Cytochrome P450 monooxygenase systems: Diversity and plasticity for adaptive stress response.

Superfamily of cytochromes P450 (CYPs) is composed of heme-thiolate-containing monooxygenase enzymes, which play crucial roles in the biosynthesis, bioactivation, and detoxification of a variety of organic compounds, both endogenic and exogenic. Majority of CYP monooxygenase systems are multi-component and contain various redox partners, cofactors and auxiliary proteins, which contribute to their diversity in both prokaryotes and eukaryotes. Recent progress in bioinformatics and computational biology approaches make it possible to undertake whole-genome and phylogenetic analyses of CYPomes of a variety of organisms. Considerable variations in sequences within and between CYP families and high similarity in secondary and tertiary structures between all CYPs along with dramatic conformational changes in secondary structure elements of a substrate binding site during catalysis have been reported. This provides structural plasticity and substrate promiscuity, which underlie functional diversity of CYPs. Gene duplication and mutation events underlie CYP evolutionary diversity and emergence of novel selectable functions, which provide the involvement of CYPs in high adaptability to changing environmental conditions and dietary restrictions. In our review, we discuss the recent advancements and challenges in the elucidating the evolutionary origin and mechanisms underlying the CYP monooxygenase system diversity and plasticity. Our review is in the view of hypothesis that diversity of CYP monooxygenase systems is translated into the broad metabolic profiles, and this has been acquired during the long evolutionary time to provide structural plasticity leading to high adaptative capabilities to environmental stress conditions.

Characterization of a cytochrome P450 that catalyzes the O-demethylation of lignin-derived benzoates.

Cytochromes P450 (P450s) are a superfamily of heme-containing enzymes possessing a broad range of monooxygenase activities. One such activity is O-demethylation, an essential and rate-determining step in emerging strategies to valorize lignin that employ carbon-carbon bond cleavage. We recently identified PbdA, a P450 from Rhodococcus jostii RHA1, and PbdB, its cognate reductase, which catalyze the O-demethylation of para-methoxylated benzoates (p-MBAs) to initiate growth of RHA1 on these compounds. PbdA had the highest affinity (Kd = 3.8 ± 0.6 µM) and apparent specificity (kcat/KM = 20 000 ± 3 000 M-1 s-1) for p-MBA. The enzyme also O-demethylated two related lignin-derived aromatic compounds with remarkable efficiency: veratrate and isovanillate. PbdA also catalyzed the hydroxylation and dehydrogenation of p-EB even though RHA1 did not grow on this compound. Atomic-resolution structures of PbdA in complex with p-MBA, p-EB and veratrate revealed a cluster of three residues that form hydrogen bonds with the substrates' carboxylate: Ser87, Ser237 and Arg84. Substitution of these residues resulted in lower affinity and O-demethylation activity on p-MBA as well as increased affinity for the acetyl analogue, p-methoxyacetophenone. The S87A and S237A variants of PbdA also catalyzed the O-demethylation of an aldehyde analogue of p-MBA, p-methoxy-benzaldehyde, while the R84M variant did not, despite binding this compound with high affinity. These results suggest that Ser87, Ser237 and Arg84 are not only important determinants of specificity but also help to orientate that substrate correctly in the active site. This study facilitates the design of biocatalysts for lignin valorization.

Comparative Genomics Uncovers the Evolutionary Dynamics of Detoxification and Insecticide Target Genes Across 11 Phlebotomine Sand Flies.

Sand flies infect more than 1 million people annually with Leishmania parasites and other bacterial and viral pathogens. Progress in understanding sand fly adaptations to xenobiotics has been hampered by the limited availability of genomic resources. To address this gap, we sequenced, assembled, and annotated the transcriptomes of 11 phlebotomine sand fly species. Subsequently, we leveraged these genomic resources to generate novel evolutionary insights pertaining to their adaptations to xenobiotics, including those contributing to insecticide resistance. Specifically, we annotated over 2,700 sand fly detoxification genes and conducted large-scale phylogenetic comparisons to uncover the evolutionary dynamics of the five major detoxification gene families: cytochrome P450s (CYPs), glutathione-S-transferases (GSTs), UDP-glycosyltransferases (UGTs), carboxyl/cholinesterases (CCEs), and ATP-binding cassette (ABC) transporters. Using this comparative approach, we show that sand flies have evolved diverse CYP and GST gene repertoires, with notable lineage-specific expansions in gene groups evolutionarily related to known xenobiotic metabolizers. Furthermore, we show that sand flies have conserved orthologs of (i) CYP4G genes involved in cuticular hydrocarbon biosynthesis, (ii) ABCB genes involved in xenobiotic toxicity, and (iii) two primary insecticide targets, acetylcholinesterase-1 (Ace1) and voltage gated sodium channel (VGSC). The biological insights and genomic resources produced in this study provide a foundation for generating and testing hypotheses regarding the molecular mechanisms underlying sand fly adaptations to xenobiotics.

Understanding Cytochrome P450 Enzyme Substrate Inhibition and Prospects for Elimination Strategies.

Cytochrome P450 (CYP450) enzymes, which are widely distributed and pivotal in various biochemical reactions, catalyze diverse processes such as hydroxylation, epoxidation, dehydrogenation, dealkylation, nitrification, and bond formation. These enzymes have been applied in drug metabolism, antibiotic production, bioremediation, and fine chemical synthesis. Recent research revealed that CYP450 catalytic kinetics deviated from the classic Michaelis-Menten model. A notable substrate inhibition phenomenon that affects the catalytic efficiency of CYP450 at high substrate concentrations was identified. However, the substrate inhibition of various reactions catalyzed by CYP450 enzymes have not been comprehensively reviewed. This review describes CYP450 substrate inhibition examples and atypical Michaelis-Menten kinetic models, and provides insight into mechanisms of these enzymes. We also reviewed 3D structure and dynamics of CYP450 with substrate binding. Outline methods for alleviating substrate inhibition in CYP450 and other enzymes, including traditional fermentation approaches and protein engineering modifications. The comprehensive analysis presented in this study lays the foundation for enhancing the catalytic efficiency of CYP450 by deregulating substrate inhibition.

The tyrosine kinase inhibitor lenvatinib is oxidized by rat cytochromes P450 and affects their expression in rat liver.

Lenvatinib is an orally effective tyrosine kinase inhibitor used to treat several types of tumors, including progressive, radioiodine-refractory differentiated thyroid cancer and advanced renal cell carcinoma. Although this drug is increasingly used in therapy, its metabolism and effects on the organism are still not described in detail. Using the rat as an experimental animal model, this study aimed to investigate the metabolism of lenvatinib by rat microsomal enzymes and cytochrome P450 (CYPs) enzymes recombinantly expressed in SupersomesTM in vitro and to assess the effect of lenvatinib on rat CYP expression in vivo. Two metabolites, O-desmethyl lenvatinib, and lenvatinib N-oxide, were produced by rat CYPs in vitro. CYP2A1 and 2C12 were found to be the most effective in forming O-desmethyl lenvatinib, while CYP3A2 was found to primarily form lenvatinib N-oxide. The administration of lenvatinib to rats caused changes in the expression of mRNA and protein, as well as the activity of various CYPs, particularly in an increase in CYP1A1. Thus, the administration of lenvatinib to rats has an impact on the level of CYPs.

Deep mutational scanning of CYP2C19 in human cells reveals a substrate specificity-abundance tradeoff.

The Cytochrome P450s (CYPs) enzyme family metabolizes ∼80% of small molecule drugs. Variants in CYPs can substantially alter drug metabolism, leading to improper dosing and severe adverse drug reactions. Due to low sequence conservation, predicting variant effects across CYPs is challenging. Even closely related CYPs like CYP2C9 and CYP2C19, which share 92% amino acid sequence identity, display distinct phenotypic properties. Using Variant Abundance by Massively Parallel sequencing (VAMP-seq), we measured the steady-state protein abundance of 7,660 single amino acid variants in CYP2C19 expressed in cultured human cells. Our findings confirmed critical positions and structural features essential for CYP function and revealed how variants at conserved positions influence abundance. We jointly analyzed 4,670 variants whose abundance was measured in both CYP2C19 and CYP2C9, finding that the homologs have different variant abundances in substrate recognition sites within the hydrophobic core. We also measured the abundance of all single and some multiple WT amino acid exchanges between CYP2C19 and CYP2C9. While most exchanges had no effect, substitutions in substrate recognition site 4 (SRS4) reduced abundance in CYP2C19. Double and triple mutants showed distinct interactions, highlighting a region that points to differing thermodynamic properties between the two homologs. These positions are known contributors to substrate specificity, suggesting an evolutionary tradeoff between stability and enzymatic function. Finally, we analyzed 368 previously unannotated human variants, finding that 43% had decreased abundance. By comparing variant effects between these homologs, we uncovered regions underlying their functional differences, advancing our understanding of this versatile family of enzymes.

Genetic susceptibility and clinical features of CYP2D6 associated with systemic lupus erythematosus in a Chinese population.

OBJECTIVE: This study aims to explore possible susceptibility genes and clinical features for systemic lupus erythematosus (SLE) patients in a Chinese population. METHODS: Expanding on the results of a prior single-center observational study involving 60 systemic lupus erythematosus patients, a subsequent single-center prospective observational study was conducted on SLE patients undergoing treatment at Nanfang Hospital Affiliated to Southern Medical University from 2021 to 2023. The identification process for drug-related target genes entailed an extensive search across PharmGKB (https://www.pharmgkb.org/), the Clinical Pharmacogenetics Implementation Consortium (CPIC),and PubMed literature databases, to pinpoint common drugs and target single nucleotide polymorphisms(SNPs)for SLE. Blood samples were individually collected and genotyped using MassARRAY® high-throughput nucleic acid mass spectrometry. Genotype frequency differences were assessed through Chi-square tests against both the larger East Asian population as well as kidney transplant recipients. Data collection relied on electronic medical records, encompassing demographic details(age, gender),medication regimens(hormones, NSAIDs, hydroxychloroquine, DMARDs, biologic agents, stomach medications, calcitriol, etc.),laboratory indicators(RF, Anti-CCP antibody, ESR, CRP, anti-ANA antibodies, dsDNA antibodies, anti-SM antibodies, S m. RNP antibodies, A LT, ALB, CR, UA, WBC, PLT, HGB, Ca, K, Glu, CHOL, TG, LDL-C, HDL-C) and lupus activity scores(SLEDAI-2K). Possible disease susceptibility genes were categorized, and SPSS26 software facilitated statistical analyses. RESULTS: The research encompassed a total of 137 SLE patients along with 50 SNPs. After conducting statistical analyses, it emerged that there existed significant disparities in CYP2D6 gene (rs1065852) distribution when compared against allele mutation rates within both East Asian populations (p < .05) and kidney transplant patients(p < .05). Wild-type gene (GG) constituted 14% of cases while mutant gene (GA + AA) constituted 86%. Allele mutation rate (A63.6%) was significantly higher among SLE patients (RR = 0.802; p = .0355). Furthermore, the variant rs1065852 genotype (GA + AA) demonstrated significant associations with lower CRP levels, higher HGB levels, and higher HDL-C levels (p < 0 0.05). CONCLUSION: The metabolic enzyme CYP2D6 may be used as susceptibility gene for predicting systemic lupus erythematosus and are correlated with CRP and other indicators.

Effects of hypothermia and hypoxia on cytochrome P450-mediated drug metabolism in neonatal Göttingen minipigs.

Asphyxiated neonates often undergo therapeutic hypothermia (TH) to reduce morbidity and mortality. As perinatal asphyxia and TH impact neonatal physiology, this could also influence enzyme functionality. Therefore, this study aimed to unravel the impact of age, hypothermia and hypoxia on porcine hepatic cytochrome P450 (CYP) gene expression, protein abundance and activity. Hepatic CYP expression, protein abundance and activity were assessed in naive adult and neonatal Göttingen minipigs, alongside those from an (non-survival) in vivo study, where four conditions-control (C), therapeutic hypothermia (TH), hypoxia (H), hypoxia and TH (H + TH)-were examined. Naive neonatal Göttingen minipigs exhibited 75% lower general CYP activity and different gene expression patterns than adults. In vitro hypothermia (33°C) decreased general CYP activity in adult liver microsomes by 36%. Gene expression was not different between TH and C while hypoxia up-regulated several genes (i.e., CYP3A29 [expression ratio; ER = 5.1472] and CYP2C33 [ER = 3.2292] in the H group and CYP2C33 [ER = 2.4914] and CYP2C42 [ER = 4.0197] in the H + TH group). The medical treatment and the interventions over 24 h, along with hypoxia and TH, affected the protein abundance. These data on CYP expression, abundance and activity in young animals can be valuable in building physiologically-based pharmacokinetic models for neonatal drug dose predictions.

Establishing cell suitability for high-level production of licorice triterpenoids in yeast.

Yeast has been an indispensable host for synthesizing complex plant-derived natural compounds, yet the yields remained largely constrained. This limitation mainly arises from overlooking the importance of cell and pathway suitability during the optimization of enzymes and pathways. Herein, beyond conventional enzyme engineering, we dissected metabolic suitability with a framework for simultaneously augmenting cofactors and carbon flux to enhance the biosynthesis of heterogenous triterpenoids. We further developed phospholipid microenvironment engineering strategies, dramatically improving yeast's suitability for the high performance of endoplasmic reticulum (ER)-localized, rate-limiting plant P450s. Combining metabolic and microenvironment suitability by manipulating only three genes, NHMGR (NADH-dependent HMG-CoA reductase), SIP4 (a DNA-binding transcription factor)and GPP1 (Glycerol-1-phosphate phosphohydrolase 1), we enabled the high-level production of 4.92 g/L rare licorice triterpenoids derived from consecutive oxidation of ß-amyrin by two P450 enzymes after fermentation optimization. This production holds substantial commercial value, highlighting the critical role of establishing cell suitability in enhancing triterpenoid biosynthesis and offering a versatile framework applicable to various plant natural product biosynthetic pathways.

Brain Exposure to the Macrocyclic ALK Inhibitor Zotizalkib is Restricted by ABCB1, and Its Plasma Disposition is Affected by Mouse Carboxylesterase 1c.

Zotizalkib (TPX-0131), a fourth-generation macrocyclic anaplastic lymphoma kinase (ALK) inhibitor, is designed to overcome resistance due to secondary ALK mutations in non-small cell lung cancer (NSCLC). We here evaluated the pharmacokinetic roles of the ABCB1 (P-gp/MDR1) and ABCG2 (BCRP) efflux transporters, OATP1 influx transporters and the metabolizing enzymes CES1 and CYP3A in plasma and tissue disposition of zotizalkib after oral administration in relevant mouse models. Zotizalkib was efficiently transported by hABCB1 in vitro. In vivo, a significant ∼9-fold higher brain-to-plasma ratio was observed in Abcb1a/b-/- and Abcb1a/b;Abcg2-/- compared to wild-type mice. No change in brain disposition was observed in Abcg2-/- mice, suggesting that mAbcb1a/b markedly restricts the brain accumulation of zotizalkib. ABCB1-mediated efflux of zotizalkib was completely inhibited by elacridar, a dual ABCB1/ABCG2 inhibitor, increasing brain exposure without any signs of acute CNS-related toxicities. In Oatp1a/b-/- mice, no marked changes in plasma exposure or tissue-to-plasma ratios were observed, indicating that zotizalkib is not a substantial in vivo substrate for mOatp1a/b. Zotizalkib may further be metabolized by CYP3A4 but only noticeably at low plasma concentrations. In Ces1-/- mice, a 2.5-fold lower plasma exposure was seen compared to wild-type, without alterations in tissue distribution. This suggests increased plasma retention of zotizalkib by binding to the abundant mouse plasma Ces1c. Notably, the hepatic expression of human CES1 did not affect zotizalkib plasma exposure or tissue distribution. The obtained pharmacokinetic insights may be useful for the further development and optimization of therapeutic efficacy and safety of zotizalkib and related compact macrocyclic ALK inhibitors.

Expression reduction and a variant of a P450 gene mediate chlorpyrifos resistance in Tetranychus urticae Koch.

INTRODUCTION: Understanding how insects and mites develop resistance to chlorpyrifos is crucial for effective field management. Although extensive research has demonstrated that T. urticae exhibits high resistance to chlorpyrifos, the specific resistance mechanism remains elusive. Investigating this mechanism could provide valuable insights for pest control strategies. OBJECTIVES: This study aimed to reveal the mechanism of chlorpyrifos resistance in T. urticae. METHODS: The expression level of the CYP392D8 gene in T. urticae strains were analyzed using real- time quantitative PCR and western blot techniques. Functional validation of CYP392D8 was conducted through RNAi and heterogeneous expression. The production of chlorpyrifos-oxon in both resistant and susceptible strains were quantified using LC-MS/MS. Furthermore, the metabolic capabilities of CYP392D8 variants were verified using HPLC-MS and molecular docking. RESULTS: The results showed the expression of CYP392D8 was reduced in some Chinese resistant populations and mites with knocked down CYP392D8 showed decreased susceptibility to chlorpyrifos. Chlorpyrifos-oxon, the active metabolite of chlorpyrifos, was generated when chlorpyrifos was incubated with recombinant CYP392D8 protein in vitro. And a higher efficiency of chlorpyrifos-oxon formation was observed with the CYP392D8-S variant from susceptible mites compared to the CYP392D8-R variant from resistant mites. After treatment with low doses of chlorpyrifos, susceptible mite extracts produced substantial amounts of chlorpyrifos-oxon, while resistant mites only showed trace amounts. In addition, molecular docking studies showed that CYP392D8-S had a higher binding capacity to chlorpyrifos than the CYP392D8-R variant. CONCLUSION: This study reveals a mechanism of insecticide resistance due to the bioactivation reduction in combination with the sequence variation in a pest mite. These findings provide an important theoretical bias for management strategies of mites in the field and comprehensive control.

Development of a high throughput cytochrome P450-ligand binding assay.

Human cytochrome P450 enzymes are membrane-embedded monooxygenases responsible for xenobiotic metabolism, steroidogenesis, fatty acid metabolism, and vitamin metabolism. Their active sites can accommodate diverse small molecules and understanding these interactions is key to decoding enzymatic functionality and designing drugs. The most common method for characterizing small molecule binding is quantifying absorbance changes that typically occur when substrates enter the active site near the heme iron. Traditionally such titrations are monitored by a spectrophotometer, requiring significant manual time, protein, and increasing solvents. This assay was adapted for semi-automated high throughput screening, increasing throughput 50-fold while requiring less protein and keeping solvent concentrations constant. This 384-well assay was validated for both type I and II shifts typically observed for substrates and heme-coordinating inhibitors, respectively. This assay was used to screen a library of ∼100 diverse imidazole-containing compounds which can coordinate with the heme iron if compatible with the overall active site. Three human cytochrome P450 enzymes were screened: drug-metabolizing CYP2A6 and CYP2D6 and sterol-metabolizing CYP8B1. Each bound different sets of imidazole compounds with varying Kd values, providing a unique binding fingerprint. As a final validation, the Kd values were used to generate pharmacophores to compare to experimental structures. Applications for the high-throughput assay include 1) facilitating generation of pharmacophores for enzymes where structures are not available, 2) screening to identify ligands for P450 orphans, 3) screening for inhibitors of P450s drug targets, 4) screening potential new drugs to avoid and/or control P450 metabolism, and 5) efficient validation of computational predictions.

Biodegradation of non-steroidal anti-inflammatory drug loxoprofen by a hyper lignin-degrading fungus Phanerochaete sordida YK-624 under non-ligninolytic conditions.

Loxoprofen has been widely used as a non-steroidal anti-inflammatory drug globally and it can also persist in the environment. Although it is known to be a non-toxic drug, its presence may still pose a potential risk to organisms in the environment. Here, the hyper lignin-degrading fungus Phanerochaete sordida YK-624 was used to study the degradation of loxoprofen. This fungus showed excellent loxoprofen biodegradation ability with 90.4% and 93.4% after one day of incubation at lower concentrations of 0.01 and 0.005 mM, respectively. And at a higher concentration of 0.1 mM, a significant removal of 94.2% was also observed after 10 days of incubation. In this study, four metabolites were isolated and determined by HR-ESI-MS and NMR. Furthermore, LC/MS analysis suggested the presence of intermediate hydroxy loxoprofen. In addition, loxoprofen-OH was also identified as a metabolite of loxoprofen through comparison with the synthesized compounds. In this metabolism of loxoprofen, cytochrome P450 may play a significant role. Interestingly, P. sordida YK-624 showed enantioselectivity in the degradation process of loxoprofen. By these results, three degradation pathways of loxoprofen by P. sordida YK-624 were hypothesized. To the best of our knowledge, this is the first report describing the potential degradation mechanisms of loxoprofen by a white-rot fungus.

STAT5B, Akt and p38 Signaling Activate FTZ-F1 to Regulate the Xenobiotic Tolerance-Related Gene SlCyp9a75b in Spodoptera litura.

Cytochrome P450 monooxygenases in insects have been verified to implicated in insecticide and phytochemical detoxification metabolism. However, the regulation of P450s, which are modulated by signal-regulated transcription factors (TFs), is less well studied in insects. Here, we found that the Malpighian tubule specific P450 gene SlCYP9A75b in Spodoptera litura is induced by xenobiotics. The transgenic Drosophila bioassay and RNAi results indicated that this P450 gene contributes to α-cypermethrin, cyantraniliprole, and nicotine tolerance. In addition, functional analysis revealed that the MAPKs p38, PI3K/Akt, and JAK-STAT activate the transcription factor fushi tarazu factor 1 (FTZ-F1) to regulate CYP9A75b expression. These findings provide mechanistic insights into the contributions of CYP9A genes to xenobiotic detoxification and support the possible involvement of different signaling pathways and TFs in tolerance to xenobiotics in insects.

Evaluation of pharmacogenetic automated clinical decision support for clopidogrel.

Aim: Clopidogrel requires CYP2C19 activation to have antiplatelet effects. Pharmacogenetic testing to identify patients with impaired CYP2C19 function can be coupled with clinical decision support (CDS) alerts to guide antiplatelet prescribing. We evaluated the impact of alerts on clopidogrel prescribing.Materials & methods: We retrospectively analyzed data for 866 patients in which CYP2C19-clopidogrel CDS was deployed at a single healthcare system during 2015-2023.Results: Analyses included 2,288 alerts. CDS acceptance rates increased from 24% in 2015 to 63% in 2023 (p < 0.05). Adjusted analyses also showed higher acceptance rates when clopidogrel had been ordered for a percutaneous intervention (OR: 28.7, p < 0.001) and when cardiologists responded to alerts (OR: 2.11, p = 0.001).Conclusion: CDS for CYP2C19-clopidogrel was effective in reducing potential drug-gene interactions. Its influence varied by clinician specialty and medication indications.

[Box: see text].

New drug combination regimen based on pharmacokinetic characteristics-Erdafitinib combined with sertraline or duloxetine.

The aim of this study is to investigate novel strategies for reducing adverse reactions caused by erdafitinib through a drug combination based on its pharmacokinetic characteristics. The spectrum and characterizations of drugs that can inhibit the metabolism of erdafitinib are examined both in vitro and in vivo. The efficacy of combination regimens are then evaluated using subcutaneous xenograft tumor models. The results demonstrated that sertraline and duloxetine, out of more than 100 screened drugs, inhibited the metabolism of erdafitinib through mixed and non-competitive inhibition, respectively. This inhibition primarily occurred via the CYP2C9 and CYP2D6 pathways. The primary alleles of CYP2C9 and CYP2D6 not only determine the metabolic characteristics of erdafitinib but also influence the strength of drug-drug interactions. Co-administration of sertraline or duloxetine with erdafitinib in rats and mice resulted in nearly a three-fold increase in the blood exposure of erdafitinib and its major metabolite M6. When sertraline or duloxetine was combined with 1/3 of the erdafitinib dosage, the anti-proliferative and pro-apoptotic effects on SNU-16 xenografts were comparable to those of the original full dose of erdafitinib. However, the combination regimen significantly mitigated hyperphosphatemia, retinal damage, intestinal villus damage, and gut microbiome dysbiosis. This study utilized pharmacokinetic methods to propose a new formulation of erdafitinib combined with sertraline or duloxetine. The findings suggest that this combination has potential for clinical co-administration based on a database analysis, thereby providing a novel strategy for anti-tumor treatment with fibroblast growth factor receptor (FGFR) inhibitors.

RNAi targeting Nav and CPR via leaf delivery reduces adult emergence and increases the susceptibility to λ-cyholthin in Tuta absoluta (Meyrick).

The tomato leafminer, Tuta absoluta (Meyrick), one of the most economically destructive pests of tomato, causes severe yields losses of tomato production globally. Rapid evolution of insecticide resistance requires the development of alternative control strategy for this pest. RNA interference (RNAi) represents a promising, innovative control strategy against key agricultural insect pests, which has recently been licensed for Colorado Potato Beetle control. Here two essential genes, voltage-gated sodium channel (Nav) and NADPH-cytochrome P450 reductase (CPR) were evaluated as targets for RNAi using an ex vivo tomato leaf delivery system. Developmental stage-dependent expression profiles showed TaNav was most abundant in adult stages, whereas TaCPR was highly expressed in larval and adult stages. T. absoluta larvae feeding on tomato leaflets treated with dsRNA targeting TaNav and TaCPR showed significant knockdown of gene expression, leading to reduction in adult emergence. Additionally, tomato leaves treated with dsRNA targeting these two genes were significantly less damaged by larval feeding and mining. Furthermore, bioassay with LC30 doses of λ-cyholthin showed that silencing TaNav and TaCPR increased T. absoluta mortality about 32.2 and 17.4%, respectively, thus indicating that RNAi targeting TaNav and TaCPR could increase the susceptibility to λ-cyholthin in T. absoluta. This study demonstrates the potential of using RNAi targeting key genes, like TaNav and TaCPR, as an alternative technology for the control of this most destructive tomato pests in the future.

Multiresolution molecular dynamics simulations reveal the interplay between conformational variability and functional interactions in membrane-bound cytochrome P450 2B4.

Cytochrome P450 2B4 (CYP 2B4) is one of the best-characterized CYPs and serves as a key model system for understanding the mechanisms of microsomal class II CYPs, which metabolize most known drugs. The highly flexible nature of CYP 2B4 is apparent from crystal structures that show the active site with either a wide open or a closed heme binding cavity. Here, we investigated the conformational ensemble of the full-length CYP 2B4 in a phospholipid bilayer, using multiresolution molecular dynamics (MD) simulations. Coarse-grained MD simulations revealed two predominant orientations of CYP 2B4's globular domain with respect to the bilayer. Their refinement by atomistic resolution MD showed adaptation of the enzyme's interaction with the lipid bilayer, leading to open configurations that facilitate ligand access to the heme binding cavity. CAVER analysis of enzyme tunnels, AquaDuct analysis of water routes, and Random Acceleration Molecular Dynamics simulations of ligand dissociation support the conformation-dependent passage of molecules between the active site and the protein surroundings. Furthermore, simulation of the re-entry of the inhibitor bifonazole into the open conformation of CYP 2B4 resulted in binding at a transient hydrophobic pocket within the active site cavity that may play a role in substrate binding or allosteric regulation. Together, these results show how the open conformation of CYP 2B4 facilitates the binding of substrates from and release of products to the membrane, whereas the closed conformation prolongs the residence time of substrates or inhibitors and selectively allows the passage of smaller reactants via the solvent and water channels.

High Burden of Ileus and Pneumonia in Clozapine-Treated Individuals With Schizophrenia: A Finnish 25-Year Follow-Up Register Study.

OBJECTIVE: The authors used longitudinal biobank data with up to 25 years of follow-up on over 2,600 clozapine users to derive reliable estimates of the real-world burden of clozapine adverse drug events (ADEs). METHODS: A total of 2,659 participants in the FinnGen biobank project had a schizophrenia diagnosis and clozapine purchases with longitudinal electronic health record follow-up for up to 25 years after clozapine initiation. Diseases and health-related events enriched during clozapine use were identified, adjusting for disease severity. The incidence and recurrence of ADEs over years of clozapine use, their effect on clozapine discontinuation and deaths, and their pharmacogenetics were studied. RESULTS: Median follow-up time after clozapine initiation was 12.7 years. Across 2,157 diseases and health-related events, 27 were enriched during clozapine use, falling into five disease categories: gastrointestinal hypomotility, seizures, pneumonia, other acute respiratory tract infections, and tachycardia, along with a heterogeneous group including neutropenia and type 2 diabetes, among others. Cumulative incidence estimates for ileus (severe gastrointestinal hypomotility) and pneumonia were 5.3% and 29.5%, respectively, 20 years after clozapine initiation. Both events were significantly associated with increased mortality among clozapine users (ileus: odds ratio=4.5; pneumonia: odds ratio=2.8). Decreased genotype-predicted CYP2C19 and CYP1A2 activities were associated with higher pneumonia risk. CONCLUSIONS: Clozapine-induced ileus and pneumonia were notably more frequent than has previously been reported and were associated with increased mortality. Two CYP genes influenced pneumonia risk. Pneumonia and ileus call for improved utilization of available preventive measures.

CYP7B1 deficiency impairs myeloid cell activation in autoimmune disease of the central nervous system.

Dysregulation of cholesterol metabolism underlies neurodegenerative disease and is increasingly implicated in neuroinflammatory diseases, such as multiple sclerosis (MS). Cytochrome P450 family 7 subfamily B member 1 (CYP7B1) is a key enzyme in alternative cholesterol metabolism. A recessive mutation in the gene CYP7B1 is known to cause a neurodegenerative disease, hereditary spastic paraplegia type 5 and oxysterol accumulation. However, the role of CYP7B1 in neuroinflammation has been little revealed. In this study, we induced experimental autoimmune encephalomyelitis (EAE), as a murine model of MS, using CYP7B1 homozygous knockout (KO) mice. We found that CYP7B1 deficiency can significantly attenuate EAE severity. CYP7B1 deficiency is sufficient to reduce leukocyte infiltration into the central nervous system, suppress proliferation of pathogenic CD4+ T cells, and decrease myeloid cell activation during EAE. Additionally, live-animal imaging targeting translocator protein expression, an outer mitochondrial membrane protein biomarker of neuroinflammation, showed that CYP7B1 deficiency results in suppressed neuroinflammation. Using human monocyte-derived microglia-like cellular disease model and primary microglia of CYP7B1 KO mice, we also found that activation of microglia of CYP7B1 deficiency was impaired. These cumulative results suggest that CYP7B1 can regulate neuroinflammation, thus providing potential new targets for therapeutic intervention.