Ensemble Modeling of Substrate Binding to Cytochromes P450:  Analysis of Catalytic Differences between CYP1A Orthologs^,

Abstract

A novel application of modeling and docking approaches involving ensembles of homology models is used to understand structural bases underlying subtle catalytic differences between related cytochromes P450 (CYPs). Mammalian CYP1A1s and fish CYP1As are orthologous enzymes with similar substrate preferences. With some substrates (3,3',4,4'-tetrachlorobiphenyl, TCB) oxidation rates differ by orders of magnitude, while others (e.g., benzo[a]pyrene; B[a]P) are oxidized at similar rates but with somewhat differing regiospecificity. These two environmental chemical substrates (TCB and B[a]P) as well as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were docked to multiple models of rat, human, scup, and/or killifish CYP1As, based on multiple templates, retaining multiple poses from each model, giving ensembles of docked poses for each species. With TCB, more poses were observed closer to the heme in ensembles of rat or human CYP1A1 than of killifish CYP1A. Analysis of interacting residues suggested that differences in TCB pose distributions are due primarily to Leu387 and Val230 in killifish CYP1A. In silico mutations L387V and V230G enabled TCB to dock closer to the heme in killifish CYP1A. Mutating additional interacting residues (Ala127, Thr233, Asn317, and Tyr386) of killifish CYP1A to the corresponding residues of human CYP1A1 resulted in TCB pose distributions nearly identical with those of human CYP1A1. Docking of TCDD to sets of consensus models of killifish, rat, and human CYP1As showed species differences similar to those with TCB, but with further structural constraints possibly contributing to slower oxidation of TCDD. Docking B[a]P to sets of consensus models of the human and fish CYP1As yielded frequencies of substrate orientations correlating with known regiospecificities for metabolism of B[a]P by these enzymes. The results demonstrate the utility of this ensemble modeling method, which can account for uncertainty inherent in homology modeling and docking by producing statistical distributions of ligand positions.

Can we use docking and scoring for hit-to-lead optimization?

“…Docking in extra precision mode takes about 10 times longer than in standard precision mode [19]. All our tests showed no correlation between the XP score and the experimental IC 50 . Figure 2 plots XP score vs. IC 50 for the KDR structure 1YWN.…”

“…Using a scaling factor of 1.0 for both protein and ligand was worse than using a scaling factor of 1.0 for the protein and 0.8 for ligands when testing Glide and Emodel scores. A scaling factor of 0.8 for protein atoms and 1.0 for ligands led to slightly worse results when using a 10 lM cutoff but better results when using a 10 nM cutoff in IC 50 . A scaling factor of 0.8 for protein atoms also reduced the number of compounds discarded by Glide filters and increased chances of retrieving more actives.…”

“…In the literature, it has been suggested that another possibility of improving the performance of scoring functions might be achieved by considering scoring of ensembles of the same compound instead of just one orientation for better rank ordering compounds [49,50]. Molecular mechanics Poisson-Bolzmann surface area (MM-PBSA) and molecular mechanics generalized Born surface area (MM-GBSA) methods have been used to estimate binding free energy in a reasonable time [51].…”

Backbone Flexibility Controls the Activity and Specificity of a Protein−Protein Interface: Specificity in Snake Venom Metalloproteases

“…57 This was shown for protein-ligand interactions, 58 protein-protein docking 59,60 and even protein design. 10,28,29,[61][62][63] But it is very difficult to access such ensembles by experimental methods.…”

EZR1: A Novel Family of Highly Expressed Retroelements Induced by TCDD and Regulated by a NF-κB-Like Factor in Embryos of Zebrafish (Danio rerio)

“…CYP1A is highly induced in TCDD-treated zebrafish embryos. 4 Access of planar halogenated aromatic hydrocarbons to the active site of CYP1As is restricted, 41 resulting in uncoupling of oxidative metabolism and production of reactive oxygen species (ROS). 42,43 Interaction of TCDD with the highly induced CYP1A in zebrafish embryos likely results in production of ROS, important effector molecules in the activation of NF-jB.…”