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PubMed2003

Tight binding of deoxyribonucleotide triphosphates to human thymidine kinase 2 expressed in Escherichia coli. Purification and partial characterization of its dimeric and tetrameric forms.

Human thymidine kinase 2 (hTK2) phosphorylates pyrimidine deoxyribonucleosides to the corresponding nucleoside monophosphates, using a nucleotide triphosphate as a phosphate donor. In this study, hTK2 was cloned and expressed at high levels in Escherichia coli as a fusion protein with maltose-binding protein. Induction of a heat-shock response by ethanol and coexpression of plasmid-encoded GroEL/ES chaperonins at 28 degrees C minimized the nonspecific aggregation of the hybrid protein and improved the recovery of three homooligomeric forms of the properly folded enzyme, i.e., dimer > tetramer > hexamer. The dimer and the tetramer were isolated in stable and highly purified forms after proteolytic removal of the fusion partner. Both oligomers contained a substoichiometric amount of deoxyribonucleotide triphosphates (dTTP > dCTP > dATP), known to be strong feedback inhibitors of the enzyme. Steady-state kinetic studies were consistent with the presence of endogenous inhibitors, and both oligomeric forms revealed a lag phase of at least approximately 5 min, which was abolished on preincubation with substrate (dThd or dCyd). The rather similar kinetic properties of the two oligomeric forms indicate that the basic functional unit is a dimer. Molecular docking experiments with a modeled hTK2 three-dimensional structure accurately predicted the binding positions at the active site of the natural substrates (dThd, dCyd, and ATP) and inhibitors (dTTP and dCTP), with highly conserved orientations obtained for all ligands. The calculated relative nonbonded interaction energies are in agreement with the biochemical data and show that the inhibitor complexes have lower stabilization energies (higher affinity) than the substrates.

Binding Sites↗

PubMed2005

Binding site of activators of the cystic fibrosis transmembrane conductance regulator in the nucleotide binding domains.

The use of substances that could activate the defective chloride channels of the mutant cystic fibrosis transmembrane conductance regulator (CFTR) has been suggested as possible therapy for cystic fibrosis. Using epithelia formed by cells stably transfected with wildtype or mutant (G551D, G1349D) CFTR, we estimated the apparent dissociation constant, K(D), of a series of CFTR activators by measuring the increase in the apical membrane current. Modification of apparent K(D) of CFTR activators by mutations of the nucleotide-binding domains (NBDs) suggests that the binding site might be in these regions. The human NBD structure was predicted by homology with murine NBD1. An NBD1-NBD2 complex was constructed by overlying monomers to a bacterial ABC transporter NBD dimer in the "head-to-tail" conformation. Binding sites for CFTR activators were predicted by molecular docking. Comparison of theoretical binding free energy estimated in the model to free energy estimated from the apparent dissociation constants, K(D), resulted in a remarkably good correlation coefficient for one of the putative binding sites, located in the interface between NBD1 and NBD2.

ATP-Binding Cassette Transporters↗

PubMed2005

Structural analysis of inhibition mechanisms of aurintricarboxylic acid on SARS-CoV polymerase and other proteins.

We recently published experimental results that indicated Aurintricarboxylic Acid (ATA) could selectively inhibit SARS-CoV replication inside host cells by greater than 1000 times. This inhibition suggested that ATA could be developed as potent anti-viral drug. Here, to extend our experimental observation, we have incorporated protein structural studies (with positive/negative controls) to investigate the potential binding modes/sites of ATA onto RNA-dependent RNA polymerase (RdRp) from SARS-CoV and other pathogenic positive-strand RNA-viruses, as well as other proteins in SARS-CoV based on the fact that ATA binds to Ca2+-activated neutral protease (m-calpain), the protein tyrosine phosphatase (PTP) and HIV integrase which have existing crystal structures. Eight regions with homologous 3D-conformation were derived for 10 proteins of interest. One of the region, Rbinding (754-766 in SARS-CoV's RdRp), located in the palm sub-domain mainly constituted of anti-parallel beta-strand-turn-beta-strand hairpin structures that covers two of the three RdRp catalytic sites (Asp 760, Asp761), was also predicted by molecular docking method (based on free energy of binding DeltaG) to be important binding motif recognized by ATA. The existence of this strictly conserved region that incorporated catalytic residues, coupled with the homologous ATA binding pockets and their consistent DeltaG values, suggested strongly ATA may be involved in an analogous inhibition mechanism of SARS-COV's RdRp in concomitant to the case in m-calpain, PTP and HIV integrase.

Amino Acid Sequence↗

PubMed2006

Structural basis of cell surface receptor recognition by botulinum neurotoxin B.

Botulinum neurotoxins (BoNTs) are potent bacterial toxins that cause paralysis at femtomolar concentrations by blocking neurotransmitter release. A 'double receptor' model has been proposed in which BoNTs recognize nerve terminals via interactions with both gangliosides and protein receptors that mediate their entry. Of seven BoNTs (subtypes A-G), the putative receptors for BoNT/A, BoNT/B and BoNT/G have been identified, but the molecular details that govern recognition remain undefined. Here we report the crystal structure of full-length BoNT/B in complex with the synaptotagmin II (Syt-II) recognition domain at 2.6 A resolution. The structure of the complex reveals that Syt-II forms a short helix that binds to a hydrophobic groove within the binding domain of BoNT/B. In addition, mutagenesis of amino acid residues within this interface on Syt-II affects binding of BoNT/B. Structural and sequence analysis reveals that this hydrophobic groove is conserved in the BoNT/G and BoNT/B subtypes, but varies in other clostridial neurotoxins. Furthermore, molecular docking studies using the ganglioside G(T1b) indicate that its binding site is more extensive than previously proposed and might form contacts with both BoNT/B and synaptotagmin. The results provide structural insights into how BoNTs recognize protein receptors and reveal a promising target for blocking toxin-receptor recognition.

Animals↗

PubMed2005

Predicting human serum albumin affinity of interleukin-8 (CXCL8) inhibitors by 3D-QSPR approach.

A novel class of 2-(R)-phenylpropionamides has been recently reported to inhibit in vitro and in vivo interleukin-8 (CXCL8)-induced biological activities. These CXCL8 inhibitors are derivatives of phenylpropionic nonsteroidal antiinflammatory drugs (NSAIDs), high-affinity ligands for site II of human serum albumin (HSA). Up to date, only a limited number of in silico models for the prediction of albumin protein binding are available. A three-dimensional quantitative structure-property relationship (3D-QSPR) approach was used to model the experimental affinity constant (K(i)) to plasma proteins of 37 structurally related molecules, using physicochemical and 3D-pharmacophoric descriptors. Molecular docking studies highlighted that training set molecules preferentially bind site II of HSA. The obtained model shows satisfactory statistical parameters both in fitting and predicting validation. External validation confirmed the statistical significance of the chemometric model, which is a powerful tool for the prediction of HSA binding in virtual libraries of structurally related compounds.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed2000

Structural basis of functional mimicry between carbohydrate and peptide ligands of con A.

Crystallographic studies have shown independent binding sites for sugar and peptide ligands of concanavalin A, although they were considered functional mimics based on biochemical experiments. The topological correlation of 12-residue peptide with different carbohydrate ligands of concanavalin A showed similarity between trimannose and the YPY region of the peptide establishing structural mimicry. Molecular docking of trimannose and the YPY motif on the reciprocal binding sites revealed equivalent interactions and energetics implying that the peptide-binding sites may constitute additional sugar-binding subsites of concanavalin A. The binding of a mannose-rich neoglycoprotein with significantly higher affinity compared with that of the methyl alpha-d-mannopyranoside is consistent with this interpretation.

Amino Acid Motifs↗

PubMed2005

Q-SiteFinder: an energy-based method for the prediction of protein-ligand binding sites.

MOTIVATION: Identifying the location of ligand binding sites on a protein is of fundamental importance for a range of applications including molecular docking, de novo drug design and structural identification and comparison of functional sites. Here, we describe a new method of ligand binding site prediction called Q-SiteFinder. It uses the interaction energy between the protein and a simple van der Waals probe to locate energetically favourable binding sites. Energetically favourable probe sites are clustered according to their spatial proximity and clusters are then ranked according to the sum of interaction energies for sites within each cluster. RESULTS: There is at least one successful prediction in the top three predicted sites in 90% of proteins tested when using Q-SiteFinder. This success rate is higher than that of a commonly used pocket detection algorithm (Pocket-Finder) which uses geometric criteria. Additionally, Q-SiteFinder is twice as effective as Pocket-Finder in generating predicted sites that map accurately onto ligand coordinates. It also generates predicted sites with the lowest average volumes of the methods examined in this study. Unlike pocket detection, the volumes of the predicted sites appear to show relatively low dependence on protein volume and are similar in volume to the ligands they contain. Restricting the size of the pocket is important for reducing the search space required for docking and de novo drug design or site comparison. The method can be applied in structural genomics studies where protein binding sites remain uncharacterized since the 86% success rate for unbound proteins appears to be only slightly lower than that of ligand-bound proteins. AVAILABILITY: Both Q-SiteFinder and Pocket-Finder have been made available online at http://www.bioinformatics.leeds.ac.uk/qsitefinder and http://www.bioinformatics.leeds.ac.uk/pocketfinder

Algorithms↗

PubMed2005

The furoxan system: design of selective nitric oxide (NO) donor inhibitors of COX-2 endowed with anti-aggregatory and vasodilating activities.

Several NO donor 3,4-diphenylfuroxan (= 3,4-diphenyl-1,2,5-oxadiazole 2-oxide) derivatives were synthesized and tested for their COX-inhibiting activities. The products were found to be selective COX-2 inhibitors, similar to the structurally related furazans (3,4-diphenyl-1,2,5-oxadiazole), devoid of the NO release property. This behavior was confirmed by a molecular-docking study. The NO-dependent platelet anti-aggregatory and vasodilating activities of the new furoxans 5-7 were studied in vitro. These properties can be modulated by inserting an appropriate spacer between the 4-phenyl group and the furoxan ring, giving rise to new, selective COX-2 furoxan derivatives endowed with anti-aggregatory and vasodilating activities, and with potentially reduced cardiotoxicities.

Celecoxib↗

PubMed2003

Synthesis and discovery of macrocyclic polyoxygenated bis-7-azaindolylmaleimides as a novel series of potent and highly selective glycogen synthase kinase-3beta inhibitors.

Attempts to design the macrocyclic maleimides as selective protein kinase C gamma inhibitors led to the unexpected discovery of a novel series of potent and highly selective glycogen synthase kinase-3beta (GSK-3beta) inhibitors. Palladium-catalyzed cross-coupling reactions were used to synthesize the key intermediates 17 and 22 that resulted in the synthesis of novel macrocycles. All three macrocyclic series (bisindolyl-, mixed 7-azaindoleindolyl-, and bis-7-azaindolylmaleimides) were found to have submicromolar inhibitory potency at GSK-3beta with various degrees of selectivity toward other protein kinases. To gain the inhibitory potency at GSK-3beta, the ring sizes of these macrocycles may play a major role. To achieve the selectivity at GSK-3beta, the additional nitrogen atoms in the indole rings may contribute to a significant degree. Overall, the bis-7-azaindolylmaleimides 28 and 29 exhibited little or no inhibitions to a panel of 50 protein kinases. Compound 29 almost behaved as a GSK-3beta specific inhibitor. Both 28 and 29 displayed good potency in GS cell-based assay. Molecular docking studies were conducted in an attempt to rationalize the GSK-3beta selectivity of azaindolylmaleimides.

Adipocytes↗

PubMed2004

The selection and design of GPCR ligands: from concept to the clinic.

Virtual screening methods using structure-based, pharmacophore-based and descriptor based protocols may be used to identify ligands for the G-protein coupled receptor target family. A complementary approach is the synthesis and screening of compound libraries designed using privileged motifs and/or based on validated hit molecules. A virtual screening approach based on molecular docking performed with GOLD using a templated homology model and a consensus scoring procedure can identify vasopressin 1a receptor antagonists. In a separate project a library design and synthesis approach based around validated hit GPCR ligands led to the identification of potent oxytocin antagonists. Subsequent optimisation of the initial library compounds has provided compounds that are now being evaluated in the clinic for the treatment of preterm labour.

Animals↗

PubMed2006

Rational design of a nonpeptide general chemical scaffold for reversible inhibition of PDZ domain interactions.

Novel small molecules were designed to specifically target the ligand-binding pocket of a PDZ domain. Iterative molecular docking and modeling allowed the design of an indole scaffold 10a as a reversible inhibitor of ligand binding. The 10a scaffold inhibited the interaction between MAGI-3 and PTEN and showed cellular activities that are consistent with the inhibition of NHERF-1 function.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Why is quinidine an inhibitor of cytochrome P450 2D6? The role of key active-site residues in quinidine binding.

We have previously shown that Phe(120), Glu(216), and Asp(301) in the active site of cytochrome P450 2D6 (CYP2D6) play a key role in substrate recognition by this important drug-metabolizing enzyme (Paine, M. J., McLaughlin, L. A., Flanagan, J. U., Kemp, C. A., Sutcliffe, M. J., Roberts, G. C., and Wolf, C. R. (2003) J. Biol. Chem. 278, 4021-4027 and Flanagan, J. U., Maréchal, J.-D., Ward, R., Kemp, C. A., McLaughlin, L. A., Sutcliffe, M. J., Roberts, G. C., Paine, M. J., and Wolf, C. R. (2004) Biochem. J. 380, 353-360). We have now examined the effect of mutations of these residues on interactions of the enzyme with the prototypical CYP2D6 inhibitor, quinidine. Abolition of the negative charge at either or both residues 216 and 301 decreased quinidine inhibition of bufuralol 1'-hydroxylation and dextromethorphan O-demethylation by at least 100-fold. The apparent dissociation constants (K(d)) for quinidine binding to the wild-type enzyme and the E216D and D301E mutants were 0.25-0.50 microm. The amide substitution of Glu(216) or Asp(301) resulted in 30-64-fold increases in the K(d) for quinidine. The double mutant E216Q/D301Q showed the largest decrease in quinidine affinity, with a K(d) of 65 microm. Alanine substitution of Phe(120), Phe(481),or Phe(483) had only a minor effect on the inhibition of bufuralol 1'-hydroxylation and dextromethorphan O-demethylation and on binding. In contrast to the wild-type enzyme, a number of the mutants studied were found to be able to metabolize quinidine. E216F produced O-demethylated quinidine, and F120A and E216Q/D301Q produced both O-demethylated quinidine and 3-hydroxyquinidine metabolites. Homology modeling and molecular docking were used to predict the modes of quinidine binding to the wild-type and mutant enzymes; these were able to rationalize the experimental observations.

Alanine↗

PubMed2005

Evolutionary trace analysis of eukaryotic DNA topoisomerase I superfamily: identification of novel antitumor drug binding site.

The studies of novel inhibitors of DNA topoisomerase I (Topo I) have already become very promising in cancer chemotherapy. Identifying the new drug-binding residues is playing an important role in the design and optimization of Topo I inhibitors. The designed compounds may have novel scaffolds, thus will be helpful to overcome the toxicities of current camptothecin (CPT) drugs and may provide a solution to cross resistance with these drugs. Multiple sequence alignments were performed on eukaryotic DNA topoisomerase I superfamily and thus the evolutionary tree was constructed. The Evolutionary Trace method was applied to identify functionally important residues of human Topo I. It has been demonstrated that class-specific hydrophobic residues Ala351, Met428, Pro431 are located around the 7,9-position of CPT, indicating suitable substitution of hydrophobic group on CPT will increase antitumor activity. The conservative residue Lys436 in the superfamily is of particular interest and new CPT derivatives designed based on this residue may greatly increase water solubility of such drugs. It has also been demonstrated that the residues Asn352 and Arg364 were conservative in the superfamily, whose mutation will render CPT resistance. As our molecular docking studies demonstrated they did not make any direct interaction with CPT, they are important drug-binding site residues for future design of novel non-camptothecin lead compounds. This work provided a strong basis for the design and synthesis of novel highly potent CPT derivatives and virtual screening for novel lead compounds.

Amino Acid Sequence↗

PubMed2005

New racemosol derivatives as potent cyclooxygenase (COX) inhibitors.

Racemosol (1) and 10-O-demethylracemosol (2), natural products from Bauhinia malabarica Roxb., exhibit potent in vitro anti-inflammatory activities against cyclooxygenase-1 and -2 (COX-1 and -2) enzymes. To investigate the structure-activity relationship (SAR) of these molecules, we prepared and fully characterized 17 derivatives by functionalizing one, two, or all three OH group(s) of 2 (Scheme). Both the size and polarity of the substituents as well as the substitution pattern in compounds 3a-q were found to be critical for anti-inflammatory activity. The orientation of the drugs and their mode of binding were studied by molecular docking based on the known 3D structure of the complex between COX-2 and the drug SC-558. Whereas the monoacetoxy derivative 3h exhibited an equally potent inhibitory activity towards both COX-1 and -2 (Table 1), its diacetoxy congener 3i was slightly more selective toward COX-2. In vivo anti-inflammatory tests showed that 3i and 2 are slightly more active than the reference compound phenylbutazone (Table 2).

Animals↗

PubMed2006

Design, synthesis, and antiproliferative and CDK2-cyclin a inhibitory activity of novel flavopiridol analogues.

The design and synthesis of a small library of 8-amidoflavone, 8-sulfonamidoflavone, 8-amido-7-hydroxyflavone, and heterocyclic analogues of flavopiridol is reported. The potential activity of these compounds as kinase inhibitors was evaluated by cytotoxicity studies in MCF-7 and ID-8 cancer cell lines and inhibition of CDK2-Cyclin A enzyme activity in vitro. The antiproliferative and CDK2-Cyclin A inhibitory activity of these analogues was significantly lower than the activity of flavopiridol. Molecular docking simulations were carried out and these studies suggested a different binding orientation inside the CDK2 binding pocket for these analogues compared to flavopiridol.

Adenosine Triphosphate↗

PubMed2006

Finding a needle in a haystack: development of a combinatorial virtual screening approach for identifying high specificity heparin/heparan sulfate sequence(s).

We describe a combinatorial virtual screening approach for predicting high specificity heparin/heparan sulfate sequences using the well-studied antithrombin-heparin interaction as a test case. Heparan sulfate hexasaccharides were simulated in the 'average backbone' conformation, wherein the inter-glycosidic bond angles were held constant at the mean of the known solution values, irrespective of their sequence. Molecular docking utilized GOLD with restrained inter-glycosidic torsions and intra-ring conformations, but flexible substituents at the 2-, 3-, and 6-positions and explicit incorporation of conformational variability of the iduronate residues. The approach reproduces the binding geometry of the sequence-specific heparin pentasaccharide to within 2.5 A. Screening of a combinatorial virtual library of 6,859 heparin hexasaccharides using a dual filter strategy, in which predicted antithrombin affinity was the first filter and self-consistency of docking was the second, resulted in only 10 sequences. Of these, nine were found to bind antithrombin in a manner identical to the natural pentasaccharide, while a novel hexasaccharide bound the inhibitor in a unique but dramatically different geometry and orientation. This work presents the first approach on combinatorial library screening for heparin/heparan sulfate GAGs to determine high specificity sequences and opens up huge opportunities to investigate numerous other physiologically relevant GAG-protein interactions.

Antithrombins↗

PubMed2005

Structural characterization of the fusion core in syncytin, envelope protein of human endogenous retrovirus family W.

Syncytin is a captive retroviral envelope protein, possibly involved in the formation of the placental syncytiotrophoblast layer generated by trophoblast cell fusion at the maternal-fetal interface. We found that syncytin and type I viral envelope proteins shared similar structural profiling, especially in the regions of N- and C-terminal heptad repeats (NHR and CHR). We expressed the predicted regions of NHR (41aa) and CHR (34aa) in syncytin as a native single chain (named 2-helix protein) to characterize it. 2-Helix protein exists as a trimer and is highly alpha-helix, thermo-stable, and denatured by low pH. NHR and CHR could form a protease-resistant complex. The complex structure built by the molecular docking demonstrated that NHR and CHR associated in an antiparallel manner. Overall, the 2-helix protein could form a thermo-stable coiled coil trimer. The fusion core structure of syncytin was first demonstrated in endogenous retrovirus. These results support the explanation how syncytin mediates cytotrophoblast cell fusion involved in placental morphogenesis.

Amino Acid Sequence↗

PubMed2006

Screening and chromatographic assessing of a novel IgG biomimetic ligand.

A novel biomimetic ligand, N-benzyloxycarbonyl-l-tyrosine (N-cbz-l-Tyr), was screened by a combination method of molecular docking and immobilized receptor technique. Then, N-cbz-l-Tyr was immobilized on Sepharose CL-4B to prepare a specific affinity adsorbent for immunoglobulin G (IgG). Scatchard analysis of the binding isotherm for IgG on the adsorbent gave an association constant (K(a)) of 4.91 x 10(6) m(-1) and a theoretical maximum adsorption capacity of 17.3 mg IgG/mL gel. IgG with a purity of 98% was separated from human plasma by this new affinity adsorbent.

Biomimetic Materials↗