Mechanistic Insights into Voltage-Driven Biocatalysis of a Cytochrome P450 Bactosomal Film on a Self-Assembled Monolayer

Simple construction of biocatalytically active films of cytochrome P450 (CYP) bactosomes is quite useful for low-cost, stereoselective, and nicotinamide adenine dinucleotide phosphate hydride-free drug metabolism assays, biosensing, and biocatalytic applications. We report here real-time monitoring of the formation of biocatalytically active films of membrane-bound human CYP 2C9 or 3A4 expressed with CYP reductase (CPR) in <i>Escherichia coli</i> (so-called bactosomes) on a cysteamine self-assembled monolayer of gold-infused quartz crystals. The CYP 2C9+CPR-containing bactosomes exhibited oxygen reduction currents and metabolite yields greater than those of the CYP 3A4+CPR film. The electrocatalytic property correlated with the greater levels of CPR activity and the amount of CYP 2C9 in the CYP 2C9+CPR bactosomes than in the CYP 3A4+CPR bactosomes. The electron mediating role of CPR in the CYP 2C9 bactosomal film (<i>E</i>°′ = −450 mV vs Ag/AgCl) toward electrocatalytic oxygen reduction and hydroxylation of diclofenac was experimentally identified by comparing the film with bactosomes expressed with either CYP 2C9 (<i>E</i>°′ = −310 mV) or CPR (<i>E</i>°′ = −450 mV). The onset of oxygen reduction potentials correlated with the formal potentials of CYP and CYP+CPR films and revealed the electrocatalysis by CYP alone or in association with CPR. Furthermore, an ∼2-fold increase in the level of 4-hydroxydiclofenac product formation supported the favorable role of added catalase (hydrogen peroxide scavenger) in preventing damage by reactive oxygen species to the membrane-bound CYP or CYP+CPR bactosomes. The insignificant role of a peroxide shunt pathway for electrocatalysis in the case of the membrane-bound CYP film alone (unlike membrane-free isolated soluble CYP enzymes) and the electron mediation by CPR from the electrode to initiate CYP catalysis in the CYP+CPR bactosomes were discovered in this study. In conclusion, this report describes voltage-driven biocatalysis by bactosomal CYP films with new mechanistic insights into the formal potentials and electrocatalytic pathways of membrane-bound CYP films either alone or in association with CPR in the membrane

A Simple Construction of Electrochemical Liver Microsomal Bioreactor for Rapid Drug Metabolism and Inhibition Assays

In order to design a green microsomal bioreactor on suitably identified carbon electrodes, it is important to understand the direct electrochemical properties at the interfaces between various carbon electrode materials and human liver microsomes (HLM). The novelty of this work is on the investigation of directly adsorbed HLM on different carbon electrodes with the goal to develop a simple, rapid, and new bioanalytical platform of HLM useful for drug metabolism and inhibition assays. These novel biointerfaces are designed in this study by a one step adsorption of HLM directly onto polished basal plane pyrolytic graphite (BPG), edge plane pyrolytic graphite (EPG), glassy carbon (GC), or high-purity graphite (HPG) electrodes. The estimated direct electron transfer (ET) rate constant of HLM on the smooth GC surface was significantly greater than that of the other electrodes. On the other hand, the electroactive surface coverage and stability of microsomal films were greater on highly surface defective, rough EPG and HPG electrodes compared to the smooth GC and less defective hydrophobic BPG surfaces. The presence of significantly higher oxygen functionalities and flatness of the GC surface is attributed to favoring faster ET rates of the coated layer of thin HLM film compared to other electrodes. The cytochrome P450 (CYP)-specific bioactivity of the liver microsomal film on the catalytically superior, stable HPG surface was confirmed by monitoring the electrocatalytic conversion of testosterone to 6β-hydroxytestosterone and its inhibition by the CYP-specific ketoconazole inhibitor. The identification of optimal HPG and EPG electrodes to design biologically active interfaces with liver microsomes is suggested to have immense significance in the design of one-step, green bioreactors for stereoselective drug metabolite synthesis and drug metabolism and inhibition assays

Label-Free Real-Time Microarray Imaging of Cancer Protein–Protein Interactions and Their Inhibition by Small Molecules

A rapid optical microarray imaging approach for anticancer drug screening at specific cancer protein–protein interface targets with binding kinetics and validation by a mass sensor is reported for the first time. Surface plasmon resonance imager (SPRi) demonstrated a 3.5-fold greater specificity for interactions between murine double minute 2 protein (MDM2) and wild-type p53 over a nonspecific p53 mutant in a real-time microfluidic analysis. Significant percentage reflectivity changes (Δ%<i>R</i>) in the SPRi signals and molecular-level mass changes were detected for both the MDM2–p53 interaction and its inhibition by a small-molecule Nutlin-3 drug analogue known for its anticancer property. We additionally demonstrate that synthetic, inexpensive binding domains of interacting cancer proteins are sufficient to screen anticancer drugs by an array-based SPRi technique with excellent specificity and sensitivity. This imaging array, combined with a mass sensor, can be used to study quantitatively any protein–protein interaction and screen for small molecules with binding and potency evaluations