Data independent acquisition for gas chromatographic MS/MS analysis of volatile compounds.

This study presents a novel tandem mass spectrometry (MS/MS) approach utilizing a data independent acquisition (DIA) concept specifically designed with gas chromatography-electron ionization-triple quadrupole mass spectrometry (GC-EI-QqQMS). This allows compound identification based on comparison between all the experimental MS/MS product ion spectra and the simulated library data of >1,000 MS/MS transitions of 71 compounds. The simulation data were generated by using the Competitive Fragmentation Modeling (CFM-ID) 3.0 program. The approach for calculation of the DIA MS/MS library match scores was then established and applied for identification of a range of terpenoids and oxygenated compounds in perfume. The identity of each peak was confirmed using 4-241 MS/MS transitions. The established data collection and analysis methods are expected to be useful for increased confidence in volatile compound analysis.

Rapid visual Candidatus Liberibacter asiaticus detection (citrus greening disease) using simple alkaline heat DNA lysis followed by loop-mediated isothermal amplification coupled hydroxynaphthol blue (AL-LAMP-HNB) for potential local use.

An outbreak of citrus greening or Huanglongbing disease bacteria occurs in many areas. We sampled and identified an ongoing ~year 2020 orange tree endemic in northern Thailand as Candidatus Liberibacter asiaticus. We thereby developed a plant greening disease (C. Liberibacter asiaticus) detection assay using simple alkaline heat DNA lysis and loop-mediated isothermal amplification coupled hydroxynaphthol blue (AL-LAMP-HNB), and evaluated the developed assay for its feasibility as point-of-care detection on 65 plant leaf samples with 100-1×104 copies of C. Liberibacter asiaticus or mocked injection compared with commercial DNA lysis kit and PCR-GE. Our assay is sensitive to 5-8.9 copies of omp (equaling 0.0056-0.01 fg) compatible with PCR-GE limit of detection. This ultra sensitive limit of detection could allow the disease detection before clinical apparent state of disease when C. Liberibacter asiaticus infection number is few, i.e. fewer than 100 copies of C. Liberibacter asiaticus. The assay is also specific with 6 degenerate primers targeting every strain of C. Liberibacter asiaticus omp from GenBank database, rapid (40 min total assay time), inexpensive (~2-3 USD/reaction), does not require sophisticated instrumentation, and has comparable assay accuracy (93.85-100% accuracy, 100% specificity, and 89.74-100% sensitivity) to bacterial DNA extraction by a commercial kit followed by PCR and gel electrophoresis (92.31% accuracy, 100% specificity, and 87.18% sensitivity) based on the real sample tests. Hence, the technique could be used in local or laboratory resource-restricted settings. The test result could be read by naked eyes through the color change from violet (negative) to sky blue (positive) for a C. Liberibacter asiaticus-infected specimen. Furthermore, this assay uses safe chemical reagents and, thus, is safe for the users.

Analysis of low-volatility pesticides in cabbage by high temperature comprehensive two-dimensional gas chromatography.

High-temperature comprehensive two-dimensional gas chromatography (HTGC × GC) using a longitudinally modulated cryogenic system (LMCS) was developed for the analysis of low-volatility pesticides in cabbage. The method applied DB-17HT and DB-5HT as the first and second dimensional (1D and 2D) columns, respectively. Twelve pesticides, namely 6 organochlorines (4,4'-DDT, ß-endosulfan, endosulfan sulfate, endrin, heptachlor, and dicofol), 4 carbamates (metolcarb, isoprocarb, methiocarb, and carbofuran), 1 organophosphate (chlorpyrifos), and 1 pyrethroid (permethrin), were spiked into cabbage samples and prepared using QuEChERS. The applied oven temperature was up to 340 °C, enabling the elution of all the target pesticides and the matrix. The effects of initial oven temperature program, temperature ramp rate, LMCS trap temperature, and modulation period (PM) on the separation results were investigated, leading to the suitable conditions of 80 °C, 15 °C min-1, 10 °C, and 12 s, respectively. The method detection limits, signal-to-noise ratio, and recoveries of the compounds were within the ranges of 0.01-0.09 mg kg-1, 4.26-32.7, and 78-104%, respectively. Good linearity ranges within the concentration range of 0.1-1 ppm with R2 > 0.9134 were also obtained with the intra and interday precisions of the peak areas of 0.4-9.8% and 1.0-10.2%, respectively.

Rapid colorimetric loop-mediated isothermal amplification for hypersensitive point-of-care Staphylococcus aureus enterotoxin A gene detection in milk and pork products.

Staphylococcus aureus strains carrying enterotoxin A gene (sea) causes food poisoning and cannot be distinguished from non-pathogenic strains by the culture method. Here, we developed a rapid, specific and sensitive visual detection of sea using loop-mediated isothermal amplification (LAMP) combined with nanogold probe (AuNP) or styryl dye (STR). LAMP-AuNP and LAMP-STR can detect as low as 9.7 fg (3.2 sea copies) and 7.2 sea copies, respectively, which were lower than PCR (97 fg or 32 sea copies). The excellent performance of these new assays was demonstrated in food samples using crude DNA lysates. While the culture method detected 104 CFU/g in ground pork and 10 CFU/mL in milk in 5-7 days, LAMP-AuNP could detect down to 10 CFU/g for both samples in 27 minutes. Analyzing 80 pork and milk samples revealed that the LAMP-AuNP showed 100% sensitivity, 97-100% specificity and 97.5-100% accuracy, which were superior to the culture method, and comparable to PCR but without requirement of a thermal cycler. Furthermore, our LAMP-AuNP detect sea at a range below the food safety control (<100 CFU/g). The LAMP-STR quantitated sea in 10-1,000 CFU (7.2-720 copies). Our crude DNA lysis combined with LAMP-AuNP/STR present effective point-of-care detection and facilitate appropriate control strategies.

Comparative studies on picosecond-resolved fluorescence of d-amino acid oxidases from human with one from porcine kidney. Photoinduced electron transfer from aromatic amino acids to the excited flavin.

Fluorescence dynamics of human d-amino acid oxidase (hDAAO) and its five inhibitors have been studied in the picoseconds time domain, and compared with one in d-amino acid oxidase from porcine kidney (pkDAAO) reported. The fluorescence lifetimes were identified as 47 ps in the dimer, 235 ps in the monomer, which are compared with those of pkDAAO (45 ps-185 ps). The fluorescence lifetimes of the hDAAO did not change upon the inhibitor bindings despite of modifications in the absorption spectra. This indicates that the lifetimes of the complexes are too short to detect with the picosecond lifetime instrument. Numbers of the aromatic amino acids are similar between the both DAAOs. The fluorescence lifetimes of hDAAO were analysed with an ET theory using the crystal structure. The difference in the lifetimes of the dimer and monomer was well described in terms of difference in the electron affinity of the excited isoalloxazine (Iso*) between the two forms of the protein, though it is not known whether the structure of the monomer is different from the dimer. Three fastest ET donors were Tyr314, Trp52 and Tyr224 in the dimer, while Tyr314, Tyr224 and Tyr55 in the monomer, which are compared to those in pkDAAO, Tyr314, Tyr224 and Tyr228 in the dimer, and Tyr224, Tyr314 and Tyr228 in the monomer. The ET rate from Trp55 in hDAAO dimer was much faster compared to the rate in pkDAAO dimer. A rise component with negative pre-exponential factor was not observed in hDAAO, which are found in pkDAAO.

Physical quantity of residue electrostatic energy in flavin mononucleotide binding protein dimer.

The electrostatic (ES) energy of each residue was for the first time quantitatively evaluated in a flavin mononucleotide binding protein (FBP). A residue electrostatic energy (RES) was obtained as the sum of the ES energies between atoms in each residue and all other atoms in the FBP dimer using atomic coordinates obtained by a molecular dynamics (MD) simulation. ES is one of the most important energies among the interaction energies in a protein. It is determined from the RES, the residues which mainly contribute to stabilize the structure of each subunit, and the binding energy between two subunits can be estimated. The RES of all residues in subunit A (Sub A) and subunit B (Sub B) were attractive forces, even though the residues contain net negative or positive charges. This reveals that the ES energies of any of the residues can contribute to stabilize the protein structure. The total binding ES energy over all residues among the subunits was distributed between -0.2 to -1.2 eV (mean = -0.67 eV) from the MD simulation time.

Dynamics of the protein structure of T169S pyranose 2-oxidase in solution: Molecular dynamics simulation.

Pyranose 2-oxidase (P2O) from Trametes multicolor contains FAD as cofactor, and forms a tetramer. The protein structure of a mutated P2O, T169S (Thr169 is replaced by Ser), in solution was studied by means of molecular dynamics simulation and analyses of photoinduced electron transfer (ET) from Trp168 to excited isoalloxazine (Iso*), and was compared with wild type (WT) P2O. Hydrogen bonding between Iso and nearby amino acids was very similar as between T169S and WT protein. Distances between Iso and Tyr456 were extremely heterogeneous among the subunits, 1.7 (1.5 in WT) in subunit A (Sub A), 0.97 (2.2 in WT) in Sub B, 1.3 (2.1 in WT) in Sub C, 1.3 nm (2.0 in WT) in Sub D. Mean values of root of mean square fluctuation over all residues were greater by four times than those in WT. This suggests that the protein structure of T169S is much more flexible than that of WT. Electrostatic (ES) energies between Iso anion in one subunit and ionic groups in the entire protein were evaluated. It was found that more than 50% of the total ES energy in each subunit is contributed from other subunits. Reported fluorescence decays were analyzed by a method as WT, previously reported. Electron affinities of Iso* in T169S were appreciably higher than those in WT. Static dielectric constants near Iso and Trp168 were also quite higher in T169S than those in WT.

Conformational difference between two subunits in flavin mononucleotide binding protein dimers from Desulfovibrio vulgaris (MF): molecular dynamics simulation.

The structural and dynamical properties of five FMN binding protein (FBP) dimers, WT (wild type), E13K (Glu13 replaced by Lys), E13R (Glu13 replaced by Arg), E13T (Glu13 replaced by Thr) and E13Q (Glu13 replaced by Gln), were investigated using a method of molecular dynamics simulation (MDS). In crystal structures, subunit A (Sub A) and subunit B (Sub B) were almost completely equivalent in all of the five FBP dimers. However, the predicted MDS structures of the two subunits were not equivalent in solution, revealed by the distances and inter-planar angles between isoalloxazine (Iso) and aromatic amino acids (Trp32, Tyr35 and Trp106) as well as the hydrogen bonding pairs between Iso and nearby amino acids. Residue root of mean square fluctuations (RMSF) also displayed considerable differences between Sub A and Sub B and in the five FBP dimers. The dynamics of the whole protein structures were examined with the distance (RNN) between the peptide N atom of the N terminal (Met1) and the peptide N atom of the C terminal (Leu122). Water molecules were rarely accessible to Iso in all FBP dimers which are in contrast with other flavoenzymes.

Classification of the mechanisms of photoinduced electron transfer from aromatic amino acids to the excited flavins in flavoproteins.

In many flavoproteins photoinduced electron transfer (ET) efficiently takes place from aromatic amino acids such as tryptophan or tyrosine to the excited isoalloxazine, so that the fluorescence lifetimes of isoalloxazine in some flavoproteins become ultrashort. The mechanism of ET in the flavoproteins was classified into four classes from the relationship between logarithmic ET rates (ln Rate) and the donor-acceptor distances (Rc), using reported data. The physical quantity, GT, is defined as the sum of solvent reorganization energy, electrostatic energy between a donor cation and an Iso anion, the standard free energy gap between the photoproducts and reactants, and net electrostatic energy between the photoproducts and other ionic groups in the flavoproteins (NetES). When GT fluctuates around zero with Rc, the ET rate becomes fastest (faster than 1 ps(-1)) in Kakitani and Mataga rates. In the ultrafast ET processes, the ln Rate becomes a parabolic function (category 1) of Rc as in FMN binding proteins and pyranose 2-oxidase at the shorter emission wavelengths, when NetES is negligible compared to the other quantities in the GT function. In the ultrafast ET processes, the ln Rate does not display any clear function of Rc (category 2) when NetES is dominant in the GT function, because of no direct relation between NetES and Rc. ET in flavodoxin from Helicobacter pylori may be classified into category 2. When GT linearly varies with Rc around a certain positive value, the ET rates become much slower (<1 ps(-1)). In this case the ln Rate linearly decreases with Rc (category 3), as Tyr224 in d-amino acid oxidase dimers. It is also conceivable that the ln Rate decreases with much scattered function of Rc (category 4), when NetES is dominant in the GT function, as Tyr314 in d-amino acid oxidase dimers. In ET processes of category 1, ET rates decrease as Rc becomes shorter than the distance at the maximum values of ln Rates, where GT is negative. Conditions and physical meanings were discussed for the GT-negative region.

Photoinduced electron transfer modeling to simulate flavoprotein fluorescence decay.

A method of analysis is described on the photoinduced electron transfer (PET) from aromatic amino acids as tryptophans (Trp) and tyrosines (Tyr) to the excited isoalloxazine (Iso*) in FMN-binding proteins (FBP) from Desulfovibrio vulgaris (strain, Miyazaki F). Time-dependent geometrical factors as the donor-acceptor distances are determined by means of a molecular dynamics simulation (MDS) of the proteins. Fluorescence decays of the single mutated isoforms of FBP are used as experimental data. The electrostatic (ES) energy between the photoproducts and ionic groups in the proteins is introduced into the Kakitani and Mataga (KM) model, which is modeled for an electron transfer process in solution. The PET parameters contained in the KM rate are determined by means of a nonlinear least square method, according to the Marquardt algorithm. The agreement between the observed and calculated decays is quite good, but not optimal. Characteristics on PET in flavoproteins, obtained by the present method, are described. Possible improvements of the method are discussed.

Non-equivalent conformations of D-amino acid oxidase dimer from porcine kidney between the two subunits. Molecular dynamics simulation and photoinduced electron transfer.

The structural difference between two subunits of D-amino acid oxidase dimer from porcine kidney was studied by molecular dynamics simulation (MDS) and rate of photoinduced electron transfer (ET) from aromatic amino acids as tyrosines (Tyr) and tryptophanes (Trp) to the excited isoalloxazine (Iso*). The donor-acceptor distances (Rc) between isoalloxazine (Iso) and the donors were shortest in Tyr224 (0.74 nm) in Sub A at 10 °C (Sub A10), in Tyr224 (0.79 nm) in Sub B at 10 °C (Sub B10), in Tyr228 (0.85 nm) in Sub A at 30 °C (Sub A30), and in Tyr224 (0.72 nm) in Sub B at 30 °C (Sub B30). The Rcs were mostly shorter in the dimer than those in the monomer. Hydrogen bonding (H-bond) pairs between Iso and surrounding amino acids varied with the subunit and temperature. O2 of the Iso ring formed an H-bond exclusively with Thr317OG1 (side chain) in both Sub A10 and Sub A30, while it formed with Gly315N (peptide), Leu316N and Thr317N in Sub B10 and Sub B30. N3H of Iso formed an H-bond with Leu51O (peptide) in Sub A10 and Sub A30, but not in Sub B10 and Sub B30. Electron affinity of Iso* was appreciably lower in Sub A10 compared to Sub B10, while it was opposite at 30 °C. ET rate to Iso* was fastest from Tyr224 in Sub A10, while it was fastest from Tyr314 in Sub B10. The ET rate was fastest from Tyr314 in Sub A30, while it was fastest from Tyr224 in Sub B30. The greater ET rates in the dimer as compared to those in the monomer were elucidated with shorter Rc in the dimer as compared to the monomer. The static dielectric constants inside the subunits and the static dielectric constant between Iso and Tyr224 or Tyr228 were not different appreciably. A few water molecules and sometimes an amino acid were located between Iso and Tyr224, which may be the reason why the dielectric constant of the entire subunits did not differ from that between Iso and Tyr224.

Binding mode prediction of biologically active compounds from plant Salvia Miltiorrhiza as integrase inhibitor.

Integrase (IN), an essential enzyme for HIV-1 replication, has been targeted in antiretroviral drug therapy. The emergence of HIV-1 variants clinically resistant to antiretroviral agents has lead to the development of alternative IN inhibitors. In the present work, binding modes of a high potent IN inhibitor, M522 and M532, within the catalytic binding site of wild type (WT) IN were determined using molecular docking calculation. Both M522 and M532 displayed similar modes of binding within the IN putative binding pocket and exhibited favorable interactions with the catalytic Mg(2+) ions, the nearby amino acids and viral DNA through metal-ligand chelation, hydrogen bonding and π-π stacking interactions. Furthermore, the modes of action of these two compounds against the mutated Y212R, N224H and S217H PFV IN were also predicted. Although the replacement of amino acid could somehow disturb inhibitor binding mode, almost key interactions which detected in the WT complexes were fairly conserved. Detailed information could highlight the application of M522 and M532 as candidate IN inhibitors for drug development against drug resistant strains.

9,10-Dioxoanthracene-1,4-diyl bis-(4-methyl-benzene-sulfonate).

The title mol-ecule, C(28)H(20)O(8)S(2), has a T-shaped conformation. The central 9,10-anthraquinone moiety is bow-shaped with the two outer aromatic rings being inclined to one another by 13.99 (11)°. The benzenesulfonate rings are inclined to one another by 47.35 (12)°, and by 34.51 (11) and 17.88 (11)° to the bridging aromatic ring of the 9,10-anthraquinone moiety. In the crystal, C-H⋯O interactions link the mol-ecules into ribbons in [100].

Structural basis for the temperature-induced transition of D-amino acid oxidase from pig kidney revealed by molecular dynamic simulation and photo-induced electron transfer.

The structural basis for the temperature-induced transition in the D-amino acid oxidase (DAAO) monomer from pig kidney was studied by means of molecular dynamic simulations (MDS). The center to center (Rc) distances between the isoalloxazine ring (Iso) and all aromatic amino acids (Trp and Tyr) were calculated at 10 °C and 30 °C. Rc was shortest in Tyr224 (0.82 and 0.88 nm at 10 and 30 °C, respectively), and then in Tyr228. Hydrogen bonding (H-bond) formed between the Iso N1 and Gly315 N (peptide), between the Iso N3H and Leu51 O (peptide) and between the Iso N5 and Ala49 N (peptide) at 10 °C, whilst no H-bond was formed at the Iso N1 and Iso N3H at 30 °C. The H-bond of Iso O4 with Leu51 N (peptide) at 10 °C switched to that with Ala49 N (peptide) at 30 °C. The reported fluorescence lifetimes (228 and 182 ps at 10 and 30 °C, respectively) of DAAO were analyzed with Kakitani and Mataga (KM) ET theory. The calculated fluorescence lifetimes displayed an excellent agreement with the observed lifetimes. The ET rate was fastest from Tyr224 to the excited Iso (Iso*) at 10 °C and from Tyr314 at 30 °C, despite the fact that the Rc was shortest between Iso and Tyr224 at both temperatures. This was explained by the electrostatic energy in the protein. The differences in the observed fluorescence lifetimes at 10 and 30 °C were ascribed to the differences in electron affinity of the Iso* at both temperatures, in which the free energies of the electron affinity of Iso* at 10 and 30 °C were -8.69 eV and -8.51 eV respectively. The other physical quantities related to ET did not differ appreciably at both temperatures. The electron affinities at both temperatures were calculated with a semi-empirical molecular orbital method (MO) of PM6. Mean calculated electron affinities over 100 snapshots with 0.1 ps intervals were -7.69 eV at 10 °C and -7.59 eV at 30 °C. The difference in the calculated electron affinities, -0.11 eV, was close to the observed difference in the free energies, -0.18 eV. The present quantitative analysis predicts that the highest ET rate can occur from a donor with longer donor-acceptor distance, which was explained by differences in electrostatic energy.

Simultaneous analyses of photoinduced electron transfer in the wild type and four single substitution isomers of the FMN binding protein from Desulfovibrio vulgaris, Miyazaki F.

The mechanism of photoinduced electron transfer (PET) from the aromatic amino acids (Trp32, Tyr35 and Trp106) to the excited flavin mononucleotide (FMN) in the wild type (WT) and four single amino acid substitution isomers (E13T, E13Q, W32A and W32Y) of FMN binding protein (FBP) from the Desulfovibrio vulgaris (Miyazaki F) were simultaneously analyzed (Method A) with the Marcus-Hush (MH) theory and Kakitani-Mataga (KM) theory using ultrafast fluorescence dynamics of these proteins. In addition, the PET mechanism of the WT, E13T and E13Q FBP systems (Method B) were also analyzed with both MH and KM theories. The KM theory could describe all of the experimental fluorescence decays better than the MH theory by both Methods A and B. The PET rates were found to largely depend on the electrostatic energies between photo-products, isoalloxazine (Iso) anion and the PET donor cations, and the other ionic groups, and hence on static dielectric constants. The dielectric constant (ε(0)(DA)) around the PET donors and acceptor was separately determined from those (ε(0)(j), j = WT, E13T, E13Q, W32Y and W32A) in the domain between the Iso anion or the donor cations and the other ionic groups in the proteins. The values of ε(0)(DA) were always lower than those of ε(0)(j), which is reasonable because no amino acid exists between the PET donors and acceptor in all systems. The values of the dielectric constants ε(0)(j) (j = WT, E13T and E13Q) were similar to those obtained previously from the analysis of the crystal structures and the average lifetimes of these FBP proteins. Energy gap law in the FBP systems was examined. An excellent parabolic function of the logarithms of the PET rates was obtained against the total free energy gap. The PET in these FBP isomers mostly took place in the so-called normal region, and partly in the inverted region.

Postprocessing of protein-ligand docking poses using linear response MM-PB/SA: application to Wee1 kinase inhibitors.

Prediction of the binding strength of untested ligands is a central issue in structure-based drug design. In order to rapidly screen large compound databases, simple scoring schemes are often used in target-based virtual screening. The resulting scores often correlate poorly with biological affinities. More rigorous scoring methods, such as MM-PB/SA, correlate better with biological data by considering solvation effects and protein flexibility in the calculation of the binding free energy of a ligand. Here we describe the performance of a modified MM-PB/SA method on 222 Wee1 kinase inhibitors (48 pyridopyrimidine and 174 pyrrolocarbazole derivatives). Docking of these inhibitors into the available Wee1 kinase crystal structure yielded a consistent binding mode, and the derived MM-PB/SA models showed a significant correlation between calculated and experimental data (r(2) values between 0.64 and 0.67). Further study of these models on external test sets of Wee1 kinase inhibitors and structurally related decoys showed that a model based on a single kinase-inhibitor conformation can discriminate the active inhibitors from decoys. We also tested whether the linear interaction energy method with continuum electrostatics (LIECE) yields comparable results to MM-PB/SA and whether the LIECE and MM-PB/SA models can be applied for virtual screening of compound libraries.

Homology modeling and molecular dynamics simulations of Dengue virus NS2B/NS3 protease: insight into molecular interaction.

The pathogenic West Nile virus (WNV) and Dengue virus (DV) are growing global threats for which there are no specific treatments. Both viruses possess a two component NS2B/NS3 protease which cleaves viral precursor proteins. Whereas for the WNV protease two crystal structures in complex with an inhibitor have been solved recently, no such information is available for the DV protease. Here, we report the generation of a homology model of DV NS2B/NS3 protease. Since it is known from the related WNV protease that it adopts a distinct conformation in free and in inhibitor-complexed form, a special emphasis was given to the analysis of the protease flexibility. Therefore, several models of DV NS2B/NS3 protease complexed with the peptidic inhibitor (Bz-Nle(P4)-Lys(P3)-Arg(P2)-Arg(P1)-H) were generated. The first DV protease model (DV-1) was constructed using the available crystal structure of the apo DV NS2B/NS3 protease. The second model (DV-2) was built taking the WNV NS3/NS2B protease in the inhibitor-complexed form as the template structure. Molecular dynamics simulations which were carried out for the WNV crystal structures as well as for the DV models provided an understanding of the role of NS2B for maintaining the protease in the active conformation. It was also demonstrated that NS2B is not only important for maintaining NS3 in the active form, but is also essential for establishing the interaction between residues from the S2 pocket and the peptidic inhibitor. The DV NS2B/NS3 model in the productive conformation can now be used for structure-based design purposes.

Receptor-based 3D-QSAR studies of checkpoint Wee1 kinase inhibitors.

One hundred and seventy-four pyrrolo[3,4-c]carbazole-1,3(2H,6H)-dione derivatives reported as inhibitors of the kinase Wee1 were used for a molecular docking and three-dimensional quantitative structure-activity relationship (3D-QSAR) study. Due to the availability of the three-dimensional structure of the Wee1 kinase a receptor-based alignment strategy was applied. Six available Wee1-inhibitor crystal structures were analyzed using the docking program GOLD resulting in a good reproduction of the experimentally derived position and interaction of the cocrystallized inhibitors. Since only a low correlation between docking scores and inhibitory activities was obtained for the series of 174 inhibitors a receptor-based 3D-QSAR study was performed, dividing the data set into 144 training set molecules and an external test set of 30 compounds. Besides the ligand alignment derived from the docking study we tested several other alignment procedures as basis for the 3D-QSAR analysis. The most predictive model was obtained using the alignment from the GOLD docking study. The CoMFA model was found to be robust (q(LOO)(2)=0.764 and r(2)=0.870). The predictive ability of the model was further examined by carrying out leave-20%-out and leave-50%-out cross-validation (q(2)=0.747 for leave-20%-out and 0.737 for leave-50%-out) and predicting the activities of 30 inhibitors used as external test set (r(pred)(2)=0.790). The graphical analysis of the CoMFA contour plot together with the key residues of the binding pocket provided important insight into the relevant interactions of the inhibitors. The results not only provide information about the essential features of potent Wee1 inhibitors but also show the advantage of using receptor-based alignment for 3D-QSAR analysis.

Quantum mechanical study of photoinduced charge transfer in FMN binding protein.

CT interactions between Iso* and nearby aromatic amino acids in FBP were investigated by a semiempirical MO method. Atomic coordinates of lumiflavin as Iso, 3-methylindole as Trp, and 4-methylphenol as Tyr, used for MO calculations, were obtained from crystal, 20 NMR structures and 40 MD structures (20 ps time intervals). Geometries of Iso-Trp32, Iso-Trp106 and Iso-Tyr35 systems were optimized by the PM3 method. The interaction energies (kcal/mol) of crystal structure were -16.9 in the Iso-Trp32 system, -7.4 in the Iso-Trp106 system and 1.4 in the Iso-Tyr35 system. The interaction energies (kcal/mol) of NMR structures were -16.5 +/- 0.28 in the Iso-Trp32 system, -10.6 +/- 0.14 in the Iso-Trp106 system, and 0.97 +/- 0.09 in the Iso-Tyr35 system. The interaction energies (kcal/mol) of MD structures were -24.3 +/- 0.19 in the Iso-Trp32 system, -10.2 +/- 0.49 in the Iso-Trp106 system, and 0.285 +/- 0.037 in the Iso-Tyr35 system. CT interaction from the aromatic amino acids to Iso* was judged from negative charge at Iso*. The charge in the Iso-Trp32 system was -0.490 in crystals, -0.439 +/- -0.099 in NMR structures, -0.454 +/- 0.048 in MD structures. The charge in the Iso and Trp106 system was -0.011 +/- 0.004 in MD structures, but negligible in other structures. CT interactions in Iso-Tyr35 system were also negligible. The ET rate obtained with Kakitani and Mataga theory and MD decreased as the magnitude of the interaction energy decreased. Correlation between the ET rate and CT interaction in FBP was examined. The interaction energy (Y) was approximated with ln(ET rate) (X) by a function, Y = 0.0036X(3) + 0.0306X(2) - 1.7822X - 21.177.