Synthesis, biological evaluation, in silico modeling and crystallization of novel small monocationic molecules with potent antiproliferative activity by dual mechanism.

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.

ChoK-ing the Pathogenic Bacteria: Potential of Human Choline Kinase Inhibitors as Antimicrobial Agents.

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.

Genetic diseases of the Kennedy pathways for membrane synthesis.

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.

Molecular basis for the interaction between human choline kinase alpha and the SH3 domain of the c-Src tyrosine kinase.

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.

Computation-aided rational design of a halophilic choline kinase for cytidine diphosphate choline production in high-salt condition.

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.

Identification of a Unique Inhibitor-Binding Site on Choline Kinase α.

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.

Molecular structure and differential function of choline kinases CHKα and CHKß in musculoskeletal system and cancer.

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 CHKß, 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 CHKß 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.

Plasmodium falciparum Choline Kinase Inhibition Leads to a Major Decrease in Phosphatidylethanolamine Causing Parasite Death.

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.

Design, synthesis, crystallization and biological evaluation of new symmetrical biscationic compounds as selective inhibitors of human Choline Kinase α1 (ChoKα1).

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.

Evolutionary Ancestry of Eukaryotic Protein Kinases and Choline Kinases.

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.

In Silico Exploration for New Antimalarials: Arylsulfonyloxy Acetimidamides as Prospective Agents.

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.

New more polar symmetrical bipyridinic compounds: new strategy for the inhibition of choline kinase α1.

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.

Structural and enzymatic characterization of the choline kinase LicA from Streptococcus pneumoniae.

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.

Pharmacophore-Based Virtual Screening to Discover New Active Compounds for Human Choline Kinase α1.

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.

Choline kinase active site provides features for designing versatile inhibitors.

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.

Composite aromatic boxes for enzymatic transformations of quaternary ammonium substrates.

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.

Discovery of a new binding site on human choline kinase α1: design, synthesis, crystallographic studies, and biological evaluation of asymmetrical bispyridinium derivatives.

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.

Antiplasmodial activity and mechanism of action of RSM-932A, a promising synergistic inhibitor of Plasmodium falciparum choline kinase.

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.

Determination of potential scaffolds for human choline kinase α1 by chemical deconvolution studies.

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.

Kinetic and mechanistic characterisation of Choline Kinase-α.

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.