Cytochrome P450 BM3 (CYP102A1) mutant M11 is able to metabolize a wide range of drugs and drug-like compounds. Among these, M11 was recently found to be able to catalyze formation of human metabolites of mefenamic acid and other nonsteroidal anti-inflammatory drugs (NSAIDs). Interestingly, single active-site mutations such as V87I were reported to invert regioselectivity in NSAID hydroxylation. In this work, we combine crystallography and molecular simulation to study the effect of single mutations on binding and regioselective metabolism of mefenamic acid by M11 mutants. The heme domain of the protein mutant M11 was expressed, purified, and crystallized, and its X-ray structure was used as template for modeling. A multistep approach was used that combines molecular docking, molecular dynamics (MD) simulation, and binding free-energy calculations to address protein flexibility. In this way, preferred binding modes that are consistent with oxidation at the experimentally observed sites of metabolism (SOMs) were identified. Whereas docking could not be used to retrospectively predict experimental trends in regioselectivity, we were able to rank binding modes in line with the preferred SOMs of mefenamic acid by M11 and its mutants by including protein flexibility and dynamics in free-energy computation. In addition, we could obtain structural insights into the change in regioselectivity of mefenamic acid hydroxylation due to single active-site mutations. Our findings confirm that use of MD and binding free-energy calculation is useful for studying biocatalysis in those cases in which enzyme binding is a critical event in determining the selective metabolism of a substrate.

Ionotropic glutamate receptors (iGluRs) are responsible for most of the fast excitatory communication between neurons in our brain. The GluD2 receptor is a puzzling member of the iGluR family: It is involved in synaptic plasticity, plays a role in human diseases, e.g. ataxia, binds glycine and D-serine with low affinity, yet no ligand has been discovered so far that can activate its ion channel. In this study, we show that the hinge region connecting the two subdomains of the GluD2 ligand-binding domain is responsible for the low affinity of D-serine, by analysing GluD2 mutants with electrophysiology, isothermal titration calorimetry and molecular dynamics calculations. The hinge region is highly variable among iGluRs and fine-tunes gating activity, suggesting that in GluD2 this region has evolved to only respond to micromolar concentrations of D-serine.

Cytochrome P450 102A1 from Bacillus megaterium (BM3) is a fatty acid hydroxylase that has one of the highest turnover rates of any mono-oxygenase. Recent studies have shown how mutants of BM3 can produce metabolites of known drug compounds similar to those observed in humans. Single-point mutations in the binding pocket change the regioselective metabolism of fenamic acids from aromatic hydroxylation to aliphatic hydroxylation. This study is concerned with the individual contribution from accessibility and reactivity for drug metabolism with a future goal to develop fast methods for prediction. For a BM3 M11 mutant as well as the M11 V87F and M11 V87I mutants, we studied the metabolism of the nonsteroidal anti-inflammatory drugs (NSAIDs) mefenamic acid, meclofenamic acid, tolfenamic acid, and diclofenac. Density functional theory (DFT; B3LYP and B3LYP-D3) calculations for all possible reactions were performed using a porphyrin model reacting with the four substrates. Molecular dynamics (MD) simulations were used to determine the potential sites of metabolism that are accessible. Finally, we combine reactivity and accessibility for each potential site to interpret the experimentally determined metabolism. Generally, the 3 and 5 positions (on the ring containing the acidic substituent) and the 2′, 3′, and 4′ positions are most reactive, whereas 4, 5, 3′, and 4′ are most accessible. Combining reactivity and accessibility show that the 5, 3′, and 4′ positions are predicted to be most prone to be metabolized, in agreement with experimentally observed data. Reactivity seems to be the dominant factor in the CYP-mediated metabolism of these NSAIDs, which is consistent with previously published methods based solely on reactivity.

Many drug compounds are oxidized by cytochrome P450 (CYP) enzymes to form reactive metabolites. This study presents density functional theory calculations of the CYP-mediated metabolism of acetaminophen and a series of related compounds that can form reactive metabolites by hydrogen abstraction. The substitution pattern affects the activation barrier for hydrogen abstraction by up to 30 kJ/mol. A correlation (R(2) = 0.72) between the transition-state energies and the corresponding substrate radical energies has been established. Using this correlation is significantly less time-demanding than using the porphyrin model to determine the activation energies. We have used this correlation on monosubstituted phenols to rationalize the effect of the various substituents in the drug compounds. In addition to facilitating a chemical interpretation, the approach is sufficiently fast and reliable to be used as an in silico method in the design of new compounds with improved metabolic stability.

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