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Candida C14-demethylase (P450DM) in ref. 8 is the only one fungal C14-demethylase whose amino acid sequence is known
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6) Candida C14-demethylase (P450DM) in ref. 8 is the only one fungal C14-demethylase whose amino acid sequence is known.
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9
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0010517370
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note
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7) The amino acid sequence of P450DM was taken from that reported by Lai et al (ref.8). The sequence and coordinates of P450CAM were obtained from Brookhaven Protein Databank (ID-code: 3CPP, ref. 9). The sequence alignment was initially done by the GCG program package(ref. 10). Then it was manually optimized with the reference of the sequence alignment by Morris et al (ref. 11). Two large insertions (1-80 and 390-442 colored in blue) were speculated to be membrane bound regions due to the hydrophobicity so that they were omitted to model. The location is, however, predicted to be close to each other on the surface of the modeled P450DM molecule. The homology modeling of P450DM was performed with the program MOLOC (ref. 12). The inhibitor docking with the P450DM model was simulated by the following steps. Firstly, we investigated the geometry of heme coordinating His residues among 20 heme proteins of which structures were well clarified by X-ray crystallography. The average distance between Fe(III) and a coordinating N atom of imidazole and the average angle of N atom (heme)-Fe(III)-N atom (imidazole) were 2.1 Å and 90°, respectively. They were used for placing azole ring of Ro09-2056 in the C-2 position. Next the lowest energy conformation from a stochastic conformational analysis of the inhibitor itself was manually placed in the active site without any significant repulsion for the P450DM model as a initial position. 100 conformations were generated by molecular dynamics (MD) calculation at 1000 K and consequence energy minimization. The energetically stable conformation was selected as a hypothetical bound conformation. MD calculation and generation of water accessible surfaces were performed by the INSIGHT II and DISCOVER software package (ref. 13). All computational work was done on Silicon Graphics Indigo2 workstation.
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10
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Genetic Computer Group Inc., Wisconsin, 1991
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10) Genetic Computer Group Inc., Wisconsin, 1991.
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Molecular Simulations Inc., San Diego, CA
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13) Insight II, Molecular Simulations Inc., San Diego, CA 1995.
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19
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0010440623
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All new compounds gave spectroscopic data in agreement with the assigned structures
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17) All new compounds gave spectroscopic data in agreement with the assigned structures.
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20
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0010490656
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Synthesis of compound 5: see ref. 5
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18) Synthesis of compound 5: see ref. 5)
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21
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0010445634
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note
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19) Antifungal activity of Ro09-2435 (C.albicans CY 3003) with 10% mouse serum; IC80=150μg/ml.
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