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PubMed2001

Biophysical characterization of recombinant human Bcl-2 and its interactions with an inhibitory ligand, antimycin A.

Apoptosis is an essential physiological process, regulated by the family of Bcl-2-related proteins. However, the molecular mechanism by which Bcl-2 regulates apoptosis still remains elusive. Here we report the functional studies of recombinant human Bcl-2 with the deletion of 22 residues at the C-terminal membrane-anchoring region (rhBcl-2Delta22). Characterization of rhBcl-2Delta22 showed that the recombinant protein is homogeneous and monodisperse in nondenaturing solutions, stable at room temperature in the presence of a metal chelator, and an alpha-helical protein with unfolding of secondary structure at a T(m) of 62.8 degrees C. Optimal membrane pore formation by rhBcl-2Delta22 required negatively charged phospholipids. The existence of a hydrophobic groove in rhBcl-2Delta22 was demonstrated by the fluorescence enhancement of the hydrophobic ANS probe with which a pro-apoptotic Bak BH3 peptide competed. The respiratory inhibitor antimycin A also bound to the hydrophobic groove of rhBcl-2Delta22 with a K(d) of 0.82 microM. The optimal binding conformation of antimycin A was predicted from molecular docking of antimycin A with the hBcl-2 model created by homology modeling. Antimycin A selectively induces apoptosis in cells overexpressing Bcl-2, suggesting that hydrophobic groove-binding compounds may act as selective apoptotic triggers in tumor cells.

Amino Acid Sequence↗

PubMed2006

The heterodimeric sweet taste receptor has multiple potential ligand binding sites.

The sweet taste receptor is a heterodimer of two G protein coupled receptors, T1R2 and T1R3. This discovery has increased our understanding at the molecular level of the mechanisms underlying sweet taste. Previous experimental studies using sweet receptor chimeras and mutants show that there are at least three potential binding sites in this heterodimeric receptor. Receptor activity toward the artificial sweeteners aspartame and neotame depends on residues in the amino terminal domain of human T1R2. In contrast, receptor activity toward the sweetener cyclamate and the sweet taste inhibitor lactisole depends on residues within the transmembrane domain of human T1R3. Furthermore, receptor activity toward the sweet protein brazzein depends on the cysteine rich domain of human T1R3. Although crystal structures are not available for the sweet taste receptor, useful homology models can be developed based on appropriate templates. The amino terminal domain, cysteine rich domain and transmembrane helix domain of T1R2 and T1R3 have been modeled based on the crystal structures of metabotropic glutamate receptor type 1, tumor necrosis factor receptor, and bovine rhodopsin, respectively. We have used homology models of the sweet taste receptors, molecular docking of sweet ligands to the receptors, and site-directed mutagenesis of the receptors to identify potential ligand binding sites of the sweet taste receptor. These studies have led to a better understanding of the structure and function of this heterodimeric receptor, and can act as a guide for rational structure-based design of novel non-caloric sweeteners, which can be used in the fighting against obesity and diabetes.

Amino Acid Sequence↗

PubMed2003

A homology model of penicillin acylase from Alcaligenes faecalis and in silico evaluation of its selectivity.

A three-dimensional model of the relatively unknown penicillin acylase from Alcaligenes faecalis (PA-AF) was built up by means of homology modeling based on three different crystal structures of penicillin acylase from various sources. An in silico selectivity study was performed to compare this homology model to the structure of the Escherichia coli enzyme (PA-EC) in order to find any selectivity differences between the two enzymes. The program GRID was applied in combination with the principal component analysis technique to identify the regions of the active sites where the PAs potentially engage different interactions with ligands. These differences were further analyzed and confirmed by molecular docking simulations. The PA-AF homology model provided the structural basis for the explanation of the different enantioselectivities of the enzymes previously demonstrated experimentally and reported in the literature. Different substrate selectivities were also predicted for PA-AF compared to PA-EC. Since no crystallographic data are available for PA-AF to date, the three-dimensional homology model represents a useful and efficient tool for fully exploiting this attractive and efficient biocatalyst, particularly in enantioselective acylations of amines.

Alcaligenes faecalis↗

PubMed2006

Synthesis of novel 2-(2'-cyclopentyl)- and 2-(2'-cyclohexyl) substituted 1-naphthol derivatives with anticyclooxygenase activity.

Eight novel 2-(2'-cyclopentyl)- and 2-(2'-cyclohexyl) substituted 1-naphthol derivatives were synthesized in good yield starting from 1-hydroxy-2-naphthoic acid. Two of them, 2-((1-(hydroxymethyl)cyclopentyl)methyl)naphthalene-1-ol (8) and 2-((1-(hydroxymethyl)cyclohexyl)methyl)-naphthalene-1-ol (9) showed anticyclooxygenase activity on COX-2 with IC(50) values of 19.90 microM and 7.77 microM, respectively and 9 also inhibited COX-1 (5.55 microM), while the other six were inactive on both isozymes. Molecular docking experiments indicated that the orientation of the active naphthols is different from that of the inactive ones. Two evidences playing important roles for the inhibition by the active compounds, are 1) C-1 and C-3' hydroxyl groups formed hydrogen bonds with COX-2/COX-1 Val523/Ile523 and Arg120, respectively, 2) hydrogen at C-5 of the naphthalene nucleus was attracted rather close to the phenolic group of Tyr385 due to van der Waals interaction.

Animals↗

PubMed2005

Humanization of an anti-human TNF-alpha antibody by variable region resurfacing with the aid of molecular modeling.

The murine monoclonal antibody Z12 is of therapeutic interest for its neutralizing biological activity against human tumor necrosis factor-alpha (hTNF-alpha). We attempted to humanize Z12 with variable domain resurfacing guided by computer modeling. First, the genes of heavy and light chain variable region (VH, VL) of Z12 were cloned and the whole three-dimensional structure of Fv fragment was constructed by using homology-based modeling and molecular docking methods. Then the complex model of Fv interacting with hTNF-alpha whose crystal structure derived from PDB database was gained with computer-guided docking program. Based on this model, a humanized version was designed. The humanized Fab antibody was constructed, expressed and purified in the pComb3H vector system and it showed unaltered binding affinity to the antigen as determined by ELISA and atomic force microscopy (AFM). The method described here can be used to humanize other anti-hTNF-alpha antibodies.

Amino Acid Sequence↗

PubMed2006

Homology-model-guided site-specific mutagenesis reveals the mechanisms of substrate binding and product-regulation of adenosine kinase from Leishmania donovani.

Despite designating catalytic roles of Asp299 and Arg131 during the transfer of gamma-phosphate from ATP to Ado (adenosine) [R. Datta, Das, Sen, Chakraborty, Adak, Mandal and A. K. Datta (2005) Biochem. J. 387, 591-600], the mechanisms that determine binding of substrate and cause product inhibition of adenosine kinase from Leishmania donovani remained unclear. In the present study, employing homology-model-guided site-specific protein mutagenesis, we show that Asp16 is indispensable, since its replacement with either valine or arginine resulted in a >200-fold increase in K(m) (Ado) with a 1000-fold decrease in k(cat)/K(m), implying its critical importance in Ado binding. Even glutamate replacement was not tolerated, indicating the essentiality of Asp16 in the maintenance of steric complementarity of the binding pocket. Use of 2'or 3'-deoxygenated Ado as substrates indicated that, although both the hydroxy groups play important roles in the formation of the enzyme-Ado complex, the binding energy (DeltaDeltaG(B)) contribution of the former was greater than the latter, suggesting possible formation of a bidentate hydrogen bond between Asp16 and the adenosyl ribose. Interestingly, AMP-inhibition and AMP-binding studies revealed that, unlike the R131A mutant, which showed abrogated AMP-binding and insensitivity towards AMP inhibition despite its unaltered K(m) (Ado), all the Asp16 mutants bound AMP efficiently and displayed AMP-sensitive catalytic activity, suggesting disparate mechanisms of binding of Ado and AMP. Molecular docking revealed that, although both Ado and AMP apparently occupied the same binding pocket, Ado binds in a manner that is subtly different from AMP binding, which relies heavily on hydrogen-bonding with Arg131 and thus creates an appropriate environment for competition with Ado. Hence, besides its role in catalysis, an additional novel function of the Arg131 residue as an effector of product-mediated enzyme regulation is proposed.

Adenosine Kinase↗

PubMedDate not supplied

[Optical biosensor study and molecular modeling of interactions between cytochrome P450cam and cytochrome b5].

The formation of complexes of cytochrome P450cam (P450cam) with full-length cytochrome b5 (d-b5) and its tryptic water-soluble fragment (t-b5) was analyzed using a two-channel IAsys+ optical biosensor. It was found that t-b5 can form complexes with P450cam, while d-b5 does not interact with P450cam. The involvement of amine groups of P450cam in the complex formation was demonstrated. The temperature dependence of t-b5(im)/P450cam complex formation was measured. The association rate constant (k(on)) increased with temperature, while the dissociation rate constant (k(off)) practically remained unchanged. It was concluded that hydrophobic interactions play a key role in the complex formation, while electrostatic interactions are significant for complex stabilization. Based on temperature dependence the activation energy, enthalpy and entropy of complex formation were calculated. It was shown that the entropy component plays a key role in t-b5(im)/P450cam interaction. Computer modeling of P450cam/t-b5 and P450cam/d-b5 interactions was carried out. Using the method of molecular docking some hypotheses of protein-protein complexes were advanced and the best ones were selected based on geometric complementarity, calculated binding energy and probability of electron tunneling between proteins. The computer modeling has shown that only P450cam and t-b5 can form the stable complex. These results are in good agreement with the experimental data obtained with the optical biosensor.

Amino Acid Sequence↗

PubMed2005

Beta-2-glycoprotein specificity of human anti-phospholipid antibody resides on the light chain: a novel mechanism for acquisition of cross-reactivity by an autoantibody.

We have recently shown that the anti-cardiolipin activity of human anti-phospholipid antibody UK4 (lambda) resides on its heavy chain. We now show that UK4 possesses strong reactivity to the plasma-protein beta2-Glycoprotein I (beta2-GPI) also. Utilizing chain shuffling experiments involving an unrelated anti-p185 antibody 4D5 (kappa) with no reactivity to beta2-GPI, we now demonstrate that both the constructs possessing the auto-antibody-derived light chain exhibited significant binding to beta2-GPI. However, the construct possessing UK4 heavy chain in association with 4D5 light chain, exhibited no anti-beta2-GPI activity. Furthermore, there was a low increase (approximately 10%) in the binding of UK4 to cardiolipin in the presence of beta2-GPI. The results demonstrate that anti-beta2-GPI activity resides on UK4 light chain and, importantly, this activity could be transferred to a novel antibody construct via the light chain alone. Computer-generated models of the three-dimensional structures of UK4 and its hybrids, suggest predominant interaction of UK4 light chain with domain IV of beta2-GPI. Molecular docking experiments highlight a number of potential sites on beta2-GPI for interaction of UK4 and indicate as to how beta2-GPI recognition may occur primarily via the autoantibody light chain. The study provides first demonstration of the occurrence of anti-phospholipid and anti-beta2-GPI activities separately on heavy and light chains of an autoantibody. The possible mechanisms that such antibodies may employ to recognise their antigens, are discussed.

Antibodies, Antiphospholipid↗

PubMed2004

Comparative structural analysis and kinetic properties of lactate dehydrogenases from the four species of human malarial parasites.

Parasite lactate dehydrogenase (pLDH) is a potential drug target for new antimalarials owing to parasite dependence on glycolysis for ATP production. The pLDH from all four species of human malarial parasites were cloned, expressed, and analyzed for structural and kinetic properties that might be exploited for drug development. pLDH from Plasmodium vivax, malariae, and ovale exhibit 90-92% identity to pLDH from Plasmodium falciparum. Catalytic residues are identical. Resides I250 and T246, conserved in most LDH, are replaced by proline in all pLDH. The pLDH contain the same five-amino acid insert (DKEWN) in the substrate specificity loops. Within the cofactor site, pLDH from P. falciparum and P. malariae are identical, while pLDH from P. vivax and P. ovale have one substitution. Homology modeling of pLDH from P. vivax, ovale, and malariae with the crystal structure of pLDH from P. falciparum gave nearly identical structures. Nevertheless, the kinetic properties and sensitivities to inhibitors targeted to the cofactor binding site differ significantly. Michaelis constants for pyruvate and lactate differ 8-9-fold; Michaelis constants for NADH, NAD(+), and the NAD(+) analogue 3-acetylpyridine adenine dinucleotide differ up to 4-fold. Dissociation constants for the inhibitors differ up to 21-fold. Molecular docking studies of the binding of the inhibitors to the cofactor sites of all four pLDH predict similar orientations, with the docked ligands positioned at the nicotinamide end of the cofactor site. pH studies indicate that inhibitor binding is independent of pH in the pH 6-8 range, suggesting that differences in dissociation constants for a specific inhibitor are not due to altered active site pK values among the four pLDH.

Amino Acid Sequence↗

PubMed2004

Homology modeling and molecular dynamics study of GSK3/SHAGGY-like kinase.

Although the GSK3/SHAGGY-like kinase is a highly conserved serine/threonine kinase implicated in many signaling pathways in eukaryotes, the lack of knowledge of its three-dimensional (3D) structure has hindered efforts to understand the binding specificities of substrate and catalytic mechanism. To understand the structure-activity relationships, the protein 3D structure was built by using homology modeling based on the known X-ray diffraction structure of Glycogen synthase kinase-3beta (Gsk3beta) and the model structure was further refined using unrestrained molecular dynamics simulations. The research indicates that the general 3D organization of the GSK3/SHAGGY-like kinase is a typical kinase family and comprises an N-terminal domain of beta-sheet and a larger C-terminal domain mainly constituted by alpha-helix. In order to understand the molecular interactions between the natural substrate-ATP and GSK3/SHAGGY-like kinase, a 3D model of the complex ATP-GSK3/SHAGGY-like kinase is developed by molecular docking program, which is helpful to guide the experimental realization and the new mutant designs as well. One important finding is that the identification of the key binding-site residue of Lys69 which plays an important role in the catalysis of GSK3/SHAGGY-like kinase and this is in consistent with experimental observation.

Adenosine Triphosphate↗

PubMed2002

Interaction of designed sulfated flavanoids with antithrombin: lessons on the design of organic activators.

Recently, we designed (-)-epicatechin sulfate (ECS), the first small nonsaccharide molecule, as an activator of antithrombin for the accelerated inhibition of factor Xa, a key proteinase of the coagulation cascade (Gunnarsson, G. T.; Desai, U. R. J. Med. Chem. 2002, 45, 1233-1243). Although sulfated flavanoid ECS was found to bind antithrombin with an affinity ( approximately 10.7 microM) comparable to the reference trisaccharide DEF ( approximately 4.5 microM), it accelerated the inhibition of factor Xa only 10-fold as compared to the approximately 300-fold observed with DEF. To determine whether this conformational activation of the inhibitor is dependent on the structure of the organic activator and to probe the basis for the deficiency in activation, we studied the interaction of similar sulfated flavanoids with antithrombin. (+)-Catechin sulfate (CS), a chiral stereoisomer of ECS, bound plasma antithrombin with a 3-fold higher affinity (K(D) = 3.5 microM) and a 2-fold higher second-order rate constant for factor Xa inhibition (k(ACT) = 6750 M(-1) s(-1)). On the contrary, the K(D) and k(ACT) were found to be lower approximately 7.4- and approximately 2.4-fold, respectively, for its racemic counterpart, (+/-)-catechin sulfate. Dependence of the equilibrium dissociation constant on the ionic strength of the medium at pH 6.0 and 7.4 suggests that nonionic interactions contribute a major proportion ( approximately 55-73%) of the total binding energy, and only 1-2 ion pairs, in comparison to the expected approximately 4 ion pairs for the reference trisaccharide, are formed in the interaction. Competitive binding experiments indicate that activator CS does not compete with a saccharide ligand that binds antithrombin in the pentasaccharide binding site, while it competes with full-length low-affinity heparin. A molecular docking study suggests plausible binding of CS in the extended heparin binding site, which is adjacent to the binding domain for the reference trisaccharide DEF. In combination, the results demonstrate that although conformational activation of antithrombin with small sulfated flavanoids is dependent on the structure of the activator, the designed activators do not bind in the pentasaccharide binding site in antithrombin resulting in weak activation. The mechanistic investigation highlights plausible directions to take in the rational design of specific high-affinity organic antithrombin activators.

Antithrombins↗

PubMed2004

Elucidation of the mechanism of inhibition of cyclooxygenases by acyl-coenzyme A and acylglucuronic conjugates of ketoprofen.

Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit the cyclooxygenase (COX) isoforms which accounts for their clinical effects. The differential inhibition of COX-1 and COX-2 is not sufficient to explain the absence of a correlation between in vitro and in vivo effects, especially for 2-aryl-propionates, thus indicating the participation of metabolites. Conjugates to glucuronic acid and to coenzyme-A are mainly produced, and have been shown to be chemically reactive. Therefore, we studied the interaction of the ketoprofen metabolites with the COX enzymes. After incubation with bovine pulmonary artery endothelial cells (BPAEC), COX-1 was inhibited stereoselectively by S-ketoprofen acylglucuronide, and more significantly by CoA-thioester. After washing-out the medium, COX-1 activity was essentially recovered, indicating a reversible inhibition. In LPS-stimulated J774.2 cells, COX activity (mainly inducible COX-2) was inhibited reversibly and stereospecifically by S-ketoprofen glucuronide, whereas it disappeared totally and was not recovered after incubation with CoA-thioester. Correspondingly, inhibition of purified COX-2 with this compound was observed to be rapid and irreversible. Using an anti-ketoprofen antibody, COX immunoprecipitated from cells exhibited adduct formation for COX-2 but not for COX-1. This was observed after incubation with CoA-thioester, and, surprisingly, also with glucuronide. Molecular docking gave support to explain this discrepancy: the glucuronide was found to establish a strong interaction with Y115 located in the membrane binding domain, whereas the thioester was preferentially bound to the active site of the enzyme. Overall, our results suggest a contribution of CoA-thioester metabolites of carboxylic NSAIDs to their pharmacological action by irreversibly and selectively inhibiting COX-2.

Acyl Coenzyme A↗

PubMed2004

Macrocyclic diacylglycerol-bis-lactones as conformationally constrained analogues of diacylglycerol-lactones. Interactions with protein kinase C.

A series of macrocyclic diacylglycerol (DAG)-bis-lactones were investigated as extreme conformationally constrained analogues of DAG-lactones in order to seek more potent protein kinase C (PKC) ligands with higher binding affinities and less lipophilicity than previous compounds. The additional constraint achieved the desired objective as exemplified by the macrocyclic DAG-bis-lactone 57, which exhibited a 6-fold higher binding affinity for PKCalpha (K(i) = 6.07 nM) than the corresponding nonmacrocyclic 3-alkylidene DAG-lactone 6. A structure-activity relationship (SAR) analysis of the macrocyclic DAG-bis-lactones demonstrated a parabolic relationship between activity and lipophilicity, as well as a predilection for the Z-alkylidene isomers as the preferred ligands. Molecular docking studies revealed that macrocyclic DAG-bis-lactone 57 bound to the C1b domain of PKCalpha exclusively in the sn-1 binding mode in contrast to DAG-lactone 6, which showed both sn-1 and sn-2 binding modes. It is proposed that the high potency displayed by these macrocyclic DAG-bis-lactones results from a set of more favorable hydrogen bonding and hydrophobic interactions with PKCalpha as well as from a reduced entropy penalty due to conformational restriction.

Diglycerides↗

PubMed2004

Proteome-wide identification of cellular targets affected by bisindolylmaleimide-type protein kinase C inhibitors.

Bisindolylmaleimide compounds such as GF109203X are potent inhibitors of protein kinase C (PKC) activity. Although bisindolylmaleimides are not entirely selective for PKC and are known to inhibit a few other protein kinases, these reagents have been extensively used to study the functional roles of PKC family enzymes in cellular signal transduction for more than a decade. Here, we establish a proteomics approach to gain further insights into the cellular effects of this compound class. Functional immobilization of suitable bisindolylmaleimide analogues in combination with the specific purification of cellular binding proteins by affinity chromatography led to the identification of several known and previously unknown enzyme targets. Subsequent in vitro binding and activity assays confirmed the protein kinases Ste20-related kinase and cyclin-dependent kinase 2 (CDK2) and the non-protein kinases adenosine kinase and quinone reductase type 2 as novel targets of bisindolylmaleimide inhibitors. As observed specifically for CDK2, minor chemical variation of the ligand by immobilizing the closely related bisindolylmaleimides III, VIII, and X dramatically affected target binding. These observed changes in affinity correlated with both the measured IC(50) values for in vitro CDK2 inhibition and results from molecular docking into the CDK2 crystal structure. Moreover, the conditions for affinity purification could be adapted in a way that immobilized bisindolylmaleimide III selectively interacted with either PKC alpha or ribosomal S6 protein kinase 1 only after activation of these kinases. Thus, we have established an efficient technique for the rapid identification of cellular bisindolylmaleimide targets and further demonstrate the comparative selectivity profiling of closely related kinase inhibitors within a cellular proteome.

Animals↗

PubMed2005

The study on the interaction between seryl-histidine dipeptide and proteins by circular dichroism and molecular modeling.

The selective cleavage of proteins is very important in key biological processes. Chemical (nonenzymatic) reagents such as cyanogen bromide and transition metal complexes are used extensively with great defects. In this paper, the binding of seryl-histidine dipeptide (abbreviated as SH) with bovine serum albumin (BSA) and lysozyme were investigated by the circular dichroism spectroscopy (CD) at 298K, molecular docking studies and quantum chemical calculations based on the previous results of polyacrylamide gel electrophoresis (PAGE). From the studies of CD, it showed that SH interacted strongly with BSA and lysozyme. The change percentages of the secondary structures of BSA and lysozyme were calculated. The contents of the beta-sheets decreased remarkably. It indicated that the interactions between SH and proteins could break the hydrogen bonds of beta-sheets selectively. The docking studies between SH and BSA showed that the position of the oxygen atom of the hydroxyl group of SH (O(12)) was in favor of a nucleophilic attack on carbon atom of the amide bond of a beta-sheet (C(34)) because the distance between O(12) and C(34) was 3.37A. Natural charges, natural atomic hybrid percentages and square sums of HOMO coefficients calculated by the NBO and population analysis at HF/6-31G* supported the suggested mechanism. And so SH may be an interesting agent for the therapeutic use.

Animals↗

PubMed2006

Simplified analogues of immucillin-G retain potent human purine nucleoside phosphorylase inhibitory activity.

A set of deazaguanine derivatives 1-3 targeting human purine nucleoside phosphorylase (hPNP) have been designed and synthesized. The new compounds are characterized by the presence of a structurally simplified "azasugar" motif to be more easily accessible by chemical synthesis than previous inhibitors. In the enzymatic assays, some of the new derivatives proved to be able to inhibit hPNP at low nanomolar concentration, thereby showing the same inhibitory potency in vitro as immucillin-H (IMH). Molecular docking experiments revealed a binding mode to hPNP essentially identical to that of IMH. As a result, the lower in vivo activity exhibited by 1d, compared with that exhibited by IMH, might be ascribed to differences in the pharmacokinetic, rather than pharmacodynamic, profile between these compounds. Derivatives 1a, 1d, and 2c emerged as the most active compounds within this new set and may represent interesting leads in the search for novel hPNP inhibitors.

Antineoplastic Agents↗

PubMed2006

Structural model of the BCL-w-BID peptide complex and its interactions with phospholipid micelles.

A peptide corresponding to the BH3 region of the proapoptotic protein, BID, could be bound in the cleft of the antiapoptotic protein, BCL-w. This binding induced major conformational rearrangements in both the peptide and protein components of the complex and led to the displacement and unfolding of the BCL-w C-terminal alpha-helix. The structure of BCL-w with a bound BID-BH3 peptide was determined using NMR spectroscopy and molecular docking. These studies confirmed that a region of 16 residues of the BID-BH3 peptide is responsible for its strong binding to BCL-w and BCL-x(L). The interactions of BCL-w and the BID-BH3 peptide complex with dodecylphosphocholine micelles were characterized and showed that the conformational change of BCL-w upon lipid binding occurred at the same time as the release and unfolding of the BH3 peptide.

Animals↗

PubMed2005

6-(N-benzoylamino)purine as a novel and potent inhibitor of xanthine oxidase: inhibition mechanism and molecular modeling studies.

The inhibition of xanthine oxidase (XO) activity by the purine analogue 6-(N-benzoylamino)purine was evaluated and compared with the standard inhibitor, allopurinol and the parent compound adenine. 6-(N-benzoylamino)purine is a highly potent inhibitor of XO (IC50 = 0.45 microM) and comparable to allopurinol (IC50 = 0.80 microM). Furthermore, 6-(N-benzoylamino)purine neither produced any enzymatic superoxide nor reduced XO by an electron transfer reaction unlike allopurinol. 6-(N-benzoylamino)purine (Ki = 0.0475 microM) is about 10000-fold more potent as a XO inhibitor compared to the only known purine analogue 8-bromoxanthine (Ki = 400 microM). 6-(N-Benzoylamino)purine is a competitive inhibitor of XO and the inhibition was not completely reversed even at 100 microM xanthine concentration. The calculated interaction energy [Ecomplex - (Eligand + Eprotein)] of -30.5, -22.6, and -17.2 kcal/mol, respectively, of 6-(N-benzoylamino)purine, 8-bromoxanthine and the parent compound adenine provided the rationale for the better enzyme inhibitory activity of 6-(N-benzoylamino)purine. To understand the role of the benzamido group in the inhibition process, molecular docking studies were carried out and it was revealed that the hydrogen bonding interactions involving N-7 of the purine ring and the N-H of Arg880, N-H of the purine ring and OH of Thr1010, as well as non-bonded interactions of the benzamido group of 6-(N-benzoylamino)purine with amino acid residues Gly799, Glu802, Phe914, Ala1078, Ala1079 and Glu1261 in the active site of XO play an important role in the stabilization of the E-I complex.

Adenine↗