Active Site Of Adenosine Deaminase Research Articles

Targeting ADA and HIV-1 Nef Protein with Protease Inhibitors: a Repurposing Strategy through Molecular Networking and an in silico Approach for Integrated Management of HIV Co-infected with Abdominal Tuberculosis

Background: Background: The co-infection of HIV and abdominal TB poses a worldwide danger to humanity. This is because there are more strains of bacteria that are resistant to many classes of currently available medications. According to current findings, repurposing existing available medications will result in more effective functioning than using newly designed medications. Objective: Based on this fact, we hypothesised that the PI could be repurposed; we used Food and Drug Administration (FDA)-approved PI drugs to treat HIV co-infected patients with abdominal TB, and a computational study has been conducted. Method: This comprises network analysis models to find their protein drug interaction (PDI) through a search tool for interacting chemicals (STICH) module of Cytoscape network analysis model followed by the screening of these drugs for their ADMET prediction and binding affinity with adenosine deaminase (ADA), a protein responsible for abdominal TB, and the HIV-1 Nef protein, responsible for the regulation of immune function (CD4+). Result: The network analysis showed 13 nearest binding drugs of these proteins of interest. The ADMET study result showed the pharmacologically relevant parameters that have a significant effect on the binding affinity, bioavailability, and toxicity of PI. The top three scores achieved by PI against adenosine deaminase enzyme activity (PDB ID: 1A4M) are viz., -23.7919, -23.3529, and -22.6773 for Ritonavir, Tipranavir, and Atazanavir, respectively. The top three scores achieved by PI against HIV-1 Nef protein activity (PDB ID: 6URI) are viz., -28.7321, -28.4987, and -28.3155 for Atazanavir, Tipranavir, and Simeprevir, respectively. The active site of ADA and HIV-1 Nef proteins comprises amino acid residues such as for Tipranavir: Arene-Cation interaction (Phenyl and Pyridine)- Arg B1081, and Lys B1033 (1A4M) and Arene-Cation interaction (Pyridine and Phenyl)- Lys D11 and Arg D33; Sidechain acceptor Thr B40; Sidechain donor- Asp D30; Backbone donor- Ala B37 (6URI). Atazanavir: Arene-Cation interaction (Phenyl)- Lys A254 and Lys B1033; Sidechain acceptor - Arg A251 (1A4M). Conclusion: Thus, from the computational studies carried out, we could obtain hints for optimising the molecular selectivity of the PI to provide help in the design of new compounds via the repurposing strategy of the FDA-approved PI for effective treatment of co-morbidity with HIV and abdominal TB. However, further pharmacokinetics, pharmacodynamics, preclinical, and clinical studies permit the design of the new agents without undesirable interactions.

Reynoutria Japonica from Traditional Chinese Medicine: A Source of Competitive Adenosine Deaminase Inhibitors for Anticancer.

Adenosine deaminase (ADA) is an important enzyme in purine metabolism and is known as a potential therapeutic target for the treatment of lymphoproliferative disorders and cancer. Traditional Chinese Herbal Medicine (TCHM) is widely used alone or in combination with chemotherapy to treat cancer, due to its ability to deliver a broad variety of bioactive secondary metabolites as promising sources of novel organic natural agents. In the present study, 29 varieties of medicinal plants were screened for the presence of ADA inhibitors. Extracts from Reynoutria japonica, Glycyrrhiza uralensis, Lithospermum erythrorhizon, Magnolia officinalis, Gardenia jasminoides, Stephania tetrandra, Commiphora myrrha, Raphanus sativus and Corydalis yanhusuo demonstrated strong ADA inhibition with rates greater than 50%. However, Reynoutria japonica possessed the highest ADA inhibitory activity at 95.26% and so was used in our study for isolating the ADA inhibitor to be further studied. Eight compounds were obtained and their structures were identified. The compound H1 had strong ADA inhibitory activity and was deduced to be emodin by 1H and 13C-NMR spectroscopic analysis with an IC50 of 0.629 mM. The molecular docking data showed that emodin could bind tightly to the active site of ADA. Our results demonstrated that emodin displayed a new biological activity which is ADA inhibitory activity with high cytotoxic activity against K562 leukemia cells. The bioactivity of cordycepin was significantly increased when used in combination with emodin. Emodin may represent a good candidate anti-cancer therapy and adenosine protective agent.

Modifications of flexible nonyl chain and nucleobase head group of (+)-erythro-9-(2′s-hydroxy-3′s-nonyl)adenine [(+)-EHNA] as adenosine deaminase inhibitors

A series of terminal nonyl chain and nucleobase modified analogues of (+)-EHNA (III) were synthesized and evaluated for their ability to inhibit adenosine deaminase (ADA). The constrained carbon analogues of (+)-EHNA, 7a-7h, 10a-c, 12, 13, 14 and 17a-c appeared very potent with Ki values in the low nanomolar range. Thio-analogues of (+)-EHNA 24a-e wherein 5′C of nonyl chain replaced by sulfur atom found to be less potent compared to (+)-EHNA. Docking of the representative compounds into the active site of ADA was performed to understand structure-activity relationships. Compounds 7a (Ki: 1.1nM) 7b (Ki: 5.2nM) and 26a (Ki: 5.9nM) showed suitable balance of potency, microsomal stability and demonstrated better pharmacokinetic properties as compared to (+)-EHNA and therefore may have therapeutic potential for various inflammatory diseases, hypertension and cancer.

Theoretical Study of the Structure of Adenosine Deaminase Complexes with Adenosine Analogues: I. Aza-, Deaza-, and Isomeric Azadeazaanalogues of Adenosine

The conformational models of the active site of adenosine deaminase (ADA) and its complexes in the basic state with adenosine and 13 isosteric analogues of the aza, deaza, and azadeaza series were constructed. The optimization of the conformational energy of the active site and the nucleoside bound with it in the complex was achieved in the force field of the whole enzyme (the 1ADD structure was used) within the molecular mechanics model using the AMBER 99 potentials. The stable conformational states of each of the complexes, as well as the optimal conformation of the ADA in the absence of ligand, were determined. It was proved that the conformational state that is close to the structure of the ADA complex with 1-deazaadenosine (1ADD) known from the X-ray study corresponds to one of the local minima of the potential surface. Another, a significantly deeper minimum was determined; it differs from the first minimum by the mutual orientation of side chains of amino acid residues. A similar conformational state is optimal for the ADA active site in the absence of the bound ligand. A qualitative correlation exists between the values of potential energies of the complexes in this conformation and the enzymatic activity of ADA toward the corresponding nucleosides. The dynamics of conformational conversions of the active site after the binding of substrate or its analogues, as well as the possibility of the estimation of the inhibitory properties of nucleosides on the basis of calculations, are discussed.

Enthalpy and enzyme activity of modified histidine residues of adenosine deaminase and diethyl pyrocarbonate complexes

Kinetic and thermodynamic studies have been made on the effect of diethyl pyrocarbonate as a histidine modifier on the active site of adenosine deaminase in 50 mM sodium phosphate buffer pH 6.8, at 27°C using UV spectrophotometry and isothermal titration calorimetry (ITC). Inactivation of adenosine deaminase by diethyl pyrocarbonate is correlated with modification of histidyl residues. The number of modified histidine residues complexed to active site of adenosine deaminase are equivalent to 4. The number and energy of histidine binding sets are determined by enthalpy curve, which represents triple stages. These stages are composed of 3,1 and 1 sites of histidyl modified residues at diethyl pyrocarbonate concentrations, 0.63, 1.8, 3.3 mM. The heat contents corresponding to the first, second and third sets are found to be 18 000, 22 000 and 21 900 kJ mol −1 respectively.

Probing the active site of adenosine deaminase by a pH responsive fluorescent competitive inhibitor

The adenine analog erythro-9-(2-hydroxy-3-nonyl)adenine, EHNA, a tight reversible inhibitor ( K I = 1.6 × 10 −9 M) of adenosine deaminase (EC 3.5.4.4) (ADase), was modified into the fluorescent etheno derivative ε-EHNA. The latter is a competitive inhibitor of adenosine deaminase [ K I = (2.80 ± 0.01)10 −6 M], having the fluorescent properties of ε-adenines. Affinity to the active site, monitored by both steady-state and dynamic fluorescence polarization, was confirmed by competition experiments with 2′-deoxycoformycin, the substrate adenosine and EHNA. The ε-adenine moiety of ε-EHNA librates at the shallow active site of ADase. The low absorptivity of ε-EHNA required the measurement of fluorescence excitation spectra. Computer subtraction of fluorescence excitation spectrum of ADase from that of its equimolar complex with ε-EHNA revealed the corrected excitation spectrum of ε-EHNA at the active site of the enzyme. This spectrum mimics that of ε-EHNA at pH 5.5 in buffer solution, implying its protonation at the active site of the enzyme. These results are in agreement with the presence of acidic amino acids that are essential to the catalytic mechanism.

The rate of formation of transition-state analogs in the active site of adenosine deaminase is encounter-controlled: implications for the mechanism

We have previously shown that purine riboside, when bound to adenosine deaminase, forms a complex in which C-6 of the purine is tetrahedral [Kurz, L. C., & Frieden, C. (1987) Biochemistry 26, 8450]. We now report the rates of formation of enzyme-inhibitor complexes of two types, those which do and those which do not form such tetrahedral intermediates. In both cases, the rates are encounter-controlled since the progress curves for formation of the complexes are well-described by a simple second-order approach to equilibrium and the rate constants show an inverse solvent viscosity dependence. Assuming that the formation of the intermediate-analogue complex is preceded by an initial ground-state analogue complex, the lifetime of that ground-state complex must be less than approximately 20 microseconds. All of the enzyme-inhibitor complexes studied share three characteristics: (1) the complexes generate large UV-difference spectra; (2) a substantial solvent isotope effect is found on the enzyme's affinity for the inhibitors; and (3) a new signal appears in the CD spectra of the complexes. Two of the nucleosides studied, 1-deazapurine riboside and 1-deaza-adenosine, form complexes which appear to mimic a ground-state rather than a reactive intermediate when bound to adenosine deaminase. We find that the values for the association rate constants for those inhibitors which form intermediate analogues are very similar to that for adenosine. The presence of a significant solvent isotope effect on the affinity of all inhibitors is attributable in part to a large transfer isotope effect on the free ligand and in part to an effect on the bound ligand. This complicates use of the solvent isotope effect in applications of the multiple isotope method for estimating intrinsic isotope effects and commitment factors.

Determination of the conformational preferences of adenosine at the active site of adenosine deaminase

Determination of the conformational preferences of adenosine at the active site of adenosine deaminase

A quantum chemical study of the enzymatic deamination of benzoadenine derivatives

A theoretical study is presented, where, by using both ab initio and semi-empirical methodologies, the properties of benzoadenine derivatives as substrates of adenosine deaminase are discussed. The results suggest that lin-benzoadenine and lin-benzoadenosine can be recognized with an affinity similar to that of adenosine, but only if they are introduced about 0.12 nm deeper inside the active site of the enzyme than the natural substrate adenosine. This fact implies the existence of non-linear hydrogen bonds inside the active site of adenosine deaminase. Ab initio molecular electrostatic potential values suggest that these hydrogen bonds can exist, and have stability similar to that of linear hydrogen bonds. Finally, the great rate of deamination of lin-benzoadenine, comparable with that of adenosine despite the absence of the ribose, is explained in the context of the hypothesis that the protonation at the N1 atom is the rate-determining step of the whole deamination reaction.

The protonated form of 1- N6-etheno-[erythro-9-(2-hydroxy-3-nonyl)] adenine is identified at the active site of adenosine deaminase

A novel fluorescent competitive inhibitor of adenosine deaminase (EC 3.5.4.4) (ADA), 1- N 6-etheno-[erythro-9-(2-hydroxy-3-nonyl)] adenine (ϵ-EHNA), is protonated at the active site of the enzyme. In ϵ-EHNA [ K 1 = (4.06 ± 1.00) 10 −6 M] part of the competive inhibition of EHNA is combined with spectroscopic properties of etheno-adenines. Computer subtraction of the fluorescence excitation spectrum of ADA from that of its equimolar complex with ϵ-EHNA yielded the corrected excitation spectrum of ϵ-EHNA at the active site of the enzyme. This spectrum mimics that of ϵ-EHNA at pH 5.5 in buffer solution and is suggested to indicate a shift in protonation equilibrium at the active site.

Adenosine deaminase converts purine riboside into an analogue of a reactive intermediate: a 13C NMR and kinetic study.

The 13C NMR spectra of [2-13C]- and [6-13C]purine ribosides have been obtained free in solution and bound to the active site of adenosine deaminase. The positions of the resonances of the bound ligand are shifted relative to those of the free ligand as follows: C-2, -3.7 ppm; C-6, -73.1 ppm. The binary complexes are in slow exchange with free purine riboside on the NMR time scale, and the dissociation rate constant is estimated to be 13.5 s-1 from the slow exchange broadening of the free signal. In aqueous solution, protonation of purine riboside at N-1 results in changes in 13C chemical shift relative to those of the free base as follows: C-2, -4.9 ppm; C-6, -7.9 ppm. The changes in chemical shift that occur when purine riboside binds to the enzyme indicate that the hybridization of C-6 changes from sp2 to sp3 in the binary complex with formation of a new bond to oxygen or sulfur. A change in C-2 hybridization can be eliminated as can protonation at N-1 as the sole cause of the chemical shift changes. The kinetic constants for the adenosine deaminase catalyzed hydrolysis of 6-chloro- and 6-fluoropurine riboside have been compared, and the reactivity order implies that carbon-halogen bond breaking does not occur in the rate-determining step. These observations support a mechanism for the enzyme in which formation of a tetrahedral intermediate is the most difficult chemical step. Enzymic stabilization of this intermediate may be an important catalytic strategy used by the enzyme to lower the standard free energy of the preceding transition state.

R- and S-alkylidene acetals of adenosine: Stereochemical probes for the active site of adenosine deaminase

Condensation of adenosine with unsymmetrical ketones leads to 2′,3′- O-alkylidene acetals with a new chiral center. The diastereoisomers were separated chromatographically, and the ratio of products was found to be 3:1. The configuration of the new chiral centers was determined by nmr spectrosopy. The diastereoisomers were used as stereochemical probes for the active site of adenosine deaminase. By determination of the K m values it was shown that the binding of the S-diastereoisomers is strongly decreased in comparison with the R-compounds. The data imply a close proximity of the 2′,3′-site of the ribose moiety to the active site of adenosine deaminase.