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PubMed2005

Metabolic regio- and stereoselectivity of cytochrome P450 2D6 towards 3,4-methylenedioxy-N-alkylamphetamines: in silico predictions and experimental validation.

A series of 3,4-methylenedioxy-N-alkylamphetamines (MDAAs) were automatically docked and subjected to molecular dynamics (MD) simulations in a cytochrome P450 2D6 (CYP2D6) protein model. The predicted substrate binding orientations, sites of oxidation, and relative reactivities were compared to the experimental data of wild-type and Phe120Ala mutant CYP2D6. Automated docking results were not sufficient to accurately rationalize experimental binding orientations of 3,4-methylenedioxy-N-methylamphetamine (MDMA) in the two enzymes as measured with spin lattice relaxation NMR. Nevertheless, the docking results could be used as starting structures for MD simulations. Predicted binding orientations of MDMA and sites of oxidation of the MDAAs derived from MD simulations matched well with the experimental data. It appeared the experimental results were best described in MD simulations considering the nitrogen atoms of the MDAAs in neutral form. Differences in regioselectivity and stereoselectivity in the oxidative metabolism of the MDAAs by the Phe120Ala mutant CYP2D6 were correctly predicted, and the effects of the Phe120Ala mutation could be rationalized as well.

Amphetamines↗

PubMed2001

Phalloidin synthetic analogues: structural requirements in the interaction with F-actin.

Synthetic derivatives of phalloidin have been investigated in solution by circular dichroism (CD) and NMR spectroscopy. They differ from natural phalloidin (PHD). bicyclo(Ala1-D-Thr2-Cys3-cis-4-hydroxy-Pro4-Ala5-2-mercapto-Trp6-(OH)2Leu7)(S-3 --> 6), in that they are modified at positions 2, 3, and 7. Among these synthetic analogues, structural differences and varying degrees of atropisomerism are found. By comparing the respective molecular models obtained by restrained molecular dynamics (RMD) simulations based on experimental NMR data, structural features that may be responsible for the different biological behavior become apparent. Our results indicate that the structural changes that result from an inversion of chirality of residue 3 lead to a complete loss of toxicity. Conversely, toxicity is less affected by the structural changes that stem from an inversion of chirality of residue 2. Moreover, unlike the other phallotoxins, when the thioether unit bridges to the opposite face of the main peptide ring, in contrast to the situation in other phallotoxins, large structural changes are observed as well as a total loss of activity. Molecular models of the synthetic phalloidin analogues have been used to investigate the necessary structural requirements for the interaction with F-actin. To this end, the F-actin/PHD model of M. Lorenz et al. was employed; docking experiments of our molecular models in the PHD binding site are presented.

Actins↗

PubMed2002

Transcytosis of plasma macromolecules in endothelial cells: a cell biological survey.

The modern exploration of endothelial cell biology is a largely interdisciplinary exercise. Cell biological, physiological, and more recently molecular biology approaches were used to study the pathways and the organelles involved in transcytosis of macromolecules in endothelial cell (EC). Here we discuss mainly the cell biological findings that revealed that EC have the attributes to fulfill the transport function. They are polarized cells, heterogeneous, and, thus, structurally and functionally adapted to the vascular bed in which they reside. The structural heterogeneity involves the number and distribution of plasmalemmal vesicles (caveolae), their generated channels, and the organization of intercellular junctions. The closely related functional heterogeneity comprises the degree of permeability for plasma molecules that vary as a function of organ. The EC are endowed with the cellular machinery to perform (1) endocytosis, that is to take up plasma proteins and the molecules they carry to be used for themselves (cholesterol-carrying low density lipoproteins, fatty acid carrying albumin, iron carrying transferrin, etc.), and (2) transcytosis, which implies to transport plasma proteins to the subjacent cells and tissues. The possible pathways for transport of molecules are transcellular, via caveolae and channels, and paracellular via intercellular junctions. Most of the results obtained, so far, indicate that transcytosis of albumin, low-density lipoproteins, metaloproteases, and insulin, is performed by cargo-vesicles and their generated channels. The paracellular pathway can be used for water and ions; in postcapillary venules, at the level of which approximately 30% of junctions are open to a space of 6 nm, small molecules may take this route. Recent data obtained by molecular biology techniques revealed that caveolae are endowed with the molecular machinery for fusion/fission, docking, and movement across cells. Moreover, the various and numerous molecules that have been detected in the caveolae membrane and the different functions assumed by this differentiated microdomain strengthen the postulate that there are at least two or more types of vesicles molecularly tailored for the local physiological requirements.

Animals↗

PubMed2005

Birch PR-10c interacts with several biologically important ligands.

PR-10c is a unique member of PR-10 proteins in birch, since it is the only one known to be post-translationally modified by glutathione and is not constitutively expressed in pollen. Both reduced and S-glutathiolated forms of PR-10c show low ribonuclease activity. However, the major function of the protein is apparently not yet resolved. Our protein-ligand interaction studies with saturation transfer difference (STD) NMR revealed that PR-10c interacts with several biologically important molecules, including cytokinin, flavonoid glycosides, sterols and emodin. Competition study with deoxycholate and kinetin revealed no statistically significant binding interference, indicating that these ligands have different binding sites in PR-10c. Ligand docking studies with a molecular model of PR-10c support the STD NMR results of ligand binding and binding epitopes, suggesting that there are three potential binding sites in PR-10c: two in the hydrophobic cavity and one in the glycine-rich loop. Our docking calculations suggested that only kinetin interacts with the glycine-rich loop, the binding occurring through its adenine moiety. Clear ligand specificity could be observed in the binding of nucleotide derivatives. S-glutathiolation of PR-10c did not affect kinetin binding. The present results suggest that birch PR-10c is a multifunctional protein, which has diverse roles in plant stress responses.

Betula↗

PubMed2004

Ligand-based structural hypotheses for virtual screening.

The majority of drug targets for small molecule therapeutics are proteins whose three-dimensional structure is not known to sufficient resolution to permit structure-based design. All three-dimensional QSAR approaches have a requirement for some hypothesis of ligand conformation and alignment, and predictions of molecular activity critically depend on this ligand-based binding site hypothesis. The molecular similarity function used in the Surflex docking system, coupled with quantitative pressure to minimize overall molecular volume, forms an effective objective function for generating hypotheses of bioactive conformations of sets of small molecules binding to their cognate proteins. Results are presented, assessing utility of the method for ligands of the serotonin, histamine, muscarinic, and GABA(A) receptors. The Surflex similarity module (Surflex-Sim) was able, in each case, to distinguish true ligands from random compounds using models constructed from just two or three known ligands. True positive rates of 60% were achieved with false positive rates of 0-3%; the theoretical enrichment rates were over 150-fold compared with random screening. The methods are practically applicable for rational design of ligands and for high-throughput virtual screening and offer competitive performance to many structure-based docking algorithms.

Algorithms↗

PubMed2002

Efficient docking of peptides to proteins without prior knowledge of the binding site.

Reliability in docking of ligand molecules to proteins or other targets is an important challenge for molecular modeling. Applications of the docking technique include not only prediction of the binding mode of novel drugs, but also other problems like the study of protein-protein interactions. Here we present a study on the reliability of the results obtained with the popular AutoDock program. We have performed systematical studies to test the ability of AutoDock to reproduce eight different protein/ligand complexes for which the structure was known, without prior knowledge of the binding site. More specifically, we look at factors influencing the accuracy of the final structure, such as the number of torsional degrees of freedom in the ligand. We conclude that the Autodock program package is able to select the correct complexes based on the energy without prior knowledge of the binding site. We named this application blind docking, as the docking algorithm is not able to "see" the binding site but can still find it. The success of blind docking represents an important finding in the era of structural genomics.

Algorithms↗

PubMed2006

Towards predictive ligand design with free-energy based computational methods?

The accurate prediction of ligand-biopolymer binding affinities is of general interest to medicinal chemistry, as well as to the broader field of molecular recognition. The ability to predict computationally the thermodynamics of these molecular recognition processes has been relatively weak until recently, however, continued developments on several fronts are extending the scope of applicability of these methods. The rapid growth in the number of protein-ligand structures has initially led to the development of a range of empirical scoring functions based on relatively simple descriptions of intermolecular interactions. These methods have had some success in ranking binding affinities when tuned to particular protein systems or in rather qualitative estimates of molecular fit in fast docking calculations. However, they are too unreliable for more detailed, quantitative, assessment and comparison of binding affinities. Physics-based free energy calculations are in principle more general and have the potential to be significantly more accurate. These approaches have seen steady development over many years and rely on carefully calibrated molecular energy functions (force-fields), simulations of the systems with explicit solvent, and the coming-of-age of continuum solvation models. In addition to the initially developped Free Energy Perturbation (FEP) and Thermodynamic Integration (TI) methods, new approaches include the Molecular Mechanics-Poisson-Boltzmann Surface Area (MM-PBSA) and the Linear Interaction Energy (LIE) approaches. This review concentrates on MM-PBSA and LIE, and their variants. The routine application of these calculations is becoming possible because of enhanced computational hardware and the development of a range of computational chemistry tools. This review addresses: i) the basic principles behind free energy calculations ii) recent methodological advances iii) comparisons of predicted and experimentally determined affinities iv) the uncertainties and limitations of both the computational and experimental data v) areas where progress can be made vi) the practicality of applying the methods at the different stages of the drug discovery and optimization process.

Animals↗

PubMed2004

A structural and dynamic model for the interaction of interleukin-8 and glycosaminoglycans: support from isothermal fluorescence titrations.

Binding of interleukin-8 (IL-8) to glycosaminoglycans (GAGs) on the surface of endothelial cells is crucial for the recruitment of neutrophils to an inflammatory site. Deriving structural knowledge about this interaction from in silico docking experiments has proved difficult because of the high flexibility and the size of GAGs. Therefore, we developed a docking method that takes into account ligand and protein flexibility by running approximately 15,000 molecular dynamics simulations of the docking event with different initial orientations of the binding partners. The method was shown to successfully reproduce the residues of basic fibroblast growth factor involved in GAG binding. Docking of a heparin hexasaccharide to IL-8 gave an interaction interface involving the basic residues His18, Lys20, Arg60, Lys64, Lys67, and Arg68. By subjecting IL-8 single-site mutants, in which these amino acids were replaced by alanine, to isothermal fluorescence titrations, the affinities for heparin were determined to be wtIL-8 > IL-8(H18A) >> IL-8(R68A) > IL-8(K67A) >> IL-8(K20A) > IL-8(R60A) >> IL-8(K64A). A comparison with the binding energies calculated from the model revealed high values for wtIL-8 and the H18A mutant and significantly lower but similar energies for the remaining mutants. Connecting the two fully sulfated hexasaccharides bound to each of the two IL-8 monomers in the dimeric chemokine by an N-acetylated dodecasaccharide gave a complex structure in which the GAG molecule aligned in a parallel fashion to the N-terminal alpha-helices of IL-8 like a horseshoe. A 5-ns molecular dynamics simulation of this complex confirmed its structural stability and revealed a reorientation in both binding sites where a disaccharide became the central binding unit. Isothermal fluorescence titration experiments using differently sulfated heparin disaccharides confirmed that a single disaccharide can indeed bind IL-8 with high affinity.

Amino Acid Substitution↗

PubMed2003

Identification of the binding sites and selectivity of sarpogrelate, a novel 5-HT2 antagonist, to human 5-HT2A, 5-HT2B and 5-HT2C receptor subtypes by molecular modeling.

The aim of the present study was to investigate the binding sites interactions and the selectivity of sarpogrelate to human 5-HT(2) receptor family (5-HT(2A), 5-HT(2B) and 5-HT(2C) receptor subtypes) using molecular modeling. Rhodopsin (RH) crystal structures were used as template to build structural models of the human serotonin-2A and -2C receptors (5-HT(2A)R, 5-HT(2C)R), whereas for 5-HT(2B)R, we used our previously published three-dimensional (3D) models based on bacteriorhodopsin (BR). Sarpogrelate, a novel 5-HT(2)R antagonist, was docked to the receptors. Molecular dynamics (MD) simulations produced the strongest interaction for 5-HT(2A)R/sarpogrelate complex. Upon binding, sarpogrelate constraints aromatic residues network (Trp(3.28), Phe(5.47), Trp(6.48), Phe(6.51), Phe(6.52) in 5-HT(2A)R; Phe(3.35), Phe(6.51), Trp(7.40) in 5-HT(2B)R; Trp(3.28), Phe(3.35), Phe(5.47), Trp(6.48), Phe(6.51), Phe(6.52) in 5-HT(2C)R) in a stacked configuration, preventing activation of the receptor. The models suggest that the structural origin of the selectivity of sarpogrelate to 5-HT(2A)R vs both 5-HT(2B)R and 5-HT(2C)R comes from the following results: (1) The tight interaction between the antagonist and the transmembrane domain (TMD) 3. Asp(3.32) neutralizes the cationic head and interacts simultaneously with carboxylic group hydrogen of the antagonist molecule. (2) Due to steric hindrance, Ser(5.46) (vs Ala(5.46) in 5HT(2B) and 5HT(2C)) prevents sarpogrelate to enter deeply inside the hydrophobic core of the helix bundle and to interact with Pro(5.50). (3) The side chain of Ile(4.56) (vs Ile(4.56) in 5HT(2B)R and Val(4.56) in 5HT(2C)R) constraints sarpogrelate to adjust its position by translating toward the strongly attractive Asp(3.32). These results are in good agreement with binding affinities (pKi) of sarpogrelate for 5-HT(2) receptor family expressed in transfected cell.

Amino Acid Sequence↗

PubMed2000

Homology model directed alignment selection for comparative molecular field analysis: application to photosystem II inhibitors.

The use of a computational docking protocol in conjunction with a protein homology model to derive molecular alignments for Comparative Molecular Field Analysis (CoMFA) was examined. In particular, the DOCK program and a model of the herbicidal target site, photosystem II (PSII), was used to derive alignments for two PSII inhibitor training sets, a set of benzo- and napthoquinones and a set of butenanilides. The protein design software in the QUANTA molecular modeling package was used to develop a homology model of spinach PSII based on the reported amino acid sequence and the X-ray crystal structure of the purple bacterium reaction center. The model is very similar to other reported PSII protein homology models. DOCK was then used to derive alignments for CoMFA modeling by docking the inhibitors in the PSII binding pocket. The molecular alignments produced from docking yielded highly predictive CoMFA models. As a comparison, the more traditional atom-atom alignments of the same two training sets failed to produce predictive CoMFA models. The general utilities of this application for homology model refinement and as an alternative scoring method are discussed.

Amino Acid Sequence↗

PubMed1997

A mechanism for toxin insertion into membranes is suggested by the crystal structure of the channel-forming domain of colicin E1.

BACKGROUND: Channel-forming colicins, including colicin E1, are a sub-family of bacteriocins. The toxic action of colicin E1 is derived from its ability to form a voltage-gated channel, which causes depolarization of the cytoplasmic membrane of sensitive Escherichia coli cells. In this process, the toxin-like colicin E1 molecule must undergo a substantial structural transition from a soluble state, in which it binds the target cell, to a membrane-bound state. Details of the structural changes that accompany this conversion may be directly applicable to other channel-forming toxins, as well as to the mechanism by which proteins insert into or cross membranes. RESULTS: The structure of the 190-residue channel-forming domain of colicin E1 in its soluble form has been solved at 2.5 A resolution. This structure contains 10alpha helices arranged in three layers (A-C) with a central hydrophobic helical hairpin in layer B, which is proposed to anchor the membrane-bound form in the bilayer. The extended N-terminal helix I provides a connection to the rest of the colicin E1 molecule, and the loop I-II may act as a hinge for re-orientation of the domain for membrane binding. A set of conserved positively charged residues on layer C may provide the docking surface on the molecule for membrane attachment. A large internal cavity between layers B and C may allow these layers to disengage, suggesting a mechanism for unfolding the molecule on the membrane that involves the perturbation of the interhelical hydrophobic interactions in layer C. CONCLUSION: On the basis of the structure of the colicin E1 channel-forming domain, its comparison with the structure of the colicin A domain and the known requirement for initial electrostatic and subsequent hydrophobic interactions, molecular details of the docking, unfolding and insertion of the channel-forming domain into the membrane are proposed. The model for docking and initial interaction with the membrane positions the hydrophobic hairpin 'anchor' approximately parallel to the membrane surface. Hydrophobic interactions in the docking layer may then be displaced by interactions with the membrane, spreading the helices on the surface and exposing the hydrophobic hairpin for insertion into the membrane.

Amino Acid Sequence↗

PubMedDate not supplied

Antitumor activity of imidazothioxanthones in murine and human tumor models in vitro and in vivo.

BACKGROUND: A new series of imidazothioxanthones has recently been synthesized as potential anticancer agents with the aim of overcoming drug resistance. The route of synthesis and DNA-binding properties of the compounds were reported previously. This paper describes the general structure-activity relationships for the class of imidazothioxanthones in panels of human and murine tumor cell lines in vitro, and the in vivo activity against human and murine solid tumors of the most potent compound, N-[3-(Dimethylamino)propylo]-11-oxo-11H-benzothiopyrano [3',2':2,3]pyrido[1,2-a]imidazo-2-carboxamide (10a). In addition, the interaction between compound 10a and DNA is also considered in terms of molecular mechanics methods and flexible docking techniques. MATERIALS AND METHODS: The cytotoxicity of compounds 10a, 11-oxo-N-[2-(pyrrolidino)ethylo]-11H-benzothiopyrano [3',2':2,3]pyrido[1,2-a]imidazo-2-carboxamide, 11-oxo-N-[2-(piperidino)ethylo]-11H-benzothiopyrano [3',2':2,3]pyrido[1,2-a]imidazo-2-carboxamide and N-[2-(morpholino)ethylo]-11-oxo-11H-benzothiopyrano [3',2':2,3]pyrido[1,2-a]imidazo-2-carboxamide (10c-10e) was assessed in human tumor cell lines and xenografts using the sulforhodamine B assay, MTT assay and the clonogenic assay. The human ovarian xenograft, PXN/109TC, two human breast carcinomas, MT-1 and MCF-7, and the murine colon adenocarcinoma, MAC15A were used for the in vivo testing of compound 10a. In addition, the interaction between compound 10a and DNA is also considered in terms of molecular mechanics methods and flexible docking techniques. RESULTS: Two compounds, 10a and 10c, showed cytotoxic activity below 10 mM in the NCI in vitro screen of 60 human tumor cell lines. The IC50 value of compound 10a was 6.8 mM and that of 10c, 8.3 mM. In addition, both compounds possessed differential activity against leukemia, colon and mammary cancer. The activity pattern was confirmed in two further screens using monolayer and clonogenic, assays. In vivo antitumor studies showed that 10a was active against the human mammary carcinoma MT-1 and murine colon cancer MAC15A. Marginal activity was observed in human ovarian cancer model PXN/109T/C and the compound was inactive in human mammary cancer MCF-7. CONCLUSION: The results warrant further in vivo testing of 10a in additional human solid tumor models. The molecular modeling showed that the planarity of the chromophore and the side-chain conformation could assist the insertion of compound 10a between the base pairs of the double helix. On the other hand, docking to the nucleotide sequence GGAATTGCCTCA suggested that the molecule could also act as a minor groove binder.

Adenocarcinoma↗

PubMed1998

A set of van der Waals and coulombic radii of protein atoms for molecular and solvent-accessible surface calculation, packing evaluation, and docking.

We analyze the contact distance distributions between nonbonded atoms in known protein structures. A complete set of van der Waals (VDW) radii for 24 protein atom types and for crystal-bound water is derived from the contact distance distributions of these atoms with a selected group of apolar atoms. In addition, a set of Coulombic radii for polar atoms is derived from their contacts with water. The contact distance distributions and the two sets of radii are derived in a systematic and self-consistent manner using an iterative procedure. The Coulombic radii for polar atoms are, on average, 0.18 A smaller than their VDW radii. The VDW radius of water is 1.7 A, which is 0.3 A larger than its Coulombic radius. We show that both the VDW and the Coulombic radii of polar atoms are needed in calculating the molecular and solvent-accessible surfaces of proteins. The VDW radii are needed to generate the apolar portions of the surface and the Coulombic radii for the polar portions. The fact that polar atoms have two apparent sizes implies that a hydrophobic cavity has to be larger than a polar cavity in order to accommodate the same number of water molecules. Most surface area calculations have used only one radius for each polar atom. As a result, unreal cavities, grooves, or pockets may be generated if the Coulombic radii of polar atoms are used. On the other hand, if the VDW radii of polar atoms are used, the details of the polar regions of the surface may be lost. The accuracy of the molecular and the solvent-accessible surfaces of proteins can be improved if the radii of polar atoms are allowed to change depending on the nature of their contacting neighbors. The surface of a protein at a protein-protein interface differs from that in solution in that it has to be generated using at least two kinds of probes, one representing a typical apolar atom and the other a typical polar atom. This observation has important implications for docking, which relies on surface complementarity at the interface.

Evaluation Studies as Topic↗

PubMed2005

Modelling the interaction of steroid receptors with endocrine disrupting chemicals.

BACKGROUND: The organic polychlorinated compounds like dichlorodiphenyltrichloroethane with its metabolites and polychlorinated biphenyls are a class of highly persistent environmental contaminants. They have been recognized to have detrimental health effects both on wildlife and humans acting as endocrine disrupters due to their ability of mimicking the action of the steroid hormones, and thus interfering with hormone response. There are several experimental evidences that they bind and activate human steroid receptors. However, despite the growing concern about the toxicological activity of endocrine disrupters, molecular data of the interaction of these compounds with biological targets are still lacking. RESULTS: We have used a flexible docking approach to characterize the molecular interaction of seven endocrine disrupting chemicals with estrogen, progesterone and androgen receptors in the ligand-binding domain. All ligands docked in the buried hydrophobic cavity corresponding to the hormone steroid pocket. The interaction was characterized by multiple hydrophobic contacts involving a different number of residues facing the binding pocket, depending on ligands orientation. The EDC ligands did not display a unique binding mode, probably due to their lipophilicity and flexibility, which conferred them a great adaptability into the hydrophobic and large binding pocket of steroid receptors. CONCLUSION: Our results are in agreement with toxicological data on binding and allow to describe a pattern of interactions for a group of ECD to steroid receptors suggesting the requirement of a hydrophobic cavity to accommodate these chlorine carrying compounds. Although the affinity is lower than for hormones, their action can be brought about by a possible synergistic effect.

Amino Acid Sequence↗

PubMed2003

Molecular evaluation using in silico protein interaction profiles.

MOTIVATION: To find a correlation between the activities and structures of molecules is one of the most important subjects for molecular evaluation study. Traditional quantitative structure-activity relationship (QSAR) methodologies represent those attempts using physicochemical descriptors. Creating a new molecular description factor based on the results of a computational docking study will add new dimensions to molecular evaluation. RESULTS: We propose a new molecular description factor analysis system called the Comparative Molecular Interaction Profile Analysis (CoMIPA) system in which the AutoDock program is used for docking evaluation of small molecule compound-protein complexes. Interaction energies are calculated, and the data sets obtained are called interaction profiles (IPFs). Using the IPF as a scoring indicator, the system could be a powerful tool to cluster the interacting properties between small molecules and bio macromolecules such as ligand-receptor bindings. Further development of the system will enable us to predict the adverse effects of a drug candidate.

Algorithms↗

PubMed2004

Evans Blue is an inhibitor of nuclear factor-kappa B (NF-kappaB)-DNA binding.

Nuclear factor-kappa B (NF-kappaB) is an important transcription factor, involved in many immune and inflammatory responses. It is critical in HIV gene expression as it has kappa B binding sites in the HIV-1 long-terminal repeat. Hence, targeting NF-kappaB to prevent its DNA binding holds a significant therapeutic potential. In this context, we report Evans Blue as a novel inhibitor of NF-kappaB-DNA binding. Evans Blue was found to be inhibiting DNA binding of NF-kappaB at a low concentration of 100 microM. Further, molecular modeling studies using docking and generation of electrostatic potential maps predicted a possible binding mode of EB to the DNA binding region of NF-kappaB, consistent with the experimental activity.

Cell Nucleus↗

PubMed2003

Purine and deazapurine nucleosides: synthetic approaches, molecular modelling and biological activity.

A number of ligands for the adenosine binding sites has been obtained by using nucleoside convergent and divergent synthesis. Most of our nucleosides have been synthesized by coupling 2,6-dichloropurine (1), 2,6-dichloro-1-deazapurine (2), 2,6-dichloro-3-deazapurine (3) with ribose, 2- and 3-deoxyribose and 2,3-dideoxyribose derivatives. The use of these versatile synthons allowed the introduction of various substituents in 2- and/or 6-positions. The glycosylation site and the anomeric configuration of the obtained nucleosides were assigned on the basis of spectroscopic studies and confirmed by molecular models. A series of potent adenosine receptor ligands has been obtained by using divergent approaches, mostly starting from guanosine. Substitutions in 2, 6, 8, and 5' position of adenosine molecule led to ligands selective for the different adenosine receptor subtypes. Furthermore, we investigated the molecular bases of the different behavior of 2- and 8-alkynyl adenosines, by means of NMR experiments and molecular modeling studies. With docking experiments, we demonstrated that the two class of molecules should have different binding modes that explain their different degree of affinity and the shift of their activity from agonistic (2-substituted derivatives) to antagonistic (8-substituted derivatives).

Animals↗

PubMed2006

Molecular basis of substrate recognition in D-3-hydroxybutyrate dehydrogenase from Pseudomonas putida.

D-3-Hydroxybutyrate dehydrogenase from Pseudomonas putida (EC 1.1.1.30) belongs to the family of short-chain dehydrogenases/reductases (SDRs). It catalyzes the reversible and stereospecific oxidation of D-3-hydroxybutyrate (D-3-HB) to acetoacetate with the aid of NAD(+) as coenzyme. This study contributes to understanding the mechanism and the high specificity of this enzyme towards its negatively charged and hydrophilic substrate. Sequence comparison of 44 bacterial HBDHs shows the residues Gln91, His141, Lys149, Lys192, and Gln193 to be strictly conserved. Site-directed mutagenesis of these amino acids to alanine and subsequent kinetic characterization of the mutated enzymes provides insight into the importance of these residues for substrate recognition and catalysis. Docking studies and molecular-dynamics simulations based on a three-dimensional structure model of a complex between P. putida HBDH and its coenzyme obtained by comparative molecular modeling were performed and provided deeper insight into the binding of the ligands at the molecular level. They show the residues Gln91, His141, Gln193, and, in particular, Lys149 to be involved in a hydrogen-bonding network with the carboxylate group of the substrate.

3-Hydroxybutyric Acid↗