A virtual high throughput screen for high affinity cytochrome P450cam substrates. Implications for in silico prediction of drug metabolism

Structure-based virtual screening techniques require reliable scoring functions to discriminate potential substrates effectively. In this study we compared the performance of GOLD, PMF, DOCK and FlexX scoring functions in FlexX flexible docking to cytochrome P450cam binding site. Crystal structures of protein-substrate complexes were most effectively reproduced by the FlexX/PMF method. On the other hand, the FlexX/GOLD approach provided the best correlation between experimental binding constants and predicted scores. Binding modes selected by the FlexX/PMF approach were rescored by GOLD to obtain a reliable measure of binding energetics. The effectiveness of the FlexX/PMF/GOLD method was demonstrated by the correct classification of 32 out of the 33 experimentally studied compounds and also in a virtual HTS test on a library of 10,000 compounds. Although almost all the available functions were developed to be general, our study on cytochrome P450cam substrates suggests that careful selection or even tailoring the scoring function might increase the prediction power of virtual screens significantly. The FlexX/PMF/GOLD methodology was tested on cytochrome P450 3A4 substrates and inhibitors. This preliminary study revealed that the combined function was able to recognise 334 out of the 345 compounds bound to 3A4.     

Detection and Characterization of Boric Acid and Borate Ion Binding to Cytochrome c Using Multiple Quantum Filtered NMR

The application of multiple quantum filtered (MQF) NMR to the identification and characterization of the binding of ligands containing quadrupolar nuclei to proteins is demonstrated. Using relaxation times measured by MQF NMR multiple binding of boric acid and borate ion to ferri and ferrocytochrome c was detected. Borate ion was found to have two different binding sites. One of them was in slow exchange, k_diss = 20 ± 3 s⁻¹ at 5°C and D₂O solution, in agreement with previous findings by ¹H NMR (G. Taler et al., 1998, Inorg. Chim. Acta 273, 388–392). The triple quantum relaxation of the borate in this site was found to be governed by dipolar interaction corresponding to an average B–H distance of 2.06 ± 0.07 Å. Other, fast exchanging sites for borate and boric acid could be detected only by MQF NMR. The binding equilibrium constants at these sites at pH 9.7 were found to be 1800 ± 200 M⁻¹ and 2.6 ± 1.5 M⁻¹ for the borate ion and boric acid, respectively. Thus, detection of binding by MQF NMR proved to be sensitive to fast exchanging ligands as well as to very weak binding that could not be detected using conventional methods.     

固定化肌红蛋白的氧化还原反应和类酶活性

用魔芋多糖（KGM）将肌红蛋白（Mb）固定在玻碳电极（GC）表面，制备了Mb—KGM膜修饰电极．结果表明，包埋在KGM中的Mb可以与电极发生直接电子传递．Mb—KGM膜修饰电极在水／有机混合溶液中，表现出可逆的直接电子传递过程．式电势（E^0=-0．434V）表明该电化学响应是MbFe（Ⅲ）／Fe（Ⅱ）电对的氧化还原．在一定范围内，溶液pH值增加，Mb氧化还原式电势负移，且呈线性关系，说明Mb的电子传递过程伴随有质子的转移．在乙醇等亲水性有机溶剂与水的混合溶液中，Mb表现出类似细胞色素P450的催化活性，能快速地催化氯代乙烷（六氯乙烷、五氯乙烷和四氯乙烷等）脱氯．Mb—KGM膜修饰电极具有较好的稳定性和重现性，可用于这些物质的定量检测．     

微多相聚氨酯大孔树脂吸附细胞色素c的研究

微多相聚氨酯大孔树脂吸附细胞色素ｃ的研究史林启＊何炳林（南开大学吸附分离功能高分子材料国家重点实验室、高分子化学研究所天津３０００７１）关键词微多相聚氨脂大孔树脂，细胞色素ｃ，吸附选择性１９９６－０７－１５收稿，１９９６－１０－２４修回国家自然科学基...     

Whole‐Cell Biotransformation of Benzene to Phenol Catalysed by Intracellular Cytochrome P450BM3 Activated by External Additives

An Escherichia coli whole‐cell biocatalyst for the direct hydroxylation of benzene to phenol has been developed. By adding amino acid derivatives as decoy molecules to the culture medium, wild‐type cytochrome P450BM3 (P450BM3) expressed in E.coli can be activated and non‐native substrates hydroxylated, without supplementing with NADPH. The yield of phenol reached 59 % when N‐heptyl‐l ‐prolyl‐l ‐phenylalanine (C7‐Pro‐Phe) was employed as the decoy molecule. It was shown that decoy molecules, especially those lacking fluorination, reached the cytosol of E. coli, thus imparting in vivo catalytic activity for the oxyfunctionalisation of non‐native substrates to intracellular P450BM3.     

A Chiral Ligand Assembly That Confers One‐Electron O2 Reduction Activity for a Cu2+‐Selective Metallohydrogel

The design of functional metallohydrogels is attractive but challenging. A rational approach is introduced for designing functional metallohydrogels using chiral ligands, a phenylalanine derivative with a pyridyl group (l /d ‐PF). Intriguingly, the as‐prepared metallohydrogel exhibits excellent O₂ binding and activating properties. Insights into the O₂ binding pathway reveals the presence of a novel [(l +d )‐PF‐Cu³⁺‐O₂^?] species, which can efficiently reduce ferric cytochrome c with the reactive O₂^? by receiving an electron from reductant ascorbic acid. This study provides helpful instructions for developing new artificial systems with specific functions through the effective combination of chiral ligands with metal ions.     

In Vitro Reconstitution of OxyC Activity Enables Total Chemoenzymatic Syntheses of Vancomycin Aglycone Variants

The bioactivity of vancomycin is enabled by three aromatic crosslinks, the biosynthesis of which has been an active area of investigation for two decades. Two cytochrome P450 enzymes, OxyB and OxyA, have been shown to introduce bisaryl ether linkages with the help of a so‐called X‐domain. The final crosslink, however, a biaryl bond thought to be installed by OxyC, has remained elusive. We report the in vitro reconstitution of the OxyC reaction and formation of the first carbon–carbon crosslink in any glycopeptide antibiotic. Using a cascade sequence, in which the peptide substrate was incubated with the Oxy enzymes in turn, we completed the chemoenzymatic synthesis of a vancomycin aglycone variant. This approach was also used to generate a new analogue carrying a thioamide linkage at residue 4, a precursor to the amidine derivative, which is effective against vancomycin‐resistant pathogens. Our results set the stage for creating therapeutic vancomycin derivatives by using the native metalloenzymes.     

Automated docking of ligands to an artificial active site: augmenting crystallographic analysis with computer modeling

The W191G cavity of cytochrome c peroxidase is useful as a model system for introducing small molecule oxidation in an artificially created cavity. A set of small, cyclic, organic cations was previously shown to bind in the buried, solvent-filled pocket created by the W191G mutation. We docked these ligands and a set of non-binders in the W191G cavity using AutoDock 3.0. For the ligands, we compared docking predictions with experimentally determined binding energies and X-ray crystal structure complexes. For the ligands, predicted binding energies differed from measured values by +/- 0.8 kcal/mol. For most ligands, the docking simulation clearly predicted a single binding mode that matched the crystallographic binding mode within 1.0 A RMSD. For 2 ligands, where the docking procedure yielded an ambiguous result, solutions matching the crystallographic result could be obtained by including an additional crystallographically observed water molecule in the protein model. For the remaining 2 ligands, docking indicated multiple binding modes, consistent with the original electron density, suggesting disordered binding of these ligands. Visual inspection of the atomic affinity grid maps used in docking calculations revealed two patches of high affinity for hydrogen bond donating groups. Multiple solutions are predicted as these two sites compete for polar hydrogens in the ligand during the docking simulation. Ligands could be distinguished, to some extent, from non-binders using a combination of two trends: predicted binding energy and level of clustering. In summary, AutoDock 3.0 appears to be useful in predicting key structural and energetic features of ligand binding in the W191G cavity.