ABSTRACT
Remdesivir, an intravenous nucleotide prodrug, has been approved for treating COVID-19 in hospitalized adults and pediatric patients. Upon administration, remdesivir can be readily hydrolyzed to form its active form GS-441524, while the cleavage of the carboxylic ester into GS-704277 is the first step for remdesivir activation. This study aims to assign the key enzymes responsible for remdesivir hydrolysis in humans, as well as to investigate the kinetics of remdesivir hydrolysis in various enzyme sources. The results showed that remdesivir could be hydrolyzed to form GS-704277 in human plasma and the microsomes from human liver (HLMs), lung (HLuMs) and kidney (HKMs), while the hydrolytic rate of remdesivir in HLMs was the fastest. Chemical inhibition and reaction phenotyping assays suggested that human carboxylesterase 1 (hCES1A) played a predominant role in remdesivir hydrolysis, while cathepsin A (CTSA), acetylcholinesterase (AchE) and butyrylcholinesterase (BchE) contributed to a lesser extent. Enzymatic kinetic analyses demonstrated that remdesivir hydrolysis in hCES1A (SHUTCM) and HLMs showed similar kinetic plots and much closed Km values to each other. Meanwhile, GS-704277 formation rates were strongly correlated with the CES1A activities in HLM samples from different individual donors. Further investigation revealed that simvastatin (a therapeutic agent for adjuvant treating COVID-19) strongly inhibited remdesivir hydrolysis in both recombinant hCES1A and HLMs. Collectively, our findings reveal that hCES1A plays a predominant role in remdesivir hydrolysis in humans, which are very helpful for predicting inter-individual variability in response to remdesivir and for guiding the rational use of this anti-COVID-19 agent in clinical settings.
Subject(s)
Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Carboxylesterase/metabolism , Acetylcholinesterase/chemistry , Acetylcholinesterase/metabolism , Adenosine Monophosphate/chemistry , Adenosine Monophosphate/metabolism , Alanine/chemistry , Alanine/metabolism , Butyrylcholinesterase/chemistry , Butyrylcholinesterase/metabolism , Carboxylesterase/chemistry , Cathepsin A/chemistry , Cathepsin A/metabolism , Humans , Hydrolysis/drug effects , Kinetics , Liver/metabolism , Microsomes, Liver/metabolism , Simvastatin/pharmacologyABSTRACT
The hunt for potential lead/drug molecules from different resources, especially from natural resources, for possible treatment of COVID-19 is ongoing. Several compounds have already been identified, but only a few are good enough to show potential against the virus. Among the identified druggable target proteins of SARS-CoV-2, this study focuses on non-structural RNA-dependent RNA polymerase protein (RdRp), a well-known enzyme for both viral genome replication and viral mRNA synthesis, and is therefore considered to be the primary target. In this study, the virtual screening followed by an in-depth docking study of the Compounds Library found that natural compound Cyclocurcumin and Silybin B have strong interaction with RdRp and much better than the remdesivir with free binding energy and inhibition constant value as êzÅ-6.29 kcal/mol and 58.39 µMêzÅ, and êzÅ-7.93kcal/mol and 45.3 µMêzÅ, respectively. The finding indicated that the selected hits (Cyclocurcumin and Silybin B) could act as non-nucleotide anti-polymerase agents, and can be further optimized as a potential inhibitor of RdRp by benchwork experiments.
Subject(s)
Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Antiviral Agents/metabolism , Biological Products/metabolism , COVID-19/metabolism , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Drug Discovery/methods , Molecular Docking Simulation/methods , Phytochemicals/metabolism , SARS-CoV-2/enzymology , Adenosine Monophosphate/chemistry , Adenosine Monophosphate/metabolism , Alanine/chemistry , Alanine/metabolism , Antiviral Agents/chemistry , Biological Products/chemistry , COVID-19/virology , Catalytic Domain , Coronavirus RNA-Dependent RNA Polymerase/antagonists & inhibitors , Coronavirus RNA-Dependent RNA Polymerase/chemistry , Curcumin/analogs & derivatives , Curcumin/chemistry , Curcumin/metabolism , Databases, Protein , Drug Evaluation, Preclinical/methods , Humans , Hydrogen Bonding , Hydrophobic and Hydrophilic Interactions , Phytochemicals/chemistry , Protein Binding , Silybin/chemistry , Silybin/metabolismABSTRACT
The outbreak of a novel febrile respiratory disease called COVID-19, caused by a newfound coronavirus SARS-CoV-2, has brought a worldwide attention. Prioritizing approved drugs is critical for quick clinical trials against COVID-19. In this study, we first manually curated three Virus-Drug Association (VDA) datasets. By incorporating VDAs with the similarity between drugs and that between viruses, we constructed a heterogeneous Virus-Drug network. A novel Random Walk with Restart method (VDA-RWR) was then developed to identify possible VDAs related to SARS-CoV-2. We compared VDA-RWR with three state-of-the-art association prediction models based on fivefold cross-validations (CVs) on viruses, drugs and virus-drug associations on three datasets. VDA-RWR obtained the best AUCs for the three fivefold CVs, significantly outperforming other methods. We found two small molecules coming together on the three datasets, that is, remdesivir and ribavirin. These two chemical agents have higher molecular binding energies of - 7.0 kcal/mol and - 6.59 kcal/mol with the domain bound structure of the human receptor angiotensin converting enzyme 2 (ACE2) and the SARS-CoV-2 spike protein, respectively. Interestingly, for the first time, experimental results suggested that navitoclax could be potentially applied to stop SARS-CoV-2 and remains to further validation.
Subject(s)
Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Angiotensin-Converting Enzyme 2/chemistry , Antiviral Agents/chemistry , Ribavirin/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Adenosine Monophosphate/chemistry , Alanine/chemistry , Aniline Compounds/chemistry , Drug Evaluation, Preclinical , Genome, Viral , Molecular Docking Simulation , SARS-CoV-2/genetics , Sulfonamides/chemistryABSTRACT
BACKGROUND: The latest coronavirus SARS-CoV-2, discovered in China and rapidly spread Worldwide. COVID-19 affected millions of people and killed hundreds of thousands worldwide. There are many ongoing studies investigating drug(s) suitable for preventing and/or treating this pandemic; however, there are no specific drugs or vaccines available to treat or prevent SARS-CoV-2 as of today. METHODS: Fifty-eight fragrance materials, which are classified as allergen fragrance molecules, were selected and used in this study. Docking simulations were carried out using four functional proteins; the Covid19 Main Protase (MPro), Receptor binding domain (RBD) of spike protein, Nucleocapsid, and host Bromodomain protein (BRD2), as target macromolecules. Three different software, AutoDock, AutoDock Vina (Vina), and Molegro Virtual Docker (MVD), running a total of four different docking protocol with optimized energy functions were used. Results were compared with the five molecules reported in the literature as potential drugs against COVID-19. Virtual screening was carried out using Vina, molecules satisfying our cut-off (- 6.5 kcal/mol) binding affinity was confirmed by MVD. Selected molecules were analyzed using the flexible docking protocol of Vina and AutoDock default settings. RESULTS: Ten out of 58 allergen fragrance molecules were selected for further docking studies. MPro and BRD2 are potential targets for the tested allergen fragrance molecules, while RBD and Nucleocapsid showed weak binding energies. According to AutoDock results, three molecules, Benzyl Cinnamate, Dihydroambrettolide, and Galaxolide, had good binding affinities to BRD2. While Dihydroambrettolide and Galaxolide showed the potential to bind to MPro, Sclareol and Vertofix had the best calculated binding affinities to this target. When the flexible docking results analyzed, all the molecules tested had better calculated binding affinities as expected. Benzyl Benzoate and Benzyl Salicylate showed good binding affinities to BRD2. In the case of MPro, Sclareol had the lowest binding affinity among all the tested allergen fragrance molecules. CONCLUSION: Allergen fragrance molecules are readily available, cost-efficient, and shown to be safe for human use. Results showed that several of these molecules had comparable binding affinities as the potential drug molecules reported in the literature to target proteins. Thus, these allergen molecules at correct doses could have significant health benefits.
Subject(s)
Allergens/chemistry , Allergens/immunology , COVID-19 Drug Treatment , COVID-19/immunology , Odorants , Perfume/chemistry , SARS-CoV-2/chemistry , SARS-CoV-2/immunology , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/chemistry , Adenosine Monophosphate/metabolism , Alanine/analogs & derivatives , Alanine/chemistry , Alanine/metabolism , Allergens/administration & dosage , Allergens/therapeutic use , Benzopyrans/chemistry , Benzopyrans/metabolism , Benzyl Compounds/chemistry , Benzyl Compounds/metabolism , Cinnamates/chemistry , Cinnamates/metabolism , Coronavirus 3C Proteases/chemistry , Coronavirus 3C Proteases/metabolism , Coronavirus Nucleocapsid Proteins/chemistry , Coronavirus Nucleocapsid Proteins/metabolism , Diterpenes/chemistry , Diterpenes/metabolism , Drug Evaluation, Preclinical , Humans , Ligands , Molecular Docking Simulation , Perfume/administration & dosage , Perfume/therapeutic use , Phosphoproteins/chemistry , Phosphoproteins/metabolism , SARS-CoV-2/drug effects , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Transcription Factors/chemistry , Transcription Factors/metabolismABSTRACT
Nucleotide analogs targeting viral RNA polymerase have been proved to be an effective strategy for antiviral treatment and are promising antiviral drugs to combat the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. In this study, we developed a robust in vitro nonradioactive primer extension assay to quantitatively evaluate the efficiency of incorporation of nucleotide analogs by SARS-CoV-2 RNA-dependent RNA polymerase (RdRp). Our results show that many nucleotide analogs can be incorporated into RNA by SARS-CoV-2 RdRp and that the incorporation of some of them leads to chain termination. The discrimination values of nucleotide analogs over those of natural nucleotides were measured to evaluate the incorporation efficiency of nucleotide analog by SARS-CoV-2 RdRp. In agreement with the data published in the literature, we found that the incorporation efficiency of remdesivir-TP is higher than that of ATP and incorporation of remdesivir-TP caused delayed chain termination, which can be overcome by higher concentrations of the next nucleotide to be incorporated. Our data also showed that the delayed chain termination pattern caused by remdesivir-TP incorporation is different for different template sequences. Multiple incorporations of remdesivir-TP caused chain termination under our assay conditions. Incorporation of sofosbuvir-TP is very low, suggesting that sofosbuvir may not be very effective in treating SARS-CoV-2 infection. As a comparison, 2'-C-methyl-GTP can be incorporated into RNA efficiently, and the derivative of 2'-C-methyl-GTP may have therapeutic application in treating SARS-CoV-2 infection. This report provides a simple screening method that should be useful for evaluating nucleotide-based drugs targeting SARS-CoV-2 RdRp and for studying the mechanism of action of selected nucleotide analogs.
Subject(s)
Antiviral Agents/pharmacology , Coronavirus RNA-Dependent RNA Polymerase/genetics , Drug Evaluation, Preclinical/methods , Nucleotides/pharmacology , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/chemistry , Adenosine Monophosphate/genetics , Adenosine Monophosphate/pharmacology , Alanine/analogs & derivatives , Alanine/chemistry , Alanine/genetics , Alanine/pharmacology , Antiviral Agents/chemistry , Coronavirus RNA-Dependent RNA Polymerase/antagonists & inhibitors , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Nucleotides/chemistry , RNA , RNA, Viral/biosynthesis , Viral Nonstructural ProteinsABSTRACT
Most recently, an outbreak of severe pneumonia caused by the infection of SARS-CoV-2, a novel coronavirus first identified in Wuhan, China, imposes serious threats to public health. Upon infecting host cells, coronaviruses assemble a multi-subunit RNA-synthesis complex of viral non-structural proteins (nsp) responsible for the replication and transcription of the viral genome. Therefore, the role and inhibition of nsp12 are indispensable. A cryo-EM structure of RdRp from SARs-CoV-2 was used to identify novel drugs from Northern South African medicinal compounds database (NANPDB) by using computational virtual screening and molecular docking approaches. Considering Remdesivir as the control, 42 compounds were shortlisted to have docking score better than Remdesivir. The top 5 hits were validated by using molecular dynamics simulation approach and free energy calculations possess strong inhibitory properties than the Remdesivir. Thus, this study paved a way for designing novel drugs by decoding the architecture of an important enzyme and its inhibition with compounds from natural resources. This disclosing of necessary knowledge regarding the screening and the identification of top hits could help to design effective therapeutic candidates against the coronaviruses and design robust preventive measurements.
Subject(s)
Antiviral Agents/pharmacology , Betacoronavirus/drug effects , Betacoronavirus/enzymology , Biological Products/pharmacology , Coronavirus Infections/virology , Pneumonia, Viral/virology , RNA-Dependent RNA Polymerase/antagonists & inhibitors , Viral Nonstructural Proteins/antagonists & inhibitors , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/chemistry , Adenosine Monophosphate/pharmacology , Alanine/analogs & derivatives , Alanine/chemistry , Alanine/pharmacology , Antiviral Agents/chemistry , Betacoronavirus/genetics , Biological Products/chemistry , COVID-19 , Catalytic Domain/genetics , Computer Simulation , Coronavirus Infections/epidemiology , Coronavirus RNA-Dependent RNA Polymerase , Databases, Pharmaceutical , Drug Evaluation, Preclinical , Genome, Viral , Host Microbial Interactions/drug effects , Humans , Ligands , Molecular Docking Simulation , Pandemics , Phylogeny , Pneumonia, Viral/epidemiology , RNA-Dependent RNA Polymerase/chemistry , RNA-Dependent RNA Polymerase/genetics , SARS-CoV-2 , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/geneticsABSTRACT
Cor pulmonale rat models were induced by a single intraperitoneal injection of monocrotaline(MCT), and the sham group received a single intraperitioneal injection of normal saline. After the model rats received intragastric administration of Qishen Yiqi droplet(QS) for 6 weeks, the contents of adenylate(ATP, ADP and AMP) in right myocardial tissues were measured by HPLC, and then the metabolism changes in myocardium of cor pulmonale rats with QS were investigated. The results showed that ATP, ADP, and AMP were well separated, with a good linearity within a certain range of concentration; and the recovery rates were within the range of 90%-108%. As compared with model group, the level of ATP was significantly elevated in high-dose treatment group; ADP contents showed an increasing trend and AMP contents showed a decreasing trend, indicating that QS could significantly improve energy metabolism system in myocardium. By using the HPLC, a qualitative and quantitative analysis method was given for the determination of ATP, ADP and AMP contents in myocardium, providing a method for energy metabolism measurement in biological samples.
Subject(s)
Adenosine Monophosphate/chemistry , Drugs, Chinese Herbal/pharmacology , Myocardium/chemistry , Pulmonary Heart Disease/drug therapy , Animals , RatsABSTRACT
High pressure NMR spectroscopy is a powerful method for identifying rare conformational states of proteins from the pressure response of their chemical shifts. Many proteins have bound adenine nucleotides at their active centers, in most cases in a complex with Mg2+-ions. The 31P NMR signals of phosphate groups of the nucleotides can be used as probes for conformational transitions in the proteins themselves. For distinguishing protein specific pressure effects from trivial pressure responses not due to the protein interaction, data of the pressure response of the free nucleotides must be available. Therefore, the pressure response of 31P chemical shifts of the adenine nucleotides AMP, ADP, and ATP and their Mg2+-complexes has been determined at pH values several units distant from the respective pK-values. It is clearly non-linear for most of the resonances. A negative first order pressure coefficient B1 was determined for all 31P resonances except Mg2+·AMP indicating an upfield shift of the resonances with pressure. The smallest and largest negative values are obtained for the α-phosphate group of ADP and ß-phosphate group of Mg2+·ATP with -0.32 and -4.59ppm/GPa, respectively. With exception of the α-phosphate group of Mg2+·AMP the second order pressure coefficients are positive leading to a saturation like behaviour. The pressure response of the adenine nucleotides is similar but not identical to that observed earlier for guanine nucleotides. The obtained data show that the chemical shift pressure response of the different phosphate groups is rather different dependent on the position of phosphate group in the nucleotide and the nucleotide used.
Subject(s)
Adenine Nucleotides/chemistry , Adenosine Diphosphate/chemistry , Adenosine Monophosphate/chemistry , Adenosine Triphosphate/chemistry , Magnesium/chemistry , Magnetic Resonance Spectroscopy , Phosphorus/chemistry , PressureABSTRACT
The homeostasis of intracellular diadenosine 5',5â³'-P(1),P(4)-tetraphosphate (Ap4A) in the yeast Saccharomyces cerevisiae is maintained by two 60% sequence-identical paralogs of Ap4A phosphorylases (Apa1 and Apa2). Enzymatic assays show that, compared to Apa1, Apa2 has a relatively higher phosphorylase activity towards Ap3A (5',5â³'-P(1),P(3)-tetraphosphate), Ap4A, and Ap5A (5',5â³'-P(1),P(5)-tetraphosphate), and Ap4A is the favorable substrate for both enzymes. To decipher the catalytic insights, we determined the crystal structures of Apa2 in the apo-, AMP-, and Ap4A-complexed forms at 2.30, 2.80, and 2.70Å resolution, respectively. Apa2 is an α/ß protein with a core domain of a twisted eight-stranded antiparallel ß-sheet flanked by several α-helices, similar to the galactose-1-phosphate uridylyltransferase (GalT) members of the histidine triad (HIT) superfamily. However, a unique auxiliary domain enables an individual Apa2 monomer to possess an intact substrate-binding cleft, which is distinct from previously reported dimeric GalT proteins. This cleft is perfectly complementary to the favorable substrate Ap4A, the AMP and ATP moieties of which are perpendicular to each other, leaving the α-phosphate group exposed at the sharp turn against the catalytic residue His161. Structural comparisons combined with site-directed mutagenesis and activity assays enable us to define the key residues for catalysis. Furthermore, multiple-sequence alignment reveals that Apa2 and homologs represent canonical Ap4A phosphorylases, which could be grouped as a unique branch in the GalT family.
Subject(s)
Nucleotidyltransferases/chemistry , Nucleotidyltransferases/metabolism , Saccharomyces cerevisiae/enzymology , Adenosine Monophosphate/chemistry , Adenosine Monophosphate/metabolism , Adenosine Triphosphate/chemistry , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Catalytic Domain , Crystallography, X-Ray , DNA Mutational Analysis , Dinucleoside Phosphates/chemistry , Dinucleoside Phosphates/metabolism , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutant Proteins/chemistry , Mutant Proteins/genetics , Mutant Proteins/metabolism , Protein Binding , Protein Conformation , Saccharomyces cerevisiae/chemistry , Sequence AlignmentABSTRACT
The activities of the bacterial RecA protein are involved in the de novo development and transmission of antibiotic resistance genes, thus allowing bacteria to overcome the metabolic stress induced by antibacterial agents. RecA is ubiquitous and highly conserved among bacteria, but has only distant homologs in human cells. Together, this evidence points to RecA as a novel and attractive antibacterial drug target. All known RecA functions require the formation of a complex formed by multiple adenosine 5'-O-triphosphate (ATP)-bound RecA monomers on single-stranded DNA. In this complex, RecA hydrolyzes ATP. Although several methods for assessing RecA's ATPase activity have been reported, these assay conditions included relatively high concentrations of enzyme and ATP and thereby restricted the RecA conformational state. Herein, we describe the validation of commercial reagents (Transcreener(®) adenosine 5'-O-diphosphate [ADP](2) fluorescence polarization assay) for the high-throughput measurement of RecA's ATPase activity with lower concentrations of ATP and RecA. Under optimized conditions, ADP detection by the Transcreener reagent provided robust and reproducible activity data (Z'=0.92). Using the Transcreener assay, we screened 113,477 small molecules against purified RecA protein. In total, 177 small molecules were identified as confirmed hits, of which 79 were characterized by IC(50) values ≤ 10 µM and 35 were active in bioassays with live bacteria. This set of compounds comprises previously unidentified scaffolds for RecA inhibition and represents tractable hit structures for efforts aimed at tuning RecA inhibitory activity in both biochemical and bacteriological assays.
Subject(s)
Adenosine Monophosphate/chemistry , Drug Evaluation, Preclinical/methods , Enzyme Inhibitors/analysis , Enzyme Inhibitors/chemistry , Fluorescent Dyes/chemistry , Rec A Recombinases/antagonists & inhibitors , Spectrometry, Fluorescence/methods , Rec A Recombinases/analysisABSTRACT
The behaviour of two cyclen-based bismacrocycles linked by aromatic spacers as receptors of adenosine monophosphate (AMP), adenosine diphosphate (ADP) and adenosine triphosphate (ATP) anions is explored. The two bismacrocycles differ from one another by the nature of their spacers, which are respectively 1,3-dimethylbenzene (BMC), or 2,6-dimethylpyridine (BPyC). Potentiometric investigations supported by (1)H and (31)P NMR measurements were performed over a wide pH range to characterize and understand the driving forces implicated in the supramolecular assemblies. A comparison is also carried out with the results presented in this work and those obtained previously with these two ligands and inorganic phosphates. The comparison exhibits the importance of pi-stacking capability of the organic anions in the binding and hydrogen-bonding network. For BPyC, NMR studies highlight two coordination schemes depending on the protonation of the nitrogen atom of the pyridinyl spacer, which acts in acidic media as a supplementary anchoring point.
Subject(s)
Adenosine Diphosphate/chemistry , Adenosine Monophosphate/chemistry , Adenosine Triphosphate/chemistry , Heterocyclic Compounds/chemistry , Macrocyclic Compounds/chemistry , Anions/chemistry , Crystallography, X-Ray , Cyclams , Ligands , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Structure , PotentiometryABSTRACT
The complex formation of uranium(VI) with four nucleotides, adenosine- (AMP), guanosine- (GMP), uridine- (UMP), and cytidine-monophosphate (CMP), has been studied in the alkaline pH range (8.5-12) by (1)H, (31)P, (13)C, and (17)O NMR spectroscopy, providing spectral integral, chemical shift, homo- and heteronuclear coupling, and diffusion coefficient data. We find that two and only two complexes are formed with all ligands in the investigated pH region independently of the total uranium(VI) and ligand concentrations. Although the coordination of the 5'-phosphate group and the 2'- and 3'-hydroxyl groups of the sugar unit to the uranyl ions is similar to that proposed earlier ("Feldman complex"), the number and the structures of the complexes are different. The uranium-to-nucleotide ratio is 6:4 in one of the complexes and 3:3 in the other one, as unambiguously determined by a combinatorial approach using a systematic variation of the ratio of two ligands in ternary uranium(VI)-nucleotide systems. The structure of the 3:3 complex has been determined by single-crystal diffraction as well, and the results confirm the structure proposed by NMR in aqueous solution. The results have important implications on the synthesis of oligonucleotides.
Subject(s)
Nucleotides/chemistry , Uranium/chemistry , Adenosine Monophosphate/chemistry , Cytidine Monophosphate/chemistry , Diffusion , Guanosine Monophosphate/chemistry , Hydrogen-Ion Concentration , Ligands , Magnetic Resonance Spectroscopy/methods , Molecular Structure , Uridine Monophosphate/chemistry , X-Ray Diffraction/methodsABSTRACT
An important role for adenosine diphosphate (ADP)-induced platelet activation and aggregation was proposed more than 40 years ago. The clinical use of clopidogrel, a prodrug of an irreversible P2Y (12) antagonist, has further proved the relevance of inhibiting signaling via the platelet-specific P2Y (12) ADP receptor in the prevention of cardiovascular events. Pharmacological studies at AstraZeneca R&D Charnwood have identified direct, selective, and competitive P2Y (12) antagonists, including cangrelor (also known as AR-C69931MX), which is suitable for intravenous administration, and AZD6140, which is suitable for oral administration. In preclinical use, these compounds predictably and effectively inhibited platelet aggregation without significant increases in bleeding time. In clinical use, these compounds may have significant advantages over current antiplatelet agents. This article summarizes preclinical and clinical data on cangrelor and AZD6140 and discusses the potential of these compounds as novel antiplatelet therapies.
Subject(s)
Adenosine Monophosphate/analogs & derivatives , Fibrinolytic Agents/therapeutic use , Membrane Proteins/antagonists & inhibitors , Platelet Aggregation Inhibitors/therapeutic use , Purinergic P2 Receptor Antagonists , Adenosine/analogs & derivatives , Adenosine/chemistry , Adenosine/pharmacology , Adenosine Diphosphate/pharmacology , Adenosine Monophosphate/administration & dosage , Adenosine Monophosphate/chemistry , Adenosine Monophosphate/pharmacology , Adenosine Monophosphate/therapeutic use , Administration, Oral , Animals , Arterial Occlusive Diseases/drug therapy , Clinical Trials as Topic , Dogs , Double-Blind Method , Drug Evaluation, Preclinical , Female , Fibrinolytic Agents/administration & dosage , Fibrinolytic Agents/pharmacology , Humans , Injections, Intravenous , Male , Platelet Aggregation/drug effects , Platelet Aggregation Inhibitors/administration & dosage , Platelet Aggregation Inhibitors/pharmacology , Pyridines/pharmacology , Rabbits , Randomized Controlled Trials as Topic , Receptors, Purinergic P2Y12 , Thrombosis/drug therapy , TicagrelorABSTRACT
AMP and adenosine are found in all cell types and can be released by cells or created extracellularly from the breakdown of ATP and ADP. We have identified an orphan G protein-coupled receptor with homology to the P2Y family of nucleotide receptors that can respond to both AMP and adenosine. Based on its ability to functionally bind the nucleotide AMP, we have named it P2Y15. Upon stimulation, P2Y15 induces both Ca2+ mobilization and cyclic AMP generation, suggesting coupling to at least two different G proteins. It is highly expressed in mast cells and is found predominantly in the tissues of the respiratory tract and kidneys, which are known to be affected by AMP, adenosine, and adenosine antagonists. Until now, the effects of AMP have been thought to depend on its dephosphorylation to adenosine but we demonstrate here that P2Y15 is a bona fide AMP receptor by showing that it binds [(32)P]AMP. Because AMP and adenosine have bronchoconstrictive effects that can be inhibited by theophylline, we tested whether theophylline and other adenosine receptor antagonists can block P2Y15. We found inhibition at a theophylline concentration well within the therapeutic dose range, indicating that P2Y15 may be a clinically important target of this drug.
Subject(s)
Adenosine Monophosphate/chemistry , Adenosine/chemistry , Calcium/metabolism , Cyclic AMP/metabolism , Receptors, Cell Surface/chemistry , Receptors, G-Protein-Coupled/chemistry , Receptors, G-Protein-Coupled/physiology , Receptors, Purinergic P2/chemistry , Receptors, Purinergic P2/physiology , Amino Acid Sequence , Animals , Cell Line , Cell Line, Tumor , Cloning, Molecular , Cyclic AMP/chemistry , DNA, Complementary/metabolism , Dose-Response Relationship, Drug , GTP-Binding Proteins/metabolism , Humans , Kinetics , Ligands , Mice , Molecular Sequence Data , Phosphorylation , Phylogeny , Protein Binding , RNA/chemistry , Rats , Receptors, Purinergic P1/chemistry , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Amino Acid , Signal Transduction , Theophylline/chemistry , Time Factors , Tissue DistributionABSTRACT
CBS domains are defined as sequence motifs that occur in several different proteins in all kingdoms of life. Although thought to be regulatory, their exact functions have been unknown. However, their importance was underlined by findings that mutations in conserved residues within them cause a variety of human hereditary diseases, including (with the gene mutated in parentheses): Wolff-Parkinson-White syndrome (gamma 2 subunit of AMP-activated protein kinase); retinitis pigmentosa (IMP dehydrogenase-1); congenital myotonia, idiopathic generalized epilepsy, hypercalciuric nephrolithiasis, and classic Bartter syndrome (CLC chloride channel family members); and homocystinuria (cystathionine beta-synthase). AMP-activated protein kinase is a sensor of cellular energy status that is activated by AMP and inhibited by ATP, but the location of the regulatory nucleotide-binding sites (which are prime targets for drugs to treat obesity and diabetes) was not characterized. We now show that tandem pairs of CBS domains from AMP-activated protein kinase, IMP dehydrogenase-2, the chloride channel CLC2, and cystathionine beta-synthase bind AMP, ATP, or S-adenosyl methionine,while mutations that cause hereditary diseases impair this binding. This shows that tandem pairs of CBS domains act, in most cases, as sensors of cellular energy status and, as such, represent a newly identified class of binding domain for adenosine derivatives.
Subject(s)
Adenosine/chemistry , Adenosine Monophosphate/chemistry , Adenosine Monophosphate/metabolism , Adenosine Triphosphate/chemistry , Adenosine Triphosphate/metabolism , Allosteric Site , Amino Acid Motifs , Animals , Binding Sites , Cloning, Molecular , DNA/metabolism , DNA, Complementary/metabolism , Dimerization , Dose-Response Relationship, Drug , Escherichia coli/metabolism , Glutathione Transferase/metabolism , Humans , Kinetics , Ligands , Liver/metabolism , Models, Molecular , Mutation , Plasmids/metabolism , Polymerase Chain Reaction , Protein Binding , Protein Isoforms , Protein Structure, Tertiary , Rats , Recombinant Fusion Proteins/metabolism , Recombinant Proteins/chemistry , Retinitis Pigmentosa/pathologyABSTRACT
The synthesis, hydrolysis, and antiviral evaluation of novel, lipophilic cycloSal-ddAMP (9a-d) and cycloSal-d4AMP (10a-d) derivatives of the antiviral purine dideoxynucleoside analogues 2', 3'-dideoxyadenosine (ddA) (2) and 2',3'-dideoxy-2', 3'-didehydroadenosine (d4A) (3) are reported. These potential pronucleotides release ddAMP (7) or d4AMP (8) selectively by a controlled, chemically induced tandem reaction. All new compounds 9 and 10a-d were synthesized in good yields using our previously reported phosphorus(III) method starting from substituted salicyl alcohols 14a-h. The phosphotriesters 9 and 10 were obtained with a stereochemical preference of 2:1 with respect to the configuration at the phosphorus center. In an 1-octanol/water mixture phosphotriesters 9 and 10 exhibited 7-43-fold higher lipophilicity than the parent nucleosides ddA (2) and d4A (3) as judged by their log P values. In hydrolysis studies, 9 and 10 decomposed under mild aqueous basic conditions releasing solely ddAMP (7) and d4AMP (8), as well as the diols 14. Further hydrolysis studies under acidic conditions showed a marked increase in stability with respect to the acid-catalyzed cleavage of the glycosyl bond. Phosphotriesters 9 and 10 exhibited antiviral potencies against wild-type HIV-1 and HIV-2 strains in human T-lymphocyte (CEM/O) cells that were, respectively, 100- and 600-fold higher than those of ddA (2) and d4A (3). Furthermore, all triesters 9 and 10 were markedly more active than the corresponding ddI compounds 11 and 12, which supports the concept of the delivery of the adenine nucleotides. Studies with adenosine deaminase (ADA) and adenosine monophosphate deaminase (AMPDA) showed that the triesters were not substrates for enzymatic deamination. The studies reported herein demonstrate conclusively that the cycloSal triesters deliver exclusively the nucleotides ddAMP and d4AMP, not only under chemical-simulated hydrolysis but also under intracellular conditions fulfilling the adenosine deaminase bypass premise.
Subject(s)
Adenosine Monophosphate/analogs & derivatives , Anti-HIV Agents/chemical synthesis , Deoxyadenine Nucleotides/chemistry , Organophosphates/chemical synthesis , Prodrugs/chemical synthesis , 3T3 Cells , AMP Deaminase/chemistry , Adenosine Deaminase/chemistry , Adenosine Monophosphate/administration & dosage , Adenosine Monophosphate/chemistry , Animals , Anti-HIV Agents/chemistry , Anti-HIV Agents/pharmacology , Cell Line , Cell Transformation, Viral/drug effects , Deoxyadenine Nucleotides/administration & dosage , Dideoxynucleotides , Drug Delivery Systems , Drug Evaluation, Preclinical , HIV-1/drug effects , HIV-2/drug effects , Humans , Hydrogen-Ion Concentration , Hydrolysis , Kinetics , Mice , Organophosphates/chemistry , Organophosphates/pharmacology , Prodrugs/chemistry , Prodrugs/pharmacology , Structure-Activity Relationship , Virus Replication/drug effectsABSTRACT
Our study was designed to examine how components of complex mixtures can inhibit the binding of other components to receptor sites in the olfactory system of the spiny lobster Panulirus argus. Biochemical binding assays were used to study how two- to six-component mixtures inhibit binding of the radiolabeled odorants taurine, L-glutamate and adenosine-5'-monophosphate to a tissue fraction rich in dendritic membrane of olfactory receptor neurons. Our results indicate that binding inhibition by mixtures can be large and is dependent on the nature of the odorant ligand and on the concentration and composition of the mixture. The binding inhibition by mixtures of structurally related components was generally predicted using a competitive binding model and binding inhibition data for the individual components. This was not the case for binding inhibition by most mixtures of structurally unrelated odorants. The binding inhibition for these mixtures was generally smaller than that for one or more of their components, indicating that complex binding interactions between components can reduce their ability to inhibit binding. The magnitude of binding inhibition was influenced more by the mixture's precise composition than by the number of components in it, since mixtures with few components were sometimes more inhibitory than mixtures with more components. These findings raise the possibility that complex binding interactions between components of a mixture and their receptors may shape the output of olfactory receptor neurons to complex mixtures.
Subject(s)
Nephropidae/physiology , Olfactory Receptor Neurons/metabolism , Receptors, Odorant/metabolism , Adenosine Monophosphate/chemistry , Adenosine Monophosphate/metabolism , Animals , Excitatory Amino Acid Antagonists/pharmacology , Glutamic Acid/metabolism , Purinergic P1 Receptor Antagonists , Receptors, Glutamate/metabolism , Receptors, Neurotransmitter/antagonists & inhibitors , Receptors, Neurotransmitter/metabolism , Receptors, Purinergic P1/metabolism , Taurine/metabolismABSTRACT
5-Nitroindole (NI), a mutagenic nitroarene, was assayed for cytotoxic effects on rat hepatocytes. After incubation with 25-100 microM NI, the adenylate energy charge of the hepatocytes decreased significantly as a result of the decrease in ATP and the increase in AMP. ATP depletion correlated well with the effects of NI on mitochondrial electron transfer and energy transduction in hepatocytes. Thus, NI (a) inhibited the antimycin-sensitive hepatocyte respiration; (b) inhibited NADH oxidation by disrupted hepatocyte mitochondria; (c) inhibited L-malate-L-glutamate oxidation by ADP-supplemented mitochondria; (d) in the absence of ADP, stimulated the same substrates and also succinate oxidation by mitochondria; (e) released the latent ATPase activity of mitochondrial F1F0-ATP synthase; (f) shifted the redox level of reduced cytochromes (c + c1) and b towards the oxidized state; (g) inhibited NADH oxidation by disrupted mitochondria in the vicinity of the NADH-dehydrogenase flavoprotein; (h) inhibited Ca2+ uptake by mitochondria using L-malate-L-glutamate as an energy source; (i) inhibited valinomycin-induced, endogenously energized K+ uptake, with little effect on the ATP-induced uptake; and (j) inhibited the MgATP-dependent contraction of Ca(2+)-swollen mitochondria. NI inhibited lipid peroxidation in hepatocytes and also in substrate-supplemented liver microsomes and mitochondria, thus ruling out hydroperoxides as a cause of NI cytotoxicity. Long-term incubation with NI produced loss of hepatocyte viability, as indicated by lactate dehydrogenase leakage.
Subject(s)
Adenosine Monophosphate/chemistry , Indoles/toxicity , Lipid Peroxidation/drug effects , Liver/drug effects , Oxidative Phosphorylation/drug effects , Adenosine Triphosphate/analysis , Animals , Calcium/metabolism , Cells, Cultured , Liver/metabolism , Male , Mitochondria, Liver/chemistry , Mitochondria, Liver/drug effects , Nitrofurans/pharmacology , Proton-Translocating ATPases/metabolism , Rats , Rats, Wistar , SpectrophotometryABSTRACT
A streptavidin-biotin system was utilized to prepare an antibody-polyadenylic acid conjugate which was subsequently attached to commercially available magnetic beads, Dynabeads oligo(dT)25. Biotinylated polyadenylic acid was combined with streptavidin and the resulting polyadenylic acid-streptavidin was conjugated with an antibody-biotin derivative. The immobilized antibody-polyadenylic acid conjugate was separated from the reaction mixture by hybridization with complementary oligonucleotide immobilized on the surface of Dynabeads oligo(dT)25. The immobilized antibody-polyadenylic acid can be released from the carrier, utilizing low-ionic-strength buffers. The system is intended to be utilized in cell sorting, using immobilized antibodies against cell surface antigens. Dissociation of antibody-containing conjugate from magnetic beads is essential for the isolation of viable cells via positive cell sorting.