues. Since, there is no reported ligand co-crystallized structure of ChoK the present study provides valuable clues on the binding conformation of the ligand and its interactions with the active site. more...

Choline kinase, responsible for the phosphorylation of choline to phosphocholine as the first step of the CDP-choline pathway for the biosynthesis of phosphatidylcholine, has been recognized as a new target for anticancer therapy. Crystal structures of human choline kinase in its apo, ADP and phosphocholine-bound complexes, respectively, reveal the molecular details of the substrate binding sites. ATP binds in a cavity where residues from both the N and C-terminal lobes contribute to form a cleft, while the choline-binding site constitutes a deep hydrophobic groove in the C-terminal domain with a rim composed of negatively charged residues. Upon binding of choline, the enzyme undergoes conformational changes independently affecting the N-terminal domain and the ATP-binding loop. From this structural analysis and comparison with other kinases, and from mutagenesis data on the homologous Caenorhabditis elegans choline kinase, a model of the ternary ADP.phosphocholine complex was built that reveals the molecular basis for the phosphoryl transfer activity of this enzyme. more...

A homology model of human choline kinase (CK-alpha) based on the X-ray crystallographic structure of C. elegans choline kinase (CKA-2) is presented. Molecular dynamics simulations performed on CK-alpha confirm the quality of the model, and also support the putative ATP and choline binding sites. A good correlation between the MD results and reported CKA-2 mutagenesis assays has been found for the main residues involved in catalytic activity. Preliminary docking studies performed on the CK-alpha model indicate that inhibitors can bind to the binding sites of both substrates (ATP and choline). A possible reason for inhibition of choline kinase by Ca(2+) ion is also proposed. more...

Choline kinase alpha (ChoKalpha) is a metabolic enzyme involved in the synthesis of phosphatidylcholine, recently implicated in cancer onset since it is overexpressed in a variety of human cancers such as mammary, lung, colorectal and prostate adenocarcinomas. Furthermore, overexpression of ChoKalpha in human HEK293T cells confers them oncogenic properties with the induction of tumors after subcutaneous injection in nude mice. ChoKalpha levels in tumor samples have been analyzed using polyclonal antibodies and Western blotting. These techniques have considerable limitations and do not allow for a precise and efficient evaluation of the real significance of ChoK overexpression in human carcinogenesis. We developed a set of monoclonal antibodies with high specificity and sensitivity against ChoKalpha, and characterized their properties. We provide evidence that the newly generated MoAbs against ChoKalpha have potential use in cancer diagnosis by conventional immunohistochemistry techniques. more...

Generation of phosphocholine by choline kinase is important for phosphatidylcholine biosynthesis via Kennedy pathway and phosphatidylcholine biosynthesis is essential for intraerythrocytic growth of malaria parasite. A putative gene (Gene ID PF14_0020) in chromosome 14, having highest sequence homology with choline kinase, has been identified by BLAST searches from P. falciparum genome sequence database. This gene has been PCR amplified, cloned, over-expressed and characterized. Choline kinase activity of the recombinant protein (PfCK) was validated as it catalyzed the formation of phosphocholine from choline in presence of ATP. The K(m) values for choline and ATP are found to be 145+/-20 microM and 2.5+/-0.3 mM, respectively. PfCK can phosphorylate choline efficiently but not ethanolamine. Southern blotting indicates that PfCK is a single copy gene and it is a cytosolic protein as evidenced by Western immunoblotting and confocal microscopy. A model structure of PfCK was constructed based on the crystal structure of choline kinase of C. elegans to search the structural homology. Consistent with the homology modeling predictions, CD analysis indicates that the alpha and beta content of PfCK are 33% and 14%, respectively. Since choline kinase plays a vital role for growth and multiplication of P. falciparum during intraerythrocytic stages, we can suggest that this well characterized PfCK may be exploited in the screening of new choline kinase inhibitors to evaluate their antimalarial activity. more...

Choline kinase (ChoK) is a cytosolic enzyme present in various tissues, which catalyzes the phosphorylation of choline to form phosphorylcholine (PCho) in the presence of ATP and magnesium. ChoK is important for the generation of two major membrane phospholipids, phosphatidylcholine (PC) and sphingomyelin (SM) and subsequently for the cell division. ChoK plays a vital role in cell signaling pathways and regulation of cell growth along with PCho involved in malignant transformation through ras oncogenes in different cancers such as breast, lung, colon, prostate, neuroblastoma, hepatic lymphomas, meningiomas and diverse murine tumours. The Ras effectors serine/threonine kinase (Raf-1), the Ral-GDP dissociation stimulator (Ral-GDS) and the phosphatidylinositol 3-kinase (PI3K) are involved in the activation of ChoK during tumorigenesis. ChoK gene induction seems to be associated with certain cell stress or cell defense. Nowadays, RNAi appear to be one of the most promising routes in the cancer therapy. The anticancer potential of both stable expression of siRNAs and their high sequence specificity by RNAi mediated suppression of oncogenic ras in human pancreatic carcinoma, human melanomas and ovarian cancer has been observed. It has an important role in sequence specific post-transcriptional gene silencing mechanism. Presently, the crystal structure of Caenorhabditis elegans choline kinase A-2 (ChoKA-2) is available, which may be useful for comparative modeling of human ChoK and further modeling studies. The present review aims at the general overview of importance, expression, structure, progress in molecular modeling, active site analysis and inhibitors of ChoK. It also highlights the recent role of ChoK in various types of Ras-dependent and Ras-independent carcinogenesis. more...

The Saccharomyces cerevisiae CKI1-encoded choline kinase catalyzes the committed step in phosphatidylcholine synthesis via the Kennedy pathway. The enzyme is phosphorylated on multiple serine residues, and some of this phosphorylation is mediated by protein kinase A. In this work we examined the hypothesis that choline kinase is also phosphorylated by protein kinase C. Using choline kinase as a substrate, protein kinase C activity was dose- and time-dependent and dependent on the concentrations of choline kinase (K(m) = 27 microg/ml) and ATP (K(m) = 15 microM). This phosphorylation, which occurred on a serine residue, was accompanied by a 1.6-fold stimulation of choline kinase activity. The synthetic peptide SRSSSQRRHS (V5max/K(m) = 17.5 mm(-1) micromol min(-1) mg(-1)) that contains the protein kinase C motif for Ser25 was a substrate for protein kinase C. A Ser25 to Ala (S25A) mutation in choline kinase resulted in a 60% decrease in protein kinase C phosphorylation of the enzyme. Phosphopeptide mapping analysis of the S25A mutant enzyme confirmed that Ser25 was a protein kinase C target site. In vivo the S25A mutation correlated with a decrease (55%) in phosphatidylcholine synthesis via the Kennedy pathway, whereas an S25D phosphorylation site mimic correlated with an increase (44%) in phosphatidylcholine synthesis. Although the S25A (protein kinase C site) mutation did not affect the phosphorylation of choline kinase by protein kinase A, the S30A (protein kinase A site) mutation caused a 46% reduction in enzyme phosphorylation by protein kinase C. A choline kinase synthetic peptide (SQRRHSLTRQ) containing Ser30 was a substrate (V(max)/K(m) = 3.0 mm(-1) micromol min(-1) mg(-1)) for protein kinase C. Comparison of phosphopeptide maps of the wild type and S30A mutant choline kinase enzymes phosphorylated by protein kinase C confirmed that Ser30 was also a target site for protein kinase C. more...

Choline kinase (CK) catalyzes the first phosphorylation reaction in the CDP-choline pathway for the biosynthesis of phosphatidylcholine (PC), yielding phosphocholine (P-Cho) from choline and ATP in the presence of Mg(2+). This enzyme exists in mammalian cells as at least three isoforms that are encoded by two separate genes termed ck-alpha and ck-beta. Each isoform is not active in its monomeric form. The active enzyme consists of either their homo- or hetero-dimeric (or oligomeric) forms. In recent years, the roles of CK in cell growth and cell stress/defense mechanisms have been intensely investigated. These functions of CK do not seem to be directly related to the net PC biosynthesis but predict another important role of this enzyme in certain cell physiology. This review summarizes briefly the recent progress of mammalian CK study which will include the gene structure of each isoform and its possible transcriptional regulation, the active configuration of the enzyme, induction of the particular isoform in chemically induced cell stress, and the possible role of this enzyme as well as of its reaction product, P-Cho, in cell growth and other cellular physiology. more...

Choline kinase catalyzes the phosphorylation of choline by ATP, the first committed step in the CDP-choline pathway for phosphatidylcholine biosynthesis. To begin to elucidate the mechanism of catalysis by this enzyme, choline kinase A-2 from Caenorhabditis elegans was analyzed by systematic mutagenesis of highly conserved residues followed by analysis of kinetic and structural parameters. Specifically, mutants were analyzed with respect to K(m) and k(cat) values for each substrate and Mg(2+), inhibitory constants for Mg(2+) and Ca(2+), secondary structure as monitored by circular dichroism, and sensitivity to unfolding in guanidinium hydrochloride. The most severe impairment of catalysis occurred with the modification of Asp-255 and Asn-260, which are located in the conserved Brenner's phosphotransferase motif, and Asp-301 and Glu-303, in the signature choline kinase motif. For example, mutation of Asp-255 or Asp-301 to Ala eliminated detectable catalytic activity, and mutation of Asn-260 and Glu-303 to Ala decreased k(cat) by 300- and 10-fold, respectively. Additionally, the K(m) for Mg(2+) for mutants N260A and E303A was approximately 30-fold higher than that of wild type. Several other residues (Ser-86, Arg-111, Glu-125, and Trp-387) were identified as being important: Catalytic efficiencies (k(cat)/K(m)) for the enzymes in which these residues were mutated to Ala were reduced to 2-25% of wild type. The high degree of structural similarity among choline kinase A-2, aminoglycoside phosphotransferases, and protein kinases, together with the results from this mutational analysis, indicates it is likely that these conserved residues are located at the catalytic core of choline kinase. more...

The genomes of Treponema denticola and Treponema pallidum contain a gene, licCA, which is predicted to encode a fusion protein containing choline kinase and CTP:phosphocholine cytidylyltransferase activities. Because both organisms have been reported to contain phosphatidylcholine, this raises the possibility that they use a CDP-choline pathway for the biosynthesis of phosphatidylcholine. This report shows that phosphatidylcholine is a major phospholipid in T. denticola, accounting for 35-40% of total phospholipid. This organism readily incorporated [14C]choline into phosphatidylcholine, indicating the presence of a choline-dependent biosynthetic pathway. The licCA gene was cloned, and recombinant LicCA had choline kinase and CTP:phosphocholine cytidylyltransferase activity. The licCA gene was disrupted in T. denticola by erythromycin cassette mutagenesis, resulting in a viable mutant. This disruption completely blocked incorporation of either [14C]choline or 32Pi into phosphatidylcholine. The rate of production of another phospholipid in T. denticola, phosphatidylethanolamine, was elevated considerably in the licCA mutant, suggesting that the elevated level of this lipid compensated for the loss of phosphatidylcholine in the membranes. Thus it appears that T. denticola does contain a licCA-dependent CDP-choline pathway for phosphatidylcholine biosynthesis. more...

Choline kinase catalyzes the ATP-dependent phosphorylation of choline, the first committed step in the CDP-choline pathway for the biosynthesis of phosphatidylcholine. The 2.0 A crystal structure of a choline kinase from C. elegans (CKA-2) reveals that the enzyme is a homodimeric protein with each monomer organized into a two-domain fold. The structure is remarkably similar to those of protein kinases and aminoglycoside phosphotransferases, despite no significant similarity in amino acid sequence. Comparisons to the structures of other kinases suggest that ATP binds to CKA-2 in a pocket formed by highly conserved and catalytically important residues. In addition, a choline binding site is proposed to be near the ATP binding pocket and formed by several structurally flexible loops. more...

The Saccharomyces cerevisiae CKI-encoded choline kinase is phosphorylated on a serine residue and stimulated by protein kinase A. We examined the hypothesis that amino acids Ser(30) and Ser(85) contained in a protein kinase A sequence motif in choline kinase are target sites for protein kinase A. The synthetic peptides SQRRHSLTRQ (V(max)/K(m) = 10.8 microm(-1) nmol min(-1) mg(-1)) and GPRRASATDV (V(max)/K(m) = 0.15 microm(-1) nmol min(-1) mg(-1)) containing the protein kinase A motif for Ser(30) and Ser(85), respectively, within the choline kinase protein were substrates for protein kinase A. Choline kinase with Ser(30) to Ala (S30A) and Ser(85) to Ala (S85A) mutations were constructed alone and in combination by site-directed mutagenesis and expressed in a cki1Delta eki1Delta double mutant that lacks choline kinase activity. The mutant enzymes were expressed normally, but the specific activity of choline kinase in cells expressing the S30A, S85A, and S30A,S85A mutant enzymes was reduced by 44, 8, and 60%, respectively, when compared with the control. In vivo labeling experiments showed that the extent of phosphorylation of the S30A, S85A, and S30A,S85A mutant enzymes was reduced by 70, 17, and 83%, respectively. Phosphorylation of the S30A, S85A, and S30A,S85A mutant enzymes by protein kinase A in vitro was reduced by 60, 7, and 96%, respectively, and peptide mapping analysis of the mutant enzymes confirmed the phosphorylation sites in the enzyme. The incorporation of (3)H-labeled choline into phosphocholine and phosphatidylcholine in cells bearing the S30A, S85A, and S30A,S85A mutant enzymes was reduced by 56, 27, and 81%, respectively, and by 58, 33, and 84%, respectively, when compared with control cells. These data supported the conclusion that phosphorylation of choline kinase on Ser(30) and Ser(85) by protein kinase A regulates PC synthesis by the CDP-choline pathway. more...

Choline/ethanolamine kinase (CK/EK) is the first enzyme in phosphatidylcholine/phosphatidylethanolamine biosynthesis in all animal cells. The highly purified CKs from mammalian sources and their recombinant gene products so far were all shown to have EK activity also, indicating that both activities reside on the same protein. CK/EK in most animal cells exists as several isoforms, for two of which (alpha and beta) their cDNAs have been cloned from both the rat and mouse, and they are found to be separate gene products. The physiological significance for the existence of more than one CK/EK enzyme, however, remains to be clarified. In this study, we isolated mouse genes encoding both types of CK/EK isozyme and determined their entire structure. The 5'-flanking promoter regions were found to have quite different features from each other, indicating that their expression could be under distinct control. Comparison of the nucleotide sequence between the corresponding coding exons showed the best homology (75%) residing on exon VIII. A search of the database resulted in the possible existence of 17 different origins of eukaryotic CK and/or EK, each of which presumably contained the entire amino acid sequence. Multialignment of their putative amino acid sequences led to an identification of the novel consensus sequence possibly required for the expression of either CK or EK activity, which corresponded to the sequence within exons VII and VIII of CK/EK-alpha and -beta genes from the mouse. This sequence was localized in close proximity to the C-terminal region of the general (Brenner's) phosphotransferase concensus sequence which was also completely conserved in all of the putative eukaryotic CK/EK proteins. The results demonstrated that, while both CK/EK-alpha and -beta genes were composed of 11 major exons, the size of their genes was quite different: 40 kb for CK/EK-alpha, whereas it was only 3.5 kb for CK/EK-beta. more...

By means of peptide sequence information, several cDNA clones encoding a 42 kDa choline/ethanolamine kinase were isolated from a rat kidney cDNA library. Eight clones were sequenced with all of them resulting in identical overlapping nucleotide sequences. Four of them possessed entire open reading frame which could encode 394 amino acids with a calculated molecular size of 45 100. The predicted amino acid sequence contained all of the internal peptide fragment sequences derived from the purified 42 kDa enzyme. When the open reading frame was introduced into pGEX-2T vector and transfected into E. coli cells, a significant choline/ethanolamine kinase activity did appear in the cell lysate. A homology comparison with the previously reported choline kinase cDNAs (CKR1 and CKR2) from rat liver showed 66%-68% in entire nucleotide sequences and 57%-59% in amino acid sequences, indicating that the cloned cDNA here must be a novel CK/EK gene product. (c) 1998 Elsevier Science B. V. more...

Choline kinase, the initial enzyme of the CDP-choline pathway, mediates the conversion of choline to phosphorylcholine and is localized in the supernatant fraction of cells. The enzyme also catalyzes the phosphorylation of ethanolamine, functioning as the initial enzyme of the CDP-ethanolamine pathway as well. Yeast choline kinase is encoded by a single structural gene, CKI, which was cloned by the genetic complementation of the choline kinase mutation cki. The deduced sequence comprises 582 amino acid residues with a molecular mass of 66316 Da and bears local sequence similarity to various protein kinases and bacterial antibiotic phosphotransferases. The expression of yeast choline kinase is transcriptionally repressed by myo-inositol and choline in a coordinate manner with other phospholipid-synthesizing enzymes in yeast. more...

While steady-state kinetic parameters (metabolite pools, Km and activation energies) are partially known for the enzymes involved in phosphatidylcholine synthesis and degradation in mammalian brain, they are not available for the nervous system of lower vertebrates or invertebrates. Since the extent of evolutionary development of an enzyme is not known a priori, we evaluated the kinetic and thermodynamic parameters of choline kinase, CTP:phosphocholine cytidylyltransferase, choline phosphotransferase and glycerophosphorylcholine phosphodiesterase in squid (Loligo pealei) optic lobe, dogfish (Mustelus canis) and rat brain. For all these enzyme activities, basic similarities in Km and inhibitor effect were found. The same was true for the activation energies Ea, with the exception of squid choline kinase and dogfish cytidylyltransferase. Treatment of microsomal membranes with phospholipase C sharply inhibited cytidylyltransferase activity in all three animal species. In dogfish brain, glycerophosphorylcholine phosphodiesterase activity was undetectable. Our results are consistent with the notion that the kinetic properties of the enzyme activities leading to the preservation of the phosphatidylcholine membranous pool may have appeared early in metazoan evolution and been fully conserved in mammals. more...