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1.
Mol Pharm ; 19(9): 3394-3404, 2022 09 05.
Article in English | MEDLINE | ID: mdl-36001090

ABSTRACT

Adenosine (ADO) is an endogenous metabolite with immense potential to be repurposed as an immunomodulatory therapeutic, as preclinical studies have demonstrated in models of epilepsy, acute respiratory distress syndrome, and traumatic brain injury, among others. The currently licensed products Adenocard and Adenoscan are formulated at 3 mg/mL of ADO for rapid bolus intravenous injection, but the systemic administration of the saline formulations for anti-inflammatory purposes is limited by the nucleoside's profound hemodynamic effects. Moreover, concentrations that can be attained in the airway or the brain through direct instillation or injection are limited by the volumes that can be accommodated in the anatomical space (<5 mL in humans) and the rapid elimination by enzymatic and transport mechanisms in the interstitium (half-life <5 s). As such, highly concentrated formulations of ADO are needed to attain pharmacologically relevant concentrations at sites of tissue injury. Herein, we report a previously uncharacterized crystalline form of ADO (rcADO) in which 6.7 mg/mL of the nucleoside is suspended in water. Importantly, the crystallinity is not diminished in a protein-rich environment, as evidenced by resuspending the crystals in albumin (15% w/v). To the best of our knowledge, this is the first report of crystalline ADO generated using a facile and organic solvent-free method aimed at localized drug delivery. The crystalline suspension may be suitable for developing ADO into injectable formulations for attaining high concentrations of the endogenous nucleoside in inflammatory locales.


Subject(s)
Adenosine Kinase , Adenosine , Adenosine/chemistry , Adenosine/metabolism , Adenosine Kinase/chemistry , Anti-Inflammatory Agents , Enzyme Inhibitors/therapeutic use , Humans , Nucleosides
2.
Adv Mater ; 33(45): e2102271, 2021 Nov.
Article in English | MEDLINE | ID: mdl-34554618

ABSTRACT

Long-term accumulation of adenosine (Ado) in tumor tissues helps to establish the immunosuppressive tumor microenvironment and to promote tumor development. Regulation of Ado metabolism is particularly pivotal for blocking Ado-mediated immunosuppression. The activity of adenosine kinase (ADK) for catalyzing the phosphorylation of Ado plays an essential role in regulating Ado metabolism. Specifically, accumulated Ado in the tumor microenvironment occupies the active site of ADK, inhibiting the phosphorylation of Ado. Phosphate can protect ADK from inactivation and restore the activity of ADK. Herein, calcium phosphate-reinforced iron-based metal-organic frameworks (CaP@Fe-MOFs) are designed to reduce Ado accumulation and to inhibit Ado-mediated immunosuppressive response in the tumor microenvironment. CaP@Fe-MOFs are found to regulate the Ado metabolism by promoting ADK-mediated phosphorylation and relieving the hypoxic tumor microenvironment. Moreover, CaP@Fe-MOFs can enhance the antitumor immune response via Ado regulation, including the increase of T lymphocytes and dendritic cells and the decrease of regulatory T lymphocytes. Finally, CaP@Fe-MOFs are used for cancer treatment in mice, alleviating the Ado-mediated immunosuppressive response and achieving tumor suppression. This study may offer a general strategy for blocking the Ado-mediated immunosuppression in the tumor microenvironment and further for enhancing the immunotherapy efficacy in vivo.


Subject(s)
Adenosine/metabolism , Calcium Phosphates/chemistry , Immunosuppressive Agents/chemistry , Metal-Organic Frameworks/chemistry , Adenosine Kinase/chemistry , Adenosine Kinase/metabolism , Animals , Catalytic Domain , Cell Line, Tumor , Humans , Immunity/drug effects , Immunosuppression Therapy/methods , Immunosuppressive Agents/pharmacology , Immunosuppressive Agents/therapeutic use , Metal-Organic Frameworks/pharmacology , Metal-Organic Frameworks/therapeutic use , Mice , Neoplasms/drug therapy , Neoplasms/immunology , Neoplasms/pathology , Phosphorylation , T-Lymphocytes, Regulatory/cytology , T-Lymphocytes, Regulatory/metabolism , Transplantation, Heterologous , Tumor Microenvironment
3.
Metab Brain Dis ; 36(4): 653-667, 2021 04.
Article in English | MEDLINE | ID: mdl-33496919

ABSTRACT

Epilepsy disease is characterized by the neuronal dysfunction or abnormal neuronal activity of the brain which is regulated by astrocytes. These are glial cells and found to be the major regulators of the brain which are guided by the occurrence of adenosine kinase (ADK) enzyme in the central nervous system (CNS). During the normal physiological environment, ADK maintains the level of adenosine in the CNS. Dysfunction of ADK levels results in accumulation of adenosine levels in the CNS that leads to the pathophysiology of the brain such as astrogliosis which is a pathological hallmark of epileptic seizures. Vicine, an alkaloid glycoside in bitter gourd juice (Momordica charantia) is found to be toxic to the human system if the bitter gourd juice is consumed more. This compound inhibits ADK enzyme activity to lead epilepsy and seizure. Here, the toxic effect of vicine targeting ADK using computational predictions was investigated. The 3-dimensional structure of ADK has been constructed using I-Tasser, which has been refined by ModRefiner, GalaxyRefine, and 3D refine and it was endorsed using PROCHECK, ERRAT, and VADAR. 3D structure of the ligand molecule has been obtained from PubChem. Molecular docking has been achieved using AutoDock 4.2 software, from which the outcome showed the effective interaction between vicine and ADK, which attains binding free energy (∆G) of - 4.13 kcal/mol. Vicine molecule interacts with the active region ARG 149 of ADK and inhibits the functions of ADK that may cause imbalance in energy homeostasis. Also, pre-ADMET results robustly propose in which vicine possesses toxicity, and meanwhile, from the Ames test, it was shown as mutagenic. Hence, the results from our study suggest that vicine was shown to be toxic that suppresses the ADK activity to undergo pathological conditions in the neuronal junctions to lead epilepsy.


Subject(s)
Adenosine Kinase/toxicity , Alkaloids/toxicity , Drug Development/methods , Glucosides/toxicity , Glycosides/toxicity , Nervous System Diseases/chemically induced , Pyrimidinones/toxicity , Adenosine Kinase/chemistry , Alkaloids/chemistry , Animals , Glucosides/chemistry , Glycosides/chemistry , Humans , Mice , Molecular Docking Simulation/methods , Momordica charantia , Protein Structure, Secondary , Pyrimidinones/chemistry , Rats , Toxins, Biological/chemistry , Toxins, Biological/toxicity
4.
Nucleic Acids Res ; 49(1): 491-503, 2021 01 11.
Article in English | MEDLINE | ID: mdl-33290549

ABSTRACT

RNA modifications can regulate the stability of RNAs, mRNA-protein interactions, and translation efficiency. Pseudouridine is a prevalent RNA modification, and its metabolic fate after RNA turnover was recently characterized in eukaryotes, in the plant Arabidopsis thaliana. Here, we present structural and biochemical analyses of PSEUDOURIDINE KINASE from Arabidopsis (AtPUKI), the enzyme catalyzing the first step in pseudouridine degradation. AtPUKI, a member of the PfkB family of carbohydrate kinases, is a homodimeric α/ß protein with a protruding small ß-strand domain, which serves simultaneously as dimerization interface and dynamic substrate specificity determinant. AtPUKI has a unique nucleoside binding site specifying the binding of pseudourine, in particular at the nucleobase, by multiple hydrophilic interactions, of which one is mediated by a loop from the small ß-strand domain of the adjacent monomer. Conformational transition of the dimerized small ß-strand domains containing active site residues is required for substrate specificity. These dynamic features explain the higher catalytic efficiency for pseudouridine over uridine. Both substrates bind well (similar Km), but only pseudouridine is turned over efficiently. Our studies provide an example for structural and functional divergence in the PfkB family and highlight how AtPUKI avoids futile uridine phosphorylation which in vivo would disturb pyrimidine homeostasis.


Subject(s)
Arabidopsis Proteins/chemistry , Arabidopsis/enzymology , Adenosine Kinase/chemistry , Amino Acid Sequence , Arabidopsis Proteins/metabolism , Binding Sites , Catalysis , Catalytic Domain , Consensus Sequence , Crystallography, X-Ray , Magnesium/metabolism , Models, Molecular , Phosphotransferases (Alcohol Group Acceptor)/chemistry , Protein Conformation , Pseudouridine/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Structure-Activity Relationship , Substrate Specificity
5.
Pharmacol Res Perspect ; 7(4): e00506, 2019 08.
Article in English | MEDLINE | ID: mdl-31367385

ABSTRACT

Adenosine (ADO) is an endogenous protective regulator that restores cellular energy balance in response to tissue trauma. Extracellular ADO has a half-life of the order of seconds thus restricting its actions to tissues and cellular sites where it is released. Adenosine kinase (AK, ATP:adenosine 5'-phosphotransferase, EC 2.7.1.20) is a cytosolic enzyme that is the rate-limiting enzyme controlling extracellular ADO concentrations. Inhibition of AK can effectively increase ADO extracellular concentrations at tissue sites where pathophysiological changes occur. Highly potent and selective nucleoside and non-nucleoside AK inhibitors were discovered in the late 1990s that showed in vivo effects consistent with the augmentation of the actions of endogenous ADO in experimental models of pain, inflammation, and seizure activity. These data supported clinical development of several AK inhibitors for the management of epilepsy and chronic pain. However, early toxicological data demonstrated that nucleoside and non-nucleoside chemotypes produced hemorrhagic microfoci in brain in an apparent ADO receptor-dependent fashion. An initial oral report of these important toxicological findings was presented at an international conference but a detailed description of these data has not appeared in the peer-reviewed literature. In the two decades following the demise of these early AK-based clinical candidates, interest in AK inhibition has renewed based on preclinical data in the areas of renal protection, diabetic retinopathy, cardioprotection, and neurology. This review provides a summary of the pharmacology and toxicology data for several AK inhibitor chemotypes and the resulting translational issues associated with the development of AK inhibitors as viable therapeutic interventions.


Subject(s)
Adenosine Kinase/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Adenosine/chemistry , Adenosine/metabolism , Adenosine Kinase/chemistry , Animals , Drug Development , Enzyme Inhibitors/adverse effects , Enzyme Inhibitors/chemistry , Humans , Molecular Structure
6.
Int J Mol Sci ; 20(15)2019 Jul 31.
Article in English | MEDLINE | ID: mdl-31370143

ABSTRACT

Adenosine kinase (ADK) is the first enzyme in the adenosine remediation pathway that catalyzes adenosine phosphorylation into adenosine monophosphate, thus regulating adenosine homeostasis in cells. To obtain new insights into ADK from Bombyx mori (BmADK), we obtained recombinant BmADK, and analyzed its activity, structure, and function. Gel-filtration showed BmADK was a monomer with molecular weight of approximately 38 kDa. Circular dichroism spectra indicated BmADK had 36.8% α-helix and 29.9% ß-strand structures, respectively. The structure of BmADK was stable in pH 5.0-11.0, and not affected under 30 °C. The melting temperature and the enthalpy and entropy changes in the thermal transition of BmADK were 46.51 ± 0.50 °C, 253.43 ± 0.20 KJ/mol, and 0.79 ± 0.01 KJ/(mol·K), respectively. Site-directed mutagenesis demonstrated G68, S201, E229, and D303 were key amino acids for BmADK structure and activity. In particular, S201A mutation significantly increased the α-helix content of BmADK and its activity. BmADK was located in the cytoplasm and highly expressed in the silk gland during the pre-pupal stage. RNA interference revealed the downregulation of BmADK decreased ATG-8, Caspase-9, Ec-R, E74A, and Br-C expression, indicating it was likely involved in 20E signaling, apoptosis, and autophagy to regulate silk gland degeneration and silkworm metamorphosis. Our study greatly expanded the knowledge on the activity, structure, and role of ADK.


Subject(s)
Adenosine Kinase/genetics , Bombyx/genetics , Insect Proteins/genetics , Larva/genetics , Pupa/genetics , Adenosine/chemistry , Adenosine/metabolism , Adenosine Kinase/chemistry , Adenosine Kinase/metabolism , Adenosine Monophosphate/chemistry , Adenosine Monophosphate/metabolism , Amino Acid Sequence , Animals , Binding Sites , Bombyx/enzymology , Bombyx/growth & development , Cloning, Molecular , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression Regulation, Developmental , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Insect Proteins/chemistry , Insect Proteins/metabolism , Kinetics , Larva/enzymology , Larva/growth & development , Models, Molecular , Phosphorylation , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Pupa/enzymology , Pupa/growth & development , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Substrate Specificity
7.
J Med Chem ; 62(9): 4483-4499, 2019 05 09.
Article in English | MEDLINE | ID: mdl-31002508

ABSTRACT

Mycobacterium tuberculosis adenosine kinase (MtbAdoK) is an essential enzyme of Mtb and forms part of the purine salvage pathway within mycobacteria. Evidence suggests that the purine salvage pathway might play a crucial role in Mtb survival and persistence during its latent phase of infection. In these studies, we adopted a structural approach to the discovery, structure-guided design, and synthesis of a series of adenosine analogues that displayed inhibition constants ranging from 5 to 120 nM against the enzyme. Two of these compounds exhibited low micromolar activity against Mtb with half maximal effective inhibitory concentrations of 1.7 and 4.0 µM. Our selectivity and preliminary pharmacokinetic studies showed that the compounds possess a higher degree of specificity against MtbAdoK when compared with the human counterpart and are well tolerated in rodents, respectively. Finally, crystallographic studies showed the molecular basis of inhibition, potency, and selectivity and revealed the presence of a potentially therapeutically relevant cavity unique to the MtbAdoK homodimer.


Subject(s)
Adenosine Kinase/metabolism , Adenosine/analogs & derivatives , Drug Design , Mycobacterium tuberculosis/enzymology , Protein Kinase Inhibitors/chemical synthesis , Adenosine/metabolism , Adenosine/pharmacokinetics , Adenosine Kinase/chemistry , Animals , Antitubercular Agents/chemical synthesis , Antitubercular Agents/metabolism , Antitubercular Agents/pharmacokinetics , Catalytic Domain , Female , Mice , Molecular Structure , Protein Binding , Protein Kinase Inhibitors/metabolism , Protein Kinase Inhibitors/pharmacokinetics , Structure-Activity Relationship
8.
Sci Rep ; 8(1): 11988, 2018 08 10.
Article in English | MEDLINE | ID: mdl-30097648

ABSTRACT

Adenosine Kinase (ADK) regulates the cellular levels of adenosine (ADO) by fine-tuning its metabolic clearance. The transfer of γ-phosphate from ATP to ADO by ADK involves regulation by the substrates and products, as well as by Mg2+ and inorganic phosphate. Here we present new crystal structures of mouse ADK (mADK) binary (mADK:ADO; 1.2 Å) and ternary (mADK:ADO:ADP; 1.8 Å) complexes. In accordance with the structural demonstration of ADO occupancy of the ATP binding site, kinetic studies confirmed a competitive model of auto-inhibition of ADK by ADO. In the ternary complex, a K+ ion is hexacoordinated between loops adjacent to the ATP binding site, where Asp310 connects the K+ coordination sphere to the ATP binding site through an anion hole structure. Nuclear Magnetic Resonance 2D 15N-1H HSQC experiments revealed that the binding of K+ perturbs Asp310 and residues of adjacent helices 14 and 15, engaging a transition to a catalytically productive structure. Consistent with the structural data, the mutants D310A and D310P are catalytically deficient and loose responsiveness to K+. Saturation Transfer Difference spectra of ATPγS provided evidence for an unfavorable interaction of the mADK D310P mutant for ATP. Reductions in K+ concentration diminish, whereas increases enhance the in vitro activity of mADK (maximum of 2.5-fold; apparent Kd = 10.4 mM). Mechanistically, K+ increases the catalytic turnover (Kcat) but does not affect the affinity of mADK for ADO or ATP. Depletion of intracellular K+ inhibited, while its restoration was accompanied by a full recovery of cellular ADK activity. Together, this novel dataset reveals the molecular basis of the allosteric activation of ADK by K+ and highlights the role of ADK in connecting depletion of intracellular K+ to the regulation of purine metabolism.


Subject(s)
Adenosine Kinase/metabolism , Metabolic Networks and Pathways , Potassium/metabolism , Purines/metabolism , Adenosine Kinase/chemistry , Adenosine Kinase/genetics , Amino Acids , Binding Sites , Enzyme Activation , Kinetics , Magnetic Resonance Imaging , Molecular Conformation , Mutation , Phosphorylation , Protein Binding , Purines/chemistry , Structure-Activity Relationship
9.
Biochem Biophys Res Commun ; 502(2): 250-254, 2018 07 12.
Article in English | MEDLINE | ID: mdl-29803677

ABSTRACT

Adenosine kinase (ADK) plays an important role in the growth and development of organisms. A convenient, quick, reliable, sensitive and low-cost assay for ADK activity is of great significance. Here, we found the reaction system with bromothymol blue as the pH indicator had a maximum absorption peak at 614 nm. The absorbance change in 614 nm was positively correlated with the generated hydrogen ions in the reaction catalyzed by ADK. Then, we demonstrated this assay was feasible for ADK activity. Further, we analyzed the effects of buffer, bromothymol blue concentrations on the sensitivity of the assay, and investigated the sensitivity of ADK contents and adenosine concentration on the assay. Finally, we calculated the Km and Vmax of ADK from Bombyx mori with this assay. Our results suggested this assay was quick, convenient, reliable, sensitive and economic for the activity of ADK. It is an excellent alternative for the conventional ADK assays.


Subject(s)
Adenosine Kinase/analysis , Colorimetry/methods , Adenosine Kinase/chemistry , Adenosine Kinase/metabolism , Animals , Bombyx/enzymology , Bromphenol Blue , Bromthymol Blue , Buffers , Colorimetry/statistics & numerical data , Coloring Agents , Feasibility Studies , Hydrogen-Ion Concentration , Kinetics , Spectrophotometry
10.
Biophys J ; 114(9): 2174-2179, 2018 05 08.
Article in English | MEDLINE | ID: mdl-29742410

ABSTRACT

Molecular motors are thought to generate force and directional motion via nonequilibrium switching between energy surfaces. Because all enzymes can undergo such switching, we hypothesized that the ability to generate rotary motion and torque is not unique to highly adapted biological motor proteins but is instead a common feature of enzymes. We used molecular dynamics simulations to compute energy surfaces for hundreds of torsions in three enzymes-adenosine kinase, protein kinase A, and HIV-1 protease-and used these energy surfaces within a kinetic model that accounts for intersurface switching and intrasurface probability flows. When substrate is out of equilibrium with product, we find computed torsion rotation rates up ∼140 cycles s-1, with stall torques up to ∼2 kcal mol-1 cycle-1, and power outputs up to ∼50 kcal mol-1 s-1. We argue that these enzymes are instances of a general phenomenon of directional probability flows on asymmetric energy surfaces for systems out of equilibrium. Thus, we conjecture that cyclic probability fluxes, corresponding to rotations of torsions and higher-order collective variables, exist in any chiral molecule driven between states in a nonequilibrium manner; we call this the "Asymmetry-Directionality" conjecture. This is expected to apply as well to synthetic chiral molecules switched in a nonequilibrium manner between energy surfaces by light, redox chemistry, or catalysis.


Subject(s)
Molecular Dynamics Simulation , Adenosine Kinase/chemistry , Adenosine Kinase/metabolism , Cyclic AMP-Dependent Protein Kinases/chemistry , Cyclic AMP-Dependent Protein Kinases/metabolism , HIV Protease/chemistry , HIV Protease/metabolism , Movement , Protein Conformation , Thermodynamics
11.
ACS Nano ; 9(6): 6069-76, 2015 Jun 23.
Article in English | MEDLINE | ID: mdl-26051465

ABSTRACT

The application of tissue-engineered blood vessels (TEBVs) is the main developmental direction of vascular replacement therapy. Due to few and/or dysfunctional endothelial progenitor cells (EPCs), it is difficult to successfully construct EPC capture TEBVs in diabetes. RNA has a potential application in cell protection and diabetes treatment, but poor specificity and low efficiency of RNA transfection in vivo limit the application of RNA. On the basis of an acellular vascular matrix, we propose an aptamer-siRNA chimera-modified TEBV that can maintain a satisfactory patency in diabetes. This TEBV consists of two parts, CD133-adenosine kinase (ADK) chimeras and a TEBV scaffold. Our results showed that CD133-ADK chimeras could selectively capture the CD133-positive cells in vivo, and then captured cells can internalize the bound chimeras to achieve RNA self-transfection. Subsequently, CD133-ADK chimeras were cut into ADK siRNA by a dicer, resulting in depletion of ADK. An ADK-deficient cell may act as a bioreactor that sustainably releases adenosine. To reduce nonspecific RNA transfection, we increased the proportion of HAuCl4 during the material preparation, through which the transfection capacity of polyethylenimine (PEI)/polyethylene glycol (PEG)-capped gold nanoparticles (PEI/PEG-AuNPs) was significantly decreased and the ability of TEBV to resist tensile and liquid shear stress was greatly enhanced. PEG and 2'-O-methyl modification was used to enhance the in vivo stability of RNA chimeras. At day 30 postgrafting, the patency rate of CD133-ADK chimera-modified TEBVs reached 90% in diabetic rats and good endothelialization was observed.


Subject(s)
Aptamers, Nucleotide/chemistry , Blood Vessels/cytology , Endothelial Progenitor Cells/cytology , RNA, Small Interfering/chemistry , Tissue Engineering , AC133 Antigen , Adenosine Kinase/chemistry , Adenosine Kinase/metabolism , Animals , Antigens, CD/chemistry , Cells, Cultured , Chimera , Diabetes Mellitus, Experimental/chemically induced , Diabetes Mellitus, Experimental/drug therapy , Glycoproteins/chemistry , Healthy Volunteers , Humans , Peptides/chemistry , RNA, Small Interfering/pharmacology , Rats , Rats, Wistar , Streptozocin
12.
J Med Chem ; 57(20): 8268-79, 2014 Oct 23.
Article in English | MEDLINE | ID: mdl-25259627

ABSTRACT

Adenosine kinase (ADK) from Mycobacterium tuberculosis (Mtb) was selected as a target for design of antimycobacterial nucleosides. Screening of 7-(het)aryl-7-deazaadenine ribonucleosides with Mtb and human (h) ADKs and testing with wild-type and drug-resistant Mtb strains identified specific inhibitors of Mtb ADK with micromolar antimycobacterial activity and low cytotoxicity. X-ray structures of complexes of Mtb and hADKs with 7-ethynyl-7-deazaadenosine showed differences in inhibitor interactions in the adenosine binding sites. 1D (1)H STD NMR experiments revealed that these inhibitors are readily accommodated into the ATP and adenosine binding sites of Mtb ADK, whereas they bind preferentially into the adenosine site of hADK. Occupation of the Mtb ADK ATP site with inhibitors and formation of catalytically less competent semiopen conformation of MtbADK after inhibitor binding in the adenosine site explain the lack of phosphorylation of 7-substituted-7-deazaadenosines. Semiempirical quantum mechanical analysis confirmed different affinity of nucleosides for the Mtb ADK adenosine and ATP sites.


Subject(s)
Adenosine Kinase/antagonists & inhibitors , Adenosine Kinase/chemistry , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/enzymology , Ribonucleosides/chemistry , Ribonucleosides/pharmacology , Adenine/analogs & derivatives , Adenine/chemistry , Adenosine Kinase/metabolism , Adenosine Triphosphate/metabolism , Antitubercular Agents/chemistry , Antitubercular Agents/pharmacology , Binding Sites , Crystallography, X-Ray , Drug Evaluation, Preclinical , Humans , Microbial Sensitivity Tests , Nuclear Magnetic Resonance, Biomolecular , Protein Conformation , Structure-Activity Relationship
13.
Acta Crystallogr F Struct Biol Commun ; 70(Pt 1): 34-9, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24419613

ABSTRACT

Trypanosoma brucei is a single-cellular parasite of the genus Kinetoplastida and is the causative agent of African sleeping sickness in humans. Adenosine kinase is a key enzyme in the purine-salvage pathway, phosphorylating adenosine to AMP, and also activates cytotoxic analogues such as cordycepin and Ara-A by their phosphorylation. The structures of T. brucei brucei adenosine kinase (TbAK) in its unliganded open conformation and complexed with adenosine and ADP in the closed conformation are both reported to 2.6 Šresolution. The structures give insight into the binding mode of the substrates and the conformational change induced upon substrate binding. This information can be used to guide the improvement of cytotoxic substrate analogues as potential antitrypanosomal drugs.


Subject(s)
Adenosine Kinase/chemistry , Trypanosoma brucei brucei/enzymology , Crystallization , Crystallography, X-Ray , Models, Molecular , Protein Structure, Secondary , Substrate Specificity
14.
Biomed Res Int ; 2013: 609289, 2013.
Article in English | MEDLINE | ID: mdl-23984386

ABSTRACT

Enzyme adenosine kinase is responsible for phosphorylation of adenosine to AMP and is crucial for parasites which are purine auxotrophs. The present study describes development of robust homology model of Leishmania donovani adenosine kinase to forecast interaction phenomenon with inhibitory molecules using structure-based drug designing strategy. Docking calculation using reported organic small molecules and natural products revealed key active site residues such as Arg131 and Asp16 for ligand binding, which is consistent with previous studies. Molecular dynamics simulation of ligand protein complex revealed the importance of hydrogen bonding with active site residues and solvent molecules, which may be crucial for successful development of drug candidates. Precise role of Phe168 residue in the active site was elucidated in this report that provided stability to ligand-protein complex via aromatic- π contacts. Overall, the present study is believed to provide valuable information to design a new compound with improved activity for antileishmanial therapeutics development.


Subject(s)
Adenosine Kinase/chemistry , Adenosine Kinase/metabolism , Leishmania donovani/enzymology , Molecular Dynamics Simulation , Amino Acid Sequence , Hydrogen Bonding , Molecular Sequence Data , Reproducibility of Results , Structural Homology, Protein
15.
Arch Biochem Biophys ; 537(1): 82-90, 2013 Sep 01.
Article in English | MEDLINE | ID: mdl-23831509

ABSTRACT

Monomeric adenosine kinase (AdK), a pivotal salvage enzyme of the purine auxotrophic parasite, Leishmania donovani, tends to aggregate naturally or selectively in presence of ADP, leading to inactivation. A cyclophilin (LdCyP) from the parasite reactivated the enzyme by disaggregating it. We studied the aggregation pathway of AdK with or without ADP. Transmission electron microscopy revealed that ADP-induced aggregates, as opposed to annular or torus-shaped natural aggregates, were mostly amorphous with protofibril-like structures. Interestingly, only the natural aggregates bound thioflavin T with a KD of 3.33 µM, indicating cross ß-sheet structure. Dynamic light scattering experiments indicated that monomers formed aggregates either upon prolonged storage or ADP exposure. ADP-aggregates were disaggregated by LdCyP with concomitant reactivation of the enzyme. The activity revived with decrease in the aggregate size. Displacement of ADP from the ADP-aggregated enzyme by LdCyP resulted in reactivation. CD-spectral studies suggested that, like the natural aggregates, ADP induced formation of ß-sheet structure in the ADP-aggregates. However, unlike the natural aggregate, it could be reconverted to α-helical conformation upon addition of LdCyP. Based on the results, a regulatory mechanism through interplay of ADP and/or LdCyP interaction with the enzyme is envisaged and a pathway of AdK reactivation by LdCyP-chaperone is proposed.


Subject(s)
Adenosine Kinase/chemistry , Cyclophilins/chemistry , Leishmania donovani/enzymology , Dimerization , Enzyme Activation
16.
Pharmacol Rev ; 65(3): 906-43, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23592612

ABSTRACT

Adenosine kinase (ADK; EC 2.7.1.20) is an evolutionarily conserved phosphotransferase that converts the purine ribonucleoside adenosine into 5'-adenosine-monophosphate. This enzymatic reaction plays a fundamental role in determining the tone of adenosine, which fulfills essential functions as a homeostatic and metabolic regulator in all living systems. Adenosine not only activates specific signaling pathways by activation of four types of adenosine receptors but it is also a primordial metabolite and regulator of biochemical enzyme reactions that couple to bioenergetic and epigenetic functions. By regulating adenosine, ADK can thus be identified as an upstream regulator of complex homeostatic and metabolic networks. Not surprisingly, ADK dysfunction is involved in several pathologies, including diabetes, epilepsy, and cancer. Consequently, ADK emerges as a rational therapeutic target, and adenosine-regulating drugs have been tested extensively. In recent attempts to improve specificity of treatment, localized therapies have been developed to augment adenosine signaling at sites of injury or pathology; those approaches include transplantation of stem cells with deletions of ADK or the use of gene therapy vectors to downregulate ADK expression. More recently, the first human mutations in ADK have been described, and novel findings suggest an unexpected role of ADK in a wider range of pathologies. ADK-regulating strategies thus represent innovative therapeutic opportunities to reconstruct network homeostasis in a multitude of conditions. This review will provide a comprehensive overview of the genetics, biochemistry, and pharmacology of ADK and will then focus on pathologies and therapeutic interventions. Challenges to translate ADK-based therapies into clinical use will be discussed critically.


Subject(s)
Adenosine Kinase/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Molecular Targeted Therapy , Signal Transduction/drug effects , Adenosine Kinase/chemistry , Adenosine Kinase/genetics , Adenosine Kinase/metabolism , Animals , Drug Design , Energy Metabolism/drug effects , Epigenesis, Genetic/drug effects , Humans
17.
Acta Crystallogr D Biol Crystallogr ; 69(Pt 1): 126-36, 2013 Jan.
Article in English | MEDLINE | ID: mdl-23275171

ABSTRACT

In adult schistosomes, the enzyme adenosine kinase (AK) is responsible for the incorporation of some adenosine analogues, such as 2-fluoroadenosine and tubercidin, into the nucleotide pool, but not others. In the present study, the structures of four complexes of Schistosoma mansoni AK bound to adenosine and adenosine analogues are reported which shed light on this observation. Two differences in the adenosine-binding site in comparison with the human counterpart (I38Q and T36A) are responsible for their differential specificities towards adenosine analogues, in which the Schistosoma enzyme does not tolerate bulky substituents at the N7 base position. This aids in explaining experimental data which were reported in the literature more than two decades ago. Furthermore, there appears to be considerable plasticity within the substrate-binding sites that affects the side-chain conformation of Ile38 and causes a previously unobserved flexibility within the loop comprising residues 286-299. These results reveal that the latter can be sterically occluded in the absence of ATP. Overall, these results contribute to the body of knowledge concerning the enzymes of the purine salvage pathway in this important human parasite.


Subject(s)
Adenosine Kinase/chemistry , Adenosine/chemistry , Schistosoma mansoni/enzymology , Adenosine/analogs & derivatives , Adenosine/metabolism , Adenosine Kinase/genetics , Adenosine Kinase/metabolism , Animals , Crystallization , Crystallography, X-Ray , Humans , Schistosoma mansoni/genetics , Schistosoma mansoni/metabolism , Sequence Alignment , Substrate Specificity/genetics
18.
PLoS Negl Trop Dis ; 5(5): e1164, 2011 May.
Article in English | MEDLINE | ID: mdl-21629723

ABSTRACT

BACKGROUND: The essential purine salvage pathway of Trypanosoma brucei bears interesting catalytic enzymes for chemotherapeutic intervention of Human African Trypanosomiasis. Unlike mammalian cells, trypanosomes lack de novo purine synthesis and completely rely on salvage from their hosts. One of the key enzymes is adenosine kinase which catalyzes the phosphorylation of ingested adenosine to form adenosine monophosphate (AMP) utilizing adenosine triphosphate (ATP) as the preferred phosphoryl donor. METHODS AND FINDINGS: Here, we present the first structures of Trypanosoma brucei rhodesiense adenosine kinase (TbrAK): the structure of TbrAK in complex with the bisubstrate inhibitor P(1),P(5)-di(adenosine-5')-pentaphosphate (AP5A) at 1.55 Å, and TbrAK complexed with the recently discovered activator 4-[5-(4-phenoxyphenyl)-2H-pyrazol-3-yl]morpholine (compound 1) at 2.8 Å resolution. CONCLUSIONS: The structural details and their comparison give new insights into substrate and activator binding to TbrAK at the molecular level. Further structure-activity relationship analyses of a series of derivatives of compound 1 support the observed binding mode of the activator and provide a possible mechanism of action with respect to their activating effect towards TbrAK.


Subject(s)
Adenosine Kinase/chemistry , Enzyme Activators/chemistry , Enzyme Inhibitors/chemistry , Trypanosoma brucei rhodesiense/enzymology , Adenosine Kinase/metabolism , Amino Acid Sequence , Binding Sites , Crystallography, X-Ray , Enzyme Activators/metabolism , Enzyme Inhibitors/metabolism , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Conformation , Sequence Alignment , Trypanosoma brucei rhodesiense/chemistry
19.
BMC Biochem ; 12: 22, 2011 May 17.
Article in English | MEDLINE | ID: mdl-21586167

ABSTRACT

BACKGROUND: Two isoforms of the enzyme adenosine kinase (AdK), which differ at their N-terminal ends, are found in mammalian cells. However, there is no information available regarding the unique functional aspects or regulation of these isoforms. RESULTS: We show that the two AdK isoforms differ only in their first exons and the promoter regions; hence they arise via differential splicing of their first exons with the other exons common to both isoforms. The expression of these isoforms also varied greatly in different rat tissues and cell lines with some tissues expressing both isoforms and others expressing only one of the isoforms. To gain insights into cellular functions of these isoforms, mutants resistant to toxic adenosine analogs formycin A and tubercidin were selected from Chinese hamster (CH) cell lines expressing either one or both isoforms. The AdK activity in most of these mutants was reduced to <5% of wild-type cells and they also showed large differences in the expression of the two isoforms. Thus, the genetic alterations in these mutants likely affected both regulatory and structural regions of AdK. We have characterized the molecular alterations in a number of these mutants. One of these mutants lacking AdK activity was affected in the conserved NxxE motif thereby providing evidence that this motif involved in the binding of Mg2+ and phosphate ions is essential for AdK function. Another mutant, FomR-4, exhibiting increased resistance to only C-adenosine analogs and whose resistance was expressed dominantly in cell-hybrids contained a single mutation leading to Ser191Phe alteration in AdK. We demonstrate that this mutation in AdK is sufficient to confer the novel genetic and biochemical characteristics of this mutant. The unusual genetic and biochemical characteristics of the FomR-4 mutant suggest that AdK in this mutant might be complexed with the enzyme AMP-kinase. Several other AdK mutants were altered in surface residues that likely affect its binding to the adenosine analogs and its interaction with other cellular proteins. CONCLUSIONS: These AdK mutants provide important insights as well as novel tools for understanding the cellular functions of the two isoforms and their regulation in mammalian cells.


Subject(s)
Adenosine Kinase/metabolism , Adenosine Kinase/chemistry , Adenosine Kinase/genetics , Amino Acid Sequence , Animals , Base Sequence , Cell Line , Cricetinae , Cricetulus , Exons , Formycins/toxicity , Gene Expression Regulation , Humans , Mice , Molecular Sequence Data , Mutation , Promoter Regions, Genetic , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Structure, Tertiary , Rats , Tubercidin/toxicity
20.
J Med Chem ; 54(5): 1401-20, 2011 Mar 10.
Article in English | MEDLINE | ID: mdl-21319802

ABSTRACT

Adenosine kinase (AK) catalyzes the phosphorylation of adenosine (Ado) to AMP by means of a kinetic mechanism in which the two substrates Ado and ATP bind the enzyme in a binary and/or ternary complex, with distinct protein conformations. Most of the described inhibitors have Ado-like structural motifs and are nonselective, and some of them (e.g., the tubercidine-like ligands) are characterized by a toxic profile. We have cloned and expressed human AK (hAK) and searched for novel non-substrate-like inhibitors. Our efforts to widen the structural diversity of AK inhibitors led to the identification of novel non-nucleoside, noncompetitive allosteric modulators characterized by a unique molecular scaffold. Among the pyrrolobenzoxa(thia)zepinones (4a-qq) developed, 4a was identified as a non-nucleoside prototype hAK inhibitor. 4a has proapoptotic efficacy, slight inhibition of short-term RNA synthesis, and cytostatic activity on tumor cell lines while showing low cytotoxicity and no significant adverse effects on short-term DNA synthesis in cells.


Subject(s)
Adenosine Kinase/antagonists & inhibitors , Antineoplastic Agents/chemical synthesis , Models, Molecular , Oxazepines/chemical synthesis , Pyrroles/chemical synthesis , Adenosine Kinase/chemistry , Allosteric Regulation , Allosteric Site , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , DNA/biosynthesis , Drug Screening Assays, Antitumor , Humans , Mice , Oxazepines/chemistry , Oxazepines/pharmacology , Pyrroles/chemistry , Pyrroles/pharmacology , RNA/biosynthesis , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/chemistry , Stereoisomerism , Structure-Activity Relationship
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