Picturing the Membrane‐assisted Choreography of Cytochrome P450 with Lipid Nanodiscs

Cytochrome P450, a family of monooxygenase enzymes, is organized as a catalytic metabolon, and requires enzymatic partners as well as environmental factors that tune its complex dynamic activity. P450 and its reducing counterparts are membrane‐bound proteins which are believed to dynamically interact to form functional complexes. Increasing experimental evidence signifies the role (s) of protein‐lipid interactions in P450’s catalytic function and efficiency. The challenges posed by the membrane have severely limited high‐resolution understanding of the molecular interfaces of these interactions. Nevertheless, recent NMR studies have provided piercing insights into the dynamic structural interactions that enable the function of P450. In this review, we will discuss different biomimetic approaches relevant to unveil molecular interplays at the membrane, focusing on our recent work on lipid‐nanodiscs. We also highlight the need to expand the use of nanodiscs, and the power of a combination of cutting‐edge solution and solid‐state NMR techniques, to study the dynamic structures of P450 as well as other membrane‐proteins.Cytochrome P450 is organized as a catalytic metabolon. Experimental evidence discloses the role (s) of protein‐lipid interactions in P450’s catalytic function. In this review, the authors discuss biomimetic approaches relevant to unveil molecular interplays at the membrane interface, focusing on their recent work on lipid‐nanodiscs, and highlight the need to expand the use of nanodiscs to other membrane‐protein research areas.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/146408/1/cphc201800444_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/146408/2/cphc201800444.pd

Cytochromeâ P450â Induced Ordering of Microsomal Membranes Modulates Affinity for Drugs

Although membrane environment is known to boost drug metabolism by mammalian cytochromeâ P450s, the factors that stabilize the structural folding and enhance protein function are unclear. In this study, we use peptideâ based lipid nanodiscs to â trapâ the lipid boundaries of microsomal cytochromeâ P450 2B4. We report the first evidence that CYP2B4 is able to induce the formation of raft domains in a biomimetic compound of the endoplasmic reticulum. NMR experiments were used to identify and quantitatively determine the lipids present in nanodiscs. A combination of biophysical experiments and molecular dynamics simulations revealed a sphingomyelin binding region in CYP2B4. The proteinâ induced lipid raft formation increased the thermal stability of P450 and dramatically altered ligand binding kinetics of the hydrophilic ligand BHT. These results unveil membrane/protein dynamics that contribute to the delicate mechanism of redox catalysis in lipid membrane.Redox catalysis in the lipid membrane: A novel application of peptide nanodiscs shows that cytochromeâ P450 2B4 is able to induce the formation of lipid raft domains in a biomimetic compound of the endoplasmic reticulum (ER). The proteinâ induced lipid rafts increase the thermal stability cytochromeâ P450 and dramatically alter the ligandâ binding kinetics of the hydrophilic ligand BHT.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/142960/1/anie201713167.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/142960/2/anie201713167_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/142960/3/anie201713167-sup-0001-misc_information.pd

Symposium Report The Role of Protein-Protein and Protein-Membrane Interactions on P450 Function

ABSTRACT This symposium summary, sponsored by the ASPET, was held at Experimental Biology 2015 on March 29, 2015, in Boston, Massachusetts. The symposium focused on: 1) the interactions of cytochrome P450s (P450s) with their redox partners; and 2) the role of the lipid membrane in their orientation and stabilization. Two presentations discussed the interactions of P450s with NADPH-P450 reductase (CPR) and cytochrome b 5 . First, solution nuclear magnetic resonance was used to compare the protein interactions that facilitated either the hydroxylase or lyase activities of CYP17A1. The lyase interaction was stimulated by the presence of b 5 and 17a-hydroxypregnenolone, whereas the hydroxylase reaction was predominant in the absence of b 5 . The role of b 5 was also shown in vivo by selective hepatic knockout of b 5 from mice expressing CYP3A4 and CYP2D6; the lack of b 5 caused a decrease in the clearance of several substrates. The role of the membrane on P450 orientation was examined using computational methods, showing that the proximal region of the P450 molecule faced the aqueous phase. The distal region, containing the substrate-access channel, was associated with the membrane. The interaction of NADPH-P450 reductase (CPR) with the membrane was also described, showing the ability of CPR to "helicopter" above the membrane. Finally, the endoplasmic reticulum (ER) was shown to be heterogeneous, having ordered membrane regions containing cholesterol and more disordered regions. Interestingly, two closely related P450s, CYP1A1 and CYP1A2, resided in different regions of the ER. The structural characteristics of their localization were examined. These studies emphasize the importance of P450 protein organization to their function

Prescription appropriateness of anti-diabetes drugs in elderly patients hospitalized in a clinical setting: evidence from the REPOSI Register

Diabetes is an increasing global health burden with the highest prevalence (24.0%) observed in elderly people. Older diabetic adults have a greater risk of hospitalization and several geriatric syndromes than older nondiabetic adults. For these conditions, special care is required in prescribing therapies including anti- diabetes drugs. Aim of this study was to evaluate the appropriateness and the adherence to safety recommendations in the prescriptions of glucose-lowering drugs in hospitalized elderly patients with diabetes. Data for this cross-sectional study were obtained from the REgistro POliterapie-Società Italiana Medicina Interna (REPOSI) that collected clinical information on patients aged ≥ 65 years acutely admitted to Italian internal medicine and geriatric non-intensive care units (ICU) from 2010 up to 2019. Prescription appropriateness was assessed according to the 2019 AGS Beers Criteria and anti-diabetes drug data sheets.Among 5349 patients, 1624 (30.3%) had diagnosis of type 2 diabetes. At admission, 37.7% of diabetic patients received treatment with metformin, 37.3% insulin therapy, 16.4% sulfonylureas, and 11.4% glinides. Surprisingly, only 3.1% of diabetic patients were treated with new classes of anti- diabetes drugs. According to prescription criteria, at admission 15.4% of patients treated with metformin and 2.6% with sulfonylureas received inappropriately these treatments. At discharge, the inappropriateness of metformin therapy decreased (10.2%, P < 0.0001). According to Beers criteria, the inappropriate prescriptions of sulfonylureas raised to 29% both at admission and at discharge. This study shows a poor adherence to current guidelines on diabetes management in hospitalized elderly people with a high prevalence of inappropriate use of sulfonylureas according to the Beers criteria

TIME-DEPENDENT INHIBITION OF CYTOCHROME P450: INTEGRATING ADVANCED BIOPHYSICAL MEASUREMENTS INTO PREDICTIVE ANALYSES

The implicit assumption made in extrapolating in vitro data to predict in vivo condition is that the qualitative behaviors of enzymes are for the most part independent of their external biochemical environments. However, cytochrome P450 superfamily (CYP), the enzyme responsible for the metabolism of drugs and xenobiotic, presents such a complex biochemical landscape that simplistic approaches usually lead to unreliable predictions. This is the case of time-dependent inhibition (TDI), which result in the inactivation of CYP over time, which is catalytically incompetent and needs to be replenished by newly synthetized protein. TDI kinetics are often characterized by mechanisms of metabolic intermediate complex (MIC) formation, shown for compounds containing amines or methylenedioxyphenyl (MDP) moieties.At present, in vitro TDI analyses lack the resolution for quantitative prediction of clinical outcomes. The central hypothesis of this proposal is that understanding the factors that are important in TDI will allow us to predict when TDI will have the potential for clinical adverse reactions. Chapter 1 of this dissertation will provide a brief introduction to the mechanistic landscape of MICs formation as well as the rationale behind the numerical approach that our group developed. In Chapter 2 the emission spectroscopy of Cytochrome P450 will be characterized as a novel tool to determine binding affinities of several drugs in solution and lipid environments. Chapter 3 will explore the application of our numerical strategy to the TDI of CYP3A4 as mediated by podophyllotoxin, a drug containing a MDP moiety. In Chapter 4 we will further test the reliability of our method studying the inhibition of CYP2D6 by paroxetine, a potent serotonin inhibitor. Finally, in Chapter 5 we discuss the potential future directions in the study of cytochrome P450 time-dependent inhibition

Oxidative Status of Ultra-Processed Foods in the Western Diet

Ultra-processed foods (UPFs) have gained substantial attention in the scientific community due to their surging consumption and potential health repercussions. In addition to their well-established poor nutritional profile, UPFs have been implicated in containing various dietary oxidized sterols (DOxSs). These DOxSs are associated with a spectrum of chronic diseases, including cardiometabolic conditions, cancer, diabetes, Parkinson’s, and Alzheimer’s disease. In this study, we present a comprehensive database documenting the presence of DOxSs and other dietary metabolites in >60 UPFs commonly consumed as part of the Western diet. Significant differences were found in DOxS and phytosterol content between ready-to-eat (RTE) and fast foods (FFs). Biomarker analysis revealed that DOxS accumulation, particularly 25-OH and triol, can potentially discriminate between RTEs and FFs. This work underscores the potential utility of dietary biomarkers in early disease detection and prevention. However, an essential next step is conducting exposure assessments to better comprehend the levels of DOxS exposure and their association with chronic diseases

Mechanism-Based Inhibition of CYP3A4 by Podophyllotoxin: Aging of an Intermediate Is Important for in Vitro/in Vivo Correlations

An in vitro observation of time-dependent inhibition (TDI) of metabolic enzymes often results in removing a potential drug from the drug pipeline. However, the accepted method for predicting TDIs of the important drug metabolizing cytochrome P450 enzymes often overestimates the drug interaction potential. Better models that take into account the complexities of the cytochrome P450 enzyme system will lead to better predictions. Herein we report the use of our previously described models for complex kinetics of podophyllotoxin. Spectral characterization of the kinetics indicates that an intermediate MI complex is formed, which slowly progresses to an essentially irreversible MI complex. The intermediate MI complex can release free enzyme during the time course of a typical 30 min TDI experiment. This slow rate of MI complex conversion results in an overprediction of the kinact value if this process is not included in the analysis of the activity versus time profile. In vitro kinetic experiments in rat liver microsomes predicted a lack of drug interaction between podophyllotoxin and midazolam. In vivo rat pharmacokinetic studies confirmed this lack of drug interaction