Molecular Docking and Dynamics Investigations for Identifying Potential Inhibitors of the 3-Chymotrypsin-like Protease of SARS-CoV-2: Repurposing of Approved Pyrimidonic Pharmaceuticals for COVID-19 Treatment.

This study demonstrates the inhibitory effect of 42 pyrimidonic pharmaceuticals (PPs) on the 3-chymotrypsin-like protease of SARS-CoV-2 (3CLpro) through molecular docking, molecular dynamics simulations, and free binding energies by means of molecular mechanics-Poisson Boltzmann surface area (MM-PBSA) and molecular mechanics-generalized Born surface area (MM-GBSA). Of these tested PPs, 11 drugs approved by the US Food and Drug Administration showed an excellent binding affinity to the catalytic residues of 3CLpro of His41 and Cys145: uracil mustard, cytarabine, floxuridine, trifluridine, stavudine, lamivudine, zalcitabine, telbivudine, tipiracil, citicoline, and uridine triacetate. Their percentage of residues involved in binding at the active sites ranged from 56 to 100, and their binding affinities were in the range from -4.6 ± 0.14 to -7.0 ± 0.19 kcal/mol. The molecular dynamics as determined by a 200 ns simulation run of solvated docked complexes confirmed the stability of PP conformations that bound to the catalytic dyad and the active sites of 3CLpro. The free energy of binding also demonstrates the stability of the PP-3CLpro complexes. Citicoline and uridine triacetate showed free binding energies of -25.53 and -7.07 kcal/mol, respectively. Therefore, I recommend that they be repurposed for the fight against COVID-19, following proper experimental and clinical validation.

A Procedure to Design One-Pot Multi-enzyme System for Industrial CDP-Choline Production.

Fermentation and chemical methods for industrial cytidine diphosphate choline (CDP-choline) catalytic production both suffer from several disadvantages such as relatively low efficiency and productivity. To overcome these problems, we applied the concept of synthetic biology to develop a new one-pot multi-enzyme system to produce CDP-choline from orotic acid. Enzymes from different sources were selected and optimized as building blocks of the system, and parameters such as oxygen supply were also optimized. This system shows a titer of 37.6 ± 1.1 mM and a reaction rate of 1.6 mM L-1 h-1, both increase 66 % from traditional processes. It also has an efficiency of energy of 25.4%, improves 2-folds. This new one-pot CDP-choline-producing system has a potential for industrial use, and the procedure to design one-pot multi-enzyme system can be applied to build other one-pot system producing energy-rich compounds.

A sandwich ELISA-like detection of C-reactive protein in blood by citicoline-bovine serum albumin conjugate and aptamer-functionalized gold nanoparticles nanozyme.

C-reactive protein (CRP) level in blood is associated with the risk of developing cardiovascular events in higher-risk populations. We present a sandwich ELISA-like assay for the determination of CRP in blood by citicoline-bovine serum albumin (citicoline-BSA) conjugate and aptamer-functionalized gold nanoparticles (aptamer-AuNPs) nanozyme. The CRP in the blood sample was selectively adsorbed to the ELISA plate coated by citicoline-BSA, and then incubated with added aptamer-AuNPs. AuNPs exhibited peroxidase activity and oxidized 3,3'5,5'-tetramethylbenzidine from colorless to blue, achieving the measurement at 652 nm. The amplified signal increased linearly in a wide range from 0.1 to 200 ng mL-1 and with a detection limit of 8 pg mL-1. Finally, the method was further tested using rat blood from an isoproterenol-induced myocardial infarction experimental model to confirm its applicability. The developed method could directly determine CRP in blood sample after dilution with high accuracy and sensitivity. This method has many advantages, such as easiness to prepare materials, good stability between batches, high specificity, low detection limit, low-cost, easiness to operate with simple instruments, the most remarkable of which is its excellent lot-to-lot stability over the classical ELISA.

Spectrofluorimetric approach for determination of citicoline in the presence of co-formulated piracetam through fluorescence quenching of eosin Y.

A simple, sensitive, and precise spectrofluorimetric method has been developed and validated for quantitation of citicoline in its pharmaceutical formulations. The proposed method based on quantitative quenching effect of citicoline on the native fluorescence of Eosin Y via developing of a binary complex reaction between the cited drug and Eosin Y in acidic medium using acetate buffer pH = 3.6. The quenching of the fluorescence of eosin was measured at 540 nm after excitation at 518 nm. Calibration graph was achieved in the range of 300-3000 ng/mL with 0.9996 as correlation coefficient and 291.0 and 93.86 ng/mL as quantitation and detection limits, respectively. The developed method considered as the first developed spectrofluorimetric one for quantitation of citicoline with high sensitivity and validated according to ICH guidelines. The selectivity of the proposed method was investigated by studying the interference of piracetam as co-formulated drug with CIT in pharmaceutical formulation, therefore the developed method could be used for routine quality control of citicoline in its pharmaceutical formulations either alone or in combination with piracetam.

Structural and functional evidence for citicoline binding and modulation of 20S proteasome activity: Novel insights into its pro-proteostatic effect.

Citicoline or CDP-choline is a drug, made up by a cytidine 5'-diphosphate moiety and choline, which upon adsorption is rapidly hydrolyzed into cytidine 5'-diphosphate and choline, easily bypassing the blood-brain barrier. Once in the brain, these metabolites are used to re-synthesize citicoline in neurons and in the other cell histo-types which uptake them. Citicoline administration finds broad therapeutic application in the treatment of glaucoma as well as other retinal disorders by virtue of its safety profile and neuro-protective and neuroenhancer activity, which significantly improves the visual function. Further, though supported by limited clinical studies, this molecule finds therapeutic application in neurodegenerative disease, delaying the cognitive decline in Alzheimer's Disease (AD) and Parkinson's Disease (PD) subjects. In this work we show that citicoline greatly affects the proteolytic activity of the 20S proteasome on synthetic and natural substrates, functioning as a bimodal allosteric modulator, likely binding at multiple sites. In silico binding simulations identify several potential binding sites for citicoline on 20S proteasome, and their topology envisages the possibility that, by occupying some of these pockets, citicoline may induce a conformational shift of the 20S proteasome, allowing to sketch a working hypothesis for the structural basis of its function as allosteric modulator. In addition, we show that over the same concentration range citicoline affects the distribution of assembled proteasome populations and turn-over of ubiquitinated proteins in SH-SY5Y and SK-N-BE human neuroblastoma cells, suggesting its potential role as a regulator of proteostasis in nervous cells.

Sophisticated polycaprolactone/gelatin nanofibrous nerve guided conduit containing platelet-rich plasma and citicoline for peripheral nerve regeneration: In vitro and in vivo study.

Peripheral nerve injury (PNI) is a devastating condition that may result in loss of sensory function, motor function, or both. In the present study, we construct an electrospun nerve guide conduit (NGC) based on polycaprolactone (PCL) and gelatin filled with citicoline bearing platelet-rich plasma (PRP) gel as a treatment for PNI. The NGCs fabricated from PCL/Gel polymeric blend using the electrospinning technique. The characterizations demonstrated that the fabricated nanofibers were straight with the diameter of 708 ±â€¯476 nm, the water contact angle of 78.30 ±â€¯2.52°, the weight loss of 41.60 ±â€¯6.94% during 60 days, the tensile strength of 5.31 ±â€¯0.97 MPa, and the young's modulus of 3.47 ±â€¯0.10 GPa. The in vitro studies revealed that the PCL/Gel/PRP/Citi NGC was biocompatible and hemocompatible. The in vivo studies conducted on sciatic nerve injury in rats showed that the implantation of PCL/Gel/PRP/Citi NGC induced regeneration of nerve tissue, demonstrated with histopathological assessments. Moreover, the sciatic function index (SFI) value of -30.3 ±â€¯3.5 and hot plate latency time of 6.10 ±â€¯1.10 s revealed that the PCL/Gel/PRP/Citi NGCs recovered motor and sensory functions. Our findings implied that the fabricated NGC exhibited promising physicochemical and biological activates favorable for PNI treatment.

Simvastatin-chitosan-citicoline conjugates nanoparticles as the co-delivery system in Alzheimer susceptible patients.

The main goal of this study was the preparation and characterization of a chitosan-based system for co-delivery of simvastatin and citicoline to overcome simvastatin unwanted side effects in Alzheimer's disease. This conjugated complex was synthesized in three steps, and 1HNMR, FTIR, and UV-Vis spectroscopy confirmed its success. The simvastatin conjugation rate to chitosan was 1.67 times more than citicoline. X-ray diffraction results showed that the crystalline property of both drugs converted to an amorphous state during the synthesis of the conjugated form. Further, SEM images revealed that the developed nanoparticles have a spherical shape with a size between 100 and 300 nm. Another characterization test was RBC hemolysis, with the lowest value at 6.04% and the highest value at 89.56% and became much lower after preparing nanoparticles using the ionotropic technique. TEM characterized the nanoparticles and showed that the gelation technique stabilized the particles.

Dendrimer grafted albumin nanoparticles for the treatment of post cerebral stroke damages: A proof of concept study.

Stroke is the second largest disease of mortality. The biggest hurdle in designing effective brain drug delivery systems is offered by the blood-brain barrier (BBB), which is highly impermeable to many drugs. Albumin nanoparticles (NP) have gained attention due to their multiple ligand binding sites and long circulatory half-life. Citicoline (CIT) is reported to enhance the acetylcholine secretion in the brain and also helps in membrane repair and regeneration. However, the poor BBB permeation of CIT results in lower levels of CIT in the brain. This demands the development of a suitable delivery platform to completely realize the therapeutic benefit of CIT in stroke therapy. This investigation reports the synthesis and characterization of second generation (2.0 G) dendrimer Amplified Albumin (dAA) biopolymer by FTIR, MALDI-TOF, and surface charge (mV). Further, the synthesized biopolymer has been utilized to develop a CIT nanoformulation using a commercially translatable one-pot process. Release of CIT from biopolymer was performed within an acetate buffer at pH 5 and Phosphate buffer at pH 7.4. Further, we investigated the ability of biopolymer to permeate BBB by in vitro permeability assay in bEnd.3 cells. MTT assay of CIT-dAA-NP, CIT-ANP, and 2.0 G PAMAM dendrimers was performed in bEnd.3 cells. Therapeutic efficacy of the synthesized biopolymer was determined by VEGF gene expression within an in vitro hypoxia model in PC12 cells. Thus, this investigation resulted in biopolymers that can be used to deliver any therapeutic agent by altering the permeability of the BBB. Also, cationization by dendrimer grafting is one such strategy that may be used to cationize any other negatively charged polymer, such as albumin. The synthesized biopolymer is not limited to deliver molecules to the brain, but can also be used to increase the loading of negatively-charged drug molecules, siRNA, or any other oligonucleotide.

Citicoline: A Superior Form of Choline?

Medicines containing citicoline (cytidine-diphosphocholine) as an active principle have been marketed since the 1970s as nootropic and psychostimulant drugs available on prescription. Recently, the inner salt variant of this substance was pronounced a food ingredient in the major world markets. However, in the EU no nutrition or health claim has been authorized for use in commercial communications concerning its properties. Citicoline is considered a dietetic source of choline and cytidine. Cytidine does not have any health claim authorized either, but there are claims authorized for choline, concerning its contribution to normal lipid metabolism, maintenance of normal liver function, and normal homocysteine metabolism. The applicability of these claims to citicoline is discussed, leading to the conclusion that the issue is not a trivial one. Intriguing data, showing that on a molar mass basis citicoline is significantly less toxic than choline, are also analyzed. It is hypothesized that, compared to choline moiety in other dietary sources such as phosphatidylcholine, choline in citicoline is less prone to conversion to trimethylamine (TMA) and its putative atherogenic N-oxide (TMAO). Epidemiological studies have suggested that choline supplementation may improve cognitive performance, and for this application citicoline may be safer and more efficacious.

Cytidine 5'-Diphosphocholine (Citicoline) in Glaucoma: Rationale of Its Use, Current Evidence and Future Perspectives.

Cytidine 5'-diphosphocholine or citicoline is an endogenous compound that acts in the biosynthetic pathway of phospholipids of cell membranes, particularly phosphatidylcholine, and it is able to increase neurotrasmitters levels in the central nervous system. Citicoline has shown positive effects in Parkinson's disease and Alzheimer's disease, as well as in amblyopia. Glaucoma is a neurodegenerative disease currently considered a disease involving ocular and visual brain structures. Neuroprotection has been proposed as a valid therapeutic option for those patients progressing despite a well-controlled intraocular pressure, the main risk factor for the progression of the disease. The aim of this review is to critically summarize the current evidence about the effect of citicoline in glaucoma.

Efficient synthesis of cytidine diphosphate choline (CDP-choline) and its analogs.

An efficient P(V)-N activation approach for the synthesis of cytidine diphosphate choline (CDP-choline) and related ribo- and deoxyribonucleotide analogs has been established.

Plasmodium falciparum CTP:phosphocholine cytidylyltransferase possesses two functional catalytic domains and is inhibited by a CDP-choline analog selected from a virtual screening.

Phosphatidylcholine is the major lipid component of the malaria parasite membranes and is required for parasite multiplication in human erythrocytes. Plasmodium falciparum CTP:phosphocholine cytidylyltransferase (PfCCT) is the rate-limiting enzyme of the phosphatidylcholine biosynthesis pathway and thus considered as a potential antimalarial target. In contrast to its mammalian orthologs, PfCCT contains a duplicated catalytic domain. Here, we show that both domains are catalytically active with similar kinetic parameters. A virtual screening strategy allowed the identification of a drug-size molecule competitively inhibiting the enzyme. This compound also prevented phosphatidylcholine biosynthesis in parasites and exerted an antimalarial effect. This study constitutes the first step towards a rationalized design of future new antimalarial agents targeting PfCCT.

Regulatory networks, genes and glycerophospholipid biosynthesis pathway in schistosomiasis: a systems biology view for pharmacological intervention.

Understanding network topology through embracing the global dynamical regulation of genes in an active state space rather than traditional one-gene-one trait approach facilitates the rational drug development process. Schistosomiasis, a neglected tropical disease, has glycerophospholipids as abundant molecules present on its surface. Lack of effective clinical solutions to treat pathogens encourages us to carry out systems-level studies that could contribute to the development of an effective therapy. Development of a strategy for identifying drug targets by combined genome-scale metabolic network and essentiality analyses through in silico approaches provides tantalizing opportunity to investigate the role of protein/substrate metabolism. A genome-scale metabolic network model reconstruction represents choline-phosphate cytidyltransferase as the rate limiting enzyme and regulates the rate of phosphatidylcholine (PC) biosynthesis. The uptake of choline was regulated by choline concentration, promoting the regulation of phosphocholine synthesis. In Schistosoma, the change in developmental stage could result from the availability of choline, hampering its developmental cycle. There are no structural reports for this protein. In order to inhibit the activity of choline-phosphate cytidyltransferase (CCT), it was modeled by homology modeling using 1COZ as the template from Bacillus subtilis. The transition-state stabilization and catalytic residues were mapped as 'HXGH' and 'RTEGISTT' motif. CCT catalyzes the formation of CDP-choline from phosphocholine in which nucleotidyltransferase adds CTP to phosphocholine. The presence of phosphocholine permits the parasite to survive in an immunologically hostile environment. This feature endeavors development of an inhibitor specific for cytidyltransferase in Schistosoma. Flavonolignans were used to inhibit this activity in which hydnowightin showed the highest affinity as compared to miltefosine.

Evolutionary and mechanistic insights into substrate and product accommodation of CTP:phosphocholine cytidylyltransferase from Plasmodium falciparum.

The enzyme CTP:phosphocholine cytidylyltransferase (CCT) is essential in the lipid biosynthesis of Plasmodia (Haemosporida), presenting a promising antimalarial target. Here, we identified two independent gene duplication events of CCT within Apicomplexa and characterized a truncated construct of Plasmodium falciparum CCT that forms a dimer resembling the molecular architecture of CCT enzymes from other sources. Based on biophysical and enzyme kinetics methods, our data show that the CDP-choline product of the CCT enzymatic reaction binds to the enzyme considerably stronger than either substrate (CTP or choline phosphate). Interestingly, in the presence of Mg²âº , considered to be a cofactor of the enzyme, the binding of the CTP substrate is attenuated by a factor of 5. The weaker binding of CTP:Mg²âº , similarly to the related enzyme family of aminoacyl tRNA synthetases, suggests that, with lack of Mg²âº , positively charged side chain(s) of CCT may contribute to CTP accommodation. Thermodynamic investigations by isothermal titration calorimetry and fluorescent spectroscopy studies indicate that accommodation of the choline phosphate moiety in the CCT active site is different when it appears on its own as one of the substrates or when it is linked to the CDP-choline product. A tryptophan residue within the active site is identified as a useful internal fluorescence sensor of enzyme-ligand binding. Results indicate that the catalytic mechanism of Plasmodium falciparum CCT may involve conformational changes affecting the choline subsite of the enzyme.

Modulation of monoaminergic transporters by choline-containing phospholipids in rat brain.

Choline-containing phospholipids were proposed as cognition enhancing agents, but evidence on their activity is controversial. CDP-choline (cytidine-5´-diphosphocholine, CDP) and choline alphoscerate (L-alpha-glycerylphosphorylcholine, GPC) represent the choline-containing phospholipids with larger clinical evidence in the treatment of sequelae of cerebrovascular accidents and of cognitive disorders. These compounds which display mainly a cholinergic profile interfere with phospholipids biosynthesis, brain metabolism and neurotransmitter systems. Dated preclinical studies and clinical evidence suggested that CDP-choline may have also a monoaminergic profile. The present study was designed to assess the influence of treatment for 7 days with choline-equivalent doses (CDP-choline: 325 mg/Kg/day; GPC: 150 mg/Kg/day) of these compounds on brain dopamine (DA), and serotonin (5-HT) levels and on DA plasma membrane transporter (DAT), vesicular monoamine transporters (VMAT1 and VMAT2), serotonin transporter (SERT), and norepinephrine transporter (NET) in the rat. Frontal cortex, striatum and cerebellum were investigated by HPLC with electrochemical detection, immunohistochemistry, Western blot analysis and ELISA techniques. CDP-choline did not affect DA levels, which increased after GPC administration in frontal cortex and cerebellum. GPC increased also 5-HT levels in frontal cortex and striatum. DAT was stimulated in frontal cortex and cerebellum by both CDP and GPC, whereas VMAT2, SERT, NET were unaffected. VMAT1 was not detectable. The above data indicate that CDP-choline and GPC possess a monoaminergic profile and interfere to some extent with brain monoamine transporters. This activity on a relevant drug target, good tolerability and safety of CDP-choline and GPC suggests that these compounds may merit further investigations in appropriate clinical settings.

A rapid LC-ESI-MS/MS method for the quantitation of choline, an active metabolite of citicoline: Application to in vivo pharmacokinetic and bioequivalence study in Indian healthy male volunteers.

A rapid, simple, and sensitive high performance liquid chromatography-tandem mass spectrometry method (LC-ESI-MS/MS) was developed and validated for the determination and pharmacokinetic investigation of choline (CL), active metabolite of citicoline in human plasma using metformin (MF) as IS. The chromatographic separation was performed on a reversed-phase Phenomenx Gemini C18 column with a mobile phase of methanol:water (containing 10mM ammonium formate) (9:1, v/v). The calibration curves were linear over the range of 0.05-5µg/ml. The validated LC-ESI-MS/MS method was successfully applied for the evaluation of pharmacokinetic parameters and bioequivalence study of test and reference control release (CR) tablet preparation of citicoline 1000mg after a single oral administration to all 12 healthy male volunteers.

Citicoline (CDP-choline): What role in the treatment of complications of infectious diseases.

A dysregulated host immune response, as opposed to the intrinsic virulence of a microbial pathogen induces a large part of the pathology seen in infectious diseases. However, current therapies are designed to target the pathogen rather than the underlying pathogenic mechanisms responsible for the manifestation of the pathology. Recent studies have highlighted the role of endothelial cell alteration in the pathology induced in sepsis and cerebral malaria. The endothelial onslaught described, is similar to that seen during ischemia reperfusion in stroke. Protecting endothelial cell membranes during sepsis and cerebral malaria, using citicoline in the same way as in stroke, has thus emerged as a new strategy that needs to be evaluated urgently. Citicoline is a natural compound that is registered for use in ischemic stroke, head trauma and neurological disorders. It enters the phosphatidylcholine synthesis pathway as a rate-limiting step and is involved in the modulation of a large number of metabolic pathways and neurotransmitter levels, and also in the biosynthesis of phospholipids in neuronal membranes. This short review highlights the potential role of citicoline as part of adjunct therapy in the treatment of infectious diseases.

A robust quantitative solid phase immunoassay for the acute phase protein C-reactive protein (CRP) based on cytidine 5'-diphosphocholine coupled dendrimers.

C-reactive protein (CRP) is an important acute phase protein, being used as a sensitive indicator of inflammation and infection and is also associated with the risk of cardiovascular problems. The present paper describes a robust and sensitive ELISA for CRP, based on the affinity of CRP for phosphocholine. In this design synthetic globular polymers (dendrimers) are used as scaffolds for the multivalent display of phosphocholine molecules. CRP present in a sample binds to the phosphocholine moiety presented at high density in the coating layer and is detectable by specific antibodies. The ELISA was applied to determination of pig and human CRP using commercially available antibodies against human CRP. The assay was shown to be more sensitive than previously published immunoassays employing albumin-coupled cytidine diphosphocholine. The coating was stable for at least 30 days at room temperature and the assay showed high intra- and interassay reproducibility. Results were compared with an immunoturbidimetric method and with a commercial ELISA kit and there was very good agreement with the immunoturbidimetric method, however not with the commercial assay, probably due to a calibration discrepancy. The assay is applicable to other species by providing an adequate detection antibody having the desired species specificity.

Localization of the phosphoethanolamine methyltransferase of the human malaria parasite Plasmodium falciparum to the Golgi apparatus.

Phosphatidylcholine is the most abundant phospholipid in the membranes of Plasmodium falciparum, the agent of severe human malaria. The synthesis of this phospholipid occurs via two routes, the CDP-choline pathway, which uses host choline as a precursor, and the plant-like serine decarboxylase-phosphoethanolamine methyltransferase (SDPM) pathway, which uses host serine as a precursor. Although various components of these pathways have been identified, their cellular locations remain unknown. We have previously reported the identification and characterization of the phosphoethanolamine methyltransferase, Pfpmt, of P. falciparum and shown that it plays a critical role in the synthesis of phosphatidylcholine via the SDPM pathway. Here we provide the first evidence that the transmethylation step of the SDPM pathway occurs in the parasite Golgi apparatus. We show that the level of Pfpmt protein in the infected erythrocyte is regulated in a stage-specific fashion, with high levels detected during the trophozoite stage at the peak of parasite membrane biogenesis. Confocal microscopy revealed that Pfpmt is not cytoplasmic. Immunoelectron microscopy revealed that Pfpmt localizes to membrane structures that extend from the nuclear membrane but that it only partially co-localizes with the endoplasmic reticulum marker BiP. Using transgenic parasites expressing green fluorescent protein targeted to different cellular compartments, a complete co-localization was detected with Rab6, a marker of the Golgi apparatus. Together these studies provide the first evidence that the transmethylation step of the SDPM pathway of P. falciparum occurs in the Golgi apparatus and indicate an important role for this organelle in parasite membrane biogenesis.